Journal articles
Potter PGW, Washer S, Jeffries AR, Holley JE, Gutowski NJ, Dempster E, Beall C (In Press). Analysis of the transcriptome and DNA methylome in response to acute and recurrent low glucose in human primary astrocytes.
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Analysis of the transcriptome and DNA methylome in response to acute and recurrent low glucose in human primary astrocytes
ABSTRACTAims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure.MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform.Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following LG exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures.Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be mediated by metabolic adaptations to better preserve intracellular and/or ER ATP levels, but this requires further investigation.
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Weightman Potter P, Vlachaki Walker J, Robb J, Chilton J, Williamson R, Randall A, Ellacott K, Beall C (In Press). Basal fatty acid oxidation increases after recurrent low glucose in human primary astrocytes. Diabetologia
Cruz AM, Malekizadeh Y, Vlachaki Walker JM, Weightman Potter PG, Pye K, Shaw SJ, Ellacott KLJ, Beall C (In Press). Brain permeable AMPK activator R481 raises glycemia by autonomic nervous system activation and amplifies the counterregulatory response to hypoglycemia in rats.
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Brain permeable AMPK activator R481 raises glycemia by autonomic nervous system activation and amplifies the counterregulatory response to hypoglycemia in rats
ABSTRACTAMP-activated protein kinase (AMPK) is a critical cellular and whole body energy sensor activated by energy stress, including hypoglycemia, which is frequently experienced by people with diabetes. Previous studies using direct delivery of an AMPK activator to the ventromedial hypothalamus (VMH) in rodents increased hepatic glucose production. Moreover, recurrent glucoprivation in the hypothalamus leads to blunted AMPK activation and defective hormonal responses to subsequent hypoglycemia. These data suggest that amplifying AMPK activation may prevent or reduce frequency hypoglycemia in diabetes. We used a novel brain-permeable AMPK activator, R481, which potently increased AMPK phosphorylation in vitro. R481 significantly increased peak glucose levels during glucose tolerance tests in rats, which were attenuated by treatment with AMPK inhibitor SBI-0206965 and completely abolished by blockade of the autonomic nervous system. This occurred without altering insulin sensitivity measured by hyperinsulinemic-euglycemic clamps. Endogenous insulin secretion was not altered by R481 treatment. During hyperinsulinemic-hypoglycemic clamp studies, R481 treatment reduced exogenous glucose requirements and amplified peak glucagon levels during hypoglycemia. These data demonstrate that peripheral administration of the brain permeable AMPK activator R481 amplifies the counterregulatory response to hypoglycemia in rats, which could have clinical relevance for prevention of hypoglycemia.
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Weightman Potter PG, Walker JMV, Robb JL, Chilton JK, Williamson R, Randall A, Ellacott KLJ, Beall C (In Press). Human primary astrocytes increase basal fatty acid oxidation following recurrent low glucose to maintain intracellular nucleotide levels.
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Human primary astrocytes increase basal fatty acid oxidation following recurrent low glucose to maintain intracellular nucleotide levels
ABSTRACTHypoglycemia is a major barrier to good glucose control in type 1 diabetes and frequent exposure to hypoglycemia can impair awareness to subsequent bouts of hypoglycemia. The neural changes that occur to reduce a person’s awareness of hypoglycemia are poorly defined. Moreover, the molecular mechanisms by which glial cells contribute to hypoglycemia sensing and glucose counterregulation require further investigation. To test whether glia, specifically astrocytes, could detect changes in glucose, we utilized human primary astrocytes (HPA) and U373 astrocytoma cells and exposed them to recurrent low glucose (RLG) in vitro. This allowed measurement, with high specificity and sensitivity, of changes in cellular metabolism following RLG. We report that the AMP-activated protein kinase (AMPK) is activated over a pathophysiologically-relevant glucose concentration range. We observed an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and hallmarks of mitochondrial stress including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG, nor were glucose uptake or glycogen levels. Taken together, these data indicate that astrocyte mitochondria are dysfunctional following recurrent low glucose exposure, which could have implications for hypoglycemia glucose counterregulation and/or hypoglycemia awareness.
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MacDonald AJ, Holmes FE, Beall C, Pickering AE, Ellacott KLJ (In Press). Regulation of food intake by astrocytes in the brainstem dorsal vagal complex.
Abstract:
Regulation of food intake by astrocytes in the brainstem dorsal vagal complex
Food intake is controlled by the coordinated action of numerous brain regions but a complete understanding remains elusive. of these brain regions the brainstem dorsal vagal complex (DVC) is the first site for integration of visceral synaptic and hormonal cues that act to inhibit food intake. The DVC consists of three nuclei: the nucleus of the solitary tract (NTS), area postrema (AP) and dorsal motor nucleus of the vagus (DMX). Targeted chemogenetic activation of appetite-responsive NTS neuronal populations causes short term decreases in food intake. Astrocytes are a class of glial cell which provide metabolic and structural support to neurons and play an active role in modulating neurotransmission. Within the hypothalamic arcuate nucleus (ARC) astrocytes are regulated by both positive and negative energy balance and express receptors for hormones that influence satiety and hunger. Chemogenetic activation of these ARC astrocytes alters food intake. Since NTS astrocytes respond to vagal stimulation, we hypothesised that they may be involved in mediating satiety. Here we show that NTS astrocytes show plastic alterations in morphology following excess food consumption and that chemogenetic activation of DVC astrocytes causes a decrease in food intake, by recruiting an appetite-inhibiting circuit, without producing aversion. These findings are the first using genetically-targeted manipulation of DVC astrocytes to demonstrate their role in the brain’s regulation of food intake.
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Forteath C, Mordi I, Nisr R, Gutierrez-Lara EJ, Alqurashi N, Phair IR, Cameron AR, Beall C, Bahr I, Mohan M, et al (2023). Amino acid homeostasis is a target of metformin therapy.
Mol Metab,
74Abstract:
Amino acid homeostasis is a target of metformin therapy.
OBJECTIVE: Unexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect. METHODS: We used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material. RESULTS: in cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective doses in vivo and in vitro observed in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells. CONCLUSIONS: Metformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.
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MacDonald AJ, Pye KR, Beall C, Ellacott KLJ (2023). Impact of chemogenetic activation of dorsal vagal complex astrocytes in mice on adaptive glucoregulatory responses.
Journal of Neuroendocrinology,
35(8).
Abstract:
Impact of chemogenetic activation of dorsal vagal complex astrocytes in mice on adaptive glucoregulatory responses
AbstractThe dorsal vagal complex (DVC) regulates diverse aspects of physiology including food intake and blood glucose homeostasis. Astrocytes play an active role in regulating DVC function and, by extension, physiological parameters. DVC astrocytes in ex vivo slices respond to low tissue glucose. The response of neurons to low glucose is conditional on intact astrocyte signalling in slice preparations, suggesting astrocytes are primary sensors of glucose deprivation (glucoprivation). Based on these published findings we hypothesised that in vivo DVC astrocyte manipulation with chemogenetics would be sufficient to alter physiological responses that control blood glucose. We found that 2‐h after systemic 2‐DG‐induced glucoprivation there were no observable changes in morphology of glial fibrillary acidic protein (GFAP)‐immunoreactive DVC cells, specifically those in the nucleus of the solitary tract (NTS). Chemogenetic activation of DVC astrocytes was sufficient to suppress nocturnal food intake by reducing both meal size and meal number and this manipulation also suppressed 2‐DG‐induced glucoprivic food intake. Chemogenetic activation of DVC astrocytes did not increase basal blood glucose nor protect against insulin‐induced hypoglycaemia. In male mice, chemogenetic DVC astrocyte activation did not alter glucose tolerance. In female mice, the initial glucose excursion was reduced in a glucose tolerance test, suggesting enhanced glucose absorption. Based on our data and published work, we propose that DVC astrocytes may play an indispensable homeostatic role, that is, are necessary to maintain the function of glucoregulatory neuronal circuitry, but alone their bulk activation is not sufficient to result in adaptive glucoregulatory responses. It is possible that there are state‐dependent effects and/or DVC astrocyte subsets that have this specialised role, but this was unresolvable using the experimental approaches employed here.
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Weightman Potter PG, Ellacott KLJ, Randall AD, Beall C (2022). Glutamate Prevents Altered Mitochondrial Function Following Recurrent Low Glucose in Hypothalamic but Not Cortical Primary Rat Astrocytes.
Cells,
11(21), 3422-3422.
Abstract:
Glutamate Prevents Altered Mitochondrial Function Following Recurrent Low Glucose in Hypothalamic but Not Cortical Primary Rat Astrocytes
Astrocytes contribute to glutamatergic signalling, which is required for hypoglycaemia counterregulation and is impaired by recurrent insulin-induced hypoglycaemia. This study examined the glutamate response of astrocytes when challenged with acute and recurrent low glucose (RLG) exposure. The metabolic responses of cortical (CRTAS) and hypothalamic (HTAS) primary rat astrocytes were measured in acute and recurrent low glucose using extracellular flux analyses. RLG caused mitochondrial adaptations in both HTAS and CRTAS, many of which were attenuated by glutamate exposure during low glucose (LG) treatments. We observed an increase in capacity of HTAS to metabolise glutamine after RLG exposure. Demonstrating astrocytic heterogeneity in the response to LG, CRTAS increased cellular acidification, a marker of glycolysis in LG, whereas this decreased in HTAS. The directional change in intracellular Ca2+ levels of each cell type, correlated with the change in extracellular acidification rate (ECAR) during LG. Further examination of glutamate-induced Ca2+ responses in low glucose treated CRTAS and HTAS identified sub-populations of glucose-excited- and glucose-inhibited-like cells with differing responses to glutamate. Lastly, release of the gliotransmitter ATP by HTAS was elevated by RLG, both with and without concurrent glutamate exposure. Therefore, hypothalamic astrocytes adapt to RLG by increasing glutamate uptake and oxidation in a manner that prevents RLG-induced mitochondrial adaptations.
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Morrissey NA, Beall C, Ellacott KLJ (2021). Absence of the mitochondrial translocator protein 18 kDa in mice does not affect body weight or food intake responses to altered energy availability.
Journal of Neuroendocrinology,
33(9).
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Absence of the mitochondrial translocator protein 18 kDa in mice does not affect body weight or food intake responses to altered energy availability
AbstractChanges in mitochondrial function in a variety of cells/tissues are critical for orchestrating systemic energy homeostasis and are linked to the development of obesity and many of its comorbidities. The mitochondrial translocator protein of 18 kDa (TSPO) is expressed in organs throughout the body, including the brain, liver, adipose tissue, gonads and adrenal glands, where it is implicated in regulating steroidogenesis and cellular metabolism. Prior work from our group and others has shown that, in rodents, TSPO levels are altered in adipose tissue by obesity and that modulation of TSPO activity may impact systemic glucose homeostasis. Furthermore, in vitro studies in a variety of cell types have implicated TSPO in mediating cellular energetics and substrate utilisation. Although mice with germline global TSPO deficiency (TSPO−/−) have no reported changes in body weight under standard husbandry conditions, we hypothesised that, given the roles of TSPO in regulating mitochondrial function and cellular metabolic flexibility, these animals may have alterations in their systemic response to altered energy availability, either nutritional excess or insufficiency. In agreement with published work, compared to wild‐type (TSPO+/+) littermates, TSPO−/− mice of both sexes did not exhibit differences in body weight on standard chow. Furthermore, following a 12‐hour overnight fast, there was no difference in weight loss or compensatory food intake during re‐feeding. Five weeks of feeding a high‐fat diet (HFD) did not reveal any impact of the absence of TSPO on body weight gain in either male or female mice. Basal blood glucose levels and glucose clearance in a glucose tolerance test were influenced by feeding a HFD diet but not by genotype. In conclusion, in the paradigms examined, germline global deletion of TSPO did not change the physiological response to deviations in systemic energy availability at the whole organism level.
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Weightman Potter PG, Washer SJ, Jeffries AR, Holley JE, Gutowski NJ, Dempster EL, Beall C (2021). Attenuated Induction of the Unfolded Protein Response in Adult Human Primary Astrocytes in Response to Recurrent Low Glucose.
FRONTIERS IN ENDOCRINOLOGY,
12 Author URL.
Cruz AM, Partridge KM, Malekizadeh Y, Vlachaki Walker JM, Weightman Potter PG, Pye KR, Shaw SJ, Ellacott KLJ, Beall C (2021). Brain Permeable AMP-Activated Protein Kinase Activator R481 Raises Glycaemia by Autonomic Nervous System Activation and Amplifies the Counterregulatory Response to Hypoglycaemia in Rats.
Frontiers in Endocrinology,
12Abstract:
Brain Permeable AMP-Activated Protein Kinase Activator R481 Raises Glycaemia by Autonomic Nervous System Activation and Amplifies the Counterregulatory Response to Hypoglycaemia in Rats
AimWe evaluated the efficacy of a novel brain permeable “metformin-like” AMP-activated protein kinase activator, R481, in regulating glucose homeostasis.Materials and MethodsWe used glucose sensing hypothalamic GT1-7 neuronal cells and pancreatic αTC1.9 α-cells to examine the effect of R481 on AMPK pathway activation and cellular metabolism. Glucose tolerance tests and hyperinsulinemic-euglycemic and hypoglycemic clamps were used in Sprague-Dawley rats to assess insulin sensitivity and hypoglycemia counterregulation, respectively.ResultsIn vitro, we demonstrate that R481 increased AMPK phosphorylation in GT1-7 and αTC1.9 cells. In Sprague-Dawley rats, R481 increased peak glucose levels during a glucose tolerance test, without altering insulin levels or glucose clearance. The effect of R481 to raise peak glucose levels was attenuated by allosteric brain permeable AMPK inhibitor SBI-0206965. This effect was also completely abolished by blockade of the autonomic nervous system using hexamethonium. During hypoglycemic clamp studies, R481 treated animals had a significantly lower glucose infusion rate compared to vehicle treated controls. Peak plasma glucagon levels were significantly higher in R481 treated rats with no change to plasma adrenaline levels. In vitro, R481 did not alter glucagon release from αTC1.9 cells, but increased glycolysis. Non brain permeable AMPK activator R419 enhanced AMPK activity in vitro in neuronal cells but did not alter glucose excursion in vivo.ConclusionsThese data demonstrate that peripheral administration of the brain permeable “metformin-like” AMPK activator R481 increases blood glucose by activation of the autonomic nervous system and amplifies the glucagon response to hypoglycemia in rats. Taken together, our data suggest that R481 amplifies the counterregulatory response to hypoglycemia by a central rather than a direct effect on the pancreatic α-cell. These data provide proof-of-concept that central AMPK could be a target for future drug development for prevention of hypoglycemia in diabetes.
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MacDonald AJ, Yang YHC, Cruz AM, Beall C, Ellacott KLJ (2021). Brain-Body Control of Glucose Homeostasis—Insights from Model Organisms.
Frontiers in Endocrinology,
12Abstract:
Brain-Body Control of Glucose Homeostasis—Insights from Model Organisms
Tight regulation of blood glucose is essential for long term health. Blood glucose levels are defended by the correct function of, and communication between, internal organs including the gastrointestinal tract, pancreas, liver, and brain. Critically, the brain is sensitive to acute changes in blood glucose level and can modulate peripheral processes to defend against these deviations. In this mini-review we highlight select key findings showcasing the utility, strengths, and limitations of model organisms to study brain-body interactions that sense and control blood glucose levels. First, we discuss the large platform of genetic tools available to investigators studying mice and how this field may yet reveal new modes of communication between peripheral organs and the brain. Second, we discuss how rats, by virtue of their size, have unique advantages for the study of CNS control of glucose homeostasis and note that they may more closely model some aspects of human (patho)physiology. Third, we discuss the nascent field of studying the CNS control of blood glucose in the zebrafish which permits ease of genetic modification, large-scale measurements of neural activity and live imaging in addition to high-throughput screening. Finally, we briefly discuss glucose homeostasis in drosophila, which have a distinct physiology and glucoregulatory systems to vertebrates.
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Potter PW, Randall A, Ellacott K, Beall C (2021). Rat primary hypothalamic, but not cortical, astrocytes increase use of glutamate to fuel metabolism after recurrent low glucose. Endocrine Abstracts
Hanna L, Kawalek TJ, Beall C, Ellacott KLJ (2020). Changes in neuronal activity across the mouse ventromedial nucleus of the hypothalamus in response to low glucose: Evaluation using an extracellular multi‐electrode array approach.
Journal of Neuroendocrinology,
32(3).
Abstract:
Changes in neuronal activity across the mouse ventromedial nucleus of the hypothalamus in response to low glucose: Evaluation using an extracellular multi‐electrode array approach
AbstractThe hypothalamic ventromedial nucleus (VMN) is involved in maintaining systemic glucose homeostasis. Neurophysiological studies in rodent brain slices have identified populations of VMN glucose‐sensing neurones: glucose‐excited (GE) neurones, cells which increased their firing rate in response to increases in glucose concentration, and glucose‐inhibited (GI) neurones, which show a reduced firing frequency in response to increasing glucose concentrations. To date, most slice electrophysiological studies characterising VMN glucose‐sensing neurones in rodents have utilised the patch clamp technique. Multi‐electrode arrays (MEAs) are a state‐of‐the‐art electrophysiological tool enabling the electrical activity of many cells to be recorded across multiple electrode sites (channels) simultaneously. We used a perforated MEA (pMEA) system to evaluate electrical activity changes across the dorsal‐ventral extent of the mouse VMN region in response to alterations in glucose concentration. Because intrinsic (ie, direct postsynaptic sensing) and extrinsic (ie, presynaptically modulated) glucosensation were not discriminated, we use the terminology ‘GE/presynaptically excited by an increase (PER)’ and ‘GI/presynaptically excited by a decrease (PED)’ in the present study to describe responsiveness to changes in extracellular glucose across the mouse VMN. We observed that 15%‐60% of channels were GE/PER, whereas 2%‐7% were GI/PED channels. Within the dorsomedial portion of the VMN (DM‐VMN), significantly more channels were GE/PER compared to the ventrolateral portion of the VMN (VL‐VMN). However, GE/PER channels within the VL‐VMN showed a significantly higher basal firing rate in 2.5 mmol l‐1 glucose than DM‐VMN GE/PER channels. No significant difference in the distribution of GI/PED channels was observed between the VMN subregions. The results of the present study demonstrate the utility of the pMEA approach for evaluating glucose responsivity across the mouse VMN. pMEA studies could be used to refine our understanding of other neuroendocrine systems by examining population level changes in electrical activity across brain nuclei, thus providing key functional neuroanatomical information to complement and inform the design of single‐cell neurophysiological studies.
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Cruz AM, Beall C (2020). Extracellular ATP Increases Glucose Metabolism in Skeletal Muscle Cells in a P2 Receptor Dependent Manner but Does Not Contribute to Palmitate-Induced Insulin Resistance. Frontiers in Physiology, 11
Robb JL, Morrissey NA, Weightman Potter PG, Smithers HE, Beall C, Ellacott KLJ (2020). Immunometabolic Changes in Glia – a Potential Role in the Pathophysiology of Obesity and Diabetes. Neuroscience, 447, 167-181.
Robb JL, Hammad NA, Weightman Potter PG, Chilton JK, Beall C, Ellacott KLJ (2020). The metabolic response to inflammation in astrocytes is regulated by nuclear factor‐kappa B signaling.
Glia,
68(11), 2246-2263.
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The metabolic response to inflammation in astrocytes is regulated by nuclear factor‐kappa B signaling
AbstractInflammation and metabolism are intrinsically linked with inflammatory stimuli inducing metabolic changes in cells and, in turn, metabolic capacity determining cellular inflammatory responses. Although well characterized in peripheral immune cells there is comparatively less known about these “immunometabolic” responses in astrocytes. In this study, we tested the hypothesis that the astrocytic inflammatory response driven by nuclear factor‐kappa B (NF‐κB) signaling is dependent on glycolytic metabolism. Using mouse primary cortical astrocyte cultures, we assessed changes in cellular metabolism after exposure to lipopolysaccharide (LPS), with cytokine ELISAs and immunoblotting being used to measure inflammatory responses. Results indicate temporally distinct metabolic adaptations to pro‐inflammatory stimulation in astrocytes: 3 hr LPS treatment increased glycolysis but did not alter mitochondrial metabolism, while following 24 hr of LPS treatment we observed increased oxidative phosphorylation, and decreased glycolytic capacity and glucose uptake, partly due to reduced glucose transporter 1 expression. Inhibition of NF‐κB signaling with the IKK‐beta inhibitor TPCA‐1 prevented the LPS induced changes to glycolysis and oxidative phosphorylation. Furthermore, TPCA‐1 treatment altered both glycolysis and oxidative phosphorylation independently from inflammatory stimulation, indicating a role for NF‐κB signaling in regulation of basal metabolism in astrocytes. Inhibition of glycolysis with 2‐deoxyglucose significantly attenuated LPS‐induced cytokine release and NF‐κB phosphorylation, indicating that intact glycolysis is required for the full inflammatory response to LPS. Together our data indicate that astrocytes display immunometabolic responses to acute LPS stimulation which may represent a potential therapeutic target for neuroinflammatory disorders.
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CRUZ AML, MALEKIZADEH Y, VLACHAKI WALKER JM, SHAW SJ, ELLACOTT KL, BEALL C (2019). 119-OR: Amplified Glucagon Response to Hypoglycemia following AMP-Activated Protein Kinase (AMPK) Activator R481 Treatment in Healthy Rats. Diabetes, 68(Supplement_1).
WEIGHTMAN POTTER PG, WASHER SJ, JEFFRIES A, DEMPSTER EL, BEALL C (2019). 401-P: Acute Low Glucose Alters Human Primary Astrocyte Expression of Endoplasmic Reticulum Stress and Mitochondrial Associated Genes, Which is Blunted after Recurrent Low Glucose. Diabetes, 68(Supplement_1).
MacDonald AJ, Robb JL, Morrissey NA, Beall C, Ellacott KLJ (2019). Astrocytes in neuroendocrine systems: an overview.
J Neuroendocrinol,
31(5).
Abstract:
Astrocytes in neuroendocrine systems: an overview.
A class of glial cell, astrocytes, is highly abundant in the central nervous system (CNS). In addition to maintaining tissue homeostasis, astrocytes regulate neuronal communication and synaptic plasticity. There is an ever-increasing appreciation that astrocytes are involved in the regulation of physiology and behaviour in normal and pathological states, including within neuroendocrine systems. Indeed, astrocytes are direct targets of hormone action in the CNS, via receptors expressed on their surface, and are also a source of regulatory neuropeptides, neurotransmitters and gliotransmitters. Furthermore, as part of the neurovascular unit, astrocytes can regulate hormone entry into the CNS. This review is intended to provide an overview of how astrocytes are impacted by and contribute to the regulation of a diverse range of neuroendocrine systems: energy homeostasis and metabolism, reproduction, fluid homeostasis, the stress response and circadian rhythms.
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Author URL.
MacDonald AJ, Holmes FE, Beall C, Pickering AE, Ellacott KLJ (2019). Regulation of food intake by astrocytes in the brainstem dorsal vagal complex.
Glia,
68(6), 1241-1254.
Abstract:
Regulation of food intake by astrocytes in the brainstem dorsal vagal complex
AbstractA role for glial cells in brain circuits controlling feeding has begun to be identified with hypothalamic astrocyte signaling implicated in regulating energy homeostasis. The nucleus of the solitary tract (NTS), within the brainstem dorsal vagal complex (DVC), integrates vagal afferent information from the viscera and plays a role in regulating food intake. We hypothesized that astrocytes in this nucleus respond to, and influence, food intake. Mice fed high‐fat chow for 12 hr during the dark phase showed NTS astrocyte activation, reflected in an increase in the number (65%) and morphological complexity of glial‐fibrillary acidic protein (GFAP)‐immunoreactive cells adjacent to the area postrema (AP), compared to control chow fed mice. To measure the impact of astrocyte activation on food intake, we delivered designer receptors exclusively activated by designer drugs (DREADDs) to DVC astrocytes (encompassing NTS, AP, and dorsal motor nucleus of the vagus) using an adeno‐associated viral (AAV) vector (AAV‐GFAP‐hM3Dq_mCherry). Chemogenetic activation with clozapine‐N‐oxide (0.3 mg/kg) produced in greater morphological complexity in astrocytes and reduced dark‐phase feeding by 84% at 4 hr postinjection compared with vehicle treatment. hM3Dq‐activation of DVC astrocytes also reduced refeeding after an overnight fast (71% lower, 4 hr postinjection) when compared to AAV‐GFAP‐mCherry expressing control mice. DREADD‐mediated astrocyte activation did not impact locomotion. hM3Dq activation of DVC astrocytes induced c‐FOS in neighboring neuronal feeding circuits (including in the parabrachial nucleus). This indicates that NTS astrocytes respond to acute nutritional excess, are involved in the integration of peripheral satiety signals, and can reduce food intake when activated.
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Logie L, Lees Z, Allwood JW, McDougal G, Beall C, Rena G (2018). Regulation of hepatic glucose production and AMPK by AICAR but not metformin depends on drug uptake through the equilibrative nucleoside transporter 1 (ENT1). Diabetes, Obesity and Metabolism, (In press)
Vlachaki Walker JM, Robb JL, Cruz AM, Malhi A, Weightman Potter PG, Ashford MLJ, McCrimmon RJ, Ellacott KLJ, Beall C (2017). AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner.
Diabetes, Obesity and Metabolism,
19(7), 997-1005.
Abstract:
AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner
Aim: to test the hypothesis that, given the role of AMP-activated protein kinase (AMPK) in regulating intracellular ATP levels, AMPK may alter ATP release from astrocytes, the main sources of extracellular ATP (eATP) within the brain. Materials and Methods: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild-type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes. Results: in U373 cells, A-769662 (100 μM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased eATP levels, and this was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types. Conclusions: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 μM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.
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Beall C, Hanna L, Ellacott KLJ (2017). CNS Targets of Adipokines.
Compr Physiol,
7(4), 1359-1406.
Abstract:
CNS Targets of Adipokines.
Our understanding of adipose tissue as an endocrine organ has been transformed over the last 20 years. During this time, a number of adipocyte-derived factors or adipokines have been identified. This article will review evidence for how adipokines acting via the central nervous system (CNS) regulate normal physiology and disease pathology. The reported CNS-mediated effects of adipokines are varied and include the regulation of energy homeostasis, autonomic nervous system activity, the reproductive axis, neurodevelopment, cardiovascular function, and cognition. Due to the wealth of information available and the diversity of their known functions, the archetypal adipokines leptin and adiponectin will be focused on extensively. Other adipokines with established CNS actions will also be discussed. Due to the difficulties associated with studying CNS function on a molecular level in humans, the majority of our knowledge, and as such the studies described in this paper, comes from work in experimental animal models; however, where possible the relevant data from human studies are also highlighted. © 2017 American Physiological Society. Compr Physiol 7:1359-1406, 2017.
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Dadak S, Beall C, Vlachaki Walker JM, Soutar MPM, McCrimmon RJ, Ashford MLJ (2017). Oleate induces K ATP channel-dependent hyperpolarization in mouse hypothalamic glucose-excited neurons without altering cellular energy charge. Neuroscience, 346, 29-42.
Jeffery N, Richardson S, Beall C, Harries LW (2017). The species origin of the cellular microenvironment influences markers of beta cell fate and function in EndoC-βH1 cells. Experimental Cell Research, 361(2), 284-291.
Cameron AR, Morrison VL, Levin D, Mohan M, Forteath C, Beall C, McNeilly AD, Balfour DJK, Savinko T, Wong AKF, et al (2016). Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status.
Circ Res,
119(5), 652-665.
Abstract:
Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status.
RATIONALE: the diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood. OBJECTIVE: Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties. METHODS AND RESULTS: in primary hepatocytes from healthy animals, metformin and the IKKβ (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α-dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1β, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/β activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02-0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22-2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11). CONCLUSION: We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups. CLINICAL TRIAL REGISTRATION: Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.
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Yavari A, Stocker CJ, Ghaffari S, Wargent ET, Steeples V, Czibik G, Pinter K, Bellahcene M, Woods A, Martínez de Morentin PB, et al (2016). Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function.
Cell Metab,
23(5), 821-836.
Abstract:
Chronic Activation of γ2 AMPK Induces Obesity and Reduces β Cell Function.
Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.
Abstract.
Author URL.
Haythorne E, Hamilton DL, Findlay JA, Beall C, McCrimmon RJ, Ashford MLJ (2016). Chronic exposure to KATP channel openers results in attenuated glucose sensing in hypothalamic GT1-7 neurons. Neuropharmacology, 111, 212-222.
Walker JV, Ashford MLJ, McCrimmon RJ, Beall C (2016). Exposure of astrocytes to recurrent low glucose delays AMPK phosphorylation and alters ATP and lactate release.
DIABETIC MEDICINE,
33, 54-54.
Author URL.
Beall C, Walker JMV, Ashford MLJ, McCrimmon RJ (2015). A critical role for AMP-activated protein kinase in regulation of gliotransmitter release from astrocytes.
DIABETIC MEDICINE,
32, 62-62.
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Cameron AR, Morrison V, McNeilly AD, Forteath C, Beall C, Stewart CA, Balfour DJK, Sutherland CD, Sakamoto K, Fagerholm SC, et al (2015). Anti-inflammatory effects of metformin.
DIABETIC MEDICINE,
32, 54-55.
Author URL.
Cameron A, Forteath C, Beall C, Rena G (2015). Anti-inflammatory effects of metformin and their relationship to the therapeutic action of the drug. Endocrine Abstracts
Walker JV, Ashford M, McCrimmon R, Beall C (2015). Characterisation of the astrocytic response to acute and recurrent hypoglycaemia. Endocrine Abstracts
Walker JMV, Gabriel J, Ashford MLJ, McCrimmon RJ, Beall C (2015). Modulation of astrocytic lactate release during hypoglycaemia by AMP-activated protein kinase and noradrenaline.
DIABETIC MEDICINE,
32, 62-63.
Author URL.
Hamilton DL, Beall C, Jeromson S, Chevtzoff C, Cuthbertson DJ, Ashford MLJ (2014). Kv1.3 inhibitors have differential effects on glucose uptake and AMPK activity in skeletal muscle cell lines and mouse ex vivo skeletal muscle.
J Physiol Sci,
64(1), 13-20.
Abstract:
Kv1.3 inhibitors have differential effects on glucose uptake and AMPK activity in skeletal muscle cell lines and mouse ex vivo skeletal muscle.
Knockout of Kv1.3 improves glucose homeostasis and confers resistance to obesity. Additionally, Kv1.3 inhibition enhances glucose uptake. This is thought to occur through calcium release. Kv1.3 inhibition in T-lymphocytes alters mitochondrial membrane potential, and, as many agents that induce Ca(2+) release or inhibit mitochondrial function activate AMPK, we hypothesised that Kv1.3 inhibition would activate AMPK and increase glucose uptake. We screened cultured muscle with a range of Kv1.3 inhibitors for their ability to alter glucose uptake. Only Psora4 increased glucose uptake in C2C12 myotubes. None of the inhibitors had any impact on L6 myotubes. Magratoxin activated AMPK in C2C12 myotubes and only Pap1 activated AMPK in the SOL. Kv1.3 inhibitors did not alter cellular respiration, indicating a lack of effect on mitochondrial function. In conclusion, AMPK does not mediate the effects of Kv1.3 inhibitors and they display differential effects in different skeletal muscle cell lines without impairing mitochondrial function.
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Beall C, Watterson KR, McCrimmon RJ, Ashford MLJ (2013). AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels.
J Bioenerg Biomembr,
45(3), 229-241.
Abstract:
AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels.
Glucose-sensing (GS) behaviour in pancreatic β-cells is dependent on ATP-sensitive K(+) channel (KATP) activity, which is controlled by the relative levels of the KATP ligands ATP and ADP, responsible for closing and opening KATP, respectively. However, the mechanism by which β-cells transfer energy status from mitochondria to KATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMP-activated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep KATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP5A blocked this action of AMPK. Using conditions that maximised AMPK-induced KATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that KATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane KATP by phosphotransfer cascades.
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Beall C, Haythorne E, Fan X, Du Q, Jovanovic S, Sherwin RS, Ashford MLJ, McCrimmon RJ (2013). Continuous hypothalamic K(ATP) activation blunts glucose counter-regulation in vivo in rats and suppresses K(ATP) conductance in vitro.
Diabetologia,
56(9), 2088-2092.
Abstract:
Continuous hypothalamic K(ATP) activation blunts glucose counter-regulation in vivo in rats and suppresses K(ATP) conductance in vitro.
AIMS/HYPOTHESIS: Acute systemic delivery of the sulfonylurea receptor (SUR)-1-specific ATP-sensitive K(+) channel (K(ATP)) opener, NN414, has been reported to amplify glucose counter-regulatory responses (CRRs) in rats exposed to hypoglycaemia. Thus, we determined whether continuous NN414 could prevent hypoglycaemia-induced defective counter-regulation. METHODS: Chronically catheterised male Sprague-Dawley rats received a continuous infusion of NN414 into the third ventricle for 8 days after implantation of osmotic minipumps. Counter-regulation was examined by hyperinsulinaemic-hypoglycaemic clamp on day 8 after three episodes of insulin-induced hypoglycaemia (recurrent hypoglycaemia [RH]) on days 5, 6 and 7. In a subset of rats exposed to RH, NN414 infusion was terminated on day 7 to wash out NN414 before examination of counter-regulation on day 8. To determine whether continuous NN414 exposure altered K(ATP) function, we used the hypothalamic glucose-sensing GT1-7 cell line, which expresses the SUR-1-containing K(ATP) channel. RESULTS: Continuous exposure to NN414 in the setting of RH increased, rather than decreased, the glucose infusion rate (GIR), as exemplified by attenuated adrenaline (epinephrine) secretion. Termination of NN414 on day 7 with subsequent washout for 24 h partially diminished the GIR. The same duration of exposure of GT1-7 cells to NN414 substantially reduced K(ATP) conductance, which was also reversed on washout of the agonist. The suppression of K(ATP) current was not associated with reduced channel subunit mRNA or protein levels. CONCLUSIONS/INTERPRETATION: These data indicate that continuous K(ATP) activation results in suppressed CRRs to hypoglycaemia in vivo, which in vitro is associated with the reversible conversion of KATP into a stable inactive state.
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Ashford M, Beall C, McCrimmon R (2012). Hypoglycaemia: exercise for the brain?.
J Neuroendocrinol,
24(10), 1365-1366.
Abstract:
Hypoglycaemia: exercise for the brain?
Low blood sugar, or hypoglycaemia, is detected by specialised sugar sensing neurones in the brain. However, the detection of hypoglycaemia is blunted after repeated hypoglycaemia and this is a result of adaptive mechanisms kicking in within the brain; mechanisms that resemble the 'training effect' in muscle. These adaptations most likely not only increase the tolerance of the brain to stress, but also perturb the detection of hypoglycaemia, further increasing the likelihood of hypoglycaemia.
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Beall C, Hamilton DL, Gallagher J, Logie L, Wright K, Soutar MP, Dadak S, Ashford FB, Haythorne E, Du Q, et al (2012). Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour.
Diabetologia,
55(9), 2432-2444.
Abstract:
Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour.
AIMS/HYPOTHESIS: Hypothalamic glucose-excited (GE) neurons contribute to whole-body glucose homeostasis and participate in the detection of hypoglycaemia. This system appears defective in type 1 diabetes, in which hypoglycaemia commonly occurs. Unfortunately, it is at present unclear which molecular components required for glucose sensing are produced in individual neurons and how these are functionally linked. We used the GT1-7 mouse hypothalamic cell line to address these issues. METHODS: Electrophysiological recordings, coupled with measurements of gene expression and protein levels and activity, were made from unmodified GT1-7 cells and cells in which AMP-activated protein kinase (AMPK) catalytic subunit gene expression and activity were reduced. RESULTS: Hypothalamic GT1-7 neurons express the genes encoding glucokinase and ATP-sensitive K(+) channel (K(ATP)) subunits K ( ir ) 6.2 and Sur1 and exhibit GE-type glucose-sensing behaviour. Lowered extracellular glucose concentration hyperpolarised the cells in a concentration-dependent manner, an outcome that was reversed by tolbutamide. Inhibition of glucose uptake or metabolism hyperpolarised cells, showing that energy metabolism is required to maintain their resting membrane potential. Short hairpin (sh)RNA directed to Ampkα2 (also known as Prkaa2) reduced GT1-7 cell AMPKα2, but not AMPKα1, activity and lowered the threshold for hypoglycaemia-induced hyperpolarisation. shAmpkα1 (also known as Prkaa1) had no effect on glucose-sensing or AMPKα2 activity. Decreased uncoupling protein 2 (Ucp2) mRNA was detected in AMPKα2-reduced cells, suggesting that AMPKα2 regulates UCP2 levels. CONCLUSIONS/INTERPRETATION: We have demonstrated that GT1-7 cells closely mimic GE neuron glucose-sensing behaviour, and reducing AMPKα2 blunts their responsiveness to hypoglycaemic challenge, possibly by altering UCP2 activity. These results show that suppression of AMPKα2 activity inhibits normal glucose-sensing behaviour and may contribute to defective detection of hypoglycaemia.
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Beall C, Ashford ML, McCrimmon RJ (2012). The physiology and pathophysiology of the neural control of the counterregulatory response.
Am J Physiol Regul Integr Comp Physiol,
302(2), R215-R223.
Abstract:
The physiology and pathophysiology of the neural control of the counterregulatory response.
Despite significant technological and pharmacological advancements, insulin replacement therapy fails to adequately replicate β-cell function, and so glucose control in type 1 diabetes mellitus (T1D) is frequently erratic, leading to periods of hypoglycemia. Moreover, the counterregulatory response (CRR) to falling blood glucose is impaired in diabetes, leading to an increased risk of severe hypoglycemia. It is now clear that the brain plays a significant role in the development of defective glucose counterregulation and impaired hypoglycemia awareness in diabetes. In this review, the basic intracellular glucose-sensing mechanisms are discussed, as well as the neural networks that respond to and coordinate the body's response to a hypoglycemic challenge. Subsequently, we discuss how the body responds to repeated hypoglycemia and how these adaptations may explain, at least in part, the development of impaired glucose counterregulation in diabetes.
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Beall C, Piipari K, Al-Qassab H, Smith MA, Parker N, Carling D, Viollet B, Withers DJ, Ashford MLJ (2010). Loss of AMP-activated protein kinase alpha2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia.
Biochem J,
429(2), 323-333.
Abstract:
Loss of AMP-activated protein kinase alpha2 subunit in mouse beta-cells impairs glucose-stimulated insulin secretion and inhibits their sensitivity to hypoglycaemia.
AMPK (AMP-activated protein kinase) signalling plays a key role in whole-body energy homoeostasis, although its precise role in pancreatic beta-cell function remains unclear. In the present study, we therefore investigated whether AMPK plays a critical function in beta-cell glucose sensing and is required for the maintenance of normal glucose homoeostasis. Mice lacking AMPK alpha2 in beta-cells and a population of hypothalamic neurons (RIPCre alpha2KO mice) and RIPCre alpha2KO mice lacking AMPK alpha1 (alpha1KORIPCre alpha2KO) globally were assessed for whole-body glucose homoeostasis and insulin secretion. Isolated pancreatic islets from these mice were assessed for glucose-stimulated insulin secretion and gene expression changes. Cultured beta-cells were examined electrophysiologically for their electrical responsiveness to hypoglycaemia. RIPCre alpha2KO mice exhibited glucose intolerance and impaired GSIS (glucose-stimulated insulin secretion) and this was exacerbated in alpha1KORIPCre alpha2KO mice. Reduced glucose concentrations failed to completely suppress insulin secretion in islets from RIPCre alpha2KO and alpha1KORIPCre alpha2KO mice, and conversely GSIS was impaired. Beta-cells lacking AMPK alpha2 or expressing a kinase-dead AMPK alpha2 failed to hyperpolarize in response to low glucose, although KATP (ATP-sensitive potassium) channel function was intact. We could detect no alteration of GLUT2 (glucose transporter 2), glucose uptake or glucokinase that could explain this glucose insensitivity. UCP2 (uncoupling protein 2) expression was reduced in RIPCre alpha2KO islets and the UCP2 inhibitor genipin suppressed low-glucose-mediated wild-type mouse beta-cell hyperpolarization, mimicking the effect of AMPK alpha2 loss. These results show that AMPK alpha2 activity is necessary to maintain normal pancreatic beta-cell glucose sensing, possibly by maintaining high beta-cell levels of UCP2.
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