Journal articles
Wakeling M, Owens NDL, Hopkinson JR, Johnson MB, Houghton JAL, Dastamani A, Flaxman CS, Wyatt RC, Hewat TI, Hopkins JJ, et al (In Press). A novel disease mechanism leading to the expression of a disallowed gene in the pancreatic beta-cell identified by non-coding, regulatory mutations controlling HK1.
Nature GeneticsAbstract:
A novel disease mechanism leading to the expression of a disallowed gene in the pancreatic beta-cell identified by non-coding, regulatory mutations controlling HK1
Gene expression is tightly regulated with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function. This silencing is largely controlled by non-coding elements and their disruption might cause human disease. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo mutations affecting a 42bp conserved region encompassed by a regulatory. element in intron 2 of Hexokinase 1 (HK1), a pancreatic beta-cell ‘disallowed’ gene. We demonstrated that these mutations resulted in expression of HK1 in the pancreatic beta-cells causing inappropriate insulin secretion and congenital hyperinsulinism. These mutations identify a regulatory region critical for cell-specific silencing. Importantly, this has revealed a new disease mechanism for non-coding mutations that cause inappropriate expression of a disallowed gene.
Abstract.
Oikarinen M, Laiho JE, Oikarinen S, Richardson SJ, Kusmartseva I, Campbell-Thompson M, Morgan NG, Pugliese A, Tauriainen S, Toniolo A, et al (In Press). Detection of enterovirus protein and RNA in multiple tissues from nPOD organ donors with type 1 diabetes.
Abstract:
Detection of enterovirus protein and RNA in multiple tissues from nPOD organ donors with type 1 diabetes
AbstractEpidemiological studies have shown an association between enterovirus (EV) infections and type 1 diabetes (T1D), and EV protein has been detected in the pancreatic islets of T1D patients. Here we correlated the detection of EVs in lymphoid tissues (spleen and pancreatic lymph nodes) and small intestinal mucosa to the virus detection in the pancreas of T1D, autoantibody-positive (aab+) and non-diabetic control organ donors of the Network for Pancreatic Organ Donors with Diabetes (nPOD) study. Formalin-fixed paraffin-embedded tissue samples were screened for insulin and EV protein using immunohistochemistry, and frozen tissue for EV genome using RT-PCR. The presence of EV protein in the pancreatic islets correlated with the presence of insulin-positive cells. Altogether 62 % of T1D and aab+ donors were positive for EV protein in pancreatic islets (only insulin-positive donors included), 40 % in duodenum and 32 % in spleen, compared to 33 %, 14 %, and 27 % of non-diabetic controls. Pancreatic lymph nodes were positive for EV protein in 60 % of T1D and aab+ cases. T1D and aab+ donors were more frequently VP1-positive in multiple organs than control donors (39 % vs. 11 %; including only insulin-positive donors). EV RNA was found in selected donors and from multiple tissue types except for duodenum, and individual T1D and aab+ donors were EV RNA-positive in multiple organs. The role of extra-pancreatic organs and their interplay with EV in T1D pathogenesis remains to be solved, but we hypothesize that these organs may serve as a reservoir for the virus which may reside in these tissues in a slow-replicating persistent form.
Abstract.
Thomas P, Leslie KA, Welters HJ, Morgan NG (In Press). Long-chain Saturated Fatty Acid Species Are Not Toxic to Human Pancreatic Β-cells and May Offer Protection Against Pro-inflammatory Cytokine Induced β-cell Death.
Abstract:
Long-chain Saturated Fatty Acid Species Are Not Toxic to Human Pancreatic Β-cells and May Offer Protection Against Pro-inflammatory Cytokine Induced β-cell Death
Abstract
. Obesity is a major risk factor for type 2 diabetes (T2D) although the causal links remain unclear. A feature shared by both conditions however is systemic inflammation and raised levels of circulating fatty acids (FFA). It is widely believed that in obese individuals genetically prone to T2D, elevated levels of plasma FFA may contribute towards the death and dysfunction of insulin-producing pancreatic β-cells in a process of (gluco)lipotoxicity. In support of this, in vitro studies have shown consistently that long-chain saturated fatty acids (LC-SFA) are toxic to rodent β-cells during chronic exposure (>24h). Conversely, shorter chain SFA and unsaturated species are well tolerated, suggesting that toxicity is dependent on carbon chain length and/or double bond configuration. Despite the wealth of evidence implicating lipotoxicity as a means of β-cell death in rodents, the evidence that a similar process occurs in humans is much less substantial. Therefore, the present study has evaluated the effects of chronic exposure to fatty acids of varying chain length and degree of saturation, on the viability of human β-cells in culture. We have also studied the effects of a combination of fatty acids and pro-inflammatory cytokines. Strikingly, we find that LC-FFA do not readily promote the demise of human β-cells and that they may even offer a measure of protection against the toxic effects of pro-inflammatory cytokines. Therefore, these findings imply that a model in which elevated circulating LC-FFA play a direct role in mediating β-cell dysfunction and death in humans, may be overly simplistic.
Abstract.
Viloria K, Nasteska D, Briant LJB, Heising S, Larner D, Fine NHF, Ashford FB, Silva Xavier GD, Ramos MJ, Manning Fox JE, et al (In Press). Vitamin D-binding protein is required for the maintenance of α-cell function and glucagon secretion.
Abstract:
Vitamin D-binding protein is required for the maintenance of α-cell function and glucagon secretion
ABSTRACTVitamin D-binding protein (DBP) or GC-globulin carries vitamin D metabolites from the circulation to target tissues. DBP expression is highly-localized to the liver and pancreatic α-cells. While DBP serum levels, gene polymorphisms and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α-cell morphology, α-cell function and glucagon secretion. Deletion of DBP led to smaller and hyperplastic α-cells, altered Na+channel conductance, impaired α-cell activation by low glucose, and reduced rates of glucagon secretion. Mechanistically, this involved reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects were also seen in β-cell and δ-cell function. Immunostaining of human pancreata revealed generalized loss of DBP expression as a feature of late-onset and longstanding, but not early-onset type 1 diabetes. Thus, DBP is a critical regulator of α-cell phenotype, with implications for diabetes pathogenesis.HIGHLIGHTSDBP expression is highly-localized to mouse and human α-cellsLoss of DBP increases α-cell number, but decreases α-cell sizeα-cells in DBP knockout islets are dysfunctional and secrete less glucagonDBP expression is decreased in α-cells of donors with late-onset or longstanding type 1 diabetes
Abstract.
Torabi F, Vadakekolathu J, Wyatt R, Leete P, Tombs MA, Richardson CC, Boocock DJ, Turner MD, Morgan NG, Richardson SJ, et al (2023). Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.
Diabet Med,
40(9).
Abstract:
Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.
AIMS: Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. METHODS: RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. RESULTS: Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. CONCLUSIONS: the results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.
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Redondo MJ, Morgan NG (2023). Heterogeneity and endotypes in type 1 diabetes mellitus.
Nat Rev Endocrinol,
19(9), 542-554.
Abstract:
Heterogeneity and endotypes in type 1 diabetes mellitus.
Despite major advances over the past decade, prevention and treatment of type 1 diabetes mellitus (T1DM) remain suboptimal, with large and unexplained variations in individual responses to interventions. The current classification schema for diabetes mellitus does not capture the complexity of this disease or guide clinical management effectively. One of the approaches to achieve the goal of applying precision medicine in diabetes mellitus is to identify endotypes (that is, well-defined subtypes) of the disease each of which has a distinct aetiopathogenesis that might be amenable to specific interventions. Here, we describe epidemiological, clinical, genetic, immunological, histological and metabolic differences within T1DM that, together, suggest heterogeneity in its aetiology and pathogenesis. We then present the emerging endotypes and their impact on T1DM prediction, prevention and treatment.
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Author URL.
Carr ALJ, Inshaw JRJ, Flaxman CS, Leete P, Wyatt RC, Russell LA, Palmer M, Prasolov D, Worthington T, Hull B, et al (2022). Circulating C-Peptide Levels in Living Children and Young People and Pancreatic β-Cell Loss in Pancreas Donors Across Type 1 Diabetes Disease Duration.
Diabetes,
71(7), 1591-1596.
Abstract:
Circulating C-Peptide Levels in Living Children and Young People and Pancreatic β-Cell Loss in Pancreas Donors Across Type 1 Diabetes Disease Duration
C-peptide declines in type 1 diabetes, although many long-duration patients retain low, but detectable levels. Histological analyses confirm that β-cells can remain following type 1 diabetes onset. We explored the trends observed in C-peptide decline in the UK Genetic Resource Investigating Diabetes (UK GRID) cohort (N = 4,079), with β-cell loss in pancreas donors from the network for Pancreatic Organ donors with Diabetes (nPOD) biobank and the Exeter Archival Diabetes Biobank (EADB) (combined N = 235), stratified by recently reported age at diagnosis endotypes (<7, 7–12, ≥13 years) across increasing diabetes durations. The proportion of individuals with detectable C-peptide declined beyond the first year after diagnosis, but this was most marked in the youngest age group (<1-year duration: age <7 years: 18 of 20 [90%], 7–12 years: 107 of 110 [97%], ≥13 years: 58 of 61 [95%] vs. 1–5 years postdiagnosis: <7 years: 172 of 522 [33%], 7–12 years: 604 of 995 [61%], ≥13 years: 225 of 289 [78%]). A similar profile was observed in β-cell loss, with those diagnosed at younger ages experiencing more rapid loss of islets containing insulin-positive (insulin+) β-cells <1 year postdiagnosis: age <7 years: 23 of 26 (88%), 7–12 years: 32 of 33 (97%), ≥13 years: 22 of 25 (88%) vs. 1–5 years postdiagnosis: <7 years: 1 of 12 (8.3%), 7–12 years: 7 of 13 (54%), ≥13 years: 7 of 8 (88%). These data should be considered in the planning and interpretation of intervention trials designed to promote β-cell retention and function.
Abstract.
Sioofy-Khojine A-B, Richardson SJ, Locke JM, Oikarinen S, Nurminen N, Laine A-P, Downes K, Lempainen J, Todd JA, Veijola R, et al (2022). Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage.
Diabetologia,
65(10), 1701-1709.
Abstract:
Detection of enterovirus RNA in peripheral blood mononuclear cells correlates with the presence of the predisposing allele of the type 1 diabetes risk gene IFIH1 and with disease stage
Abstract
. Aims/hypothesis
. Enteroviral infection has been implicated consistently as a key environmental factor correlating with the appearance of autoimmunity and/or the presence of overt type 1 diabetes, in which pancreatic insulin-producing beta cells are destroyed by an autoimmune response. Genetic predisposition through variation in the type 1 diabetes risk gene IFIH1 (interferon induced with helicase C domain 1), which encodes the viral pattern-recognition receptor melanoma differentiation-associated protein 5 (MDA5), supports a potential link between enterovirus infection and type 1 diabetes.
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. Methods
. We used molecular techniques to detect enterovirus RNA in peripheral blood samples (in separated cellular compartments or plasma) from two cohorts comprising 79 children or 72 adults that include individuals with and without type 1 diabetes who had multiple autoantibodies. We also used immunohistochemistry to detect the enteroviral protein VP1 in the pancreatic islets of post-mortem donors (n=43) with type 1 diabetes.
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. Results
. We observed enhanced detection sensitivity when sampling the cellular compartment compared with the non-cellular compartment of peripheral blood (OR 21.69; 95% CI 3.64, 229.20; p<0.0001). In addition, we show that children with autoimmunity are more likely to test positive for enterovirus RNA than those without autoimmunity (OR 11.60; 95% CI 1.89, 126.90; p=0.0065). Furthermore, we found that individuals carrying the predisposing allele (946Thr) of the common variant in IFIH1 (rs1990760, Thr946Ala) are more likely to test positive for enterovirus in peripheral blood (OR 3.07; 95% CI 1.02, 8.58; p=0.045). In contrast, using immunohistochemistry, there was no correlation between the common variant in IFIH1 and detection of enteroviral VP1 protein in the pancreatic islets of donors with type 1 diabetes.
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. Conclusions/interpretation
. Our data indicate that, in peripheral blood, antigen-presenting cells are the predominant source of enterovirus infection, and that infection is correlated with disease stage and genetic predisposition, thereby supporting a role for enterovirus infection prior to disease onset.
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. Graphical abstract
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Thomas P, Arden C, Corcoran J, Hacker C, Welters HJ, Morgan NG (2022). Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.
Nutr Diabetes,
12(1).
Abstract:
Differential routing and disposition of the long-chain saturated fatty acid palmitate in rodent vs human beta-cells.
BACKGROUND: Rodent and human β-cells are differentially susceptible to the "lipotoxic" effects of long-chain saturated fatty acids (LC-SFA) but the factors accounting for this are unclear. Here, we have studied the intracellular disposition of the LC-SFA palmitate in human vs rodent β-cells and present data that reveal new insights into the factors regulating β-cell lipotoxicity. METHODS: the subcellular distribution of the LC-SFA palmitate was studied in rodent (INS-1E and INS-1 823/13 cells) and human (EndoC-βH1) β-cells using confocal fluorescence and electron microscopy (EM). Protein expression was assessed by Western blotting and cell viability, by vital dye staining. RESULTS: Exposure of INS-1 cells to palmitate for 24 h led to loss of viability, whereas EndoC-βH1 cells remained viable even after 72 h of treatment with a high concentration (1 mM) of palmitate. Use of the fluorescent palmitate analogue BODIPY FL C16 revealed an early localisation of the LC-SFA to the Golgi apparatus in INS-1 cells and this correlated with distention of intracellular membranes, visualised under the EM. Despite this, the PERK-dependent ER stress pathway was not activated under these conditions. By contrast, BODIPY FL C16 did not accumulate in the Golgi apparatus in EndoC-βH1 cells but, rather, co-localised with the lipid droplet-associated protein, PLIN2, suggesting preferential routing into lipid droplets. When INS-1 cells were treated with a combination of palmitate plus oleate, the toxic effects of palmitate were attenuated and BODIPY FL C16 localised primarily with PLIN2 but not with a Golgi marker. CONCLUSION: in rodent β-cells, palmitate accumulates in the Golgi apparatus at early time points whereas, in EndoC- βH1 cells, it is routed preferentially into lipid droplets. This may account for the differential sensitivity of rodent vs human β-cells to "lipotoxicity" since manoeuvres leading to the incorporation of palmitate into lipid droplets is associated with the maintenance of cell viability in both cell types.
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Morgan NG, Richardson SJ, Powers AC, Saunders DC, Brissova M (2022). Images from the Exeter Archival Diabetes Biobank Now Accessible via Pancreatlas. Diabetes Care, 45(12), e174-e175.
Dhayal S, Leslie KA, Baity M, Akhbari P, Richardson SJ, Russell MA, Morgan NG (2022). Temporal regulation of interferon signalling in human EndoC-βH1 cells.
J Mol Endocrinol,
69(2), 299-313.
Abstract:
Temporal regulation of interferon signalling in human EndoC-βH1 cells.
During the development of type 1 diabetes, interferons (IFN) are elaborated from islet-infiltrating immune cells and/or from virally infected β-cells. They act via specific receptors to increase, acutely, the phosphorylation of the transcription factors STAT1 and 2. However, the longer-term impacts of chronic IFN stimulation are poorly understood and were investigated in the current study. Human EndoC-βH1 cells were treated with IFNα, IFNγ or IFNλ either acutely (
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Rodriguez-Calvo T, Chen Y-C, Verchere CB, Haataja L, Arvan P, Leete P, Richardson SJ, Morgan NG, Qian W-J, Pugliese A, et al (2021). Altered β-Cell Prohormone Processing and Secretion in Type 1 Diabetes.
Diabetes,
70(5), 1038-1050.
Abstract:
Altered β-Cell Prohormone Processing and Secretion in Type 1 Diabetes.
Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.
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Leete P, Morgan NG (2021). Footprints of Immune Cells in the Pancreas in Type 1 Diabetes; to "B" or Not to "B": is That Still the Question?.
Front Endocrinol (Lausanne),
12Abstract:
Footprints of Immune Cells in the Pancreas in Type 1 Diabetes; to "B" or Not to "B": is That Still the Question?
Significant progress has been made in understanding the phenotypes of circulating immune cell sub-populations in human type 1 diabetes but much less is known about the equivalent populations that infiltrate the islets to cause beta-cell loss. In particular, considerable uncertainties remain about the phenotype and role of B-lymphocytes in the pancreas. This gap in understanding reflects both the difficulty in accessing the gland to study islet inflammation during disease progression and the fact that the number and proportion of islet-associated B-lymphocytes varies significantly according to the disease endotype. In very young children (especially those
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Weider T, Richardson SJ, Morgan NG, Paulsen TH, Dahl-Jørgensen K, Hammerstad SS (2021). HLA Class I Upregulation and Antiviral Immune Responses in Graves Disease.
J Clin Endocrinol Metab,
106(4), e1763-e1774.
Abstract:
HLA Class I Upregulation and Antiviral Immune Responses in Graves Disease.
CONTEXT: the origin of Graves disease (GD) remains elusive. However, evidence of an association between GD and viral infections is emerging. Human leukocyte antigen (HLA) class I presents viral antigens to circulating immune cells and plays a crucial role in the defense against viral infections. OBJECTIVE: This work aimed to investigate HLA class I expression, enterovirus presence, and the viral immune response proteins signal transducer and activation of transcription 1 (STAT1) and protein kinase R (PKR) in thyroid tissue from GD patients. METHODS: We collected thyroid tissue from core needle biopsies or surgical specimens from 48 GD patients and 24 controls. Standard immunohistochemistry was used to detect HLA class I and enteroviral capsid protein 1 (VP1) on formalin-fixed and paraffin-embedded tissue. STAT1 and PKR were examined by combined immunofluorescence staining. HLA class I expression score was the main outcome measure. RESULTS: the HLA class I expression score, which takes both proportion and intensity of immunostaining into account, was significantly higher in GD patients (3.1â€
±â€
3.3) than in controls (0.5â€
±â€
0.9) (Pâ€
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Korpos É, Kadri N, Loismann S, Findeisen CR, Arfuso F, Burke GW, Richardson SJ, Morgan NG, Bogdani M, Pugliese A, et al (2021). Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.
Diabetologia,
64(7), 1626-1641.
Abstract:
Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.
AIMS/HYPOTHESIS: We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. METHODS: Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. RESULTS: TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. CONCLUSIONS/INTERPRETATION: We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.
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Chaffey JR, Young J, Leslie KA, Partridge K, Akhbari P, Dhayal S, Hill JL, Wedgwood KCA, Burnett E, Russell MA, et al (2021). Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection.
Sci Rep,
11(1).
Abstract:
Investigation of the utility of the 1.1B4 cell as a model human beta cell line for study of persistent enteroviral infection.
The generation of a human pancreatic beta cell line which reproduces the responses seen in primary beta cells, but is amenable to propagation in culture, has long been an important goal in diabetes research. This is particularly true for studies focussing on the role of enteroviral infection as a potential cause of beta-cell autoimmunity in type 1 diabetes. In the present work we made use of a clonal beta cell line (1.1B4) available from the European Collection of Authenticated Cell Cultures, which had been generated by the fusion of primary human beta-cells with a pancreatic ductal carcinoma cell, PANC-1. Our goal was to study the factors allowing the development and persistence of a chronic enteroviral infection in human beta-cells. Since PANC-1 cells have been reported to support persistent enteroviral infection, the hybrid 1.1B4 cells appeared to offer an ideal vehicle for our studies. In support of this, infection of the cells with a Coxsackie virus isolated originally from the pancreas of a child with type 1 diabetes, CVB4.E2, at a low multiplicity of infection, resulted in the development of a state of persistent infection. Investigation of the molecular mechanisms suggested that this response was facilitated by a number of unexpected outcomes including an apparent failure of the cells to up-regulate certain anti-viral response gene products in response to interferons. However, more detailed exploration revealed that this lack of response was restricted to molecular targets that were either activated by, or detected with, human-selective reagents. By contrast, and to our surprise, the cells were much more responsive to rodent-selective reagents. Using multiple approaches, we then established that populations of 1.1B4 cells are not homogeneous but that they contain a mixture of rodent and human cells. This was true both of our own cell stocks and those held by the European Collection of Authenticated Cell Cultures. In view of this unexpected finding, we developed a strategy to harvest, isolate and expand single cell clones from the heterogeneous population, which allowed us to establish colonies of 1.1B4 cells that were uniquely human (h1.1.B4). However, extensive analysis of the gene expression profiles, immunoreactive insulin content, regulated secretory pathways and the electrophysiological properties of these cells demonstrated that they did not retain the principal characteristics expected of human beta cells. Our data suggest that stocks of 1.1B4 cells should be evaluated carefully prior to their use as a model human beta-cell since they may not retain the phenotype expected of human beta-cells.
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Author URL.
Thomas P, Leslie KA, Welters HJ, Morgan NG (2021). Long-chain saturated fatty acid species are not toxic to human pancreatic beta-cells and may offer protection against pro-inflammatory cytokine induced beta-cell death.
NUTRITION & METABOLISM,
18(1).
Author URL.
Colli ML, Ramos-RodrÃguez M, Nakayasu ES, Alvelos MI, Lopes M, Hill JLE, Turatsinze J-V, Coomans de Brachène A, Russell MA, Raurell-Vila H, et al (2020). An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells.
Nat Commun,
11(1).
Abstract:
An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells.
Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.
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White MG, Maheshwari RR, Anderson SJ, Berlinguer-Palmini R, Jones C, Richardson SJ, Rotti PG, Armour SL, Ding Y, Krasnogor N, et al (2020). In Situ Analysis Reveals That CFTR is Expressed in Only a Small Minority of β-Cells in Normal Adult Human Pancreas.
J Clin Endocrinol Metab,
105(5), 1366-1374.
Abstract:
In Situ Analysis Reveals That CFTR is Expressed in Only a Small Minority of β-Cells in Normal Adult Human Pancreas.
CONTEXT: Although diabetes affects 40% to 50% of adults with cystic fibrosis, remarkably little is known regarding the underlying mechanisms leading to impaired pancreatic β-cell insulin secretion. Efforts toward improving the functional β-cell deficit in cystic fibrosis-related diabetes (CFRD) have been hampered by an incomplete understanding of whether β-cell function is intrinsically regulated by cystic fibrosis transmembrane conductance regulator (CFTR). Definitively excluding meaningful CFTR expression in human β-cells in situ would contribute significantly to the understanding of CFRD pathogenesis. OBJECTIVE: to determine CFTR messenger ribonucleic acid (mRNA) and protein expression within β-cells in situ in the unmanipulated human pancreas of donors without any known pancreatic pathology. DESIGN: in situ hybridization for CFTR mRNA expression in parallel with insulin immunohistochemical staining and immunofluorescence co-localization of CFTR with insulin and the ductal marker, Keratin-7 (KRT7), were undertaken in pancreatic tissue blocks from 10 normal adult, nonobese deceased organ donors over a wide age range (23-71 years) with quantitative image analysis. RESULTS: CFTR mRNA was detectable in a mean 0.45% (range 0.17%-0.83%) of insulin-positive cells. CFTR protein expression was co-localized with KRT7. One hundred percent of insulin-positive cells were immunonegative for CFTR. CONCLUSIONS: for the first time, in situ CFTR mRNA expression in the unmanipulated pancreas has been shown to be present in only a very small minority (
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Battaglia M, Ahmed S, Anderson MS, Atkinson MA, Becker D, Bingley PJ, Bosi E, Brusko TM, DiMeglio LA, Evans-Molina C, et al (2020). Introducing the Endotype Concept to Address the Challenge of Disease Heterogeneity in Type 1 Diabetes.
Diabetes Care,
43(1), 5-12.
Abstract:
Introducing the Endotype Concept to Address the Challenge of Disease Heterogeneity in Type 1 Diabetes.
The clinical diagnosis of new-onset type 1 diabetes has, for many years, been considered relatively straightforward. Recently, however, there is increasing awareness that within this single clinical phenotype exists considerable heterogeneity: disease onset spans the complete age range; genetic susceptibility is complex; rates of progression differ markedly, as does insulin secretory capacity; and complication rates, glycemic control, and therapeutic intervention efficacy vary widely. Mechanistic and immunopathological studies typically show considerable patchiness across subjects, undermining conclusions regarding disease pathways. Without better understanding, type 1 diabetes heterogeneity represents a major barrier both to deciphering pathogenesis and to the translational effort of designing, conducting, and interpreting clinical trials of disease-modifying agents. This realization comes during a period of unprecedented change in clinical medicine, with increasing emphasis on greater individualization and precision. For complex disorders such as type 1 diabetes, the option of maintaining the "single disease" approach appears untenable, as does the notion of individualizing each single patient's care, obliging us to conceptualize type 1 diabetes less in terms of phenotypes (observable characteristics) and more in terms of disease endotypes (underlying biological mechanisms). Here, we provide our view on an approach to dissect heterogeneity in type 1 diabetes. Using lessons from other diseases and the data gathered to date, we aim to delineate a roadmap through which the field can incorporate the endotype concept into laboratory and clinical practice. We predict that such an effort will accelerate the implementation of precision medicine and has the potential for impact on our approach to translational research, trial design, and clinical management.
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Armour SL, Anderson SJ, Richardson SJ, Ding Y, Carey C, Lyon J, Maheshwari RR, Al-Jahdami N, Krasnogor N, Morgan NG, et al (2020). Reduced Expression of the Co-regulator TLE1 in Type 2 Diabetes is Associated with Increased Islet α-Cell Number.
Endocrinology,
161(4).
Abstract:
Reduced Expression of the Co-regulator TLE1 in Type 2 Diabetes is Associated with Increased Islet α-Cell Number.
β-Cell dysfunction in type 2 diabetes (T2D) is associated with loss of cellular identity and mis-expression of alternative islet hormones, including glucagon. The molecular basis for these cellular changes has been attributed to dysregulation of core β-cell transcription factors, which regulate β-cell identity through activating and repressive mechanisms. The TLE1 gene lies near a T2D susceptibility locus and, recently, the glucagon repressive actions of this transcriptional coregulator have been demonstrated in vitro. We investigated whether TLE1 expression is disrupted in human T2D, and whether this is associated with increased islet glucagon-expressing cells. Automated image analysis following immunofluorescence in donors with (n = 7) and without (n = 7) T2D revealed that T2D was associated with higher islet α/β cell ratio (Control: 0.7 ± 0.1 vs T2D: 1.6 ± 0.4; P <. 05) and an increased frequency of bihormonal (insulin+/glucagon+) cells (Control: 0.8 ± 0.2% vs T2D: 2.0 ± 0.4%, P <. 05). In nondiabetic donors, the majority of TLE1-positive cells were mono-hormonal β-cells (insulin+/glucagon-: 98.2 ± 0.5%; insulin+/glucagon+: 0.7 ± 0.2%; insulin-/glucagon+: 1.1 ± 0.4%; P <. 001). TLE1 expression was reduced in T2D (Control: 36 ± 2.9% vs T2D: 24 ± 2.6%; P <. 05). Reduced islet TLE1 expression was inversely correlated with α/β cell ratio (r = -0.55; P <. 05). TLE1 knockdown in EndoC-βH1 cells was associated with a 2.5-fold increase in glucagon gene mRNA and mis-expression of glucagon in insulin-positive cells. These data support TLE1 as a putative regulator of human β-cell identity, with dysregulated expression in T2D associated with increased glucagon expression potentially reflecting β- to α-cell conversion.
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Leete P, Oram RA, McDonald TJ, Shields BM, Ziller C, TIGI study team, Hattersley AT, Richardson SJ, Morgan NG (2020). Studies of insulin and proinsulin in pancreas and serum support the existence of aetiopathological endotypes of type 1 diabetes associated with age at diagnosis.
Diabetologia,
63(6), 1258-1267.
Abstract:
Studies of insulin and proinsulin in pancreas and serum support the existence of aetiopathological endotypes of type 1 diabetes associated with age at diagnosis.
AIMS/HYPOTHESIS: it is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue. METHODS: Immunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (
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Khilji MS, Bresson SE, Verstappen D, Pihl C, Andersen PAK, Agergaard JB, Dahlby T, Bryde TH, Klindt K, Nielsen CK, et al (2020). The inducible β5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient β-cells and is overexpressed in type 2 diabetes pancreatic islets.
Am J Physiol Endocrinol Metab,
318(6), E892-E900.
Abstract:
The inducible β5i proteasome subunit contributes to proinsulin degradation in GRP94-deficient β-cells and is overexpressed in type 2 diabetes pancreatic islets.
Proinsulin is a misfolding-prone protein, and its efficient breakdown is critical when β-cells are confronted with high-insulin biosynthetic demands, to prevent endoplasmic reticulum stress, a key trigger of secretory dysfunction and, if uncompensated, apoptosis. Proinsulin degradation is thought to be performed by the constitutively expressed standard proteasome, while the roles of other proteasomes are unknown. We recently demonstrated that deficiency of the proinsulin chaperone glucose-regulated protein 94 (GRP94) causes impaired proinsulin handling and defective insulin secretion associated with a compensated endoplasmic reticulum stress response. Taking advantage of this model of restricted folding capacity, we investigated the role of different proteasomes in proinsulin degradation, reasoning that insulin secretory dynamics require an inducible protein degradation system. We show that the expression of only one enzymatically active proteasome subunit, namely, the inducible β5i-subunit, was increased in GRP94 CRISPR/Cas9 knockout (KO) cells. Additionally, the level of β5i-containing intermediate proteasomes was significantly increased in these cells, as was β5i-related chymotrypsin-like activity. Moreover, proinsulin levels were restored in GRP94 KO upon β5i small interfering RNA-mediated knockdown. Finally, the fraction of β-cells expressing the β5i-subunit is increased in human islets from type 2 diabetes patients. We conclude that β5i is an inducible proteasome subunit dedicated to the degradation of mishandled proinsulin.
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Akhbari P, Richardson SJ, Morgan NG (2020). Type 1 Diabetes: Interferons and the Aftermath of Pancreatic Beta-Cell Enteroviral Infection.
Microorganisms,
8(9), 1419-1419.
Abstract:
Type 1 Diabetes: Interferons and the Aftermath of Pancreatic Beta-Cell Enteroviral Infection
Enteroviruses (EVs) have long been implicated in the pathogenesis of type 1 diabetes (T1D), and accumulating evidence has associated virus-induced autoimmunity with the loss of pancreatic beta cells in T1D. Inflammatory cytokines including interferons (IFN) form a primary line of defence against viral infections, and their chronic elevation is a hallmark feature of many autoimmune diseases. IFNs play a key role in activating and regulating innate and adaptive immune responses, and to do so they modulate the expression of networks of genes and transcription factors known generically as IFN stimulated genes (ISGs). ISGs in turn modulate critical cellular processes ranging from cellular metabolism and growth regulation to endoplasmic reticulum (ER) stress and apoptosis. More recent studies have revealed that IFNs also modulate gene expression at an epigenetic as well as post-transcriptional and post-translational levels. As such, IFNs form a key link connecting the various genetic, environmental and immunological factors involved in the initiation and progression of T1D. Therefore, gaining an improved understanding of the mechanisms by which IFNs modulate beta cell function and survival is crucial in explaining the pathogenesis of virally-induced T1D. This should provide the means to prevent, decelerate or even reverse beta cell impairment.
Abstract.
Weider T, Richardson SJ, Morgan NG, Paulsen TH, Dahl-Jørgensen K, Hammerstad SS (2020). Upregulation of HLA Class I and Antiviral Tissue Responses in Hashimoto's Thyroiditis.
Thyroid,
30(3), 432-442.
Abstract:
Upregulation of HLA Class I and Antiviral Tissue Responses in Hashimoto's Thyroiditis.
Background: Hashimoto's thyroiditis (HT) is a common autoimmune disease of unknown origin. However, viral infections have been implicated as triggers for autoimmunity. Human leukocyte antigen (HLA) class I presents antigens to circulating immune cells and plays a crucial role in the defense against viral infections. This study aimed to investigate the presence of enterovirus and HLA class I expression in one of the largest HT thyroid tissue cohorts to date. In addition, viral receptors and viral immune response proteins were examined. Methods: Thyroid tissue samples from 46 HT patients were obtained using core needle biopsy. Thyroid tissue collected during neck surgery for other reasons than thyroid autoimmunity served as controls. Standard immunohistochemistry on formalin-fixed, paraffin-embedded tissue samples were used to detect HLA class I, enteroviral capsid protein 1 (VP1), and coxsackie and adenovirus receptor (CAR) in thyroid cells. A subset of the samples was examined with combined immunofluorescence staining for signal transducer and activator of transcription 1 (STAT1) and protein kinase R (PKR). Results: Significantly more HLA class I-positive samples were found in the HT group (31 out of 46 [67.4%]) than in the control group (5 out of 24 [20.8%]) (p
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Viloria K, Nasteska D, Briant LJB, Heising S, Larner DP, Fine NHF, Ashford FB, da Silva Xavier G, Ramos MJ, Hasib A, et al (2020). Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.
Cell Rep,
31(11).
Abstract:
Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.
Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.
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Jeffery N, Richardson S, Chambers D, Morgan NG, Harries LW (2019). Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.
Hum Mol Genet,
28(16), 2763-2774.
Abstract:
Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns.
Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.
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Dhayal S, Zummo FP, Anderson MW, Thomas P, Welters HJ, Arden C, Morgan NG (2019). Differential effects of saturated and unsaturated fatty acids on autophagy in pancreatic β-cells.
J Mol Endocrinol,
63(4), 285-296.
Abstract:
Differential effects of saturated and unsaturated fatty acids on autophagy in pancreatic β-cells.
Long-chain saturated fatty acids are lipotoxic to pancreatic β-cells, whereas most unsaturates are better tolerated and some may even be cytoprotective. Fatty acids alter autophagy in β-cells and there is increasing evidence that such alterations can impact directly on the regulation of viability. Accordingly, we have compared the effects of palmitate (C16:0) and palmitoleate (C16:1) on autophagy in cultured β-cells and human islets. Treatment of BRIN-BD11 β-cells with palmitate led to enhanced autophagic activity, as judged by cleavage of microtubule-associated protein 1 light chain 3-I (LC3-I) and this correlated with a marked loss of cell viability in the cells. In addition, transfection of these cells with an mCherry-YFP-LC3 reporter construct revealed the accumulation of autophagosomes in palmitate-treated cells, indicating an impairment of autophagosome-lysosome fusion. This was also seen upon addition of the vacuolar ATPase inhibitor, bafilomycin A1. Exposure of BRIN-BD11 cells to palmitoleate (C16:1) did not lead directly to changes in autophagic activity or flux, but it antagonised the actions of palmitate. In parallel, palmitoleate also improved the viability of palmitate-treated BRIN-BD11 cells. Equivalent responses were observed in INS-1E cells and in isolated human islets. Taken together, these data suggest that palmitate may cause an impairment of autophagosome-lysosome fusion. These effects were not reproduced by palmitoleate which, instead, antagonised the responses mediated by palmitate suggesting that attenuation of β-cell stress may contribute to the improvement in cell viability caused by the mono-unsaturated fatty acid.
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Bahal S, Houssen ME, Manson A, Lorenzo L, Russell MA, Morgan NG, Tahami F, Grigoriadou S (2019). Evidence that a STAT3 Mutation Causing Hyper IgE Syndrome Leads to Repression of Transcriptional Activity.
Case Reports Immunol,
2019Abstract:
Evidence that a STAT3 Mutation Causing Hyper IgE Syndrome Leads to Repression of Transcriptional Activity.
We present the case of a 19-year-old female with a mild form of Autosomal Dominant Hyper IgE syndrome (HIES) associated with a loss-of-function mutation in STAT3. Within the first years of life she developed multiple, Staphylococcus aureus associated abscesses in the neck and face requiring frequent incision and drainage. Respiratory tract infections were not a feature of the clinical phenotype and a high resolution thoracic CT scan was unremarkable. Retained dentition was noted but fungal nail disease and recurrent thrush were absent. The total IgE was 970 IU/L, Lymphocyte counts and immunoglobulin levels were normal (IgG borderline 18.5 gr/L). There was suboptimal response to test immunisation with Pneumovax II vaccine. Th17 cell phenotyping revealed low levels of IL-17 expressing cells (0.3% of total CD4 T Cells numbers). Genetic analysis identified a missense mutation, N567D, in a conserved region of the linker domain of STAT3. Functional studies in HEK293 cells reveal that this mutation potently inhibits STAT3 activity when compared to the wildtype protein. This is consistent with other reported mutations in STAT3 associated with HIES. However, surprisingly, the magnitude of inhibition was similar to another STAT3 mutation (V637M) which causes a much more severe form of the disease.
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Russell MA, Redick SD, Blodgett DM, Richardson SJ, Leete P, Krogvold L, Dahl-Jørgensen K, Bottino R, Brissova M, Spaeth JM, et al (2019). HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet β-Cells from Donors with Type 1 Diabetes.
Diabetes,
68(5), 988-1001.
Abstract:
HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet β-Cells from Donors with Type 1 Diabetes.
Type 1 diabetes studies consistently generate data showing islet β-cell dysfunction and T cell-mediated anti-β-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the β-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single β-cells. Consistent with immunohistological studies, β-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These β-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- β-cells, specifically found in islets with lymphocytic infiltrates. β-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ β-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic β-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. β-Cell expression of Class II molecules suggests that β-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.
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Basile G, Kulkarni RN, Morgan NG (2019). How, When, and Where Do Human β-Cells Regenerate?.
Curr Diab Rep,
19(8).
Abstract:
How, When, and Where Do Human β-Cells Regenerate?
PURPOSE OF REVIEW: Pancreatic β-cells play a critical role in whole-body glucose homeostasis by regulating the release of insulin in response to minute by minute alterations in metabolic demand. As such, β-cells are staunchly resilient but there are circumstances where they can become functionally compromised or physically lost due to pathophysiological changes which culminate in overt hyperglycemia and diabetes. RECENT FINDINGS: in humans, β-cell mass appears to be largely defined in the postnatal period and this early replicative and generative phase is followed by a refractory state which persists throughout life. Despite this, efforts to identify physiological and pharmacological factors which might re-initiate β-cell replication (or cause the replenishment of β-cells by neogenesis or transdifferentiation) are beginning to bear fruit. Controlled manipulation of β-cell mass in humans still represents a holy grail for therapeutic intervention in diabetes, but progress is being made which may lead to ultimate success.
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Dunne JL, Richardson SJ, Atkinson MA, Craig ME, Dahl-Jørgensen K, Flodström-Tullberg M, Hyöty H, Lloyd RE, Morgan NG, Pugliese A, et al (2019). Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence. Reply to Skog O, Klingel K, Roivainen M et al [letter].
Diabetologia,
62(6), 1100-1103.
Author URL.
King R, Hill JL, Saha B, Tong Y, Strutt BJ, Russell MA, Morgan NG, Richardson SJ, Hill DJ (2019). Offspring of Mice Exposed to a Low-Protein Diet in Utero Demonstrate Changes in mTOR Signaling in Pancreatic Islets of Langerhans, Associated with Altered Glucagon and Insulin Expression and a Lower β-Cell Mass.
Nutrients,
11(3).
Abstract:
Offspring of Mice Exposed to a Low-Protein Diet in Utero Demonstrate Changes in mTOR Signaling in Pancreatic Islets of Langerhans, Associated with Altered Glucagon and Insulin Expression and a Lower β-Cell Mass.
Low birth weight is a risk factor for gestational and type 2 diabetes (T2D). Since mammalian target of rapamycin (mTOR) controls pancreatic β-cell mass and hormone release, we hypothesized that nutritional insult in utero might permanently alter mTOR signaling. Mice were fed a low-protein (LP, 8%) or control (C, 20%) diet throughout pregnancy, and offspring examined until 130 days age. Mice receiving LP were born 12% smaller and β-cell mass was significantly reduced throughout life. Islet mTOR levels were lower in LP-exposed mice and localized predominantly to α-rather than β-cells. Incubation of isolated mouse islets with rapamycin significantly reduced cell proliferation while increasing apoptosis. mRNA levels for mTORC complex genes mTOR, Rictor and Raptor were elevated at 7 days in LP mice, as were the mTOR and Raptor proteins. Proglucagon gene expression was similarly increased, but not insulin or the immune/metabolic defense protein STING. In human and mouse pancreas STING was strongly associated with islet β-cells. Results support long-term changes in islet mTOR signaling in response to nutritional insult in utero, with altered expression of glucagon and insulin and a reduced β-cell mass. This may contribute to an increased risk of gestational or type 2 diabetes.
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Dunne JL, Richardson SJ, Atkinson MA, Craig ME, Dahl-Jørgensen K, Flodström-Tullberg M, Hyöty H, Insel RA, Lernmark Å, Lloyd RE, et al (2019). Rationale for enteroviral vaccination and antiviral therapies in human type 1 diabetes.
Diabetologia,
62(5), 744-753.
Abstract:
Rationale for enteroviral vaccination and antiviral therapies in human type 1 diabetes.
In type 1 diabetes, pancreatic beta cells are destroyed by chronic autoimmune responses. The disease develops in genetically susceptible individuals, but a role for environmental factors has been postulated. Viral infections have long been considered as candidates for environmental triggers but, given the lack of evidence for an acute, widespread, cytopathic effect in the pancreas in type 1 diabetes or for a closely related temporal association of diabetes onset with such infections, a role for viruses in type 1 diabetes remains unproven. Moreover, viruses have rarely been isolated from the pancreas of individuals with type 1 diabetes, mainly (but not solely) due to the inaccessibility of the organ. Here, we review past and recent literature to evaluate the proposals that chronic, recurrent and, possibly, persistent enteroviral infections occur in pancreatic beta cells in type 1 diabetes. We also explore whether these infections may be sustained by different virus strains over time and whether multiple viral hits can occur during the natural history of type 1 diabetes. We emphasise that only a minority of beta cells appear to be infected at any given time and that enteroviruses may become replication defective, which could explain why they have been isolated from the pancreas only rarely. We argue that enteroviral infection of beta cells largely depends on the host innate and adaptive immune responses, including innate responses mounted by beta cells. Thus, we propose that viruses could play a role in type 1 diabetes on multiple levels, including in the triggering and chronic stimulation of autoimmunity and in the generation of inflammation and the promotion of beta cell dysfunction and stress, each of which might then contribute to autoimmunity, as part of a vicious circle. We conclude that studies into the effects of vaccinations and/or antiviral drugs (some of which are currently on-going) is the only means by which the role of viruses in type 1 diabetes can be finally proven or disproven.
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Leslie KA, Russell MA, Taniguchi K, Richardson SJ, Morgan NG (2019). The transcription factor STAT6 plays a critical role in promoting beta cell viability and is depleted in islets of individuals with type 1 diabetes.
Diabetologia,
62(1), 87-98.
Abstract:
The transcription factor STAT6 plays a critical role in promoting beta cell viability and is depleted in islets of individuals with type 1 diabetes.
AIMS/HYPOTHESIS: in type 1 diabetes, selective beta cell loss occurs within the inflamed milieu of insulitic islets. This milieu is generated via the enhanced secretion of proinflammatory cytokines and by the loss of anti-inflammatory molecules such as IL-4 and IL-13. While the actions of proinflammatory cytokines have been well-studied in beta cells, the effects of their anti-inflammatory counterparts have received relatively little attention and we have addressed this. METHODS: Clonal beta cells, isolated human islets and pancreas sections from control individuals and those with type 1 diabetes were employed. Gene expression was measured using targeted gene arrays and by quantitative RT-PCR. Protein expression was monitored in cell extracts by western blotting and in tissue sections by immunocytochemistry. Target proteins were knocked down selectively with interference RNA. RESULTS: Cytoprotection achieved with IL-4 and IL-13 is mediated by the early activation of signal transducer and activator of transcription 6 (STAT6) in beta cells, leading to the upregulation of anti-apoptotic proteins, including myeloid leukaemia-1 (MCL-1) and B cell lymphoma-extra large (BCLXL). We also report the induction of signal regulatory protein-α (SIRPα), and find that knockdown of SIRPα is associated with reduced beta cell viability. These anti-apoptotic proteins and their attendant cytoprotective effects are lost following siRNA-mediated knockdown of STAT6 in beta cells. Importantly, analysis of human pancreas sections revealed that STAT6 is markedly depleted in the beta cells of individuals with type 1 diabetes, implying the loss of cytoprotective responses. CONCLUSIONS/INTERPRETATION: Selective loss of STAT6 may contribute to beta cell demise during the progression of type 1 diabetes.
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Author URL.
Viloria K, Nasteska D, Larner D, Fine N, Ashford F, Heising S, Xavier GDS, Briant L, Flaxman C, Morgan N, et al (2019). Vitamin D-binding protein is required for the maintenance of [alpha]-cell identity and function. Endocrine Abstracts
Russell MA, Pigors M, Houssen ME, Manson A, Kelsell D, Longhurst H, Morgan NG (2018). A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).
Clin Immunol,
187, 132-136.
Abstract:
A novel de novo activating mutation in STAT3 identified in a patient with common variable immunodeficiency (CVID).
Common variable immunodeficiency (CVID) is characterised by repeated infection associated with primary acquired hypogammaglobulinemia. CVID frequently has a complex aetiology but, in certain cases, it has a monogenic cause. Recently, variants within the gene encoding the transcription factor STAT3 were implicated in monogenic CVID. Here, we describe a patient presenting with symptoms synonymous with CVID, who displayed reduced levels of IgG and IgA, repeated viral infections and multiple additional co-morbidities. Whole-exome sequencing revealed a de novo novel missense mutation in the coiled-coil domain of STAT3 (c.870A>T; p.K290N). Accordingly, the K290N variant of STAT3 was generated, and a STAT3 responsive dual-luciferase reporter assay revealed that the variant strongly enhances STAT3 transcriptional activity both under basal and stimulated (with IL-6) conditions. Overall, these data complement earlier studies in which CVID-associated STAT3 mutations are predicted to enhance transcriptional activity, suggesting that such patients may respond favourably to IL-6 receptor antagonists (e.g. tocilizumab).
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Holm LJ, Krogvold L, Hasselby JP, Kaur S, Claessens LA, Russell MA, Mathews CE, Hanssen KF, Morgan NG, Koeleman BPC, et al (2018). Abnormal islet sphingolipid metabolism in type 1 diabetes.
Diabetologia,
61(7), 1650-1661.
Abstract:
Abnormal islet sphingolipid metabolism in type 1 diabetes.
AIMS/HYPOTHESIS: Sphingolipids play important roles in beta cell physiology, by regulating proinsulin folding and insulin secretion and in controlling apoptosis, as studied in animal models and cell cultures. Here we investigate whether sphingolipid metabolism may contribute to the pathogenesis of human type 1 diabetes and whether increasing the levels of the sphingolipid sulfatide would prevent models of diabetes in NOD mice. METHODS: We examined the amount and distribution of sulfatide in human pancreatic islets by immunohistochemistry, immunofluorescence and electron microscopy. Transcriptional analysis was used to evaluate expression of sphingolipid-related genes in isolated human islets. Genome-wide association studies (GWAS) and a T cell proliferation assay were used to identify type 1 diabetes related polymorphisms and test how these affect cellular islet autoimmunity. Finally, we treated NOD mice with fenofibrate, a known activator of sulfatide biosynthesis, to evaluate the effect on experimental autoimmune diabetes development. RESULTS: We found reduced amounts of sulfatide, 23% of the levels in control participants, in pancreatic islets of individuals with newly diagnosed type 1 diabetes, which were associated with reduced expression of enzymes involved in sphingolipid metabolism. Next, we discovered eight gene polymorphisms (ORMDL3, SPHK2, B4GALNT1, SLC1A5, GALC, PPARD, PPARG and B4GALT1) involved in sphingolipid metabolism that contribute to the genetic predisposition to type 1 diabetes. These gene polymorphisms correlated with the degree of cellular islet autoimmunity in a cohort of individuals with type 1 diabetes. Finally, using fenofibrate, which activates sulfatide biosynthesis, we completely prevented diabetes in NOD mice and even reversed the disease in half of otherwise diabetic animals. CONCLUSIONS/INTERPRETATION: These results indicate that islet sphingolipid metabolism is abnormal in type 1 diabetes and suggest that modulation may represent a novel therapeutic approach. DATA AVAILABILITY: the RNA expression data is available online at https://www.dropbox.com/s/93mk5tzl5fdyo6b/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%2C%20RNA%20expression.xlsx?dl=0. A list of SNPs identified is available at https://www.dropbox.com/s/yfojma9xanpp2ju/Abnormal%20islet%20sphingolipid%20metabolism%20in%20type%201%20diabetes%20SNP.xlsx?dl=0.
Abstract.
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Vecchio F, Lo Buono N, Stabilini A, Nigi L, Dufort MJ, Geyer S, Rancoita PM, Cugnata F, Mandelli A, Valle A, et al (2018). Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes.
JCI Insight,
3(18).
Abstract:
Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes.
BACKGROUND: Neutrophils and their inflammatory mediators are key pathogenic components in multiple autoimmune diseases, while their role in human type 1 diabetes (T1D), a disease that progresses sequentially through identifiable stages prior to the clinical onset, is not well understood. We previously reported that the number of circulating neutrophils is reduced in patients with T1D and in presymptomatic at-risk subjects. The aim of the present work was to identify possible changes in circulating and pancreas-residing neutrophils throughout the disease course to better elucidate neutrophil involvement in human T1D. METHODS: Data collected from 389 subjects at risk of developing T1D, and enrolled in 4 distinct studies performed by TrialNet, were analyzed with comprehensive statistical approaches to determine whether the number of circulating neutrophils correlates with pancreas function. To obtain a broad analysis of pancreas-infiltrating neutrophils throughout all disease stages, pancreas sections collected worldwide from 4 different cohorts (i.e. nPOD, DiViD, Siena, and Exeter) were analyzed by immunohistochemistry and immunofluorescence. Finally, circulating neutrophils were purified from unrelated nondiabetic subjects and donors at various T1D stages and their transcriptomic signature was determined by RNA sequencing. RESULTS: Here, we show that the decline in β cell function is greatest in individuals with the lowest peripheral neutrophil numbers. Neutrophils infiltrate the pancreas prior to the onset of symptoms and they continue to do so as the disease progresses. of interest, a fraction of these pancreas-infiltrating neutrophils also extrudes neutrophil extracellular traps (NETs), suggesting a tissue-specific pathogenic role. Whole-transcriptome analysis of purified blood neutrophils revealed a unique molecular signature that is distinguished by an overabundance of IFN-associated genes; despite being healthy, said signature is already present in T1D-autoantibody-negative at-risk subjects. CONCLUSIONS: These results reveal an unexpected abnormality in neutrophil disposition both in the circulation and in the pancreas of presymptomatic and symptomatic T1D subjects, implying that targeting neutrophils might represent a previously unrecognized therapeutic modality. FUNDING: Juvenile Diabetes Research Foundation (JDRF), NIH, Diabetes UK.
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Shields B, McDonald T, Oram R, Hill A, Hudson M, Leete P, Pearson E, Richardson S, Morgan N, Hattersley A, et al (2018). C-peptide decline in type 1 diabetes has two phases: an initial exponential fall and a subsequent stable phase. Diabetes Care
Richardson SJ, Morgan NG (2018). Enteroviral infections in the pathogenesis of type 1 diabetes: new insights for therapeutic intervention. Current Opinion in Pharmacology, 43, 11-19.
Morgan NG, Richardson SJ (2018). Fifty years of pancreatic islet pathology in human type 1 diabetes: insights gained and progress made. Diabetologia, 61(12), 2499-2506.
Kronenberg-Versteeg D, Eichmann M, Russell MA, Ru AD, Hehn B, Yusuf N, Veelen PAV, Richardson SJ, Morgan NG, Lemberg MK, et al (2018). Molecular pathways for immune recognition of preproinsulin signal peptide in type 1 diabetes.
Diabetes,
67(4), 687-696.
Abstract:
Molecular pathways for immune recognition of preproinsulin signal peptide in type 1 diabetes
The signal peptide region of preproinsulin (PPI) contains epitopes targeted by HLA-A-restricted (HLA-A0201, A2402) cytotoxic T cells as part of the pathogenesis of b-cell destruction in type 1 diabetes. We extended the discovery of the PPI epitope to disease-associated HLA-B∗1801 and HLA-B∗3906 (risk) and HLA-A∗1101 and HLA-B∗3801 (protective) alleles, revealing that four of six alleles present epitopes derived from the signal peptide region. During cotranslational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells, we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter associated with antigen processing (TAP), ER-luminal epitopes, which are TAP independent, each presented by different HLA class I molecules and N-terminal trimmed by ER aminopeptidase 1 for optimal presentation. In vivo, TAP expression is significantly upregulated and correlatedwith HLA class I hyperexpression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.
Abstract.
Colli ML, Hill JLE, Marroquà L, Chaffey J, Dos Santos RS, Leete P, Coomans de Brachène A, Paula FMM, Op de Beeck A, Castela A, et al (2018). PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.
EBioMedicine,
36, 367-375.
Abstract:
PDL1 is expressed in the islets of people with type 1 diabetes and is up-regulated by interferons-α and-γ via IRF1 induction.
BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: the expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-βH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1β, induced PDL1 expression in vitro in human islet cells and EndoC-βH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.
Abstract.
Author URL.
Ifie E, Russell MA, Dhayal S, Leete P, Sebastiani G, Nigi L, Dotta F, Marjomäki V, Eizirik DL, Morgan NG, et al (2018). Unexpected subcellular distribution of a specific isoform of the Coxsackie and adenovirus receptor, CAR-SIV, in human pancreatic beta cells. Diabetologia, 61(11), 2344-2355.
Saarimäki-Vire J, Balboa D, Russell MA, Saarikettu J, Kinnunen M, Keskitalo S, Malhi A, Valensisi C, Andrus C, Eurola S, et al (2017). An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation.
Cell Rep,
19(2), 281-294.
Abstract:
An Activating STAT3 Mutation Causes Neonatal Diabetes through Premature Induction of Pancreatic Differentiation.
Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.
Abstract.
Author URL.
Morgan NG (2017). Bringing the human pancreas into focus: new paradigms for the understanding of Type 1 diabetes.
Diabet Med,
34(7), 879-886.
Abstract:
Bringing the human pancreas into focus: new paradigms for the understanding of Type 1 diabetes.
Type 1 diabetes affects increasingly large numbers of people globally (including at least half a million children under the age of 14 years) and it remains an illness with life-long and often devastating consequences. It is surprising, therefore, that the underlying aetiology of Type 1 diabetes remains poorly understood. This is largely because the cellular and molecular processes leading to the loss of β cells in the pancreas have rarely been studied at, or soon after, the onset of disease. Where such studies have been undertaken, a number of surprises have emerged which serve to challenge conventional wisdom. In particular, it is increasingly understood that the process of islet inflammation (insulitis) is much less florid in humans than in certain animal models. Moreover, the profile of immune cells involved in the inflammatory attack on β cells is variable and this variation occurs at the level of individual patients. As a result, two distinct profiles of insulitis have now been defined that are differentially aggressive and that might, therefore, require specifically tailored therapeutic approaches to slow the progression of disease. In addition, the outcomes are also different in that the more aggressive form (termed 'CD20Hi') is associated with extensive β-cell loss and an early age of disease onset (13 years) and the retention of a higher proportion of residual β cells. In the present review, these new findings are explained and their implications evaluated in terms of future therapies.
Abstract.
Author URL.
Busse N, Paroni F, Richardson SJ, Laiho JE, Oikarinen M, Frisk G, Hyöty H, de Koning E, Morgan NG, Maedler K, et al (2017). Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes.
Oncotarget,
8(8), 12620-12636.
Abstract:
Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes.
Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family.With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.
Abstract.
Author URL.
Willcox A, Richardson SJ, Walker LSK, Kent SC, Morgan NG, Gillespie KM (2017). Germinal centre frequency is decreased in pancreatic lymph nodes from individuals with recent-onset type 1 diabetes.
Diabetologia,
60(7), 1294-1303.
Abstract:
Germinal centre frequency is decreased in pancreatic lymph nodes from individuals with recent-onset type 1 diabetes
Aims/hypothesis: Pancreatic lymph nodes (PLNs) are critical sites for the initial interaction between islet autoantigens and autoreactive lymphocytes, but the histology of PLNs in tissue from individuals with type 1 diabetes has not been analysed in detail. The aim of this study was to examine PLN tissue sections from healthy donors compared with those at risk of, or with recent-onset and longer-duration type 1 diabetes. Methods: Immunofluorescence staining was used to examine PLN sections from the following donor groups: non-diabetic (n=15), non-diabetic islet autoantibody-positive (n=5), recent-onset (≤1.5 years duration) type 1 diabetes (n=13), and longer-duration type 1 diabetes (n=15). Staining for CD3, CD20 and Ki67 was used to detect primary and secondary (germinal centre-containing) follicles and CD21 and CD35 to detect follicular dendritic cell networks. Results: the frequency of secondary follicles was lower in the recent-onset type 1 diabetes group compared with the non-diabetic control group. The presence of insulitis (as evidence of ongoing beta cell destruction) and diagnosis of type 1 diabetes at a younger age, however, did not appear to be associated with a lower frequency of secondary follicles. A higher proportion of primary B cell follicles were observed to lack follicular dendritic cell networks in the recent-onset type 1 diabetes group. Conclusions/interpretation: Histological analysis of rare PLNs from individuals with type 1 diabetes suggests a previously unrecognised phenotype comprising decreased primary B cell follicle frequency and fewer follicular dendritic cell networks in recent-onset type 1 diabetes.
Abstract.
Campbell-Thompson ML, Atkinson MA, Butler AE, Giepmans BN, von Herrath MG, Hyöty H, Kay TW, Morgan NG, Powers AC, Pugliese A, et al (2017). Re-addressing the 2013 consensus guidelines for the diagnosis of insulitis in human type 1 diabetes: is change necessary?. Diabetologia, 60(4), 753-755.
Dharmadhikari G, Stolz K, Hauke M, Morgan NG, Varki A, de Koning E, Kelm S, Maedler K (2017). Siglec-7 restores β-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.
Sci Rep,
7Abstract:
Siglec-7 restores β-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.
Chronic inflammation plays a key role in both type 1 and type 2 diabetes. Cytokine and chemokine production within the islets in a diabetic milieu results in β-cell failure and diabetes progression. Identification of targets, which both prevent macrophage activation and infiltration into islets and restore β-cell functionality is essential for effective diabetes therapy. We report that certain Sialic-acid-binding immunoglobulin-like-lectins (siglecs) are expressed in human pancreatic islets in a cell-type specific manner. Siglec-7 was expressed on β-cells and down-regulated in type 1 and type 2 diabetes and in infiltrating activated immune cells. Over-expression of Siglec-7 in diabetic islets reduced cytokines, prevented β-cell dysfunction and apoptosis and reduced recruiting of migrating monocytes. Our data suggest that restoration of human Siglec-7 expression may be a novel therapeutic strategy targeted to both inhibition of immune activation and preservation of β-cell function and survival.
Abstract.
Author URL.
Wagner FF, Lundh M, Kaya T, McCarren P, Zhang Y-L, Chattopadhyay S, Gale JP, Galbo T, Fisher SL, Meier BC, et al (2016). An Isochemogenic Set of Inhibitors to Define the Therapeutic Potential of Histone Deacetylases in β-Cell Protection.
ACS Chem Biol,
11(2), 363-374.
Abstract:
An Isochemogenic Set of Inhibitors to Define the Therapeutic Potential of Histone Deacetylases in β-Cell Protection.
Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in β-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic β-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic β-cells from inflammatory cytokines and nutrient overload in diabetes.
Abstract.
Author URL.
Morgan N, Richardson S (2016). Changing perspectives on the progression of type 1 diabetes. Practical Diabetes, 33(4), 118-120.
Leete P, Willcox A, Krogvold L, Dahl-Jørgensen K, Foulis AK, Richardson SJ, Morgan NG (2016). Differential Insulitic Profiles Determine the Extent of β-Cell Destruction and the Age at Onset of Type 1 Diabetes.
Diabetes,
65(5), 1362-1369.
Abstract:
Differential Insulitic Profiles Determine the Extent of β-Cell Destruction and the Age at Onset of Type 1 Diabetes.
Type 1 diabetes (T1D) results from a T cell-mediated destruction of pancreatic β-cells following the infiltration of leukocytes (including CD8(+), CD4(+), and CD20(+) cells) into and around pancreatic islets (insulitis). Recently, we reported that two distinct patterns of insulitis occur in patients with recent-onset T1D from the U.K. and that these differ principally in the proportion of infiltrating CD20(+) B cells (designated CD20Hi and CD20Lo, respectively). We have now extended this analysis to include patients from the Network for Pancreatic Organ Donors with Diabetes (U.S.) and Diabetes Virus Detection (DiViD) study (Norway) cohorts and confirm that the two profiles of insulitis occur more widely. Moreover, we show that patients can be directly stratified according to their insulitic profile and that those receiving a diagnosis before the age of 7 years always display the CD20Hi profile. By contrast, individuals who received a diagnosis beyond the age of 13 years are uniformly defined as CD20Lo. This implies that the two forms of insulitis are differentially aggressive and that patients with a CD20Hi profile lose their β-cells at a more rapid rate. In support of this, we also find that the proportion of residual insulin-containing islets (ICIs) increases in parallel with age at the onset of T1D. Importantly, those receiving a diagnosis in, or beyond, their teenage years retain ∼40% ICIs at diagnosis, implying that a functional deficit rather than an absolute β-cell loss may be causal for disease onset in these patients. We conclude that appropriate patient stratification will be critical for correct interpretation of the outcomes of intervention therapies targeted to islet-infiltrating immune cells in T1D.
Abstract.
Author URL.
Richardson SJ, Rodriguez-Calvo T, Gerling IC, Mathews CE, Kaddis JS, Russell MA, Zeissler M, Leete P, Krogvold L, Dahl-Jørgensen K, et al (2016). Islet cell hyperexpression of HLA class I antigens: a defining feature in type 1 diabetes.
Diabetologia,
59(11), 2448-2458.
Abstract:
Islet cell hyperexpression of HLA class I antigens: a defining feature in type 1 diabetes.
AIMS/HYPOTHESIS: Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it. METHODS: Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays. RESULTS: Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of β2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both 'classical' HLA class I isoforms (i.e. HLA-ABC) as well as a 'non-classical' HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter. CONCLUSIONS/INTERPRETATION: Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.
Abstract.
Author URL.
Corcoran J, Petrov P, Whatmore J, Winlove P, Morgan NG (2016). Molecular analysis of the uptake and disposition of long chain fatty acids (LCFA) in beta cells.
DIABETIC MEDICINE,
33, 81-81.
Author URL.
Laiho JE, Oikarinen M, Richardson SJ, Frisk G, Nyalwidhe J, Burch TC, Morris MA, Oikarinen S, Pugliese A, Dotta F, et al (2016). Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells. Journal of Clinical Virology, 77, 21-28.
Wedgwood KCA, Richardson SJ, Morgan NG, tsaneva-atanasova K (2016). Spatiotemporal dynamics of insulitis in human type 1 diabetes.
Frontiers in PhysiologyAbstract:
Spatiotemporal dynamics of insulitis in human type 1 diabetes
Type 1 diabetes (T1D) is an auto-immune disease characterised by the selective destruction ofthe insulin secreting beta cells in the pancreas during an inflammatory phase known as insulitis.Patients with T1D are typically dependent on the administration of externally provided insulinin order to manage blood glucose levels. Whilst technological developments have significantlyimproved both the life expectancy and quality of life of these patients, an understanding of themechanisms of the disease remains elusive. Animal models, such as the NOD mouse model,have been widely used to probe the process of insulitis, but there exist very few data from humansstudied at disease onset. In this manuscript, we employ data from human pancreases collectedclose to the onset of type 1 diabetes and propose a spatio-temporal computational model forthe progression of insulitis in human T1D, with particular focus on the mechanisms underlyingthe development of insulitis in pancreatic islets. This framework allows us to investigate how thetime-course of insulitis progression is affected by altering key parameters, such as the number ofthe CD20+ B cells present in the inflammatory infiltrate, which has recently been proposed toinfluence the aggressiveness of the disease. Through the analysis of repeated simulations ofour stochastic model which track the number of beta cells within an islet, we find that increasednumbers of B cells in the peri-islet space lead to faster destruction of the beta cells. We also findthat the balance between the degradation and repair of the basement membrane surrounding theislet is a critical component in governing the overall destruction rate of the beta cells and theirremaining number. Our model provides a framework for continued and improved spatio-temporalmodelling of human T1D
Abstract.
Ferru-Clément R, Spanova M, Dhayal S, Morgan NG, Hélye R, Becq F, Hirose H, Antonny B, Vamparys L, Fuchs PFJ, et al (2016). Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.
J Cell Sci,
129(12), 2368-2381.
Abstract:
Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.
Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian β-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.
Abstract.
Author URL.
Krogvold L, Edwin B, Buanes T, Frisk G, Skog O, Anagandula M, Korsgren O, Undlien D, Eike MC, Richardson SJ, et al (2015). Detection of a low-grade enteroviral infection in the islets of langerhans of living patients newly diagnosed with type 1 diabetes.
Diabetes,
64(5), 1682-1687.
Abstract:
Detection of a low-grade enteroviral infection in the islets of langerhans of living patients newly diagnosed with type 1 diabetes.
The Diabetes Virus Detection study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes for the presence of viruses. Minimal pancreatic tail resection was performed 3-9 weeks after onset of type 1 diabetes in six adult patients (age 24-35 years). The presence of enteroviral capsid protein 1 (VP1) and the expression of class I HLA were investigated by immunohistochemistry. Enterovirus RNA was analyzed from isolated pancreatic islets and from fresh-frozen whole pancreatic tissue using PCR and sequencing. Nondiabetic organ donors served as controls. VP1 was detected in the islets of all type 1 diabetic patients (two of nine controls). Hyperexpression of class I HLA molecules was found in the islets of all patients (one of nine controls). Enterovirus-specific RNA sequences were detected in four of six patients (zero of six controls). The results were confirmed in various laboratories. Only 1.7% of the islets contained VP1(+) cells, and the amount of enterovirus RNA was low. The results provide evidence for the presence of enterovirus in pancreatic islets of type 1 diabetic patients, which is consistent with the possibility that a low-grade enteroviral infection in the pancreatic islets contributes to disease progression in humans.
Abstract.
Author URL.
Marroqui L, Lopes M, dos Santos RS, Grieco FA, Roivainen M, Richardson SJ, Morgan NG, Op de Beeck A, Eizirik DL (2015). Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and β cells.
Elife,
4Abstract:
Differential cell autonomous responses determine the outcome of coxsackievirus infections in murine pancreatic α and β cells.
Type 1 diabetes (T1D) is an autoimmune disease caused by loss of pancreatic β cells via apoptosis while neighboring α cells are preserved. Viral infections by coxsackieviruses (CVB) may contribute to trigger autoimmunity in T1D. Cellular permissiveness to viral infection is modulated by innate antiviral responses, which vary among different cell types. We presently describe that global gene expression is similar in cytokine-treated and virus-infected human islet cells, with up-regulation of gene networks involved in cell autonomous immune responses. Comparison between the responses of rat pancreatic α and β cells to infection by CVB5 and 4 indicate that α cells trigger a more efficient antiviral response than β cells, including higher basal and induced expression of STAT1-regulated genes, and are thus better able to clear viral infections than β cells. These differences may explain why pancreatic β cells, but not α cells, are targeted by an autoimmune response during T1D.
Abstract.
Author URL.
Arif S, Leete P, Nguyen V, Marks K, Nor NM, Estorninho M, Kronenberg-Versteeg D, Bingley PJ, Todd JA, Guy C, et al (2015). Erratum. Blood and Islet Phenotypes Indicate Immunological Heterogeneity in Type 1 Diabetes. Diabetes 2014;63:3835-3845.
Diabetes,
64(9).
Author URL.
Taniguchi K, Russell MA, Richardson SJ, Morgan NG (2015). The subcellular distribution of cyclin-D1 and cyclin-D3 within human islet cells varies according to the status of the pancreas donor.
Diabetologia,
58(9), 2056-2063.
Abstract:
The subcellular distribution of cyclin-D1 and cyclin-D3 within human islet cells varies according to the status of the pancreas donor
Aims/hypothesis: in humans, the rate of beta cell proliferation declines rapidly during the postnatal period and remains low throughout adult life. Recent studies suggest that this may reflect the distribution of cell cycle regulators which, unusually, are located in the cytosolic compartment of beta cells in islets isolated from adults. In the present work, we examined whether the localisation of cyclin-D molecules is also cytosolic in the islet cells of pancreatic samples studied in situ. Methods: Immunohistochemical approaches were employed to examine the subcellular localisation of cyclin-D1, -D2 and -D3 in human pancreatic samples recovered either from heart-beating donors or post mortem. Immunofluorescence methods were used to reveal the cellular localisation of cyclin-D1 and -D3. Results: the distribution of cyclin-D2 was invariably cytosolic in islet cells, whereas the localisation of cyclin-D1 and -D3 varied according to the status of the donor. In pancreatic sections from heart-beating donors these molecules were primarily nuclear. By contrast, in samples collected post mortem, they were mainly cytosolic. Cyclin-D1 was detected only in beta cells whereas cyclin-D3 was detected in both alpha and beta cells. The proportion of donors who were immunopositive for cyclin-D1 declined from 71% in controls to 30% in those with type 1 diabetes. Cyclin-D3 was present in the islets of the majority of donors in both groups. Conclusions/interpretation: the subcellular localisation of cyclin-D molecules varies according to the status of the donor. Both cyclin-D1 and -D3 can be found in the nuclei of human islet cells in situ.
Abstract.
Taniguchi K, Russell MA, Richardson SJ, Morgan NG (2015). The subcellular distribution of cyclin-D1 and cyclin-D3 within human islet cells varies according to the status of the pancreas donor.
DiabetologiaAbstract:
The subcellular distribution of cyclin-D1 and cyclin-D3 within human islet cells varies according to the status of the pancreas donor
Aims/hypothesis: in humans, the rate of beta cell proliferation declines rapidly during the postnatal period and remains low throughout adult life. Recent studies suggest that this may reflect the distribution of cell cycle regulators which, unusually, are located in the cytosolic compartment of beta cells in islets isolated from adults. In the present work, we examined whether the localisation of cyclin-D molecules is also cytosolic in the islet cells of pancreatic samples studied in situ. Methods: Immunohistochemical approaches were employed to examine the subcellular localisation of cyclin-D1, -D2 and -D3 in human pancreatic samples recovered either from heart-beating donors or post mortem. Immunofluorescence methods were used to reveal the cellular localisation of cyclin-D1 and -D3. Results: the distribution of cyclin-D2 was invariably cytosolic in islet cells, whereas the localisation of cyclin-D1 and -D3 varied according to the status of the donor. In pancreatic sections from heart-beating donors these molecules were primarily nuclear. By contrast, in samples collected post mortem, they were mainly cytosolic. Cyclin-D1 was detected only in beta cells whereas cyclin-D3 was detected in both alpha and beta cells. The proportion of donors who were immunopositive for cyclin-D1 declined from 71% in controls to 30% in those with type 1 diabetes. Cyclin-D3 was present in the islets of the majority of donors in both groups. Conclusions/interpretation: the subcellular localisation of cyclin-D molecules varies according to the status of the donor. Both cyclin-D1 and -D3 can be found in the nuclei of human islet cells in situ.
Abstract.
Flanagan SE, Haapaniemi E, Russell MA, Caswell R, Allen HL, De Franco E, McDonald TJ, Rajala H, Ramelius A, Barton J, et al (2014). Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.
Nat Genet,
46(8), 812-814.
Abstract:
Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.
Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.
Abstract.
Author URL.
Arif S, Leete P, Nguyen V, Marks K, Nor NM, Estorninho M, Kronenberg-Versteeg D, Bingley PJ, Todd JA, Guy C, et al (2014). Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes.
Diabetes,
63(11), 3835-3845.
Abstract:
Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes.
Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ-positive, multiautoantibody-positive) and partially regulated (interleukin-10-positive, pauci-autoantibody-positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed "hyper-immune CD20Hi" and "pauci-immune CD20Lo." of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.
Abstract.
Author URL.
Richardson SJ, Leete P, Dhayal S, Russell MA, Oikarinen M, Laiho JE, Svedin E, Lind K, Rosenling T, Chapman N, et al (2014). Detection of enterovirus in the islet cells of patients with type 1 diabetes: what do we learn from immunohistochemistry? Reply to Hansson SF, Korsgren S, Pontén F et al [letter].
Diabetologia,
57(3), 647-649.
Author URL.
Morgan NG, Richardson SJ (2014). Enteroviruses as causative agents in type 1 diabetes: loose ends or lost cause?.
Trends Endocrinol Metab,
25(12), 611-619.
Abstract:
Enteroviruses as causative agents in type 1 diabetes: loose ends or lost cause?
Considerable evidence implies that an enteroviral infection may accelerate or precipitate type 1 diabetes (T1D) in some individuals. However, causality is not proven. We present and critically assess evidence suggesting that islet β cells can become infected with enterovirus, and argue that this may result in one of several consequences. Occasionally, a fully lytic infection may arise and this culminates in fulminant diabetes. Alternatively, an atypical persistent infection develops which can be either benign or promote islet autoimmunity. We propose a model in which the 'strength' of the β cell response to the establishment of a persistent enteroviral infection determines the final disease outcome.
Abstract.
Author URL.
Richardson SJ, Leete P, Dhayal S, Russell MA, Oikarinen M, Laiho JE, Svedin E, Lind K, Rosenling T, Chapman N, et al (2014). Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas.
Diabetologia,
57(2), 392-401.
Abstract:
Evaluation of the fidelity of immunolabelling obtained with clone 5D8/1, a monoclonal antibody directed against the enteroviral capsid protein, VP1, in human pancreas
Aims/hypothesis: Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes. Methods: Enteroviral VP1, CKB and ATP5B expression were analysed by western blotting, RT-PCR and immunocytochemistry in a range of cultured cell lines, isolated human islets and human tissue. Results: Clone 5D8/1 labelled CKB, but not ATP5B, on western blots performed under denaturing conditions. In cultured human cell lines, isolated human islets and pancreas sections from patients with type 1 diabetes, the immunolabelling of ATP5B, CKB and VP1 by 5D8/1 was readily distinguishable. Moreover, in a human tissue microarray displaying more than 80 different cells and tissues, only two (stomach and colon; both of which are potential sites of enterovirus infection) were immunopositive when stained with clone 5D8/1. Conclusions/interpretation: When used under carefully optimised conditions, the immunolabelling pattern detected in sections of human pancreas with clone 5D8/1 did not reflect cross-reactivity with either ATP5B or CKB. Rather, 5D8/1 is likely to be representative of enteroviral antigen expression. © 2013 Springer-Verlag Berlin Heidelberg.
Abstract.
Stone VM, Dhayal S, Brocklehurst KJ, Lenaghan C, Sörhede Winzell M, Hammar M, Xu X, Smith DM, Morgan NG (2014). GPR120 (FFAR4) is preferentially expressed in pancreatic delta cells and regulates somatostatin secretion from murine islets of Langerhans.
Diabetologia,
57(6), 1182-1191.
Abstract:
GPR120 (FFAR4) is preferentially expressed in pancreatic delta cells and regulates somatostatin secretion from murine islets of Langerhans.
AIMS/HYPOTHESIS: the NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/β-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas. METHODS: a KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of β-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting β-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists. RESULTS: β-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice. CONCLUSIONS/INTERPRETATION: the results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.
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Author URL.
Morgan NG, Leete P, Foulis AK, Richardson SJ (2014). Islet inflammation in human type 1 diabetes mellitus.
IUBMB Life,
66(11), 723-734.
Abstract:
Islet inflammation in human type 1 diabetes mellitus.
Type 1 diabetes mellitus (T1DM) is caused by the selective deletion of pancreatic β-cells in response to an assault mounted within the pancreas by infiltrating immune cells. However, this apparently clear and focussed annunciation conceals a stark reality in which the cellular and molecular events leading to β-cell loss remain poorly understood in humans. This reflects the difficulty of studying these processes in living individuals and the fact that, using pathological specimens, islet inflammation has been analysed in fewer than 200 recent-onset cases of T1DM worldwide, over the past century. Nevertheless, insights have been gained and the composition of the islet infiltrate is being disclosed. This is shown to be primarily lymphocytic in nature, with populations of both CD8+ and CD4+ T cells displaying an autoreactivity against specific islet antigenic peptides. The T cells are often accompanied by influent CD20+ B cells, although new data imply that the proportions of these individual cell types vary and that patients fall into at least two distinct categories having either a hyper-immune (CD20Hi) or a pauci-immune (CD20Lo) phenotype. The overall rate of β-cell decline appears to correlate with these two phenotypes such that hyper-immune patients lose β-cells more quickly and tend to develop disease at an earlier age than those with the pauci-immune profile. In this article, we review the evidence which underpins our current understanding of the aetiology of T1DM and highlight both the established features as well as areas of on-going ambiguity and debate.
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Richardson SJ, Morgan NG, Foulis AK (2014). Pancreatic pathology in type 1 diabetes mellitus.
Endocrine Pathology,
25(1), 80-92.
Abstract:
Pancreatic pathology in type 1 diabetes mellitus
Type 1 diabetes is a multifactorial disease resulting from a complex interplay between host genetics, the immune system and the environment, that culminates in the destruction of insulin-producing beta cells. The incidence of type 1 diabetes is increasing at an alarming rate, especially in children under the age of 5 (Gepts in Diabetes 14(10):619-613, 1965; Foulis et al. in Lancet 29(5):267-274, 1986; Gamble, Taylor and Cumming in British Medical Journal 4(5887):260-262 1973). Genetic predisposition, although clearly important, cannot explain this rise, and so, it has been proposed that changes in the 'environment' and/or changes in 'how we respond to our environment' must contribute to this rising incidence. In order to gain an improved understanding of the factors influencing the disease process, it is important, firstly, to focus on the organ at the centre of the illness - the pancreas. This review summarises our knowledge of the pathology of the endocrine pancreas in human type 1 diabetes and, in particular, explores the progression of this understanding over the past 25 years. © 2014 Springer Science+Business Media.
Abstract.
Richardson SJ, Morgan NG, Foulis AK (2014). Pancreatic pathology in type 1 diabetes mellitus.
Endocr Pathol,
25(1), 80-92.
Abstract:
Pancreatic pathology in type 1 diabetes mellitus.
Type 1 diabetes is a multifactorial disease resulting from a complex interplay between host genetics, the immune system and the environment, that culminates in the destruction of insulin-producing beta cells. The incidence of type 1 diabetes is increasing at an alarming rate, especially in children under the age of 5 (Gepts in Diabetes 14(10):619-613, 1965; Foulis et al. in Lancet 29(5):267-274, 1986; Gamble, Taylor and Cumming in British Medical Journal 4(5887):260-262 1973). Genetic predisposition, although clearly important, cannot explain this rise, and so, it has been proposed that changes in the 'environment' and/or changes in 'how we respond to our environment' must contribute to this rising incidence. In order to gain an improved understanding of the factors influencing the disease process, it is important, firstly, to focus on the organ at the centre of the illness-the pancreas. This review summarises our knowledge of the pathology of the endocrine pancreas in human type 1 diabetes and, in particular, explores the progression of this understanding over the past 25 years.
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Author URL.
Bjursell M, Xu X, Admyre T, Böttcher G, Lundin S, Nilsson R, Stone VM, Morgan NG, Lam YY, Storlien LH, et al (2014). The beneficial effects of n-3 polyunsaturated fatty acids on diet induced obesity and impaired glucose control do not require Gpr120.
PLoS One,
9(12).
Abstract:
The beneficial effects of n-3 polyunsaturated fatty acids on diet induced obesity and impaired glucose control do not require Gpr120.
GPR120 (Ffar4) has been postulated to represent an important receptor mediating the improved metabolic profile seen upon ingestion of a diet enriched in polyunsaturated fatty acids (PUFAs). GPR120 is highly expressed in the digestive system, adipose tissue, lung and macrophages and also present in the endocrine pancreas. A new Gpr120 deficient mouse model on pure C57bl/6N background was developed to investigate the importance of the receptor for long-term feeding with a diet enriched with fish oil. Male Gpr120 deficient mice were fed two different high fat diets (HFDs) for 18 weeks. The diets contained lipids that were mainly saturated (SAT) or mainly n-3 polyunsaturated fatty acids (PUFA). Body composition, as well as glucose, lipid and energy metabolism, was studied. As expected, wild type mice fed the PUFA HFD gained less body weight and had lower body fat mass, hepatic lipid levels, plasma cholesterol and insulin levels and better glucose tolerance as compared to those fed the SAT HFD. Gpr120 deficient mice showed a similar improvement on the PUFA HFD as was observed for wild type mice. If anything, the Gpr120 deficient mice responded better to the PUFA HFD as compared to wild type mice with respect to liver fat content, plasma glucose levels and islet morphology. Gpr120 deficient animals were found to have similar energy, glucose and lipid metabolism when fed HFD PUFA compared to wild type mice. Therefore, GPR120 appears to be dispensable for the improved metabolic profile associated with intake of a diet enriched in n-3 PUFA fatty acids.
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Russell MA, Morgan NG (2014). The impact of anti-inflammatory cytokines on the pancreatic β-cell.
Islets,
6(3).
Abstract:
The impact of anti-inflammatory cytokines on the pancreatic β-cell.
Considerable efforts have been invested to understand the mechanisms by which pro-inflammatory cytokines mediate the demise of β-cells in type 1 diabetes but much less attention has been paid to the role of anti-inflammatory cytokines as potential cytoprotective agents in these cells. Despite this, there is increasing evidence that anti-inflammatory molecules such as interleukin (IL)-4, IL-10 and IL-13 can exert a direct influence of β-cell function and viability and that the circulating levels of these cytokines may be reduced in type 1 diabetes. Thus, it seems possible that targeting of anti-inflammatory pathways might offer therapeutic potential in this disease. In the present review, we consider the evidence implicating IL-4, IL-10 and IL-13 as cytoprotective agents in the β-cell and discuss the receptor components and downstream signaling pathways that mediate these effects.
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Russell MA, Cooper AC, Dhayal S, Morgan NG (2013). Differential effects of interleukin-13 and interleukin-6 on Jak/STAT signaling and cell viability in pancreatic β-cells.
Islets,
5(2), 95-105.
Abstract:
Differential effects of interleukin-13 and interleukin-6 on Jak/STAT signaling and cell viability in pancreatic β-cells.
Pro-inflammatory cytokines are important mediators of β-cell demise in type 1 diabetes, and similar mechanisms are increasingly implicated in type 2 diabetes, where a state of chronic inflammation may persist. It is likely that the actions of anti-inflammatory cytokines are also altered in diabetes. Cytokines are released from immune cells, which may be recruited to the islets in diabetes, but they can also be produced by islet endocrine cells in response to environmental factors, including enteroviral infection. Since enteroviral infection of islet cells may influence the development of diabetes in humans, we examined the actions of two cytokines, IL-13 and IL-6, whose expression are reported to be altered in β-cells during enteroviral infection. Human and rodent islet cells were shown to express receptors for both IL-13 and IL-6, and treatment with either cytokine resulted in the rapid phosphorylation of STAT3 and STAT6. However, while β-cells were protected against a range of cytotoxic insults during exposure to IL-13, treatment with IL-6 enhanced cytotoxicity and western blotting revealed that IL-13 induced one specific isoform of phospho-STAT6 preferentially. Upon incubation with both cytokines together, the isoform of STAT6 that was upregulated by IL-13 alone was again induced, and the effects of IL-6 on β-cell viability were attenuated. Overall, the results suggest that induction of specific isoforms of STAT family transcription factors may underlie the cytoprotective actions of IL-13, and they imply that selective targeting of specific STAT-mediated signaling components could provide a means to ameliorate the loss of β-cell viability in diabetes.
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Richardson SJ, Leete P, Bone AJ, Foulis AK, Morgan NG (2013). Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1.
Diabetologia,
56(1), 185-193.
Abstract:
Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1
Aims/hypothesis: Immunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection. Methods: Antigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA. Results: VP1 immunoreactivity was present in ∼20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1. Conclusions/interpretation: the presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited. © 2012 Springer-Verlag Berlin Heidelberg.
Abstract.
Lind K, Richardson SJ, Leete P, Morgan NG, Korsgren O, Flodström-Tullberg M (2013). Induction of an Antiviral State and Attenuated Coxsackievirus Replication in Type III Interferon Treated Primary Human Pancreatic Islets. Journal of virology
Anagandula M, Richardson SJ, Oberste MS, Sioofy-Khojine A-B, Hyöty H, Morgan NG, Korsgren O, Frisk G (2013). Infection of human islets of langerhans with two strains of coxsackie B virus serotype 1: Assessment of virus replication, degree of cell death and induction of genes involved in the innate immunity pathway. Journal of Medical Virology
Caton PW, Richardson SJ, Kieswich J, Bugliani M, Holland ML, Marchetti P, Morgan NG, Yaqoob MM, Holness MJ, Sugden MC, et al (2013). Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients. Diabetologia, 1-10.
Marhfour I, Lopez XM, Lefkaditis D, Salmon I, Allagnat F, Richardson SJ, Morgan NG, Eizirik DL (2012). Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.
Diabetologia,
55(9), 2417-2420.
Abstract:
Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes
Aims/hypothesis: Endoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children). Methods: Antibodies against the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) were validated using HeLa cells treated with the ER stressor thapsigargin. These antibodies were then used to stain serial sections of paraffin-embedded pancreas from type 1 diabetic and non-diabetic individuals; samples were also immunostained for CD45, insulin and glucagon. Immunostaining intensities of the ER stress markers were quantified using a software-based, unbiased quantitative approach. Results: Islets from individuals with type 1 diabetes showed increased levels of CHOP and, at least for insulitis-positive and beta cell-containing islets, BIP. XBP-1 expression was not, however, increased. Conclusions/interpretation: Islet cells from individuals with type 1 diabetes display a partial ER stress response, with evidence of the induction of some, but not all, components of the unfolded protein response. © 2012 Springer-Verlag.
Abstract.
Lundh M, Christensen DP, Damgaard Nielsen M, Richardson SJ, Dahllöf MS, Skovgaard T, Berthelsen J, Dinarello CA, Stevenazzi A, Mascagni P, et al (2012). Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children. Diabetologia, 1-11.
Stone VM, Dhayal S, Smith DM, Lenaghan C, Brocklehurst KJ, Morgan NG (2012). The cytoprotective effects of oleoylethanolamide in insulin-secreting cells do not require activation of GPR119.
Br J Pharmacol,
165(8), 2758-2770.
Abstract:
The cytoprotective effects of oleoylethanolamide in insulin-secreting cells do not require activation of GPR119.
BACKGROUND AND PURPOSE: β-cells express a range of fatty acid-responsive G protein-coupled receptors, including GPR119, which regulates insulin secretion and is seen as a potential therapeutic target in type 2 diabetes. The long-chain unsaturated fatty acid derivative oleoylethanolamide (OEA) is an endogenous agonist of GPR119 and, under certain conditions, some long-chain unsaturated fatty acids can promote β-cell cytoprotection. It is not known, however, if OEA is cytoprotective in β-cells. The present study has examined this and determined whether GPR119 is involved. METHODS: Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1E, were exposed to fatty acids complexed with BSA. cAMP levels, insulin release and cell viability were measured. Protein expression was studied by Western blotting and receptor expression by RT-PCR. KEY RESULTS: GPR119 was expressed in both BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA per se is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate. CONCLUSIONS AND IMPLICATIONS: Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection.
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Keane DC, Takahashi HK, Dhayal S, Morgan NG, Curi R, Newsholme P (2011). Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line.
Clin Sci (Lond),
120(5), 195-206.
Abstract:
Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line.
Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to β-cell incubations at 100 μM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P
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Russell MA, Morgan NG (2011). Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion.
Clin Sci (Lond),
120(9), 403-413.
Abstract:
Conditional expression of the FTO gene product in rat INS-1 cells reveals its rapid turnover and a role in the profile of glucose-induced insulin secretion.
Common polymorphisms within the FTO (fat mass and obesity-associated) gene correlate with increased BMI (body mass index) and a rising risk of Type 2 diabetes. FTO is highly expressed in the brain but has also been detected in peripheral tissues, including the endocrine pancreas, although its function there is unclear. The aim of the present study was to investigate the role of FTO protein in pancreatic β-cells using a conditional expression system developed in INS-1 cells. INS-1 cells were stably transfected with FTO-HA (haemagluttinin) incorporated under the control of a tetracycline-inducible promoter. Induction of FTO protein resulted in localization of the tagged protein to the nucleus. The level of FTO-HA protein achieved in transfected cells was tightly regulated, and experiments with selective inhibitors revealed that FTO-HA is rapidly degraded via the ubiquitin/proteasome pathway. The nuclear localization was not altered by proteasome inhibitors, although following treatment with PYR-41, an inhibitor of ubiquitination, some of the protein adopted a perinuclear localization. Unexpectedly, modestly increased expression of FTO-HA selectively enhanced the first phase of insulin secretion when INS-1 monolayers or pseudoislets were stimulated with 20 mM glucose, whereas the second phase remained unchanged. The mechanism responsible for the potentiation of glucose-induced insulin secretion is unclear; however, further experiments revealed that it did not involve an increase in insulin biosynthesis or any changes in STAT3 (signal transducer and activator of transcription 3) expression. Taken together, these results suggest that the FTO protein may play a hitherto unrecognized role in the control of first-phase insulin secretion in pancreatic β-cells.
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Reers C, Hauge-Evans AC, Morgan NG, Willcox A, Persaud SJ, Jones PM (2011). Downregulation of proliferation does not affect the secretory function of transformed β-cell lines regardless of their anatomical configuration.
Islets,
3(3), 80-88.
Abstract:
Downregulation of proliferation does not affect the secretory function of transformed β-cell lines regardless of their anatomical configuration
Aims and objectives: Proliferation in transformed β-cell lines is high compared to primary islet cells and is accompanied by reduced insulin content and release. Our aim was to determine whether experimental reduction of proliferation restores the cells to a more authentic β-cell phenotype in terms of secretory function and to investigate the potential beneficial effect of their configuration as islet-like structures. Results: Mitosis inhibitor mitomycin c (MMC) treatment neither altered the rate of proliferation nor improved the secretory responses of MIN6 monolayer cells. The proliferative rate of MIN6 cells was not affected by pseudoislet formation, but in contrast to monolayer cells, pseudoislets responded to 20 mM glucose with a 2.6-fold increase in insulin secretion. MMC reduced proliferation in MIN6 pseudoislets, but did not further improve their secretory responsiveness. Withdrawal of doxycycline resulted in complete growth-arrest in R7T1 cells, but monolayer and pseudoislet R7T1 cells were unresponsive to glucose and remained so upon growth-arrest although insulin content was increased in growth-arrested pseudoislets. Methods: MIN6 monolayer and pseudoislet cells were treated with MMC whereas growth-arrest was induced in R7T1 monolayer and pseudoislet cells by withdrawal of doxycycline. Proliferation rates were determined by immunocytochemical measurements of BrdU incorporation and insulin secretion was assessed by radioimmunoassay. Conclusions: Secretory function of transformed β-cells is not influenced by experimental reduction of proliferation, but can be modulated by enhanced cell-cell contact within islet-like structures. These results have implications for future studies of islet cell redifferentiation and for the generation of islet-like material for transplantation therapy in Type 1 diabetes. ©2011 Landes Bioscience.
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Johnstone KA, Diakogiannaki E, Dhayal S, Morgan NG, Harries LW (2011). Dysregulation of Hnf1b gene expression in cultured beta-cells in response to cytotoxic fatty acid.
JOP,
12(1), 6-10.
Abstract:
Dysregulation of Hnf1b gene expression in cultured beta-cells in response to cytotoxic fatty acid.
CONTEXT: Increased levels of circulating fatty acids deriving from over-nutrition are thought to contribute to the progressive beta-cell failure associated with type 2 diabetes. Pancreatic beta-cells in culture are sensitive to exposure to long-chain saturated fatty acids (e.g. palmitate) which cause cytotoxicity, whereas the monounsaturated equivalents (e.g. palmitoleate) are cytoprotective. OBJECTIVES: in this study we sought to determine whether of members of the hepatocyte nuclear factor (HNF) family of transcription factors, which are mutated in familial, young-onset, monogenic beta-cell diabetes, could play a role in fatty acid-mediated cytotoxicity in cultured beta-cells. DESIGN: We used real-time PCR to determine whether hepatocyte nuclear factor gene expression was altered in response to palmitate exposure in the BRIN-BD11 beta-cell line. RESULTS: We found that the Hnf isoforms expressed in BRIN-BD11 cells are dysregulated by palmitate exposure. The expression of Hnf1b is specifically reduced by exposure to palmitate, and this response is prevented by co-incubation with palmitoleate. CONCLUSIONS: Down-regulation of Hnf1b gene expression accompanies palmitate-mediated cytotoxicity in cultured beta-cells.
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Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG (2011). Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes.
Diabetologia,
54(9), 2417-2420.
Abstract:
Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes.
AIMS/HYPOTHESIS: the enteroviral capsid protein, VP1, was recently shown to be present in some beta cells in more than 60% of patients with recent-onset type 1 diabetes but in very few age-matched controls. The rate of proliferation of islet cells was also markedly increased in the type 1 diabetic patients. As it has been suggested that enteroviruses replicate most efficiently in proliferating cells, we have investigated whether VP1 is preferentially present in proliferating beta cells in type 1 diabetes. METHODS: Combined immunoperoxidase and immunofluorescence staining was used to record the presence of enteroviral VP1, insulin and Ki67 in the islets of recent-onset type 1 diabetic patients. RESULTS: from a total of 1,175 islets, 359 (30.5%) contained insulin. VP1-producing endocrine cells were found in 72 islets (6.1% of total), all of which retained insulin. Ki67(+) endocrine cells were present in 52 (4.4%) islets, with 44 (84.6%) of these being insulin-positive. Overall, 28 of 1,175 (2.4%) islets contained both Ki67(+) cells and VP1(+) cells. Dual positivity of these markers accounted for 38.9% of the total VP1(+) islets and 53.8% of the total Ki67(+) islets. No individual islet cells were dual-positive for Ki67 and VP1. CONCLUSIONS/INTERPRETATION: Ki67(+) cells were frequently observed in islets that also contained VP1(+) cells, suggesting that the factors facilitating viral replication may also drive islet cell proliferation. However, in an individual cell, VP1 production does not require concurrent beta cell proliferation.
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Richardson SJ, Willcox A, Bone AJ, Morgan NG, Foulis AK (2011). Immunopathology of the human pancreas in type-I diabetes.
Semin Immunopathol,
33(1), 9-21.
Abstract:
Immunopathology of the human pancreas in type-I diabetes.
Type 1 diabetes is a chronic autoimmune disease characterised by the selective destruction of pancreatic beta (β) cells. The understanding of the aetiology of this disease has increased dramatically in recent years by the study of tissue recovered from patients, from analysis of the responses of isolated islet and β-cells in tissue culture and via the use of animal models. However, knowledge of the immunopathology of type 1 diabetes in humans is still relatively deficient due largely to the difficulty of accessing appropriate samples. Here we review the state of current knowledge in relation to the histopathological features of the disease in humans. We focus specifically on recent-onset type 1 diabetes cases since in such patients, evidence of the ongoing disease process is still present. We chart the progression of the disease by describing the characteristic features of the pancreas, consider the sequence of immune cell infiltration and discuss the abnormalities of MHC antigen expression. The possibility that these changes might derive from a persistent enteroviral infection of the islet beta cells is examined.
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Dhayal S, Morgan NG (2011). Pharmacological characterization of the cytoprotective effects of polyunsaturated fatty acids in insulin-secreting BRIN-BD11 cells.
Br J Pharmacol,
162(6), 1340-1350.
Abstract:
Pharmacological characterization of the cytoprotective effects of polyunsaturated fatty acids in insulin-secreting BRIN-BD11 cells.
BACKGROUND AND PURPOSE: Free fatty acids are important metabolic fuels for mammalian cells but, recently, it has become clear that they can also fulfil signalling functions, which are independent of their metabolic fate. We are investigating the ability of unsaturated free fatty acids to exert a cytoprotective response during exposure of insulin-secreting cells to toxic stimuli. The majority of earlier studies have focussed on monounsaturated fatty acids but this has now been extended to define the structural requirements of the cytoprotective effects of polyunsaturated species. EXPERIMENTAL APPROACH: Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1, were exposed to fatty acids or their derivatives complexed with BSA and the viability of the cells was analysed by flow cytometry after staining with propidium iodide. KEY RESULTS: a variety of polyunsaturated fatty acids with chain lengths between C18-C22 attenuated the cytotoxic actions of the saturated fatty acid, palmitate (C16:0) in BRIN-BD11 and INS-1 cells. These effects were dose-dependent and displayed potencies that were much higher than those achieved with monounsaturated fatty acids. Methyl esters of the polyunsaturates were also effective. The cytoprotective responses were not altered by incubation of cells with inhibitors of cyclooxygenase or lipoxygenase enzymes although they were antagonized dose-dependently by arachidonyltrifluoromethylketone (AACOCF(3)). CONCLUSIONS AND IMPLICATIONS: the results are consistent with the involvement of a specific fatty acid binding site having loose, but defined, structural criteria, in mediating the cytoprotective effects of unsaturated fatty acids. AACOCF(3) may be of value in defining this site in molecular terms.
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Dhayal S, Morgan NG (2011). Structure-activity relationships influencing lipid-induced changes in eIF2α phosphorylation and cell viability in BRIN-BD11 cells.
FEBS Lett,
585(14), 2243-2248.
Abstract:
Structure-activity relationships influencing lipid-induced changes in eIF2α phosphorylation and cell viability in BRIN-BD11 cells.
Fatty acids influence the viability of eukaryotic cells differentially such that long chain saturated molecules are poorly tolerated, whereas unsaturated species are less detrimental and can be cytoprotective. The basis for these effects is unclear but studies in yeast imply that they reflect the spatial configuration of the molecules when incorporated into the ER membrane. Using BRIN-BD11 β-cells, we show that a wide range of unsaturated free fatty acids and their methyl-esters (having differing chain length and disposition of the double bonds) elicit cytoprotection and relief of protein kinase RNA-like endoplasmic reticulum kinase-dependent ER stress. Thus, both physical properties and specific signalling events may regulate fatty acid responses in β-cells.
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Lidder P, Flanagan D, Fleming S, Russell M, Morgan N, Wheatley T, Rahamin J, Shaw S, Lewis S (2010). Combining enteral with parenteral nutrition to improve postoperative glucose control.
Br J Nutr,
103(11), 1635-1641.
Abstract:
Combining enteral with parenteral nutrition to improve postoperative glucose control.
The provision of parenteral nutrition (PN) to 'stressed' patients often results in hyperglycaemia, which may be detrimental. In animal models limited amounts of enteral nutrition (EN) improve intestinal integrity and stimulate intestinal incretin production, which may lead to improved glucose control. We set out to assess if combining EN with PN results in improved glucose homeostasis rather than PN given alone. We conducted a randomised trial in a university teaching hospital of patients undergoing a 'curative' oesophagectomy for adenocarcinoma. Differences between the two intervention groups were assessed for continuous glucose measurement, insulin sensitivity using insulin tolerance tests (ITT) and homeostasis model analysis (HOMA), the incretin glucose-dependent insulinotropic polypeptide (GIP) and intestinal permeability. The combination of PN with EN resulted in lower interstitial glucose concentrations (P = 0.002), reduced insulin resistance, improved insulin sensitivity (HOMA-insulin resistance (IR) P = 0.045; HOMA beta P = 0.037; ITT P = 0.006), improved intestinal permeability (P < 0.001) and increased GIP (P = 0.01) when compared with PN alone. The combination of EN with PN, when compared with PN alone, results in reduced glucose concentrations, reduced insulin resistance, increased incretins and improvements in intestinal permeability.
Abstract.
Author URL.
Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG (2010). Evidence of increased islet cell proliferation in patients with recent-onset type 1 diabetes. Diabetologia, 53, 2020-2028.
Russell MA, Morgan NG (2010). Expression and functional roles of guanylate cyclase isoforms in BRIN-BD11 β-cells.
Islets,
2(6), 374-382.
Abstract:
Expression and functional roles of guanylate cyclase isoforms in BRIN-BD11 β-cells.
This study has assessed the expression and functional significance of cGMP-dependent signalling components in BRIN-BD11 cells. RT-PCR analysis revealed the expression of two subunits of soluble guanylate cyclase (sGC) suggesting the presence of an α2/β1 heterodimer. The expression of three particulate guanylate cyclases (pGC) was also detected (GC-A, GC-B and GC-C), as well as two cGMP-selective PDE isoforms (PDE5A and PDE9). Stimulation of BRIN-BD11 cells with agonists selective for sGC (NO, YC-1 and BAY 41-2272), GC-A (atrial natriuretic peptide (ANP) or GC-C (guanylin) caused an elevation in cGMP, and in the case of sGC, this was blocked by the selective inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). The stimulatory effects of each activator on cGMP levels were further potentiated by the PDE5A inhibitor, zaprinast. Treatment of cells with sGC activators induced a loss of viability and increased insulin secretion. However these effects were not attenuated by ODQ suggesting that they were independent of a rise in cGMP. A modest increase in β-cell death and insulin secretion were also observed in guanylin and ANP treated cells, although the latter only reduced cell viability in the presence of a PDE5A inhibitor. Taken together, the data reveal that BRIN-BD11 cells express several functionally active enzymes capable of modulating cGMP levels, and they imply that signalling through these proteins may impact upon β-cell viability. The results further suggest that pGC isozymes can also regulate insulin secretion but that the pool of cGMP controlling insulin release is small relative to the global cGMP concentration in the cell.
Abstract.
Author URL.
Persaud SJ, Arden C, Bergsten P, Bone AJ, Brown J, Dunmore S, Harrison M, Hauge-Evans A, Kelly C, King A, et al (2010). Pseudoislets as primary islet replacements for research: Report on a symposium at King's College London, London UK.
Islets,
2(4), 236-239.
Abstract:
Pseudoislets as primary islet replacements for research: Report on a symposium at King's College London, London UK
Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed 'pseudoislets' largely recapitulates the function of primary islet β-cells. the Diabetes research Group at King's College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on "pseudoislets as primary islet replacements for research", which was funded by the UK National Centre for the replacement, refinement and reduction of Animals in research (NC3rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. this short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research. © 2010 Landes Bioscience.
Abstract.
Garin I, Edghill EL, Akerman I, Rubio-Cabezas O, Rica I, Locke JM, Maestro MA, Alshaikh A, Bundak R, Del Castillo G, et al (2010). Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis.
Proceedings of the National Academy of Sciences of the United States of America,
107(7), 3105-3110.
Abstract:
Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis
Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, andalteredmRNAstability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (-3.2 SD score vs.-2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.
Abstract.
Dhayal S, Morgan NG (2010). The significance of GPR119 agonists as a future treatment for type 2 diabetes.
Drug News Perspect,
23(7), 418-424.
Abstract:
The significance of GPR119 agonists as a future treatment for type 2 diabetes.
GPR119 is a G protein-coupled receptor that is expressed on only a limited number of tissues, including pancreatic β-cells and enteroendocrine cells in the small intestine, and that appears to be involved in the regulation of metabolic homeostasis. The protein was originally defined as an orphan receptor, but it has subsequently been shown to bind a variety of lipid-derived ligands, as well as a range of small synthetic molecules. There is still debate as to the identity of its principal endogenous ligand, but certain lysophospholipids species, various fatty acyl-ethanolamides and N-oleoyldopamine have all been proposed as potential agonists. GPR119 is coupled to the signal transducer Gαs and activation of the receptor leads to increased adenylate cyclase activity via Gαs and a rise in intracellular cAMP. This then potentiates glucose-induced insulin secretion or promotes the release of intestinal incretin hormones, according to cell type. Both mechanisms ultimately lead to a rise in insulin secretion (either directly or indirectly) and improved glucose control. Thus, GPR119 may represent an important new therapeutic target for the design of insulin secretagogues able to promote improvements in blood glucose control in patients with type 2 diabetes. Accordingly, a range of lead compounds are in development as potential therapeutic agents.
Abstract.
Author URL.
Morgan NG, Dhayal S (2010). Unsaturated fatty acids as cytoprotective agents in the pancreatic beta-cell.
Prostaglandins Leukot Essent Fatty Acids,
82(4-6), 231-236.
Abstract:
Unsaturated fatty acids as cytoprotective agents in the pancreatic beta-cell.
It is widely accepted that, in type 2 diabetes, elevated levels of free fatty acids and glucose contribute to a state of glucolipotoxicity in which beta-cell function declines and, ultimately, cell viability is compromised. This suggests that beta-cells do not readily tolerate chronic elevations in fatty acid levels. In vitro studies suggest, however, that beta-cells respond differentially to long chain fatty acids, such that saturated species are lipotoxic whereas long chain mono-unsaturated fatty acids can provide cytoprotection. This difference does not appear to be mediated by a mutual metabolic antagonism between saturated and unsaturated species (although differential alterations in neutral lipid disposition may occur in response to these fatty acids) and the mechanisms remain unclear. This review summaries the current understanding of the actions of mono-unsaturated fatty acids in beta-cells and highlights areas of controversy as well as key unresolved issues which require to be addressed.
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Author URL.
Richardson SJ, Willcox A, Hilton DA, Tauriainen S, Hyoty H, Bone AJ, Foulis AK, Morgan NG (2010). Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue.
J Clin Virol,
49(3), 180-185.
Abstract:
Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue.
BACKGROUND: the detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. OBJECTIVES: the aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. STUDY DESIGN: dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. RESULTS: Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. CONCLUSIONS: the dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.
Abstract.
Author URL.
Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG (2009). Analysis of islet inflammation in human type 1 diabetes.
Clin Exp Immunol,
155(2), 173-181.
Abstract:
Analysis of islet inflammation in human type 1 diabetes.
The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post-mortem from 29 patients (mean age 11.7 years) with recent-onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8(+) cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68(+)) were also present during both early and later insulitis, although in fewer numbers. CD20(+) cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138(+) plasma cells were infrequent at all stages of insulitis. CD4(+) cells were present in the islet infiltrate in all patients but were less abundant than CD8(+) or CD68(+) cells. Forkhead box protein P3(+) regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8(+) cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20(+) cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3(+) T cells do not appear to be required for beta cell death.
Abstract.
Author URL.
Morgan NG (2009). Fatty acids and beta-cell toxicity.
Curr Opin Clin Nutr Metab Care,
12(2), 117-122.
Abstract:
Fatty acids and beta-cell toxicity.
PURPOSE OF REVIEW: the rising incidence of type 2 diabetes is due, in part, to the detrimental effects of certain fatty acids on pancreatic beta-cell function and viability. The present review examines recent advances in the understanding of the molecular mechanisms by which fatty acids influence the life and death of beta cells. RECENT FINDINGS: There are important differences in the cytotoxic potential of fatty acids, with long-chain saturated molecules being the most potent. By contrast, monounsaturates and polyunsaturates are relatively well tolerated and, in some cases, are actively cytoprotective. The mechanisms underlying the toxicity of the saturates may reflect a decrease in protein processing, which drives the accumulation of unfolded proteins in the endoplasmic reticulum. This triggers an apoptotic response by virtue of enhanced endoplasmic reticulum stress and induction of CHOP-10 synthesis. Alterations in the regulatory control of other proapoptotic genes via changes in microRNA synthesis may also contribute. The cytoprotection deriving from incubation with long-chain mono-unsaturates is probably receptor mediated and involves antagonistic actions on the effector arm of the endoplasmic reticulum stress pathway. SUMMARY: the findings have implications for the development of new therapeutic agents designed to minimize beta-cell dysfunction and the loss of beta-cell viability in type 2 diabetes.
Abstract.
Author URL.
Morgan NG, Dhayal S (2009). G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell.
Biochem Pharmacol,
78(12), 1419-1427.
Abstract:
G-protein coupled receptors mediating long chain fatty acid signalling in the pancreatic beta-cell.
It is increasingly clear that some of the effects of both free and derivatised long chain fatty acids in pancreatic beta-cells are mediated by a group of G-protein coupled receptors. Some of these display close structural homology while others are more divergent. This Commentary reviews the expression and functional roles of three such molecules, GPR40, GPR119 and GPR120. GPR40 is the best characterised of this group and appears to mediate the acute stimulatory effects of long chain fatty acids on insulin secretion. GPR40 has also been proposed as a potential mediator of fatty acid toxicity but this is more controversial. GPR119 is also involved in stimulation of insulin secretion and responds primarily to ethanolamide derivatives of long chain fatty acids and also to some lysophospholipids rather than to free fatty acids. It may represent a useful target for the development of new insulin secretagogues aimed to enhance insulin release in patients with type 2 diabetes. GPR120 is the most enigmatic of the lipid-responsive cell-surface receptors and its function remains to be established. It has been proposed to play a cytoprotective role in certain other cell types but it is unclear whether it fulfils a similar function in beta-cells.
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Kaminski A, Welters HJ, Kaminski ER, Morgan NG (2009). Human and rodent pancreatic beta-cells express IL-4 receptors and IL-4 protects against beta-cell apoptosis by activation of the PI3K and JAK/STAT pathways.
Biosci Rep,
30(3), 169-175.
Abstract:
Human and rodent pancreatic beta-cells express IL-4 receptors and IL-4 protects against beta-cell apoptosis by activation of the PI3K and JAK/STAT pathways.
Secretion of pro-inflammatory cytokines is associated with loss of pancreatic beta-cell viability and cell death. IL-4 (interleukin-4) has been reported to mediate a protective effect against the loss of pancreatic beta-cells, and IL-4 receptors have been found in rat pancreatic beta-cells at both the RNA and the protein level. The aim of the present study was to investigate IL-4 receptor expression in human islet cells and to examine the signalling pathways by which IL-4 exerts its effects using the rat beta-cell lines, BRIN-BD11 and INS-1E. By means of immunohistochemistry, it was demonstrated that IL-4 receptors are present on human islet cells. Using a flow cytometric method for evaluating cell death, it was confirmed that incubating beta-cells with IL-4 attenuated cell death induced by IL-1beta and interferon-gamma by approx. 65%. This effect was abrogated by the presence of the PI3K (phosphoinositide 3-kinase) inhibitor, wortmannin, suggesting that activation of the PI3K pathway is involved. In support of this, Western blotting revealed that incubation of cells with IL-4 resulted in increased phosphorylation of Akt (also called protein kinase B), a downstream target of PI3K. Increased tyrosine phosphorylation of STAT6 (signal transducer and activator of transcription 6) also occurred in response to IL-4 and a selective JAK3 (Janus kinase 3) inhibitor reduced the cytoprotective response. Both effects were prevented by overexpression of the tyrosine phosphatase, PTP-BL (protein tyrosine phosphatase-BL). We conclude that IL-4 receptors are functionally competent in pancreatic beta-cells and that they signal via PI3K and JAK/STAT pathways. These findings may have implications for future therapeutic strategies for the management of diabetes.
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Author URL.
Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG (2009). Islet-associated macrophages in type 2 diabetes.
Diabetologia,
52(8), 1686-1688.
Author URL.
Morgan NG, Diakogiannaki E, Russell MA (2009). The incubation and monitoring of cell viability in primary rat islets of Langerhans and pancreatic beta-cell lines.
Methods Mol Biol,
560, 53-64.
Abstract:
The incubation and monitoring of cell viability in primary rat islets of Langerhans and pancreatic beta-cell lines.
This chapter describes a method to measure the viability of isolated intact islets of Langerhans from rat pancreas and considers the use of isolated islets and of pancreatic beta-cell lines to study cell viability following culture. The islet isolation method is based on the use of preparations of collagenase to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding acini. The islets can be obtained in relatively pure form and are suitable for use in hormone secretion assays as well as for measurement of cell viability. They can be cultured if required and viability maintained for periods of days to weeks. Beta-cell lines are useful for study of the control of cell viability although their secretory capacity is usually altered compared to primary islet cells. Islet cell death can be estimated in a number of ways using either direct or more indirect means. Some methods will distinguish between apoptotic and necrotic death while others offer a more generic index of changes in viability.
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Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG (2009). The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.
Diabetologia,
52(6), 1143-1151.
Abstract:
The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.
AIMS/HYPOTHESIS: Evidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls. METHODS: Serial sections of paraffin-embedded pancreatic autopsy samples from 72 recent-onset type 1 diabetes patients and up to 161 controls were immunostained for insulin, glucagon, vp1, double-stranded RNA activated protein kinase R (PKR) and MHC class I. RESULTS: vp1-immunopositive cells were detected in multiple islets of 44 out of 72 young recent-onset type 1 diabetic patients, compared with a total of only three islets in three out of 50 neonatal and paediatric normal controls. vp1 staining was restricted to insulin-containing beta cells. Among the control pancreases, vp1 immunopositivity was also observed in some islets from ten out of 25 type 2 diabetic patients. A strong correlation was established between islet cell vp1 positivity and PKR production in insulin-containing islets of both type 1 and type 2 diabetic patients, consistent with a persistent viral infection of the islets. CONCLUSIONS/INTERPRETATION: Immunoreactive vp1 is commonly found in the islets of recent-onset type 1 diabetes patients, but only rarely in normal paediatric controls. vp1 immunostaining was also observed in some islets of type 2 diabetes patients, suggesting that the phenomenon is not restricted to type 1 diabetes patients.
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Diakogiannaki E, Welters HJ, Morgan NG (2008). Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids.
J Endocrinol,
197(3), 553-563.
Abstract:
Differential regulation of the endoplasmic reticulum stress response in pancreatic beta-cells exposed to long-chain saturated and monounsaturated fatty acids.
Exposure of pancreatic beta-cells to long-chain fatty acids leads to the activation of some components of the endoplasmic reticulum (ER) stress pathway and this mechanism may underlie the ability of certain fatty acids to promote beta-cell death. We have studied ER stress in BRIN-BD11 beta-cells exposed to either the saturated fatty acid palmitate (C16:0) or the monounsaturated palmitoleate (C16:1). Palmitate (0.025-0.25 mM) induced the expression of various markers of the RNA-dependent protein kinase-like ER eukaryotic initiation factor 2 alpha (eIF2 alpha) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2 alpha; ATF4, activating transcription factor 4 and C/EBP homologous protein (CHOP-10)) although it failed to promote the expression of the ER chaperone GRP78. By contrast, palmitoleate did not induce any markers of the ER stress pathway even at concentrations as high as 1 mM. When palmitate and palmitoleate were added in combination, a marked attenuation of the ER stress response occurred. Under these conditions, the levels of phospho-eIF2 alpha, ATF4 and CHOP-10 were reduced to less than those found in control cells. Palmitoleate also attenuated the ER stress response to the protein glycosylation inhibitor, tunicamycin, and improved the viability of the cells exposed to this agent. Exposure of the BRIN-BD11 cells to the protein phosphatase inhibitor, salubrinal, in the absence of fatty acids resulted in increased eIF2 alpha phosphorylation but this was abolished by co-incubation with palmitoleate. We conclude that saturated fatty acids activate components of the PERK-dependent ER stress pathway in beta-cells, ultimately leading to increased apoptosis. This effect is antagonised by monounsaturates that may exert their anti-apoptotic actions by regulating the activity of one or more kinase enzymes involved in mediating the phosphorylation of eIF2 alpha.
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Author URL.
Farn RD, Morgan NG, Ramsden CA (2008). Preparation of analogues of efaroxan and KU14R as potential imidazoline receptor subtype 3 ligands.
Journal of Heterocyclic Chemistry,
45(3), 887-896.
Abstract:
Preparation of analogues of efaroxan and KU14R as potential imidazoline receptor subtype 3 ligands
(Chemical Equation Presented) the preparation and characterization of new analogues of the imidazoline insulin secretagogue efaroxan 1 are described. These include 1,2,4-triazole, 1,3,4-oxadiazole, pyrimidine, 1,4,5,6- tetrahydropyrimidine and 1,2-dihydro-1,2,4,5-tetrazine analogues. Bromination of 2,3-dihydro-2-ethylbenzo[b]furan-2-carbonitrile 19 gives the 3,3-dibromo analogue 28, which is readily hydrolysed to the corresponding ketone 29. Reduction of this ketone gives the alcohol 31 as a mixture of diastereoisomers that is converted to the fluoride 32 using diethylaminosulfur trifluoride.
Abstract.
Ravanan R, Wong SF, Morgan NG, Mathieson PW, Smith RM (2007). Inhalation of glutamic acid decarboxylase 65-derived peptides can protect against recurrent autoimmune but not alloimmune responses in the non-obese diabetic mouse.
Clin Exp Immunol,
148(2), 368-372.
Abstract:
Inhalation of glutamic acid decarboxylase 65-derived peptides can protect against recurrent autoimmune but not alloimmune responses in the non-obese diabetic mouse.
Systemic administration of islet-derived antigens has been shown to protect against diabetes in the non-obese diabetic (NOD) mouse by the induction of antigen-specific regulatory T cells. Bystander regulation to related and unrelated islet-derived antigens (intramolecular and intermolecular recognition) in this context is recognized. We tested if intranasal administration of glutamic acid decarboxylase 65 (GAD 65)-derived peptides could protect against both autoimmune and, through bystander regulation, alloimmune responses in a NOD mouse model. Spontaneously diabetic female NOD mice underwent islet transplantation from either C57Bl/6 or NOD islet donors. Islet recipients were treated with intranasal GAD 65-derived peptides or control (ovalbumin) peptide pre- and post-transplantation. In-vitro analysis of the effect of inhalation was defined using lymph node proliferation assays and supernatant analysis for cytokines. GAD 65-derived peptide inhalation resulted in significant protection against recurrent autoimmune disease, with the generation of an interleukin (IL)-10-producing immune phenotype in a syngeneic islet transplant model. This phenotype, however, was not robust enough to protect against alloimmune responses. Inhalation of GAD-derived peptides induces an immunoregulatory response that protects against recurrent autoimmune, but not alloimmune responses in the NOD mouse.
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Author URL.
Newsholme P, Keane D, Welters HJ, Morgan NG (2007). Life and death decisions of the pancreatic beta-cell: the role of fatty acids.
Clin Sci (Lond),
112(1), 27-42.
Abstract:
Life and death decisions of the pancreatic beta-cell: the role of fatty acids.
Both stimulatory and detrimental effects of NEFAs (non-esterified fatty acids) on pancreatic beta-cells have been recognized. Acute exposure of the pancreatic beta-cell to high glucose concentrations and/or saturated NEFAs results in a substantial increase in insulin release, whereas chronic exposure results in desensitization and suppression of secretion, followed by induction of apoptosis. Some unsaturated NEFAs also promote insulin release acutely, but they are less toxic to beta-cells during chronic exposure and can even exert positive protective effects. Therefore changes in the levels of NEFAs are likely to be important for the regulation of beta-cell function and viability under physiological conditions. In addition, the switching between endogenous fatty acid synthesis or oxidation in the beta-cell, together with alterations in neutral lipid accumulation, may have critical implications for beta-cell function and integrity. Long-chain acyl-CoA (formed from either endogenously synthesized or exogenous fatty acids) controls several aspects of beta-cell function, including activation of specific isoenzymes of PKC (protein kinase C), modulation of ion channels, protein acylation, ceramide formation and/or NO-mediated apoptosis, and transcription factor activity. In this review, we describe the effects of exogenous and endogenous fatty acids on beta-cell metabolism and gene and protein expression, and have explored the outcomes with respect to insulin secretion and beta-cell integrity.
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Diakogiannaki E, Dhayal S, Childs CE, Calder PC, Welters HJ, Morgan NG (2007). Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic beta-cells.
J Endocrinol,
194(2), 283-291.
Abstract:
Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic beta-cells.
Long-chain saturated and monounsaturated fatty acids differ in their propensity to induce beta-cell death in vitro with palmitate (C16:0) being cytotoxic, whereas palmitoleate (C16:1n-7) is cytoprotective. We now show that this cytoprotective capacity extends to a poorly metabolised C16:1n-7 derivative, methyl-palmitoleate (0.25 mM palmitate alone: 92 +/- 4% death after 18 h; palmitate plus 0.25 mM methyl-palmitoleate: 12 +/- 2%; P < 0.001). Palmitoleate and its methylated derivative also acted as mitogens in cultured beta-cells (5-bromo-2-deoxyuridine incorporation - control: 0.15 +/- 0.01 units; 0.25 mM palmitoleate: 0.22 +/- 0.01 units; P < 0.05). It has been proposed that alterations in neutral lipid synthesis (particularly triacylglycerol (TAG) formation) might mediate the differential responses to saturated and unsaturated fatty acids and we have examined this proposition. Palmitate and palmitoleate both promoted beta-cell phospholipid remodelling and increased TAG formation (control: 0.9 +/- 0.1 nmol TAG/10(6) cells; 0.25 mM palmitate: 1.55 +/- 0.07; 0.25 mM palmitoleate: 1.4 +/- 0.05; palmitate plus palmitoleate: 2.3 +/- 0.1). By contrast, methyl-palmitoleate failed to influence TAG levels (0.25 mM methyl-palmitoleate alone: 0.95 +/- 0.06 nmol TAG/10(6) cells; methyl-palmitoleate plus palmitate: 1.5 +/- 0.05) or its fatty acid composition in beta-cells exposed to palmitate. The results suggest that monounsaturated fatty acids can promote cell viability and mitogenesis by a mechanism that does not require their metabolism and is independent of alterations in TAG formation.
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Author URL.
Kaminski A, Kaminski ER, Morgan NG (2007). Pre-incubation with interleukin-4 mediates a direct protective effect against the loss of pancreatic beta-cell viability induced by proinflammatory cytokines.
Clin Exp Immunol,
148(3), 583-588.
Abstract:
Pre-incubation with interleukin-4 mediates a direct protective effect against the loss of pancreatic beta-cell viability induced by proinflammatory cytokines.
Loss of pancreatic beta-cells in type I diabetes is associated with an increase in T helper 1 (Th1) proinflammatory cytokines in the islet milieu, with a concomitant reduction in Th2 anti-inflammatory cytokines. In animal models, manoeuvres designed to polarize Th1 responses towards Th2, particularly involving interleukin (IL)-4, have been shown to protect against insulitis and diabetes. The aim of this study was to determine whether IL-4 can exert a direct effect on beta-cell viability. The rat pancreatic beta-cell line, BRIN-BD11, was used. IL-4R mRNA expression was assayed by reverse transcription-polymerase chain reaction and DNA sequencing and protein expression measured using anti-IL-4R antibodies and confocal microscopy. Cells were pretreated in vitro with IL-4, incubated with IL-1beta and interferon (IFN)-gamma and DNA fragmentation and nitrite production analysed by flow cytometry and Griess assay, respectively. Expression of type I (IL-4R alpha and common gamma-chain) and type II (IL-4R alpha, IL-13R alpha-1) IL-4R mRNA transcripts, together with cell surface expression of IL-4R, was demonstrated. Pre-incubation with IL-4 reduced significantly cell death induced by IL-1beta alone or by a combination of IL-1beta and IFN-gamma, although this was not accompanied by a reduced production of nitrite. The protective effect of IL-4 was not seen when all three cytokines were added simultaneously. These results demonstrate, for the first time, expression of IL-4 receptor components on rat pancreatic beta-cells and reveal a direct protective effect on the loss of viability mediated by proinflammatory cytokines when beta-cells are pre-incubated with IL-4.
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Author URL.
Diakogiannaki E, Welters HJ, Eickelmann P, Morgan NG (2007). The unsaturated fatty acid, palmitoleate, inhibits the expression of the ER-stress-associated pro-apoptotic factor CHOP-10/GADD153 in pancreatic beta-cells.
DIABETIC MEDICINE,
24, 10-11.
Author URL.
Tarasov AI, Welters HJ, Senkel S, Ryffel GU, Hattersley AT, Morgan NG, Ashcroft FM (2006). A Kir6.2 mutation causing neonatal diabetes impairs electrical activity and insulin secretion from INS-1 beta-cells.
Diabetes,
55(11), 3075-3082.
Abstract:
A Kir6.2 mutation causing neonatal diabetes impairs electrical activity and insulin secretion from INS-1 beta-cells.
ATP-sensitive K(+) channels (K(ATP) channels) couple beta-cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on beta-cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wild-type Kir6.2 had no effect on the ATP sensitivity of the K(ATP) channel, whole-cell K(ATP) current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced K(ATP) channel ATP sensitivity (the half-maximal inhibitory concentration increased from approximately 20 mumol/l to approximately 2 mmol/l), and the metabolic substrate methyl succinate failed to close K(ATP) channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the K(ATP) current and hyperpolarizing the beta-cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between K(ATP) current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion.
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Author URL.
Welters HJ, Senkel S, Klein-Hitpass L, Erdmann S, Thomas H, Harries LW, Pearson ER, Bingham C, Hattersley AT, Ryffel GU, et al (2006). Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.
J Endocrinol,
190(1), 171-181.
Abstract:
Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells.
Mutations in the gene encoding hepatocyte nuclear factor (HNF)1beta result in maturity-onset diabetes of the young-(MODY)5, by impairing insulin secretory responses and, possibly, by reducing beta-cell mass. The functional role of HNF1beta in normal beta-cells is poorly understood; therefore, in the present study, wild-type (WT) HNF1beta, or one of two naturally occurring MODY5 mutations (an activating mutation, P328L329del, or a dominant-negative form, A263insGG) were conditionally expressed in the pancreatic beta-cell line, insulin-1 (INS-1), and the functional consequences examined. Surprisingly, overexpression of the dominant-negative mutant did not modify any of the functional properties of the cells studied (including insulin secretion, cell growth and viability). By contrast, expression of WT HNF1beta was associated with a time- and dose-dependent inhibition of INS-1 cell proliferation and a marked increase in apoptosis. Induction of WT HNF1beta also inhibited the insulin secretory response to nutrient stimuli, membrane depolarisation or activation of protein kinases a and C and this correlated with a significant decrease in pancrease-duodenum homeobox-1 protein levels. The attenuation of insulin secretion was, however, dissociated from the inhibition of proliferation and loss of viability, since expression of the P328L329del mutant led to a reduced rate of cell proliferation, but failed to induce apoptosis or to alter insulin secretion. Taken together, the present results suggest that mature rodent beta-cells are sensitive to increased expression of WT HNF1beta and they imply that the levels of this protein are tightly regulated to maintain secretory competence and cell viability.
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NMorgan, Diakogiannaki E, Mordue J, Welters H (2006). Differential protective effects of palmitoleic acid and cAMP on caspase activation and cell viability in pancreatic b-cells exposed to pamitate. APOPTOSIS, 11(7), 1231-1238.
Harries LW, Ellard S, Stride A, Morgan NG, Hattersley AT (2006). Isomers of the TCF1 gene encoding hepatocyte nuclear factor-1 alpha show differential expression in the pancreas and define the relationship between mutation position and clinical phenotype in monogenic diabetes.
Hum Mol Genet,
15(14), 2216-2224.
Abstract:
Isomers of the TCF1 gene encoding hepatocyte nuclear factor-1 alpha show differential expression in the pancreas and define the relationship between mutation position and clinical phenotype in monogenic diabetes.
The generation of multiple transcripts by mRNA processing has the potential to moderate differences in gene expression both between tissues and at different stages of development. Where gene function is compromised by mutation, the presence of multiple isoforms may influence the resulting phenotype. Heterozygous mutations in the transcription factor hepatocyte nuclear factor-1 alpha (HNF1A or TCF1 gene) result in early-onset diabetes as a result of pancreatic beta-cell dysfunction. We investigated the expression of the three alternatively processed isoforms of the HNF1A gene and their impact on the phenotype associated with mutations. Real-time PCR demonstrated variation in tissue expression of HNF1A isomers: HNF1A(A), with the lowest transactivation activity compared with the truncated isoforms HNF1A(B) and HNF1A(C), is the major isomer in liver (54%) and kidney (67%) but not in adult pancreas (24%) and islets (26%). However, in fetal pancreas HNF1A(A) is the major transcript (84%), which supports developmental regulation of isomer expression. We examined whether the isomers affected by the mutation altered the diabetes phenotype in 564 subjects with 123 mutations in HNF1A. Mutations that affected only isomer HNF1A(A) (exons 8-10) were diagnosed later (25.5 years) than mutations affecting all three isomers (exons 1-6) (18.0 years) (P=0.006). This first genotype/phenotype relationship described for patients with HNF1A mutations is explained by isomer structure and not by either mutation type or functional domain. We conclude that all three isomers may be critical for beta-cell function and could play a role in both the developing and mature beta cell.
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Author URL.
Taylor JP, Jackson DA, Morgan NG, Chan SLF (2006). Rhes expression in pancreatic beta-cells is regulated by efaroxan in a calcium-dependent process.
Biochem Biophys Res Commun,
349(2), 809-815.
Abstract:
Rhes expression in pancreatic beta-cells is regulated by efaroxan in a calcium-dependent process.
The monomeric G-protein Rhes has been described to be present in pancreatic beta-cells, and a putative role in the control of insulin release has been proposed. Here, we show that treatment of beta-cells with the imidazoline insulin secretagogue efaroxan resulted in a concentration- and time-dependent increase in the expression of Rhes, which peaked after 4h of efaroxan exposure; thereafter, Rhes mRNA levels decreased. Marked stereoselectivity was displayed, with (-)-efaroxan (the selectively insulinotropic enantiomer) being much more effective than (+)-efaroxan at raising Rhes transcript levels. The mechanism by which Rhes gene expression is activated in beta-cells appears to require the influx of extracellular calcium and de novo protein synthesis, and is not directly associated with the release of insulin. The present results confirm our earlier proposal that Rhes is an imidazoline-regulated transcript in pancreatic beta-cells. Studies to understand the role of Rhes as a regulator of beta-cell function are, thus, warranted.
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Author URL.
(2004). 40th EASD Annual Meeting of the European Association for the Study of Diabetes : Munich, Germany, 5-9 September 2004.
Diabetologia,
47(Suppl 1), A1-A464.
Author URL.
Welters HJ, Smith SA, Tadayyon M, Scarpello JHB, Morgan NG (2004). Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells.
J Mol Endocrinol,
32(1), 227-235.
Abstract:
Evidence that protein kinase Cdelta is not required for palmitate-induced cytotoxicity in BRIN-BD11 beta-cells.
Chronic exposure of pancreatic beta-cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of beta-cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the delta isoform of protein kinase C (PKCdelta) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKCdelta, rottlerin. However, activation of PKCdelta with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKCdelta did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKCdelta. Since rottlerin can inhibit various other kinases in addition to PKCdelta, a range of additional kinase inhibitors was also tested. of these, only the putative Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKCdelta is not required for this response.
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Welters HJ, McBain SC, Tadayyon M, Scarpello JHB, Smith SA, Morgan NG (2004). Expression and functional activity of PPARgamma in pancreatic beta cells.
Br J Pharmacol,
142(7), 1162-1170.
Abstract:
Expression and functional activity of PPARgamma in pancreatic beta cells.
Rosiglitazone is an agonist of peroxisome proliferator activated receptor-gamma (PPARgamma) and ameliorates insulin resistance in type II diabetes. In addition, it may also promote increased pancreatic beta-cell viability, although it is not known whether this effect is mediated by a direct action on the beta cell. We have investigated this possibility. Semiquantitative real-time reverse transcription-polymerase chain reaction analysis (Taqman) revealed that freshly isolated rat islets and the clonal beta-cell line, BRIN-BD11, express PPARgamma, as well as PPARalpha and PPARdelta. The levels of expression of PPARgamma were estimated by reference to adipose tissue and were found to represent approximately 60% (islets) and 30% (BRIN-BD11) of that found in freshly isolated visceral adipose tissue. Western blotting confirmed the presence of immunoreactive PPARgamma in rat (and human) islets and in BRIN-BD11 cells. Transfection of BRIN-BD11 cells with a PPARgamma-sensitive luciferase reporter construct was used to evaluate the functional competence of the endogenous PPARgamma. Luciferase activity was modestly increased by the putative endogenous ligand, 15-deoxy-Delta12,14 prostaglandin J2 (15dPGJ2). Rosiglitazone also caused activation of the luciferase reporter construct but this effect required concentrations of the drug (50-100 microm) that are beyond the expected therapeutic range. This suggests that PPARgamma is relatively insensitive to activation by rosiglitazone in BRIN-BD11 cells. Exposure of BRIN-BD11 cells to the lipotoxic effector, palmitate, caused a marked loss of viability. This was attenuated by treatment of the cells with either actinomycin D or cycloheximide suggesting that a pathway of programmed cell death was involved. Rosiglitazone failed to protect BRIN-BD11 cells from the toxic actions of palmitate at concentrations up to 50 microm. Similar results were obtained with a range of other PPARgamma agonists. Taken together, the present data suggest that, at least under in vitro conditions, thiazolidinediones do not exert direct protective effects against fatty acid-mediated cytotoxicity in pancreatic beta cells.
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Prell GD, Martinelli GP, Holstein GR, Matulić-Adamić J, Watanabe KA, Chan SLF, Morgan NG, Haxhiu MA, Ernsberger P (2004). Imidazoleacetic acid-ribotide: an endogenous ligand that stimulates imidazol(in)e receptors.
Proc Natl Acad Sci U S A,
101(37), 13677-13682.
Abstract:
Imidazoleacetic acid-ribotide: an endogenous ligand that stimulates imidazol(in)e receptors.
We identified the previously unknown structures of ribosylated imidazoleacetic acids in rat, bovine, and human tissues to be imidazole-4-acetic acid-ribotide (IAA-RP) and its metabolite, imidazole-4-acetic acid-riboside. We also found that IAA-RP has physicochemical properties similar to those of an unidentified substance(s) extracted from mammalian tissues that interacts with imidazol(in)e receptors (I-Rs). ["Imidazoline," by consensus (International Union of Pharmacology), includes imidazole, imidazoline, and related compounds. We demonstrate that the imidazole IAA-RP acts at I-Rs, and because few (if any) imidazolines exist in vivo, we have adopted the term "imidazol(in)e-Rs."] the latter regulate multiple functions in the CNS and periphery. We now show that IAA-RP (i) is present in brain and tissue extracts that exhibit I-R activity; (ii) is present in neurons of brainstem areas, including the rostroventrolateral medulla, a region where drugs active at I-Rs are known to modulate blood pressure; (iii) is present within synaptosome-enriched fractions of brain where its release is Ca(2+)-dependent, consistent with transmitter function; (iv) produces I-R-linked effects in vitro (e.g. arachidonic acid and insulin release) that are blocked by relevant antagonists; and (v) produces hypertension when microinjected into the rostroventrolateral medulla. Our data also suggest that IAA-RP may interact with a novel imidazol(in)e-like receptor at this site. We propose that IAA-RP is a neuroregulator acting via I-Rs.
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Kaminski A, Gao H, Morgan NG (2004). Involvement of the cGMP signalling pathway in the regulation of viability in insulin-secreting BRIN-BD11 cells.
FEBS Lett,
559(1-3), 118-124.
Abstract:
Involvement of the cGMP signalling pathway in the regulation of viability in insulin-secreting BRIN-BD11 cells.
We have evaluated the hypothesis that cGMP may serve as an intracellular messenger regulating the viability of pancreatic beta-cells. A direct activator of soluble guanylyl cyclase, YC-1, caused a time- and dose-dependent loss of viability in clonal BRIN-BD11 beta-cells. This was accompanied by a rise in cGMP and was antagonised by Rp-8-pCPT-cGMPS, a selective inhibitor of protein kinase G (PKG). Reverse transcription polymerase chain reaction analysis confirmed that BRIN-BD11 cells (and human islets) express all three known isoforms of PKG (PKG-Ialpha, -Ibeta and II). Cell death induced by YC-1 was not sensitive to cell-permeable caspase inhibitors and was not accompanied by oligonucleosomal DNA fragmentation. The response was, however, inhibited by actinomycin D, suggesting that a transcription-dependent pathway of programmed cell death is involved in the actions of cGMP.
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Welters HJ, Tadayyon M, Scarpello JHB, Smith SA, Morgan NG (2004). Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure.
FEBS Lett,
560(1-3), 103-108.
Abstract:
Mono-unsaturated fatty acids protect against beta-cell apoptosis induced by saturated fatty acids, serum withdrawal or cytokine exposure.
Long-chain saturated fatty acids are cytotoxic to pancreatic beta-cells while shorter-chain saturated and long-chain unsaturated molecules are better tolerated. Mono-unsaturated fatty acids are not, however, inert since they inhibit the pro-apoptotic effects of saturated molecules. In the present work we show that the mono-unsaturates palmitoleate (C16:1) or oleate (C18:1) also cause marked inhibition of apoptosis induced by exposure of clonal BRIN-BD11 beta-cells to serum withdrawal or a combination of interleukin-1beta plus interferon-gamma. This response was dose-dependent and not accompanied by changes in NO formation. Taken together, the results suggest that mono-unsaturated fatty acids regulate a distal step common to several apoptotic pathways in pancreatic beta-cells.
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Yang Y, Zhang S, Jones G, Morgan N, El Haj AJ (2004). Phosphorylcholine-containing polymers for use in cell encapsulation.
Artif Cells Blood Substit Immobil Biotechnol,
32(1), 91-104.
Abstract:
Phosphorylcholine-containing polymers for use in cell encapsulation.
A model system for encapsulation of pancreatic islets which has potential properties for improving biocompatibility and immunosuppression was investigated. In vitro and in vivo studies have shown that phosphorylcholine-containing polymers have high biocompatibility due to low adsorption of proteins and reduced thrombus formation. Encapsulation of islets isolated from rats with a compound membrane composed of phosphorylcholine-containing polymers and cellulose acetate led to rapid insulin production and diffusion across the membrane in response to glucose challenge. The phosphorylcholine-containing polymer had a molecular weight of about 1.3 x 10(4) Da. The polymer-coated membrane excluded larger molecules such as IgG (molecular weight 150 kDa), thereby acting as a physical immuno-barrier, but allowed smaller molecules such as glucose and insulin to pass through.
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Squires PE, Hills CE, Rogers GJ, Garland P, Farley SR, Morgan NG (2004). The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.
Eur J Pharmacol,
501(1-3), 31-39.
Abstract:
The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.
beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect contributes to the insulin secretory response.
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Cooper EJ, Hudson AL, Parker CA, Morgan NG (2003). Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans.
Eur J Pharmacol,
482(1-3), 189-196.
Abstract:
Effects of the beta-carbolines, harmane and pinoline, on insulin secretion from isolated human islets of Langerhans.
It is well known that certain imidazoline compounds can stimulate insulin secretion and this has been attributed to the activation of imidazoline I(3) binding sites in the pancreatic beta-cell. Recently, it has been proposed that beta-carbolines may be endogenous ligands having activity at imidazoline sites and we have, therefore, studied the effects of beta-carbolines on insulin secretion. The beta-carbolines harmane, norharmane and pinoline increased insulin secretion two- to threefold from isolated human islets of Langerhans. The effects of harmane and pinoline were dose-dependent (EC(50): 5 and 25 microM, respectively) and these agents also blocked the inhibitory effects of the potassium channel agonist, diazoxide, on glucose-induced insulin release. Stimulation of insulin secretion by harmane was glucose-dependent but, unlike the imidazoline I(3) receptor agonist efaroxan, it increased the rate of insulin release beyond that elicited by 20 mM glucose (20 mM glucose alone: 253+/-34% vs. basal; 20 mM glucose plus 100 microM harmane: 327+/-15%; P
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Gao H, Mourtada M, Morgan NG (2003). Effects of the imidazoline binding site ligands, idazoxan and efaroxan, on the viability of insulin-secreting BRIN-BD11 cells.
JOP,
4(3), 117-124.
Abstract:
Effects of the imidazoline binding site ligands, idazoxan and efaroxan, on the viability of insulin-secreting BRIN-BD11 cells.
CONTEXT: Certain imidazoline drugs stimulate insulin secretion acutely but their longer term effects on the viability of pancreatic beta-cells are less well characterised. Indeed, some reports have suggested that imidazolines can be toxic to beta-cells while others have reported protective effects against other cytotoxic agents. OBJECTIVE: in order to address these discrepancies, the effects of two structurally related imidazolines, efaroxan and idazoxan, on the viability of clonal BRIN-BD11 beta-cells, were compared. DESIGN AND MAIN OUTCOME MEASURES: BRIN-BD11 cells were exposed to test reagents and their viability monitored by measuring cellular reducing ability and DNA fragmentation. Nitric oxide was measured indirectly via medium nitrite formation. RESULTS: Efaroxan (up to 100 micro M) did not directly affect BRIN-BD11 cell viability in the absence of other agents and it did not protect these cells against the cytotoxic effects of interleukin-1beta. Indeed, analysis of DNA fragmentation in BRIN-BD11 cells revealed that efaroxan enhanced the level of damage caused by interleukin-1beta. Idazoxan caused a time- and dose-dependent loss of BRIN-BD11 cell viability in the absence of other ligands. This was associated with marked DNA degradation but was not associated with formation of nitric oxide. The effects of idazoxan were insensitive to blockade of alpha(2)-adrenoceptors or 5-HT(1A) (5-hydroxytryptamine; serotonin) receptors. CONCLUSIONS: the results confirm that idazoxan is cytotoxic to beta-cells but show that efaroxan is better tolerated. However, since efaroxan enhanced the cytotoxic effects of interleukin-1beta, it appears that this imidazoline may sensitise BRIN-BD11 cells to the damaging effects of certain cytokines.
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McBain SC, Morgan NG (2003). Functional effects of expression of wolframin-antisense transcripts in BRIN-BD11 beta-cells.
Biochem Biophys Res Commun,
307(3), 684-688.
Abstract:
Functional effects of expression of wolframin-antisense transcripts in BRIN-BD11 beta-cells.
Wolfram syndrome is a rare condition in which the pancreatic beta-cells of patients are selectively deleted during the early years of life by a non-autoimmune-mediated mechanism. The condition is associated with mutations in the gene encoding wolframin, suggesting that this protein exerts a critical, but currently unknown, function in beta-cells. We have used an antisense strategy to modulate the expression of wolframin in insulin-secreting BRIN-BD11 cells to study its function. Stably transfected clones were established expressing full-length human wolframin antisense transcripts. These cells exhibited a dramatic reduction in cell proliferation rate and changes in morphology, although insulin secretion was not modified. The results imply that wolframin expression is required to sustain normal rates of beta-cell proliferation.
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Elliott J, Scarpello JHB, Morgan NG (2002). Differential effects of genistein on apoptosis induced by fluoride and pertussis toxin in human and rat pancreatic islets and RINm5F cells.
J Endocrinol,
172(1), 137-143.
Abstract:
Differential effects of genistein on apoptosis induced by fluoride and pertussis toxin in human and rat pancreatic islets and RINm5F cells.
Clonal pancreatic beta-cell lines have been used widely for the study of the factors involved in the regulation of apoptosis but it has not been firmly established that the response of normal islets mirrors that found in transformed beta-cells. In the present work, the role of pertussis toxin (Ptx)-sensitive G-proteins in the control of beta-cell apoptosis was studied in isolated rat and human islets of Langerhans and compared with the clonal beta-cell line, RINm5F. Annexin-V and deoxycarboxyfluoroscein diacetate staining was used to identify viable, apoptotic and necrotic cells directly, under fluorescence illumination. Treatment of human and rat islet cells with the G-protein activator fluoride (NaF; 5 mM) caused a marked increase in apoptosis that was further potentiated in islets pretreated with Ptx. The tyrosine kinase inhibitor genistein (100 microM) also increased islet cell apoptosis and the combination of 100 microM genistein and 5 mM NaF did not lead to any diminution of the apoptotic response. This latter effect was quite different from that seen in RINm5F cells where the combination of 100 microM genistein and 5 mM NaF resulted in much less apoptosis than was observed with either agent alone. In islets treated with a lower concentration of genistein (25 microM; that did not, itself, increase cell death), the drug attenuated NaF-induced apoptosis and also blocked the enhancement mediated by Ptx. These results revealed that human (and rat) islets are equipped with a Ptx-sensitive pathway that may be regulated by tyrosine phosphorylation and is anti-apoptotic. However, they also define conditions under which marked differences in response between RINm5F cells and normal islets were observed and they suggest that care should be taken when extrapolating data obtained with clonal cell lines to the situation in normal islet cells.
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Kowluru A, Morgan NG (2002). GTP-binding proteins in cell survival and demise: the emerging picture in the pancreatic beta-cell.
Biochem Pharmacol,
63(6), 1027-1035.
Abstract:
GTP-binding proteins in cell survival and demise: the emerging picture in the pancreatic beta-cell.
It is widely believed that guanine nucleotide-binding regulatory proteins (G-proteins) play central roles as "molecular switches" in a variety of cellular processes ranging from signal transduction to protein and vesicle trafficking. To achieve these regulatory functions, G-proteins form complexes with a wide range of effector molecules whose activities are altered upon interaction with the G-protein. These effector molecules can be either soluble or membrane bound, and it is likely that some are localized to secretory granules where they direct the movement, docking, and fusion of granules during exocytosis. The effector molecules regulated by G-proteins are diverse and include phospholipases, protein kinases, protein phosphatases, ion channels, adenylate cyclases, cytoskeletal elements, as well as secretory vesicle and plasma membrane-associated fusion-proteins. The majority of studies performed in the pancreatic beta-cell have focused on the role of G-proteins in the regulation of insulin secretion, whereas very little attention has been focused on their potential involvement in other cellular processes. Such studies have identified and implicated both heterotrimeric (comprising alpha, beta, and gamma subunits) and monomeric (low molecular mass) G-proteins in the regulation of insulin secretion, but intriguing recent evidence has also begun to emerge which favors the view that they may be involved in the maintenance of beta-cell viability. In the present commentary, we will review this evidence and discuss the current understanding of the role of G-proteins in the life and death of the beta-cell.
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Chan SLF, Monks LK, Gao H, Deaville P, Morgan NG (2002). Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell.
Br J Pharmacol,
136(1), 31-36.
Abstract:
Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell.
Efaroxan induces membrane depolarization by interaction with the pore forming subunit of the ATP-sensitive potassium channel, Kir6.2. However, this effect is not responsible for its full secretory activity. In this study we have used an anti-idiotypic approach to generate antibodies that recognize additional proteins that may be regulated by efaroxan in pancreatic beta-cells. Using these antisera in an expression cloning strategy we have identified a monomeric GTP-binding protein, Rhes, as a potential target for regulation by imidazoline ligands. Rhes is shown to be expressed in beta-cells and its expression is regulated by efaroxan under conditions when a structurally related molecule, KU14R, is ineffective. The results reveal that beta-cells express Rhes and suggest that changes in the expression of this molecule may regulate the sensitivity of beta-cells to imidazoline secretagogues.
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Chan SL, Mourtada M, Morgan NG (2001). Characterization of a KATP channel-independent pathway involved in potentiation of insulin secretion by efaroxan.
Diabetes,
50(2), 340-347.
Abstract:
Characterization of a KATP channel-independent pathway involved in potentiation of insulin secretion by efaroxan.
Efaroxan, like several other imidazoline reagents, elicits a glucose-dependent increase in insulin secretion from pancreatic beta-cells. This response has been attributed to efaroxan-mediated blockade of KATP channels, with the subsequent gating of voltage-sensitive calcium channels. However, increasing evidence suggests that, at best, this mechanism can account for only part of the secretory response to the imidazoline. In support of this, we now show that efaroxan can induce functional changes in the secretory pathway of pancreatic beta-cells that are independent of KATP channel blockade. In particular, efaroxan was found to promote a sustained sensitization of glucose-induced insulin release that persisted after removal of the drug and to potentiate Ca2+-induced insulin secretion from electropermeabilized islets. To investigate the mechanisms involved, we studied the effects of the efaroxan antagonist KU14R. This agent is known to selectively inhibit insulin secretion induced by efaroxan, without altering the secretory response to glucose or KCl. Surprisingly, however, KU14R markedly impaired the potentiation of insulin secretion mediated by agents that raise cAMP, including the adenylate cyclase activator, forskolin, and the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX). These effects were not accompanied by any reduction in cAMP levels, suggesting an antagonistic action of KU14R at a more distal point in the pathway of potentiation. In accord with our previous work, islets that were exposed to efaroxan for 24 h became selectively desensitized to this agent, but they still responded normally to glucose. Unexpectedly, however, the ability of either forskolin or IBMX to potentiate glucose-induced insulin secretion was severely impaired in these islets. By contrast, the elevation of cAMP was unaffected by culture of islets with efaroxan. Taken together, the data suggest that, in addition to effects on the KATP channel, imidazolines also interact with a more distal component that is crucial to the potentiation of insulin secretion. This component is not required for Ca2+-dependent secretion per se but is essential to the mechanism by which cAMP potentiates insulin release. Overall, the results indicate that the actions of efaroxan at this distal site may be more important for control of insulin secretion than its effects on the KATP channel.
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Elliott J, Scarpello JH, Morgan NG (2001). Effects of tyrosine kinase inhibitors on cell death induced by sodium fluoride and pertussis toxin in the pancreatic beta-cell line, RINm5F.
Br J Pharmacol,
132(1), 119-126.
Abstract:
Effects of tyrosine kinase inhibitors on cell death induced by sodium fluoride and pertussis toxin in the pancreatic beta-cell line, RINm5F.
1. Sodium fluoride causes apoptosis of pancreatic beta-cells and this response is enhanced by pre-treatment with pertussis toxin. In the present study, tyrosine kinase inhibitors were used to investigate the mechanisms of action of NaF and pertussis toxin in the beta-cell line, RINm5F. 2. Exposure of RINm5F cells to low concentrations of genistein or tyrphostin A25 resulted in significant inhibition of cell death induced by 5 mM NaF. Higher concentrations (>25 microM) were cytotoxic in the absence of NaF but, paradoxically, the combination of genistein and NaF induced less cell death than when each agent was used alone. 3. The increase in cell death induced by 100 microM genistein was markedly inhibited by ciprofloxacin, a drug which binds to topoisomerase II. Etoposide (which inhibits topoisomerase II but has no effect on tyrosine kinase activity) also caused an increase in RINm5F cell death. Neither etoposide nor ciprofloxacin altered the response to 5 mM NaF. 4. Pertussis toxin markedly enhanced the extent of RINm5F cell death induced by NaF and this effect was completely prevented by 25 microM genistein. The inhibition caused by genistein was not affected by ciprofloxacin but was reproduced by a structurally dissimilar tyrosine kinase inhibitor, herbimycin A. 5. The results demonstrate that RINm5F beta-cells express a pertussis toxin sensitive pathway that is anti-apoptotic. The activity of this pathway is most evident in cells exposed to pro-apoptotic stimuli where the effects of pertussis toxin can be blocked by inhibitors of tyrosine kinase enzymes. A genistein-sensitive tyrosine kinase does not appear to be involved in RINm5F cell survival under basal conditions.
Abstract.
Author URL.
Morgan NG, Chan SL (2001). Imidazoline binding sites in the endocrine pancreas: can they fulfil their potential as targets for the development of new insulin secretagogues?.
Curr Pharm Des,
7(14), 1413-1431.
Abstract:
Imidazoline binding sites in the endocrine pancreas: can they fulfil their potential as targets for the development of new insulin secretagogues?
A variety of compounds containing an imidazoline ring have the ability to stimulate insulin secretion. Many of these also improve glycaemia in experimental models of type 2 diabetes and in man, suggesting that this class may be useful in the development of new orally active anti-diabetic drugs. However, the mechanisms by which imidazolines promote insulin secretion have not been clarified. The response does not appear to be due to the binding of ligands to either of the two major types of "imidazoline receptor" defined by pharmacological criteria (I1 and I2 sites) but may result from interaction with a novel imidazoline binding site. One such site has been identified in association with the ATP-sensitive potassium (K(ATP)) channel in the beta-cell and has been designated "I3". Electrophysiological and biochemical evidence suggest that the I3 site may be intrinsic to the ion-conducting pore component, Kir6.2, of the K(ATP) channel, but the effects of imidazoline ligands on insulin secretion can be dissociated from the regulation of Kir6.2. Indeed, there is increasing evidence that some imidazolines can control exocytosis directly, both in beta-cells and in pancreatic alpha-cells. Thus, it is proposed that a further imidazoline binding site is primarily responsible for control of hormone secretion. Evidence is reviewed which suggests that this site occupies a central position within an amplification pathway that also mediates the effects of cAMP in the beta-cell. Characterisation of this site should provide the stimulus for the design of new insulin secretagogues that are devoid of K(ATP) channel-blocking properties.
Abstract.
Author URL.
Trigwell SM, Radford PM, Page SR, Loweth AC, James RF, Morgan NG, Todd I (2001). Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus.
Clin Exp Immunol,
126(2), 242-249.
Abstract:
Islet glutamic acid decarboxylase modified by reactive oxygen species is recognized by antibodies from patients with type 1 diabetes mellitus.
The generation of an autoimmune response against islet beta-cells is central to the pathogenesis of type 1 diabetes mellitus, and this response is driven by the stimulation of autoreactive lymphocytes by components of the beta-cells themselves. Reactive oxygen species (ROS) have been implicated in the beta-cell destruction which leads to type 1 diabetes and may modify beta-cell components so as to enhance their immunogenicity. We investigated the effects of oxidation reactions catalysed by copper or iron on the major beta-cell autoantigen glutamic acid decarboxylase (GAD). Lysates of purified rat islets were exposed to copper or iron sulphate with or without hydrogen peroxide or ascorbic acid. Immunostaining showed that these treatments generated high molecular weight covalently linked aggregates containing GAD. These are not formed by intermolecular disulphide bonds between cysteine residues since they cannot be resolved into monomeric form when electrophoresed under extreme reducing conditions. There was no modification of insulin or pro-insulin by ROS. The same oxidative changes to GAD could be induced in viable islet cells treated with copper sulphate and hydrogen peroxide, and thus the modifications are not an artefact of the catalysed oxidation of cell-free lysates. Sera from patients with type 1 diabetes and stiffman syndrome containing GAD antibodies reacted predominantly with the highest molecular weight modified protein band of GAD: normal human sera did not precipitate GAD. Thus, oxidatively modified aggregates of GAD react with serum antibodies of type 1 diabetes patients and some SMS patients: this is consistent with oxidative modifications of autoantigens being relevant to the pathogenesis of type 1 diabetes.
Abstract.
Author URL.
Clews J, Morgan NG, Ramsden CA (2001). Preparation of novel 2-(benzo[b]furan-2-yl)-1H-imidazolines for photoaffinity labelling and affinity isolation of imidazoline binding proteins.
JOURNAL OF HETEROCYCLIC CHEMISTRY,
38(2), 519-521.
Author URL.
Clews J, Morgan NG, Ramsden CA (2001). Preparation of the I-3 imidazoline receptor antagonist KU14R and related 2,3-dihydrobenzo[b]furan derivatives.
SYNTHESIS-STUTTGART(10), 1546-1550.
Author URL.
Loweth AC, Watts K, McBain SC, Williams GT, Scarpello JH, Morgan NG (2000). Dissociation between Fas expression and induction of apoptosis in human islets of Langerhans.
Diabetes Obes Metab,
2(1), 57-60.
Abstract:
Dissociation between Fas expression and induction of apoptosis in human islets of Langerhans.
There is increasing evidence that inappropriate induction of apoptosis in pancreatic beta-cells may precede the development of type 1 diabetes in animal models and in man. One mechanism by which this has been proposed to occur involves up-regulation of the death receptor Fas on beta-cells, resulting in apoptosis of the Fas-bearing beta-cells upon ligation of the receptor. We have examined this hypothesis in isolated human islets of Langerhans and show that--in contrast to data obtained with rodent beta-cells--expression of Fas per se is not sufficient to allow induction of apoptosis upon addition of agonistic anti-Fas serum.
Abstract.
Author URL.
Mourtada M, Elliott J, Smith SA, Morgan NG (2000). Effects of imidazoline binding site ligands on the growth and viability of clonal pancreatic beta-cells.
Naunyn Schmiedebergs Arch Pharmacol,
361(2), 146-154.
Abstract:
Effects of imidazoline binding site ligands on the growth and viability of clonal pancreatic beta-cells.
Pancreatic beta-cells express imidazoline binding sites which play a role in the regulation of insulin secretion, but it is not known whether ligands for these sites also affect other aspects of beta-cell physiology. In the present study, we have investigated the effects of a range of imidazoline reagents on the growth and viability of clonal pancreatic beta-cells (RINm5F and HIT-T15). Three imidazoline compounds (idazoxan, phentolamine and antazoline) were found to cause marked inhibition of beta-cell growth in a time and concentration dependent manner. Idazoxan was the most potent of these with as little as 0.5 microM causing a significant decrease in beta-cell viability (EC50 approximately 10 microM). All three imidazolines also decreased the viability of clonal beta-cells in parallel with their inhibitory effects on cell growth. These effects were not reproduced by any of a wide-range of other imidazoline compounds, including effective insulin secretagogues such as efaroxan and RX821002. The effects of the three ligands did not correlate with their relative potencies for binding to any of the well-characterised imidazoline binding sites nor to alpha2-adrenoceptors. In addition, the inhibitory responses were not antagonised by other imidazoline binding site ligands. The inhibitory effects of idazoxan on the growth of RINm5F and HIT-T15 beta-cells required as little as 3-h exposure to the imidazoline and were not readily reversible when the reagent was removed. Reductions in growth rate were accompanied by marked alterations in the morphology of the cells, which could be detected before loss of viability. Cells exposed to phentolamine showed the characteristic features of apoptosis in that the nuclei were condensed (as judged by acridine orange staining) and electrophoresis of DNA revealed the presence of oligonucleosomal fragmentation. These changes could not be detected in cells exposed to idazoxan despite the more profound reduction in viability induced by this agent. We conclude that a sub-group of imidazoline compounds can exert profoundly detrimental effects on the growth and viability of clonal beta-cells but that these effects do not correlate with their binding affinity at imidazoline binding sites or alpha2-adrenoceptors.
Abstract.
Author URL.
(1999). 35th Annual Meeting of the European Association for the Study of Diabetes : Brussels, Belgium, 28 September-2 October 1999.
Diabetologia,
42(Suppl 1), A1-A330.
Author URL.
Monks LK, Cosgrove KE, Dunne MJ, Ramsden CA, Morgan NG, Chan SL (1999). Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand.
FEBS Lett,
447(1), 61-64.
Abstract:
Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand.
We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP-sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino-efaroxan blocks ATP-sensitive potassium channels in human pancreatic beta-cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.
Abstract.
Author URL.
Pizzinat N, Chan SL, Remaury A, Morgan NG, Parini A (1999). Characterization of monoamine oxidase isoforms in human islets of Langerhans.
Life Sci,
65(4), 441-448.
Abstract:
Characterization of monoamine oxidase isoforms in human islets of Langerhans.
In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms a & B are present in human pancreas. Two complementary approaches indicated that both MAO a and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO a (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO a and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.
Abstract.
Author URL.
Pelé-Tounian A, Chan SL, Rondu F, Le Bihan G, Giroix MH, Lamouri A, Touboul E, Pfeiffer B, Manechez D, Renard P, et al (1999). Effect of the new imidazoline derivative S-22068 (PMS 847) on insulin secretion in vitro and glucose turnover in vivo in rats.
Eur J Pharmacol,
377(1), 81-87.
Abstract:
Effect of the new imidazoline derivative S-22068 (PMS 847) on insulin secretion in vitro and glucose turnover in vivo in rats.
We have investigated the possible mechanisms underlying the antihyperglycaemic effect of the imidazoline derivative S-22068. In vitro, in the presence of 5 mmol/l glucose, S-22068 (100 micromol/l) induced a significant and sustained increase in insulin secretion from isolated, perifused, rat islets and a marked sensitization to a subsequent glucose challenge (10 mmol/l). S-22068 (100 micromol/l was able to antagonize the stimulatory effect of diazoxide on 86Rb efflux from preloaded islets incubated in the presence of 20 mmol/l glucose. Experiments were also performed to investigate whether S-22068 can alter glucose turnover and peripheral insulin sensitivity in vivo in mildly diabetic rats and obese, insulin resistant, Zucker rats. Neither glucose production nor individual tissue glucose utilization was modified by S-22068 in either group of rats. Similar results were obtained whether the studies were performed under basal conditions or during euglycaemic/hyperinsulinemic clamps. The results suggest that S-22068 exerts part of its antihyperglycaemic effect by promoting insulin secretion without alteration of peripheral insulin sensitivity.
Abstract.
Author URL.
Parker CA, Hudson AL, Nutt DJ, Dillon MP, Eglen RM, Chan SL, Morgan NG, Crosby J (1999). Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues.
Eur J Pharmacol,
378(2), 213-221.
Abstract:
Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues.
Crude methanolic clonidine-displacing substance (CDS) extracted from bovine lung competed for radioligand binding from alpha2-adrenoceptors and I2-sites present in rat brain membranes, and from I1-sites present in rat brain and kidney membranes. There was no difference in the competition of [3H]clonidine binding to alpha2-adrenoceptors present in either rat or rabbit brain membranes by the crude CDS extract and therefore either tissue could be used to estimate the number of units of CDS present in extracts. Further purification by reverse phase high performance liquid chromatography (RP-HPLC), with UV detection, of extracts obtained from bovine lung, brain and rat brain exhibited similar three-peak profiles, previously reported. Corresponding fractions competed for radioligand binding to alpha2-adrenoceptors present in rat brain membranes, eluting between 19 and 23 min, which corresponded with the middle peak of the three-peaks. Therefore, we propose the CDS-like material eluting from all these tissues to be similar. Interestingly, CDS extracted from bovine adrenal glands under the same conditions showed a similar three-peak profile, but did not repeat the displacement of binding just at 19-23 min, but at every time point after 4 min. This suggests this tissue could represent a source of CDS in this species.
Abstract.
Author URL.
Morgan NG (1999). Imidazoline receptors: new targets for antihyperglycaemic drugs.
Expert Opin Investig Drugs,
8(5), 575-584.
Abstract:
Imidazoline receptors: new targets for antihyperglycaemic drugs.
In both animal models of Type 2 diabetes and in man, it has been evident for many years that certain imidazoline drugs can stimulate insulin secretion and improve glycaemia. This suggests that they may be useful new reagents for use in the management of Type 2 diabetes. However, despite their promise, no imidazoline compound has yet come into clinical use as an effective therapeutic agent in diabetes. This should not be taken as evidence of a flaw in the basic hypothesis, but derives, in part, from continuing ignorance about the molecular characteristics of imidazoline binding proteins, and the precise structure-activity relationships of their ligands. In this review, the mode of action of antihyperglycaemic imidazoline compounds is considered, and the possibility discussed that these agents may interact with a unique subtype of imidazoline binding site associated with ATP-sensitive potassium channels. The functional consequences of this interaction are summarised together with evidence that the binding site may actually lie within the channel complex. Additional data implicating the participation of alpha2-adrenoceptors in some actions of imidazolines are evaluated, and examples of relevant drugs having encouraging therapeutic profiles are highlighted. The possibility that some anti-diabetic imidazoline reagents may exert extra-pancreatic effects is also considered. Overall, the article aims to highlight important developments within the field but also draws attention to those areas where controversy remains.
Abstract.
Author URL.
Mourtada M, Chan SL, Smith SA, Morgan NG (1999). Multiple effector pathways regulate the insulin secretory response to the imidazoline RX871024 in isolated rat pancreatic islets.
Br J Pharmacol,
127(5), 1279-1287.
Abstract:
Multiple effector pathways regulate the insulin secretory response to the imidazoline RX871024 in isolated rat pancreatic islets.
When isolated rat islets were cultured for 18 h prior to use, the putative imidazoline binding site ligand, RX871024 caused a dose-dependent increase in insulin secretion at both 6 mM and 20 mM glucose. By contrast, a second ligand, efaroxan, was ineffective at 20 mM glucose whereas it did stimulate insulin secretion in response to 6 mM glucose. Exposure of islets to RX871024 (50 microM) for 18 h, resulted in loss of responsiveness to this reagent upon subsequent re-exposure. However, islets that were unresponsive to RX871024 still responded normally to efaroxan. The imidazoline antagonist, KU14R, blocked the insulin secretory response to efaroxan, but failed to prevent the stimulatory response to RX871024. By contrast with its effects in cultured islets, RX871024 inhibited glucose-induced insulin release from freshly isolated islets. Efaroxan did not inhibit insulin secretion under any conditions studied. In freshly isolated islets, the effects of RX871024 on insulin secretion could be converted from inhibitory to stimulatory, by starvation of the animals. Inhibition of insulin secretion by RX871024 in freshly isolated islets was prevented by the cyclo-oxygenase inhibitors indomethacin or flurbiprofen. Consistent with this, RX871024 caused a marked increase in islet PGE2 formation. Efaroxan did not alter islet PGE2 levels. The results suggest that RX871024 exerts multiple effects in the pancreatic beta-cell and that its effects on insulin secretion cannot be ascribed only to interaction with a putative imidazoline binding site.
Abstract.
Author URL.
Eglen RM, Hudson AL, Kendall DA, Nutt DJ, Morgan NG, Wilson VG, Dillon MP (1998). 'Seeing through a glass darkly': casting light on imidazoline 'I' sites.
Trends Pharmacol Sci,
19(9), 381-390.
Abstract:
'Seeing through a glass darkly': casting light on imidazoline 'I' sites.
Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.
Abstract.
Author URL.
El-Mansoury AM, Morgan NG (1998). Activation of protein kinase C modulates alpha2-adrenergic signalling in rat pancreatic islets.
Cell Signal,
10(9), 637-643.
Abstract:
Activation of protein kinase C modulates alpha2-adrenergic signalling in rat pancreatic islets.
Treatment of rat pancreatic islets with 4beta-phorbol-myristate-acetate (PMA) caused a significant reduction in the ability of the alpha2-adrenoceptor agonist noradrenaline to inhibit glucose-induced insulin secretion. This effect was most evident when low concentrations of the catecholamine were used (less than 1 microM) and was lost when the noradrenaline concentration was increased to 10 microM. The effect was probably mediated by activation of protein kinase C, because the ability of PMA to desensitise islets to noradrenaline was prevented by a selective inhibitor of calcium-dependent isoforms of the enzyme, Gö6976. The response to PMA was reproduced when islet protein kinase C was activated by a receptor-mediated mechanism involving incubation with the muscarinic agonist carbachol. In parallel with desensitisation of the inhibitory control of insulin secretion by noradrenaline, PMA treatment also reduced the ability of a low concentration of noradrenaline (0.1 microM) to inhibit islet cAMP formation. The loss of sensitivity to catecholamine, induced by PMA in rat islets, was not caused by any change in the levels of alpha2-adrenoceptor expression or in their ligand-binding affinity. It was, however, associated with a marked increase in the extent of phosphorylation of members of the Gi/Go, family of pertussis toxin-sensitive G proteins in PMA-treated islets. Immunoprecipitation of Gi alpha2 and Galpha o from 32P-labelled islets after treatment with PMA revealed that both G proteins are substrates for protein kinase C. Overall, the results indicate that activation of protein kinase C leads to phosphorylation of islet Gi and Go causing their uncoupling from alpha2-adrenoceptors. We propose that this mechanism may form an important component of a physiological system designed to limit the tendency for catecholamines to inhibit insulin secretion under conditions in which the parasympathetic innervation of the islets is activated.
Abstract.
Author URL.
Chan SL, Pallett AL, Clews J, Ramsden CA, Chapman JC, Kane C, Dunne MJ, Morgan NG (1998). Characterisation of new efaroxan derivatives for use in purification of imidazoline-binding sites.
Eur J Pharmacol,
355(1), 67-76.
Abstract:
Characterisation of new efaroxan derivatives for use in purification of imidazoline-binding sites.
The insulin secretagogue activity of certain imidazoline compounds is mediated by a binding site associated with ATP-sensitive K+ (K(ATP)) channels in the pancreatic beta-cell. We describe the effects of a series of structural modifications to efaroxan on its activity at this site. Substitution of amino-, nitro- or azide- groups onto the 5-position of the benzene ring of efaroxan did not significantly affect the functional interaction of the ligand with the islet imidazoline binding site. Modification of the imidazoline ring to an imidazole to generate 2-(2-ethyl-2,3-dihydrobenzo[b]furan-2-yl)-1H-imidazole (KU14R) resulted in loss of secretagogue activity. Indeed, this reagent appeared to act as an imidazoline antagonist since it blocked the secretory responses to imidazoline compounds and also inhibited the blockade of beta-cell K(ATP) channels by efaroxan in patch clamp experiments. Application of KU14R alone resulted in a modest reduction in K(ATP) channel opening, suggesting that it may display weak partial agonism, at least in patch-clamp experiments.
Abstract.
Author URL.
Mourtada M, Smith SA, Morgan NG (1998). Effector systems involved in the insulin secretory responses to efaroxan and RX871024 in rat islets of Langerhans.
Eur J Pharmacol,
350(2-3), 251-258.
Abstract:
Effector systems involved in the insulin secretory responses to efaroxan and RX871024 in rat islets of Langerhans.
One component of the mechanism by which imidazoline compounds promote insulin secretion involves closure of ATP-sensitive K+ channels in the beta-cell plasma membrane. Recently, however, it has also been proposed that these compounds may exert important effects on more distal effector systems. In the present work, we have investigated the contribution played by protein kinases a and C to the insulin secretory responses of isolated rat islets of Langerhans treated with efaroxan and RX871024 (1-phenyl-2-(imidazolin-2-yl) benzimidazole). Removal of extracellular Ca2+ or blockade of voltage-sensitive Ca2+ channels prevented stimulation of insulin secretion by efaroxan, confirming a critical role for increased Ca2+ influx in the secretory response. By contrast, inhibition of protein kinases a or C failed to alter efaroxan-induced insulin secretion. RX871024 dose-dependently increased insulin secretion from cultured islets incubated with 20 mM glucose. This effect was unaffected by modulation of protein kinase C, but was significantly attenuated by a selective inhibitor of protein kinase a (Rp-cAMPs). Measurements of cAMP revealed that RX871024 increased the islet cAMP content by more than 3-fold; reaching values similar in magnitude to those elicited by 50 microM 3-isobutyl-1-methyl xanthine. The results reveal that neither protein kinase a nor protein kinase C is obligatory for stimulation of insulin secretion by imidazolines. However, they suggest that a rise in cAMP may contribute to the amplified secretory response observed when cultured islets are incubated with RX871024 in the presence of a stimulatory glucose concentration.
Abstract.
Author URL.
Loweth AC, Williams GT, James RF, Scarpello JH, Morgan NG (1998). Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation.
Diabetes,
47(5), 727-732.
Abstract:
Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation.
IDDM results from a progressive loss of pancreatic beta-cells that, in humans, may be triggered by a combination of genetic and environmental factors. Recently, attention has been focused on the hypothesis that the loss of beta-cells is initiated by inappropriate induction of apoptosis. We now demonstrate that human islets of Langerhans undergo apoptosis upon exposure to interleukin-1beta. The cytokine also sharply increases the number of cells that enter apoptosis on treatment with a stimulatory anti-Fas antibody. Western blotting and immunocytochemistry clearly show for the first time that human pancreatic beta-cells normally express Fas ligand. The results suggest that human islet cells are primed to undergo apoptosis by interleukin-1beta and that this involves the close association between cell-surface Fas and its ligand.
Abstract.
Author URL.
Loweth AC, Morgan NG (1998). Methods for the study of NO-induced apoptosis in cultured cells.
Methods Mol Biol,
100, 311-320.
Author URL.
Chan SL, Morgan NG (1998). Sigma receptor ligands and imidazoline secretagogues mediate their insulin secretory effects by activating distinct receptor systems in isolated islets.
Eur J Pharmacol,
350(2-3), 267-272.
Abstract:
Sigma receptor ligands and imidazoline secretagogues mediate their insulin secretory effects by activating distinct receptor systems in isolated islets.
The effects of two potent sigma receptor agonists (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine) and DTG (N,N'-di-(o-tolyl)guanidine) on the insulin secretory responses in rat islets of Langerhans were investigated. Both sigma receptor ligands were able to potentiate the insulin secretory response of islets incubated at 6 mM glucose, in a dose-dependent manner and were also able to reverse the effects of diazoxide on insulin release. When islets were treated with efaroxan, a well-characterised imidazoline insulin secretagogue, and either (+)-3-PPP or DTG together, there was an unexpected and profound absence of stimulation of insulin release as compared to when islets were incubated with each compound alone. Experiments performed with islets where there was desensitization of DTG/sigma receptor or efaroxan/imidazoline binding site mediated responses suggest that at least two distinct receptor systems appear to be involved. The complex interactions of these two classes of drug require further investigation.
Abstract.
Author URL.
Chan SL, Pallett AL, Morgan NG (1997). Clotrimazole and efaroxan stimulate insulin secretion by different mechanisms in rat pancreatic islets.
Naunyn Schmiedebergs Arch Pharmacol,
356(6), 763-768.
Abstract:
Clotrimazole and efaroxan stimulate insulin secretion by different mechanisms in rat pancreatic islets.
It is now well established that the imidazoline insulin secretagogue efaroxan mediates its effects by inducing closure of ATP-sensitive potassium channels in the pancreatic beta-cell, leading to membrane depolarisation, Ca2+ influx and increased insulin secretion. However, a recent study has shown that efaroxan may also act as a blocker of a second class of potassium channel (the Kmaxi channel) in red blood cells, raising the possibility that its effects in islets could be mediated by interactions with both types of channel. Since the antimycotic imidazole compound clotrimazole is a highly potent blocker of Kmaxi channels, we have studied the effects of this drug on insulin secretion. Clotrimazole stimulated insulin secretion from rat islets of Langerhans incubated in the presence of 6 mM glucose, in a dose-dependent manner. Experiments performed at different glucose concentrations showed that the actions of clotrimazole were most prominent at low glucose concentrations whereas it did not enhance secretion beyond the rate induced by 20 mM glucose. The insulinotropic action of clotrimazole was temperature dependent but was independent of extracellular calcium. Clotrimazole appeared to block ATP-sensitive potassium channels in islets since, like efaroxan and glibencamide, it was able to prevent the inhibitory effects of diazoxide on glucose-induced insulin secretion. However, neither the direct stimulatory effect of clotrimazole on insulin release nor the abilty of clotrimazole to reverse the inhibitory actions of diazoxide was sensitive to blockade by the imidazoline secretagogue antagonist KU14R. Overall, the results suggest that clotrimazole exerts an insulinotropic effect in pancreatic beta-cells that is distinct from the actions of imidazoline secretagogues such as efaroxan. Clotrimazole can increase insulin secretion at sub-maximal glucose concentrations by an action which appears to be independent of membrane ion channel events.
Abstract.
Author URL.
Chan SL, Perrett CW, Morgan NG (1997). Differential expression of alpha 2-adrenoceptor subtypes in purified rat pancreatic islet A- and B-cells.
Cell Signal,
9(1), 71-78.
Abstract:
Differential expression of alpha 2-adrenoceptor subtypes in purified rat pancreatic islet A- and B-cells.
In the endocrine pancreas, alpha 2-adrenoceptor stimulation reduces glucose-induced insulin secretion from islet B-cells. There is, however, controversy with regard to the effects of alpha 2-agonists on islet A-cell function. In this paper, we have used RNA samples prepared from whole rat islets and from FACS-purified rat A- and B-cells to study alpha 2-adrenoceptor subtype expression. RNase protection assays detected transcripts encoding alpha 2a and alpha 2b subtypes in the RNA pool of rat islets. Reverse transcription-PCR revealed that transcripts for all three alpha 2-adrenoceptor subtypes are present in rat islet cells in purified A-cell RNA. In contrast, RT-PCR of islet B-cell RNA yielded products corresponding to alpha 2a and alpha 2c, with no evidence for the presence of alpha 2b. Thus, the results reveal that both islet cell types express more than one receptor subtype and suggest that the distribution of the subtypes may differ between rat islet A- and B-cells.
Abstract.
Author URL.
Loweth AC, Williams GT, Scarpello JH, Morgan NG (1997). Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15.
FEBS Lett,
400(3), 285-288.
Abstract:
Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15.
Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 microM) and dibutyryl cGMP (300 microM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10-250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.
Abstract.
Author URL.
Chan SL, Pallett AL, Clews J, Ramsden CA, Morgan NG (1997). Evidence that the ability of imidazoline compounds to stimulate insulin secretion is not due to interaction with sigma receptors.
Eur J Pharmacol,
323(2-3), 241-244.
Abstract:
Evidence that the ability of imidazoline compounds to stimulate insulin secretion is not due to interaction with sigma receptors.
Recent studies have suggested that a variety of ion channels possess a binding site for ligands such as phencyclidine (PCP), dizocilpine and certain sigma ligands and that some imidazoline compounds can also bind to this site. We have investigated whether interaction with this binding site could account for the ability of imidazolines to stimulate insulin secretion from rat islets. Neither PCP nor dizocilpine shared the insulin secretory activity of the imidazoline efaroxan in rat islets suggesting that they do not have similar actions in the pancreatic B-cell. Further, we were able to define a new antagonist, KU14R (2(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole), which selectively blocks the insulin secretory response to imidazolines. The results suggest that imidazolines do not stimulate insulin secretion by causing physical blockade of the K(+)-ATP channel in pancreatic B-cells and show that their effects are not reproduced by PCP or sigma receptor ligands.
Abstract.
Author URL.
Mourtada M, Smith SA, Morgan NG (1997). Insulin secretagogues with an imidazoline structure inhibit arginine-induced secretion from isolated glucagon secretion from isolated rat islets of Langerhans.
Biochem Biophys Res Commun,
236(1), 162-166.
Abstract:
Insulin secretagogues with an imidazoline structure inhibit arginine-induced secretion from isolated glucagon secretion from isolated rat islets of Langerhans.
It is well documented that imidazoline compounds such as efaroxan and phentolamine act as potent insulin secretagogues both in vivo and in vitro, an effect which is mediated principally by blockade of ATP-sensitive potassium channels in the pancreatic B-cell. However, little is known about the effects of these drugs on the secretion of other pancreatic hormones and, in the present work, we have investigated the effects of selective imidazoline compounds on glucagon release from isolated rat islets of Langerhans. None of several imidazoline compounds tested (efaroxan, phentolamine, idazoxan, antazoline) affected glucagon secretion from islets incubated with 4 mM glucose. However, when the rate of glucagon release was stimulated by L-arginine (20 mM) efaroxan caused a rapid, sustained and dose-dependent inhibition of the secretory response (EC50 approximately 30 microM). This effect was seen under both static incubation and islet perifusion conditions. Antazoline and phentolamine also inhibited arginine-induced glucagon secretion, whereas idazoxan (an imidazoline which does not affect insulin secretion) failed to alter glucagon release. The inhibitory effects of imidazolines on glucagon release were not secondary to changes in insulin secretion. Taken together, the results indicate that pancreatic A-cells express functional imidazoline receptors which can regulate the secretory activity of the cells.
Abstract.
Author URL.
Mourtada M, Brown CA, Smith SA, Piercy V, Chan SL, Morgan NG (1997). Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion.
Br J Pharmacol,
121(4), 799-805.
Abstract:
Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion.
1. Imidazoline alpha 2-antagonist drugs such as efaroxan have been shown to increase the insulin secretory response to sulphonylureas from rat pancreatic B-cells. We have investigated whether this reflects binding to an islet imidazoline receptor or whether alpha 2-adrenoceptor antagonism is involved. 2. Administration of (+/-)-efaroxan or glibenclamide to Wistar rats was associated with a transient increase in plasma insulin. When both drugs were administered together, the resultant increase in insulin levels was much greater than that obtained with either drug alone. 3. Use of the resolved enantiomers of efaroxan revealed that the ability of the compound to enhance the insulin secretory response to glibenclamide resided only in the alpha 2-selective-(+)-enantiomer; the imidazoline receptor-selective-(-)-enantiomer was ineffective. 4. In vitro, (+)-efaroxan increased the insulin secretory response to glibenclamide in rat freshly isolated and cultured islets of Langerhans, whereas (-)-efaroxan was inactive. By contrast, (+)-efaroxan did not potentiate glucose-induced insulin secretion but (-)-efaroxan induced a marked increase in insulin secretion from islets incubated in the presence of 6 mM glucose. 5. Incubation of rat islets under conditions designed to minimize the extent of alpha 2-adrenoceptor signalling (by receptor blockade with phenoxybenzamine; receptor down-regulation or treatment with pertussis toxin) abolished the capacity of (+)- and (+/-)-efaroxan to enhance the insulin secretory response to glibenclamide. However, these manoeuvres did not alter the ability of (+/-)-efaroxan to potentiate glucose-induced insulin secretion. 6. The results indicate that the enantiomers of efaroxan exert differential effects on insulin secretion which may result from binding to effector sites having opposite stereoselectivity. Binding of (-)-efaroxan (presumably to imidazoline receptors) results in potentiation of glucose-induced insulin secretion, whereas interaction of (+)-efaroxan with a second site leads to selective enhancement of sulphonylurea-induced insulin release.
Abstract.
Author URL.
Di Matteo MA, Loweth AC, Thomas S, Mabley JG, Morgan NG, Thorpe JR, Green IC (1997). Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms.
Apoptosis,
2(2), 164-177.
Abstract:
Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms.
We have shown that nitric oxide treatment for 30-90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24-72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.
Abstract.
Author URL.
Chan SL, Atlas D, James RF, Morgan NG (1997). The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans.
Br J Pharmacol,
120(5), 926-932.
Abstract:
The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans.
1. The effects of a rat brain extract containing clonidine-displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated. 2. CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mM glucose, in a dose-dependent manner. The magnitude of this effect was similar to that in response to the well-characterized imidazoline secretagogue, efaroxan. 3. CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose-induced insulin release, in both rat and human islets. 4. These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline-mediated actions of CDS can be blocked by specific imidazoline antagonists. 5. The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri-filtration through a 3,000 Da cut-off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide. 6. Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.
Abstract.
Author URL.
Loweth AC, Scarpello JH, Morgan NG (1996). A specific inhibitor of cytosolic phospholipase A2 activity, AACOCF3, inhibits glucose-induced insulin secretion from isolated rat islets.
Biochem Biophys Res Commun,
218(2), 423-427.
Abstract:
A specific inhibitor of cytosolic phospholipase A2 activity, AACOCF3, inhibits glucose-induced insulin secretion from isolated rat islets.
In pancreatic beta-cells, arachidonic acid accumulation results primarily from phospholipid hydrolysis by phospholipase A2, and activation of this enzyme has been shown to accompany glucose-induced insulin secretion. Inhibitors of phospholipase A2 attenuate the secretory response, although the compounds used to date have not discriminated between cytosolic and secretory isoforms of phospholipase A2. In this work, the specific cytosolic phospholipase A2 inhibitor, AACOCF3, caused a dose-dependent inhibition of glucose-induced insulin secretion from isolated rat islets and this response was significantly relieved by exogenous arachidonic acid. The results suggest that, despite the low levels of expression of cytosolic phospholipase A2 in rat islets, this enzyme contributes to the control of glucose-induced insulin secretion in rat pancreatic beta-cells.
Abstract.
Author URL.
Lacey RJ, Chan SL, Cable HC, James RF, Perrett CW, Scarpello JH, Morgan NG (1996). Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.
J Endocrinol,
148(3), 531-543.
Abstract:
Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.
Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.
Abstract.
Author URL.
Loweth AC, Williams GT, Scarpello JH, Morgan NG (1996). Heterotrimeric G-proteins are implicated in the regulation of apoptosis in pancreatic beta-cells.
Exp Cell Res,
229(1), 69-76.
Abstract:
Heterotrimeric G-proteins are implicated in the regulation of apoptosis in pancreatic beta-cells.
Recent studies have provided evidence that apoptosis of pancreatic beta-cells is important in the early etiology of both type I and type II diabetes mellitus. The mechanisms responsible for induction of apoptosis are unknown, but we present evidence that the signal transduction pathway controlling the process in pancreatic beta-cells is regulated by G-proteins. We have employed the global G-protein activator fluoride and show that this agent induces apoptosis in clonal RINm5F pancreatic beta-cells and also in the cells of normal rat islets of Langerhans. The process is time and concentration dependent and may reflect the formation of AIF4- since it was inhibited by the aluminum chelator deferoxamine. Induction of apoptosis by fluoride was confirmed by acridine orange staining of cell nuclei, by electron-microscopic examination of chromatin condensation, and by oligonucleosomal degradation of DNA. The involvement of G-proteins was confirmed by culture of beta-cells in the presence of pertussis toxin (PTX) prior to exposure to fluoride. PTX did not affect the extent of cell death under control conditions but it consistently, and markedly, enhanced the response to fluoride. The results demonstrate that apoptosis can be induced in pancreatic beta-cells by sustained activation of a G-protein-dependent signaling pathway(s) and they further suggest that a pertussis toxin-sensitive G-protein is involved in attenuation of the response. Treatment of RINm5F pancreatic beta-cells with dibutyrylcAMP resulted in a dose-dependent, saturable increase in cell death, suggesting that a sustained rise in intracellular cAMP may form part of the effector system controlling apoptosis.
Abstract.
Author URL.
Morgan NG, Pallett AL, Mourtada M, Chan SLF, Clews J, Ramsden CA (1996). Synthesis and characterisation of KU14R a novel antagonist of the pancreatic islet imidazoline binding site.
DIABETOLOGIA,
39, 456-456.
Author URL.
Cable HC, el-Mansoury A, Morgan NG (1995). Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells.
Biochem J,
307 ( Pt 1)(Pt 1), 169-174.
Abstract:
Activation of alpha-2-adrenoceptors results in an increase in F-actin formation in HIT-T15 pancreatic B-cells.
1. Alpha-2-adrenoceptor agonists, such as noradrenaline, are potent inhibitors of insulin secretion, and it has been suggested that they control a late step in the pathway of exocytosis. We have investigated whether this could be related to a change in the extent of actin polymerization in the pancreatic B-cell, since actin microfilaments are implicated in regulating the access of secretory granules to the plasma membrane prior to exocytosis. 2. Cultured HIT-T15 pancreatic B-cells responded to noradrenaline with an increase in F-actin content, as judged by a rise in the fluorescence output after probing of the cells with phalloidin (a toxin which binds specifically to F-actin) conjugated to rhodamine. The response to noradrenaline was rapid, dose-dependent and sustained and could be reproduced by the highly selective alpha-2-agonist UK14,304. Examination of HIT-T15 cells by fluorescence microscopy after treatment with rhodamine-phalloidin, revealed a significant localization of F-actin immediately adjacent to the plasma membrane. The pattern of F-actin distribution in the cells was not altered dramatically by noradrenaline, although the intensity of staining close to the plasma membrane appeared to be slightly reduced. 3. The increase in F-actin content induced by noradrenaline and UK14,304 was inhibited significantly by the alpha-2-antagonist idazoxan but not by the alpha-1-selective antagonist prazosin. Pretreatment of HIT-T15 cells with pertussis toxin did not lead to any direct alteration in F-actin content, although the toxin significantly modified the responses induced by noradrenaline and UK14,304. In each case, cells incubated for 24 h with pertussis toxin responded to the alpha-2-agonist with an enhanced fluorescence output, indicating that F-actin levels had increased still further. This did not correlate with any gross change in the distribution of F-actin as judged by fluorescence microscopy. 4. The results demonstrate that alpha-2-adrenoceptors are coupled to control of actin polymerization in HIT-T15 cells. They suggest that regulation of F-actin formation could be a component of the mechanism by which alpha-2-agonists mediate inhibition of insulin secretion.
Abstract.
Author URL.
Morgan NG, Chan SL, Brown CA, Tsoli E (1995). Characterization of the imidazoline binding site involved in regulation of insulin secretion.
Ann N Y Acad Sci,
763, 361-373.
Author URL.
LOWETH AC, SCARPELLO JHB, MORGAN NG (1995). PHOSPHOLIPASE A(2) EXPRESSION IN HUMAN AND RODENT INSULIN-SECRETING CELLS (VOL 112, PG 177, 1995).
MOLECULAR AND CELLULAR ENDOCRINOLOGY,
114(1-2), 227-228.
Author URL.
Chan SL, Brown CA, Scarpello KE, Morgan NG (1995). Pancreatic beta-cells express an imidazoline binding site that is distinct from I1 and I2 sites.
Ann N Y Acad Sci,
763, 153-156.
Author URL.
Loweth AC, Scarpello JH, Morgan NG (1995). Phospholipase A2 expression in human and rodent insulin-secreting cells.
Mol Cell Endocrinol,
112(2), 177-183.
Abstract:
Phospholipase A2 expression in human and rodent insulin-secreting cells.
The expression of different isoforms of phospholipase A2 in human and rat islets of Langerhans and in the clonal B-cell lines, HIT-T15 and RINm5F has been investigated, using polyclonal antisera specific to human cytosolic (cPLA2) or human Groups I and II secretory (sPLA2) isoforms. Abundant levels of a 100-kDa protein corresponding to cPLA2 were detected in cytosolic extracts of human islets. A 100-kDa cPLA2 was not detectable in rat islets, RINm5F or HIT-T15 cells using an anti-cPLA2 serum raised against cloned human cPLA2 cDNA, despite the antiserum being cross-reactive with cPLA2 from rat kidney. Human and rat islets were found to express a 21-kDa protein immunoreactive with Group I sPLA2 antiserum. Group II sPLA2 was not detected in human or rat islets. RIN cells did not express detectable levels of either Group I or Group II sPLA2, but HIT cells expressed variable quantities of Group II sPLA2. These differences in PLA2 expression suggest that caution should be exercised when extrapolating conclusions about lipid-derived signalling molecules from insulin-secreting cell lines to normal islets of Langerhans.
Abstract.
Author URL.
Tsoli E, Chan SL, Morgan NG (1995). The imidazoline I1 receptor agonist, moxonidine, inhibits insulin secretion from isolated rat islets of Langerhans.
Eur J Pharmacol,
284(1-2), 199-203.
Abstract:
The imidazoline I1 receptor agonist, moxonidine, inhibits insulin secretion from isolated rat islets of Langerhans.
In order to study the pharmacology of the putative imidazoline receptor involved in stimulation of insulin secretion, the potent and selective imidazoline I1 receptor agonist, moxonidine, was employed. Surprisingly, this agent caused a rapid and complete inhibition of glucose-induced insulin secretion in isolated rat islets of Langerhans. This response was reversible upon removal of the compound but was only partially attenuated under conditions of complete alpha 2 blockade, suggesting that it did not derive entirely from the weak alpha 2-adrenoceptor agonist activity of moxonidine. Furthermore, the response could not be attributed to activation of imidazoline I1 receptors since it was not reproduced by a second potent imidazoline I1 receptor agonist, cimetidine, and could not be alleviated by the imidazoline I1 receptor antagonist efaroxan. The results confirm that the imidazoline receptor involved in control of insulin secretion differs from the I1 subclass and suggest that moxonidine inhibits insulin secretion by a mechanism unrelated to imidazoline I1 receptor agonism.
Abstract.
Author URL.
Loweth AC, Scarpello JH, Morgan NG (1994). A comparison of cytosolic phospholipase A2 expression in human islets of Langerhans and rodent insulin-secreting cells.
Biochem Soc Trans,
22(4).
Author URL.
Chan SL, Brown CA, Scarpello KE, Morgan NG (1994). The imidazoline site involved in control of insulin secretion: characteristics that distinguish it from I1- and I2-sites.
Br J Pharmacol,
112(4), 1065-1070.
Abstract:
The imidazoline site involved in control of insulin secretion: characteristics that distinguish it from I1- and I2-sites.
1. The nature of the binding site mediating the insulin secretagogue activity of certain imidazoline compounds remains unclear and the pharmacology of the I1- and I2-imidazoline sites, described in many tissues, does not correlate with the observed responses to imidazolines in islets. In the present paper, we describe further results which support the concept that the islet imidazoline site may represent a novel subtype of imidazoline receptor. 2. Culture of rat isolated islets in the presence of imidazoline secretagogues (either efaroxan or phentolamine) resulted in loss of responsiveness on subsequent re-exposure to these agents. However, culture of islets with either idazoxan or UK14,304 (imidazoline ligands that do not stimulate insulin secretion) did not lead to any loss of response when the islets were subsequently exposed to efaroxan. By contrast, islets cultured with UK14,304 (a potent alpha 2-adrenoceptor agonist), displayed loss of sensitivity to noradrenaline, consistent with down-regulation of alpha 2-adrenoceptors. 3. In order to characterize the imidazoline site further, radioligand binding studies were performed in membranes from RINm5F insulinoma cells using [3H]-RX821002, an imidazoline insulin secretagogue that does not interact significantly with imidazoline sites in other tissues. [3H]-RX821002 labelled alpha 2-adrenoceptors with high affinity (2.01 +/- 0.7 nM) but also labelled a second, non-adrenoceptor site with much lower affinity. 4. Under conditions of alpha 2-adrenoceptor blockade (in the presence of adrenaline), efaroxan displaced [3H]-RX821002 binding to the low affinity site, in a dose-dependent manner. Competition studies employing additional imidazoline compounds of varying secretagogue activity revealed that the pharmacological profile of the low affinity site correlates well with that observed in secretion experiments.5. The results obtained from the down-regulation experiments with isolated islets and from the radioligand binding studies suggest that the low affinity [3H]-RX821002 binding site may represent the functional receptor responsible for the secretagogue activity of imidazoline compounds in the endocrine pancreas and that it has a pharmacological profile distinct from those of I,- and 12-sites.
Abstract.
Author URL.
Morgan NG, Cable HC, Newcombe NR, Williams GT (1994). Treatment of cultured pancreatic B-cells with streptozotocin induces cell death by apoptosis.
Biosci Rep,
14(5), 243-250.
Abstract:
Treatment of cultured pancreatic B-cells with streptozotocin induces cell death by apoptosis.
Treatment of cultured pancreatic B-cells (HIT-T15 and RINm5F) with the diabetogenic drug streptozotocin resulted in a significant increase in the number of cells that became detached from the substrate during a subsequent culture period. Examination of the detached cells by fluorescence microscopy after staining with acridine orange or by electron microscopy revealed evidence of chromatin condensation and margination. Isolation and fractionation of DNA from these cells revealed a pattern of oligonucleosomal fragmentation that was not evident in untreated cells. All of these features are characteristic of entry of the cells into apoptosis and the results suggest that the diabetogenic action of streptozotocin involves induction of apoptosis in pancreatic B-cells.
Abstract.
Author URL.
Brown CA, Chan SL, Stillings MR, Smith SA, Morgan NG (1993). Antagonism of the stimulatory effects of efaroxan and glibenclamide in rat pancreatic islets by the imidazoline, RX801080.
Br J Pharmacol,
110(3), 1017-1022.
Abstract:
Antagonism of the stimulatory effects of efaroxan and glibenclamide in rat pancreatic islets by the imidazoline, RX801080.
1. The imidazoline alpha 2-adrenoceptor antagonist, efaroxan, stimulates insulin secretion from rat isolated islets and antagonizes the ability of diazoxide to inhibit glucose-induced insulin secretion. These effects result from closure of ATP-sensitive potassium channels although the mechanisms involved have not been elucidated. 2. In the present work, we have examined the effects of a close structural analogue of efaroxan, RX801080, in rat isolated islets of Langerhans. RX801080 was found to be ineffective as a stimulator of insulin secretion and did not prevent the inhibition of insulin secretion mediated by diazoxide. 3. RX801080 acted as an antagonist of the actions of several imidazolines (efaroxan, phentolamine and midaglizole) in rat islets. It dose-dependently inhibited the ability of efaroxan to antagonize the effects of diazoxide in islets and also completely inhibited the direct stimulation of insulin secretion mediated by efaroxan. 4. RX801080 also antagonized the effects of the non-imidazoline, ATP-sensitive potassium channel blocker, glibenclamide, in rat islets. It inhibited both the capacity of glibenclamide to stimulate insulin secretion and the ability of glibenclamide to overcome the inhibitory effects of diazoxide in rat islets. 5. Antagonism of glibenclamide responses by RX801080 was not due to inhibition of binding of the sulphonylurea to its receptor on the pancreatic beta-cell. 6. The results suggest that imidazoline compounds and sulphonylureas interact with distinct binding sites on islet cells, but that these sites can interact functionally to control islet cell ATP-sensitive potassium channel activity and insulin secretion.
Abstract.
Author URL.
Lacey RJ, Cable HC, James RF, London NJ, Scarpello JH, Morgan NG (1993). Concentration-dependent effects of adrenaline on the profile of insulin secretion from isolated human islets of Langerhans.
J Endocrinol,
138(3), 555-563.
Abstract:
Concentration-dependent effects of adrenaline on the profile of insulin secretion from isolated human islets of Langerhans.
The effects of the mixed alpha/beta-agonist adrenaline on insulin secretion from isolated human islets of Langerhans were studied. In static incubation experiments, adrenaline (0.1 nmol/l to 10 mumol/l) caused a concentration-dependent inhibition of glucose-induced insulin secretion from isolated human islets. However, perifusion experiments revealed that the time-course of the secretory changes induced by adrenaline was complex. When employed at a high concentration (1 mumol/l), adrenaline caused a sustained inhibition of glucose-induced insulin secretion, which could be relieved by the addition of the alpha 2-antagonist yohimbine (10 mumol/l). By contrast, infusion of adrenaline at a lower concentration (10 nmol/l), produced a large initial potentiation of glucose-induced insulin secretion. This response was, however, short-lived and followed by sustained inhibition of secretion, which could be relieved by yohimbine (10 mumol/l). The initial stimulation of insulin secretion provoked by 10 nmol adrenaline/l was abolished when islets were incubated in the presence of the beta-antagonist, propranolol (1 mumol/l), consistent with activation of beta-adrenoceptors. In support of this, treatment of human islets with the selective beta 2-agonist clenbuterol, was also associated with marked stimulation of insulin secretion. By contrast, each of two selective beta 3-agonists tested failed to alter insulin secretion from human islets. The results indicate that human pancreatic B-cells are equipped with both alpha 2- and beta 2-adrenoceptors which can affect insulin secretion. Adrenaline interacts with both of these but the alpha 2-response is predominant and can overcome the tendency of beta 2-adrenoceptors to potentiate insulin release.
Abstract.
Author URL.
Brown CA, Morgan NG (1993). Control of insulin secretion by imidazolines in rat pancreatic islets.
Biochem Soc Trans,
21(2).
Author URL.
Brown CA, Loweth AC, Smith SA, Morgan NG (1993). Stimulation of insulin secretion by imidazoline compounds is not due to interaction with non-adrenoceptor idazoxan binding sites.
Br J Pharmacol,
108(2), 312-317.
Abstract:
Stimulation of insulin secretion by imidazoline compounds is not due to interaction with non-adrenoceptor idazoxan binding sites.
1. The potency of interaction of several imidazoline compounds with non-adrenoceptor idazoxan binding sites (NAIBS) in rat liver membranes was compared with their ability to alter insulin secretion from rat pancreatic islets. 2. NAIBS could be labelled specifically with [3H]-idazoxan in both rat liver membranes and in rat islet homogenates. Liver binding sites exhibited a KD for [3H]-idazoxan of 24 nM and a Bmax of 264 fmol mg-1 protein. 3. Binding of [3H]-idazoxan to NAIBS in rat liver membranes was displaced effectively by unlabelled idazoxan (IC50 0.1 microM) and by UK14304 (IC50 0.5 microM). However, two other imidazoline compounds efaroxan and RX821002, which are related in structure to idazoxan, were much less effective as displacers. 4. In insulin secretion experiments, the ATP-sensitive potassium channel agonist diazoxide (250 microM) was able to suppress the rise in insulin secretion induced by 20 mM glucose. Both efaroxan and RX821002 (100 microM) antagonized the inhibitory effect of diazoxide on glucose-induced insulin secretion. By contrast, neither idazoxan (100 microM) nor UK14304 (50 microM), was able to overcome significantly the inhibitory effect of diazoxide. 5. The ability of 100 microM efaroxan to antagonize the suppression of insulin secretion mediated by diazoxide, was not prevented by idazoxan (up to 100 microM) or by UK14304 (up to 50 microM). 6. The results indicate that the stimulatory effects of imidazoline compounds on insulin secretion are not due to interaction with NAIBS similar to those present in rat liver.
Abstract.
Author URL.
Chan SL, Brown CA, Morgan NG (1993). Stimulation of insulin secretion by the imidazoline alpha 2-adrenoceptor antagonist efaroxan is mediated by a novel, stereoselective, binding site.
Eur J Pharmacol,
230(3), 375-378.
Abstract:
Stimulation of insulin secretion by the imidazoline alpha 2-adrenoceptor antagonist efaroxan is mediated by a novel, stereoselective, binding site.
We have studied the stereospecificity of three responses mediated by the alpha 2-antagonist efaroxan in rat islets of Langerhans. alpha 2-Adrenergic inhibition of insulin secretion was relieved most effectively by the (+) enantiomer. In contrast, direct stimulation of insulin secretion and antagonism of the inhibitory effects of diazoxide were both preferentially mediated by the (-) enantiomer. Culture of islets in the presence of efaroxan resulted in loss of responsiveness to subsequent re-addition of the drug. On the basis of these results we propose the existence, in islets, of a novel 'non-adrenergic' binding site at which efaroxan acts as an agonist.
Abstract.
Author URL.
Lacey RJ, Scarpello JH, Morgan NG (1992). Evidence that the presence of arginine can lead to overestimation of glucagon levels measured by radioimmunoassay.
Clin Chim Acta,
210(3), 211-219.
Author URL.
Berrow NS, Milligan G, Morgan NG (1992). Immunological characterization of the guanine-nucleotide binding proteins Gi and Go in rat islets of Langerhans.
J Mol Endocrinol,
8(2), 103-108.
Abstract:
Immunological characterization of the guanine-nucleotide binding proteins Gi and Go in rat islets of Langerhans.
Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate Mr of 40,000. Antiserum I1C (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets. Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of Mr 39-40,000. Thus, at least two molecular forms of Go are present in rat islets. Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.
Abstract.
Author URL.
Berrow NS, Hurst RD, Chan SL, Morgan NG (1992). Immunoprecipitation of a pertussis toxin substrate of the G(o) family from rat islets of Langerhans.
Biosci Rep,
12(2), 95-100.
Abstract:
Immunoprecipitation of a pertussis toxin substrate of the G(o) family from rat islets of Langerhans.
Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent "G(i)" which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with 32P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-G(o) antiserum resulted in specific immunoprecipitation of a 32P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes G(o).
Abstract.
Author URL.
Chan SL, Stillings MR, Morgan NG (1991). Mechanisms involved in stimulation of insulin secretion by the hypoglycaemic alpha-adrenergic antagonist, DG-5128.
Biochem Biophys Res Commun,
176(3), 1545-1551.
Abstract:
Mechanisms involved in stimulation of insulin secretion by the hypoglycaemic alpha-adrenergic antagonist, DG-5128.
The selective alpha 2-antagonist DG-5128 provoked a dose-dependent stimulation of insulin release from isolated rat islets. DG-5128 was only weakly effective as an antagonist of noradrenaline-induced inhibition of insulin secretion but, surprisingly, was able to reverse the suppression of secretion and increase in 86Rb efflux from preloaded islets, mediated by diazoxide. These effects were not reproduced with more effective alpha-antagonists, suggesting that stimulation of insulin secretion by DG-5128 is independent of alpha-receptor blockade.
Abstract.
Author URL.
Lacey RJ, Berrow NS, Scarpello JH, Morgan NG (1991). Selective stimulation of glucagon secretion by beta 2-adrenoceptors in isolated islets of Langerhans of the rat.
Br J Pharmacol,
103(3), 1824-1828.
Abstract:
Selective stimulation of glucagon secretion by beta 2-adrenoceptors in isolated islets of Langerhans of the rat.
1. In rat isolated islets of Langerhans the selective beta 2-adrenoceptor agonist, clenbuterol (1 to 20 microM), significantly increased the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) within 2 min of incubation. 2. The cyclic AMP response to clenbuterol was inhibited in the presence of the selective beta 2 adrenoceptor antagonist, ICI 118551 (0.1 or 10 microM) but remained unchanged when the beta 1-antagonist, atenolol (0.1 microM) was administered. 3. Despite causing an elevation in cyclic AMP, clenbuterol (up to 20 microM) failed to influence insulin secretion at any glucose concentration tested, even in the presence of a phosphodiesterase inhibitor. 4. By contrast, clenbuterol elicited a dose-dependent rise in the rate of glucagon secretion; the maximal agonist-induced increase in secretion was two fold, a response equivalent to that observed with 20 mM L-arginine. 5. ICI 118551 significantly inhibited the rise in glucagon secretion induced by clenbuterol (up to 20 microM). 6. The results indicate that the rat islet a cell population is equipped with functional beta 2-adrenoceptors which influence glucagon secretion via the second messenger cyclic AMP, but that the B cells are deficient in functional beta-receptors.
Abstract.
Author URL.
Chan SL, Dunne MJ, Stillings MR, Morgan NG (1991). The alpha 2-adrenoceptor antagonist efaroxan modulates K+ATP channels in insulin-secreting cells.
Eur J Pharmacol,
204(1), 41-48.
Abstract:
The alpha 2-adrenoceptor antagonist efaroxan modulates K+ATP channels in insulin-secreting cells.
The actions of efaroxan, a highly selective and potent alpha 2-adrenoceptor antagonist, on insulin secretion, cAMP levels, 86Rb+ efflux and ATP-regulated potassium (K+ATP) channels have been studied using isolated pancreatic islets of Langerhans and RINm5F cells. In the absence of an adrenoceptor agonist, efaroxan (1-100 microM) potentiated glucose-induced secretion over the range 4-10 mM glucose, but was without effect upon the maximal rate of secretion induced by 20 mM glucose. Efaroxan did not affect cAMP levels. Suppression of insulin release by the potassium channel opener diazoxide, was partially alleviated by efaroxan and was associated with an inhibition of the diazoxide-induced increase in the rate of 86Rb+ efflux. Using isolated patches of membrane we found efaroxan to be an effective blocker of K+ATP channels, with a KI value of 12 microM and a Hill coefficient of approximately 1. These data indicate that efaroxan promotes insulin secretion, in the absence of exogenous agonists, by a mechanism that involves inhibition of ATP-regulated K+ channels.
Abstract.
Author URL.
Lacey RJ, Berrow NS, London NJ, Lake SP, James RF, Scarpello JH, Morgan NG (1990). Differential effects of beta-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man.
J Mol Endocrinol,
5(1), 49-54.
Abstract:
Differential effects of beta-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man.
The selective beta 2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to beta-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mM glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mM glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of beta-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different beta 2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to beta-agonists, whereas those of human islets are equipped with functional beta-receptors which can directly influence the rate of insulin secretion.
Abstract.
Author URL.
Hurst RD, Morgan NG (1990). Evidence for differential effects of noradrenaline and somatostatin on intracellular messenger systems in rat islets of Langerhans.
J Mol Endocrinol,
4(3), 231-237.
Abstract:
Evidence for differential effects of noradrenaline and somatostatin on intracellular messenger systems in rat islets of Langerhans.
The mechanisms involved in inhibition of insulin secretion by somatostatin and noradrenaline were compared in order to establish whether the receptors for these agents are coupled to similar effector systems in the pancreatic B cell. Both agents significantly reduced forskolin-induced adenylate cyclase activity in islet homogenates, although noradrenaline was more effective than somatostatin. The capacity of noradrenaline to inhibit insulin secretion was largely unaffected by agents that increase intracellular cyclic AMP, whereas the effect of somatostatin as an inhibitor was markedly reduced under these conditions. Both noradrenaline and somatostatin inhibited the stimulation of insulin secretion induced by K+ depolarization, but different mechanism were involved. Somatostatin significantly inhibited K(+)-stimulated 45Ca2+ efflux and influx in islets, while noradrenaline exerted only a minor influence on these processes. The data indicate that noradrenaline controls insulin secretion by a mechanism which operates beyond the level of intracellular messenger generation. In contrast, somatostatin exerts at least part of its inhibitory effect on insulin secretion by directly controlling islet cell Ca2+ influx in a manner which may be regulated by cyclic AMP.
Abstract.
Author URL.
Berrow NS, Morgan NG (1990). Evidence for the presence of low molecular mass GTP-binding proteins in rat islets of Langerhans.
Biochem Soc Trans,
18(3), 485-486.
Author URL.
Chan SL, Morgan NG (1990). Stimulation of insulin secretion by efaroxan may involve interaction with potassium channels.
Eur J Pharmacol,
176(1), 97-101.
Abstract:
Stimulation of insulin secretion by efaroxan may involve interaction with potassium channels.
The imidazoline alpha 2-antagonist efaroxan was found to stimulate insulin secretion from isolated rat pancreatic islets incubated in glucose concentrations between 4 and 12 mM. This response could not be attributed to interaction of efaroxan with either classical alpha 2-receptors or with a B-cell 'imidazoline receptor', since the effect was not reproduced by the structural analogue idazoxan. Stimulation of insulin secretion by efaroxan correlated with the ability of the drug to reverse the inhibition of secretion mediated by the potassium channel agonist diazoxide. The data suggest that the capacity of efaroxan to stimulate insulin secretion may be related to an interaction with potassium channels in the pancreatic B-cell.
Abstract.
Author URL.
Morgan NG, Hurst RD, Berrow NS, Montague W (1989). Calcium handling by stimulated islets of Langerhans.
Biochem Soc Trans,
17(1), 64-66.
Author URL.
Hurst RD, Chan SL, Morgan NG (1989). Effects of benextramine on the adrenergic inhibition of insulin secretion in isolated rat pancreatic islets.
J Mol Endocrinol,
2(2), 99-105.
Abstract:
Effects of benextramine on the adrenergic inhibition of insulin secretion in isolated rat pancreatic islets.
Insulin secretion from isolated rat islets of Langerhans in the presence of 4 mM glucose averaged 2.26 +/- 0.20 (S.E.M.) ng/islet per 90 min and was significantly (P less than 0.001; n = 30) increased to 3.28 +/- 0.21 ng/islet per 90 min by the covalent alpha-adrenoceptor antagonist benextramine (10 microM). Glucose (20 mM) also increased the secretion rate (to 6.24 +/- 6.0 ng/islet per 90 min) but, under these conditions, the response was not further enhanced by benextramine. Clonidine and noradrenaline (1 nM-10 microM) each caused dose-dependent inhibition of glucose-induced insulin secretion which was maximal at 1 microM. Benextramine, when added simultaneously with the agonist, relieved, in a dose-dependent manner, the inhibition of secretion induced by either clonidine or noradrenaline with similar sensitivity. Even after a 30-min preincubation with benextramine the antagonist failed to differentiate between noradrenaline, adrenaline and clonidine with respect to inhibition of insulin secretion. In contrast to its effects on adrenergic responses, short-term treatment with benextramine did not significantly affect muscarinic-cholinergic receptor-mediated 45Ca2+ efflux from rat islets of Langerhans perifused in Ca2+-depleted medium. These data suggest that benextramine does not differentiate between clonidine and noradrenaline in rat islets of Langerhans but that it does show preference for alpha-adrenoceptors in this tissue.
Abstract.
Author URL.
Chan SL, Morgan NG (1989). Effects of phenoxybenzamine on insulin secretion from isolated rat islets of Langerhans.
Biosci Rep,
9(2), 223-230.
Abstract:
Effects of phenoxybenzamine on insulin secretion from isolated rat islets of Langerhans.
In isolated rat islets the alpha 2-adrenergic antagonist phenoxybenzamine was found to be only partially effective at relieving the inhibition of glucose-induced insulin secretion mediated by noradrenaline. Further experiments revealed a direct inhibitory effect of phenoxybenzamine itself on the secretory response to glucose. At concentrations above 1 microM the antagonist inhibited insulin secretion in a dose-dependent manner, with greater than 50% inhibition at 50 microM. The inhibition of secretion developed rapidly in perifused islets, and was not altered when islets were also incubated with idazoxan or benextramine, suggesting that it did not reflect binding of phenoxybenzamine to the alpha 2-receptor. Paradoxically phenoxybenzamine significantly increased the basal secretion rate in the presence of 4 mM glucose. The results demonstrate that phenoxybenzamine can exert direct effects on insulin secretion which are unrelated to its alpha-antagonist properties.
Abstract.
Author URL.
Hurst RD, Morgan NG (1989). Intracellular events responsible for the inhibition of insulin secretion by somatostatin.
Biochem Soc Trans,
17(6), 1085-1086.
Author URL.
Morgan NG, Hurst RD (1988). Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans.
FEBS Lett,
227(2), 153-156.
Abstract:
Dissociation between intracellular calcium mobilization and insulin secretion in isolated rat islets of Langerhans.
The neuropeptide bombesin provoked a dose-dependent stimulation of 45Ca2+ efflux from pre-loaded islets of Langerhans. This response occurred rapidly, was not sustained and did not depend on the presence of extracellular calcium, suggesting that it resulted from the mobilization of intracellular calcium stores. Under conditions when large increases in 45Ca2+ efflux were observed, bombesin completely failed to stimulate the rate of insulin secretion. Similar results were also obtained with the muscarinic cholinergic agonist, carbachol. The data suggest that the release of calcium from intracellular pools is not sufficient to induce an increase in insulin secretion in normal islet cells.
Abstract.
Author URL.
Morgan NG, Rumford GM, Montague W (1987). Mechanisms involved in intracellular calcium mobilization in isolated rat islets of Langerhans.
Biochem J,
244(3), 669-674.
Abstract:
Mechanisms involved in intracellular calcium mobilization in isolated rat islets of Langerhans.
1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.
Abstract.
Author URL.
MORGAN NG (1987). REGULATION OF INSULIN-SECRETION BY ALPHA-2-ADRENERGIC AGONISTS.
TRENDS IN PHARMACOLOGICAL SCIENCES,
8(10), 369-370.
Author URL.
Montague W, Morgan NG, Rumford GM, Prince CA (1985). Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.
Biochem J,
227(2), 483-489.
Abstract:
Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans.
The metabolism of inositol-containing phospholipids during insulin secretion was studied in rat islets of Langerhans preincubated with [3H]inositol to label their phospholipids. Glucose (20 mM) caused a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and an accumulation of inositol trisphosphate and inositol bisphosphate. This effect was maximal at 60s, did not require the presence of extracellular Ca2+, and was abolished by mannoheptulose (15 mM), but not by noradrenaline (1 microM). Mannose (20 mM) and DL-glyceraldehyde (10 mM) produced similar effects to those of glucose, but galactose (20 mM) and KCl (30 mM) were without effect. These results are compatible with the hypothesis that an early event in the stimulus-secretion coupling mechanism in the pancreatic B-cell is the rapid breakdown of polyphosphoinositides catalysed by phospholipase C. Moreover, they suggest that the breakdown of polyphosphoinositides is linked to sugar metabolism in the B-cell. This observation is important, since it demonstrates that events in a cell other than plasma-membrane receptor occupancy can promote polyphosphoinositide hydrolysis.
Abstract.
Author URL.
Morgan NG, Rumford GM, Montague W (1985). Effects of noradrenaline on 45Ca2+ efflux from isolated rat islets of Langerhans.
Biosci Rep,
5(12), 1053-1060.
Abstract:
Effects of noradrenaline on 45Ca2+ efflux from isolated rat islets of Langerhans.
Noradrenaline caused a prompt but transient increase in the rate of 45Ca2+ efflux from isolated rat islets of Langerhans perifused in Ca2+ depleted medium. The response was modest in size and was unaffected by isosmotic replacement of NaCl with choline chloride or by inclusion of 0.5 mM dibutyryl cAMP in the perifusion medium, suggesting that it was not mediated by Na+: Ca2+ exchange nor by lowered cAMP. Despite its effect on 45Ca2+ efflux, noradrenaline treatment did not alter the kinetics of 45Ca2+ efflux in response to the muscarinic agonist, carbamylcholine, nor did it change the magnitude of the response to this agent. Simultaneous introduction of 20 mM glucose with noradrenaline prevented a rise in 45Ca2+ efflux and indeed resulted in inhibition of 45Ca2+ efflux. The data suggest that noradrenaline does not directly activate the mechanisms which regulate Ca2+ extrusion from islets cells, and they do not support a primary role for the Ca2+ efflux response in mediating adrenergic inhibition of insulin secretion.
Abstract.
Author URL.
Morgan NG, Short CD, Rumford GM, Montague W (1985). Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.
Biochem J,
231(3), 629-634.
Abstract:
Effects of the calcium-channel agonist CGP 28392 on insulin secretion from isolated rat islets of Langerhans.
The rate of insulin secretion from isolated rat islets of Langerhans was affected by a number of dihydropyridine derivatives known to interact with voltage-sensitive Ca2+ channels in excitable cells. The channel antagonists nifedipine and nitrendipine were potent inhibitors of glucose-induced insulin secretion in response to both 8 mM- and 20 mM-glucose, although they did not lower the basal secretion rate observed in the presence of 4 mM-glucose. The Ca2+-channel agonist, CGP 28392, also failed to alter the basal rate of insulin secretion. In the presence of 8 mM-glucose, however, 1 microM-CGP 28392 enhanced the insulin-secretion rate to a value approximately double that with 8 mM-glucose alone. This effect was dose-dependent, with half the maximal response elicited by 0.1 microM-CGP 28392, and full enhancement at 10 microM. The response was rapid in onset, with an increase in insulin secretion evident within 2 min of CGP 28392 infusion in perifused islets. Stimulation of insulin secretion by CGP 28392 was correlated with a rapid enhancement of glucose-stimulated 45Ca2+ uptake into islets cells, and with a transiently increased rate of 45Ca2+ efflux from pre-loaded islets. Stimulation of insulin secretion by CGP 28392 was abolished in the presence of noradrenaline, although under these conditions the rapid stimulation of 45Ca2+ influx induced by CGP 28392 was only partially inhibited. In contrast with these results, when islets were incubated in the presence of 20 mM-glucose, CGP 28392 caused a dose-dependent inhibition of insulin secretion. Half-maximal inhibition required approx. 0.2 microM-CGP 28392, with maximal effects observed at 10 microM. Under these conditions, however, the extent of insulin secretion was still only decreased by about 50%, to a value which was similar to that seen in the presence of 8 mM-glucose and CGP 28392. These results suggest that dihydropyridine derivatives can alter the activity of voltage-dependent Ca2+ channels in islet cells, and are consistent with the possibility that gating of these channels plays an important role in regulating the rate of insulin secretion after glucose stimulation.
Abstract.
Author URL.
Morgan NG, Rumford GM, Montague W (1985). Studies on the mechanism by which melittin stimulates insulin secretion from isolated rat islets of Langerhans.
Biochim Biophys Acta,
845(3), 526-532.
Abstract:
Studies on the mechanism by which melittin stimulates insulin secretion from isolated rat islets of Langerhans.
Incubation of isolated rat islets of Langerhans with melittin resulted in a dose-dependent stimulation of insulin secretion with half the maximal response occurring at 4 micrograms/ml melittin. The effect of melittin on insulin secretion was dependent on extracellular calcium, was inhibited by the phospholipase A2 inhibitor quinacrine and by the lipoxygenase inhibitor nordihydroguaiaretic acid. Stimulation of insulin secretion by melittin was associated with a calcium-dependent loss of [3H]arachidonic acid from phospholipids in islet cells prelabelled with [3H]arachidonic acid. Analysis of the islet phospholipids involved in this response revealed that the [3H]arachidonic acid was released predominantly from phosphatidylcholine. These results suggest that melittin may stimulate insulin secretion by activating phospholipase A2 in islet cells, causing the release of arachidonic acid from membrane phospholipid. The results are consistent with suggestions that the subsequent metabolism of arachidonic acid via the lipoxygenase pathway may be involved in regulating the insulin secretory response.
Abstract.
Author URL.
Morgan NG, Montague W (1985). Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.
Biochem J,
226(2), 571-576.
Abstract:
Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.
Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.
Abstract.
Author URL.
Morgan NG, Rumford GM, Montague W (1985). Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.
Biochem J,
228(3), 713-718.
Abstract:
Studies on the role of inositol trisphosphate in the regulation of insulin secretion from isolated rat islets of Langerhans.
Glucose (20 mM) and carbachol (1 mM) produced a rapid increase in [3H]inositol trisphosphate (InsP3) formation in isolated rat islets of Langerhans prelabelled with myo-[3H]inositol. The magnitude of the increase in InsP3 formation was similar when either agent was used alone and was additive when they were used together. In islets prelabelled with 45Ca2+ and treated with carbachol (1 mM), the rise in InsP3 correlated with a rapid, transient, release of 45Ca2+ from the cells, consistent with mobilization of 45Ca2+ from an intracellular pool. Under these conditions, however, insulin secretion was not increased. In contrast, islets prelabelled with 45Ca2+ and exposed to 20mM-glucose exhibited a delayed and decreased 45Ca2+ efflux, but released 7-8-fold more insulin than did those exposed to carbachol. Depletion of extracellular Ca2+ failed to modify the increase in InsP3 elicited by either glucose or carbachol, whereas it selectively inhibited the efflux of 45Ca2+ induced by glucose in preloaded islets. Under these conditions, however, glucose was still able to induce a small stimulation of the first phase of insulin secretion. These results demonstrate that polyphosphoinositide metabolism, Ca2+ mobilization and insulin release can all be dissociated in islet cells, and suggest that glucose and carbachol regulate these parameters by different mechanisms.
Abstract.
Author URL.
Morgan NG, Charest R, Blackmore PF, Exton JH (1984). Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.
Proc Natl Acad Sci U S A,
81(13), 4208-4212.
Abstract:
Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.
Treatment of isolated hepatocytes with the alpha 1-adrenergic agonist norepinephrine induced a dose-dependent increase in free cytosolic Ca2+, as judged by fluorescence increases, in cells loaded with the Ca2+ indicator (2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8 -bis [carboxymethyl]aminoquinoline (quin-2). Pretreatment with either glucagon or dibutyryl cAMP increased the rate and magnitude of the quin-2 fluorescence response in hepatocytes treated with submaximal doses of norepinephrine and increased the cell sensitivity such that a physiological concentration of norepinephrine (7.5 nM) was able to provoke a quin-2 fluorescence response. Similar enhancement of norepinephrine-induced phosphorylase activation and pyridine nucleotide reduction in isolated hepatocytes and Ca2+ efflux from the perfused liver was also observed in the presence of glucagon. These potentiated responses correlated with a cAMP-dependent increase (mediated by glucagon, dibutyryl cAMP, or forskolin) in the binding of [3H]norepinephrine or [3H]epinephrine to sites present on isolated hepatocytes bearing the characteristics of alpha 1-adrenergic receptors. The data suggest that a cAMP-dependent mechanism is involved in the regulation of alpha 1-agonist binding to liver cells and, thereby, in the control of hepatic carbohydrate metabolism in response to catecholamines.
Abstract.
Author URL.
Morgan NG, Montague W (1984). Stimulation of insulin secretion from isolated rat islets of Langerhans by melittin.
Biosci Rep,
4(8), 665-671.
Abstract:
Stimulation of insulin secretion from isolated rat islets of Langerhans by melittin.
Melittin, an amphipathic polypeptide, stimulated the secretion of insulin from rat islets of Langerhans incubated in vitro. The secretory response was dose-dependent and saturable with half the maximal response elicited by a melittin concentration of 4 micrograms/ml. The response was rapid in onset, an increase in secretion occurring within 2 min of exposure of the islets to melittin (2 micrograms/ml). An enhanced secretory rate could be maintained for at least 40 min in the presence of melittin but declined steadily when the agent was removed. Stimulation of secretion by melittin occurred in the absence of glucose and in the presence of both 4 mM and 8 mM glucose but not in the presence of 20 mM glucose. The effect of melittin on secretion was dependent on the presence of extracellular calcium but was not inhibited by norepinephrine. The data suggest that melittin may be a valuable agent for further study of the role played by the B-cell plasma membrane in the regulation of insulin secretion.
Abstract.
Author URL.
Morgan NG, Blackmore PF, Exton JH (1983). Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats.
J Biol Chem,
258(8), 5103-5109.
Abstract:
Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats.
Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.
Abstract.
Author URL.
Morgan NG, Exton JH, Blackmore PF (1983). Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects.
FEBS Lett,
153(1), 77-80.
Abstract:
Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects.
Incubation of isolated hepatocytes containing normal Ca2+ levels with angiotensin II, vasopressin or A23187 caused significant inhibition of the cAMP response to glucagon. Angiotensin II also inhibited cAMP accumulation induced by either glucagon or epinephrine in Ca2+-depleted hepatocytes. When submaximal doses of hormone were employed such that cell cAMP was elevated only 3-4-fold (approximately 2 pmol cAMP/mg wet wt cells) inhibition by angiotensin II was correlated with a decrease in phosphorylase activation. The data demonstrate that inhibition of hepatic cAMP accumulation results in reduced metabolic responses to glucagon and epinephrine and do not support the contention that the hepatic actions of glucagon are independent of cAMP.
Abstract.
Author URL.
Morgan NG, Waynick LE, Exton JH (1983). Characterisation of the alpha 1-adrenergic control of hepatic cAMP in male rats.
Eur J Pharmacol,
96(1-2), 1-10.
Abstract:
Characterisation of the alpha 1-adrenergic control of hepatic cAMP in male rats.
alpha 1-Adrenergic agonists characteristically elicit a mobilization of intracellular Ca2+ in rat liver. These agents also induced accumulation of cAMP in mature male rats (greater than 300 g body weight) and in Ca2+-depleted hepatocytes from 200 g rats although not in Ca2+-depleted cells from juvenile (less than 100 g) male rats. Comparison of these two responses revealed a similar agonist potency order in both cases, although cAMP accumulation was approximately 5-fold less sensitive to agonists. A variety of alpha-antagonists, including prazosin, phenoxybenzamine and dihydroergocryptine were equipotent as inhibitors of each response, although the alpha 1-adrenergic cAMP response was more sensitive to inhibition by WB-4101 and phentolamine. These data are discussed and a model proposed whereby in mature male rats, the same alpha 1-adrenergic receptor population becomes simultaneously coupled to two separate signal transduction mechanisms, namely Ca2+ mobilization and cAMP generation.
Abstract.
Author URL.
Morgan NG, Blackmore PF, Exton JH (1983). Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP.
J Biol Chem,
258(8), 5110-5116.
Abstract:
Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP.
The Ca2+ content of hepatocytes from juvenile male rats (80-110 g) or adult female rats (135-155 g) displayed a biphasic dose-response curve to epinephrine. Low concentrations (less than or equal to 10(-7) M) caused efflux of Ca2+ from the cells, while higher concentrations (10(-6) M and 10(-5) M) induced net Ca2+ uptake which correlated with a large beta 2-adrenergic-mediated increase in cAMP (Morgan, N. G. Blackmore, P. F. and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109). Calcium accumulation could be induced in cells from older male rats (180-230 g) by combining a Ca2+-mobilizing hormone with either exogenous cAMP or glucagon (10(-8) M). Readdition of Ca2+ in the presence of glucagon to cells treated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also resulted in enhanced Ca2+ accumulation compared with controls. Addition of vasopressin plus glucagon to the medium perfusing male rat livers also led to cell Ca2+ accumulation, as evidenced by uptake of Ca2+ from the perfusate. Incubation of hepatocytes with antimycin A, oligomycin, and carbonyl cyanide m-chlorophenylhydrazone prevented net Ca2+ accumulation suggesting that mitochondria play a role in the uptake response. This was confirmed by isolation of mitochondria from cells incubated under conditions which promote Ca2+ accumulation. Within 5 min of incubation, the Ca2+ content of these mitochondria was increased 2-fold relative to controls, an effect which was inhibited by oligomycin. These studies demonstrate that a rise in hepatic cAMP can reverse hormonally induced Ca2+ mobilization and point to a major role for the mitochondria in this effect.
Abstract.
Author URL.
Morgan NG, Shipp CC, Exton JH (1983). Studies on the mechanism of inhibition of hepatic cAMP accumulation by vasopressin.
FEBS Lett,
163(2), 277-281.
Abstract:
Studies on the mechanism of inhibition of hepatic cAMP accumulation by vasopressin.
Vasopressin elicited a dose-dependent inhibition of glucagon-induced cAMP accumulation in isolated hepatocytes. This response was not diminished by incubation of cells with the calmodulin antagonists trifluoperazine or chlorpromazine and was only slightly reduced in Ca2+-depleted hepatocytes. Half-maximal inhibition of cAMP accumulation occurred at 8 X 10(-11) M vasopressin, a dose which does not increase cytosolic Ca2+ in hepatocytes. Direct activation of adenylate cyclase by forskolin was significantly inhibited by vasopressin in Ca2+-depleted cells. It is concluded that inhibition of hormone-induced cAMP accumulation by vasopressin in liver is not dependent on cellular Ca2+ mobilisation but may involve direct inhibition of adenylate cyclase.
Abstract.
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Morgan NG, Shuman EA, Exton JH, Blackmore PF (1982). Stimulation of hepatic glycogenolysis by alpha 1- and beta 2-adrenergic agonists. Evidence against short term agonist-induced desensitization of the responses.
J Biol Chem,
257(23), 13907-13910.
Abstract:
Stimulation of hepatic glycogenolysis by alpha 1- and beta 2-adrenergic agonists. Evidence against short term agonist-induced desensitization of the responses.
Addition of alpha 1-adrenergic agonists or vasopressin to the medium perfusing rat livers was associated with a rapid efflux of Ca2+, which was rapidly reaccumulated on removal of the stimulus. The magnitudes of the respective Ca2+ efflux and influx responses were similar, suggesting that Ca2+ was being mobilized from, and then reaccumulated into, the same pool(s). Addition of combinations of alpha 1-agonists, vasopressin and angiotensin II to isolated hepatocytes revealed that the Ca2+ efflux response induced by each individual hormone could only be augmented by addition of a second hormone when submaximal doses of each were employed. This suggests that an identical pool(s) of hepatocyte Ca2+ is mobilized in response to each of these agents. No desensitization of the alpha 1-adrenergic glycogenolytic response was observed in the perfused liver, upon repeated or more prolonged (25 min) exposure to agonist, providing cell Ca2+ reaccumulation occurred before addition of each successive stimulus. Selective short term stimulation of the beta 2-adrenergic glycogenolytic response in immature male rats (less than 150 g, body weight) was not associated with desensitization to subsequent stimulation. These data demonstrate that the glycogenolytic response of liver cells does not become desensitized by successive short term stimulation with alpha 1- or beta 2-adrenergic agonists.
Abstract.
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Morgan NG, Montague W (1982). Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.
Biochem J,
204(1), 111-125.
Abstract:
Studies on the interaction of staphylococcal delta-haemolysin with isolated islets of Langerhans.
delta-Haemolysin, a small surface-active polypeptide purified from the culture media of Staphylococcus aureus, was observed to stimulate the release of insulin from isolated rat islets of Langerhans. This effect was dose-dependent and saturable, with the half-maximal response elicited by a delta-haemolysin concentration of 10 micrograms/ml. Stimulation of insulin release by delta-haemolysin (10 micrograms/ml) was not dependent on the presence of glucose in the incubation medium, but was augmented by increasing concentrations of the sugar. The release of insulin in response to delta-haemolysin could be inhibited by depletion of extracellular Ca2+ or by adrenaline (epinephrine) (10 microM) and was readily reversible when delta-haemolysin was removed from the medium. In addition, the response was potentiated by incubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2 mM). These observations suggest that delta-haemolysin induced a true activation of the beta-cell secretory mechanism. Stimulation of islets of Langerhans with delta-haemolysin was found to be associated with a modest increase in intracellular cyclic AMP levels, although the adenylate cyclase activity of islet homogenates was not increased by delta-haemolysin. delta-Haemolysin was observed to induce a dose-dependent net accumulation of 45Ca2+ by islet cells and to stimulate the efflux of 45Ca2+ from preloaded islets. The efflux of 45Ca2+ was modest in size and short-lived, but dramatically increased in medium depleted fo 40Ca2+. Incubation in the presence of verapamil augmented delta-haemolysin-induced 45Ca2+ efflux and insulin secretion. delta-Haemolysin was found to be a potent 45Ca2+-translocating ionophore in an artificial system. This response was dose-dependent and could be augmented by verapamil. In addition, phosphatidylcholine (25 micrograms/ml) was found to inhibit both delta-haemolysin induced 45Ca2+ translocation and insulin release in a precisely parallel manner. These studies suggest that the ability of delta-haemolysin to stimulate insulin release may be due, in part, to the facilitation of Ca2+ entry into the beta-cells of islets of Langerhans, mediated directly by an ionophoretic mechanism.
Abstract.
Author URL.
Morgan NG, Fitzhugh AJ, Montague W (1981). The effect of staphylococcal delta-haemolysin on the secretory activity of the pancreatic beta-cell.
Biosci Rep,
1(2), 135-140.
Abstract:
The effect of staphylococcal delta-haemolysin on the secretory activity of the pancreatic beta-cell.
The secretion of insulin from isolated rat islets of Langerhans was found to be stimulated by the surface-active staphylococcal exotoxin, delta-haemolysin. The response was dependent on the concentration of delta-haemolysin, was rapid in onset, and could be maintained for at least an hour in the presence of the agent. The rate of secretion rapidly declined on removal of delta-haemolysin and the islets remained responsive to glucose following toxin treatment. Further characterization of the interaction of this agent with the beta-cell plasma membrane may provide valuable information concerning the role played by this membrane in the regulation of insulin secretion.
Abstract.
Author URL.
Ducommun Y, Morgan NG, Montague W (1979). Subcellular distribution of protein kinase activities in mammalian islets of Langerhans [proceedings].
Biochem Soc Trans,
7(5), 913-915.
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