Publications by year
In Press
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.
Abstract:
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.
Abstract.
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 Full text.
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.
Abstract:
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.
Abstract.
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.
Abstract:
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.
Abstract.
2021
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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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.
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.
Abstract.
2020
Beall C, Cruz AM, Potter PGW, Walker JMV, Malekizadeh Y, Pye KR, Shaw SJ, Ellacott KLJ (2020). Amp-activated protein kinase (AMPK) activator R481 amplifies the glucagon response to hypoglycaemia without worsening hyperglycaemia in diabetic rats.
Author URL.
Hall B (2020). An investigation into the effect of acute and chronic KATP channel modulation on membrane conductance and cellular metabolism.
Abstract:
An investigation into the effect of acute and chronic KATP channel modulation on membrane conductance and cellular metabolism.
The ATP-sensitive potassium (KATP) channel is a vital link between cellular metabolism and electrical excitability in a variety of cell types, including pancreatic beta cells and hypothalamic neurons where they are involved in the response to changing plasma glucose levels. Blockade of KATP channels using sulfonylureas is a common therapeutic target for Type 2 diabetes (T2D) treatment. However, long-term sulfonylurea therapy is associated with a 33% chance of failure, reducing overall drug efficacy and pancreatic beta cell function, reducing their lasting effectiveness. Furthermore, gain-of-function mutations in KATP channels, leading to NDM and DEND syndrome, can drastically alter basal cellular metabolism and mitochondrial function, indicating a potential role of KATP channel activity in the regulation of glucose metabolism. In addition, DEND mutations have been shown to cause a wide array of neurological conditions, currently untreatable with sulfonylurea therapy.
The aim of these studies was to answer three main questions; what is the impact of chronic blockade on membrane conductance and KATP channel activity after removal of the drug? does the alteration of KATP channel activity to regulate cellular glucose metabolism? and lastly, what concentration does memantine block native KATP channels at? Chapter 3 presents evidence that both gliclazide (5 μM) and tolbutamide (50 μM) caused a significant augmentation of KATP-mediated membrane conductance in GT1-7 cells after 48 hours of treatment of ~2x and 1.5x, respectively. Overall, these data provide an alternative explanation to the increased rate of drug failure after long term therapy with sulfonylureas. Chapter 4 shows that the acute modulation of KATP channel activity caused little to no significant alterations in either glycolysis or mitochondrial activity, as has been previously described. However, chronic KATP channel blockade with glibenclamide increased mitochondrial ATP production and decreased basal glycolysis, potentially as a response to increased cellular excitability. Chapter 5 shows that external exposure of GT1-7 cells to memantine (100 μM) caused significant reduction in KATP-dependent whole-cell membrane conductance (~80% maximal). However, no significant effect was observed at either 10 μM or 1 μM memantine. Overall, these data suggest that memantine does block KATP channels, but at a much higher concentration than is therapeutically relevant.
Overall, this work provides new evidence that supplements our understanding of both the impacts of pharmacological treatment on KATP channel activity, as well as the role of KATP channel activity on cellular glucose metabolism. Furthermore, and most importantly, this work furthers our understanding of the effects of memantine on KATP mediated membrane conductance and its effectiveness, or lack thereof, as a novel treatment for NDM.
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Cruz A, Potter PW, Pye K, Shaw S, Ellacott K, Beall C (2020). Brain permeable AMPK activator R481 acutely raises blood glucose by activating the autonomic nervous system without altering insulin sensitivity.
Author URL.
Potter PGW, Randall A, Beall C (2020). Cellular metabolism and Ca2+ signalling are modified differentially in rat hypothalamic and cortical primary astrocytes by acute and recurrent low glucose.
Author URL.
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).
Full text.
Robb J (2020). Characterisation of immunometabolic responses in astrocytes.
Abstract:
Characterisation of immunometabolic responses in astrocytes
Astrocytes play a role in the central nervous system (CNS) inflammatory response. In many immune cell types cellular inflammation and metabolism are linked, a phenomenon termed ‘immunometabolism’. This has led to attempts to reduce chronic inflammation through manipulating cellular metabolism. Proteins of interest for this approach include transcription factor nuclear factor-kappa B (NF-κB) and mitochondrial protein ‘translocator protein (18 kDa)’ (TSPO). TSPO is of particular interest as a therapy for CNS disease as many TSPO ligands can access the CNS and have been demonstrated to have anti-inflammatory effects. However, immunometabolism has not been well described in astrocytes, and the function of TSPO is currently disputed. In this thesis, mouse primary astrocytes were used to characterise immunometabolic responses following treatment with the pro-inflammatory stimulus lipopolysaccharide (LPS). An initial increase in glycolytic metabolism was measured, prior to a shift towards oxidative phosphorylation and away from glucose metabolism, in part mediated by a decrease in glucose transporter GLUT1 expression. Pharmacological inhibition of NF-κB signalling demonstrated that this pathway is important in mediating the cellular metabolic response to inflammation in astrocytes, and may play a role in maintaining basal metabolic function in these cells. Pharmacological or genetic modulation of TSPO signalling in human astroglioma (U373) cells and/or mouse primary astrocytes demonstrated that while TSPO suppressed fatty acid oxidation and promoted glycolytic metabolism, this did not appear to acutely alter the inflammatory response of these cells after LPS treatment; however, the longer term effects remain to be explored. Together these data demonstrate that inflammation and metabolism are intrinsically linked in astrocytes and TSPO plays an important role in regulating metabolism in these cells.
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MacDonald A (2020). Defining the contribution of dorsal vagal complex astrocytes to the regulation of food intake.
Abstract:
Defining the contribution of dorsal vagal complex astrocytes to the regulation of food intake
Food intake is controlled by the coordinated action of numerous brain regions but a complete understanding of the process remains elusive. The nucleus of the solitary tract (NTS), located in the brainstem dorsal vagal complex (DVC) is the first site for integration of visceral synaptic and hormonal cues that act to inhibit food intake. NTS neurons receive synaptic input from sensory neurons of the vagus nerve that relay signals of gastrointestinal stretch and nutrient content. In response to these signals of ingestion, NTS neurons signal to higher brain centres in the hypothalamus and midbrain to inhibit hunger and promote meal termination.
A role for astrocytes in brain circuits controlling food intake has begun to be
identified. Hypothalamic astrocyte signalling has been implicated in regulating energy homeostasis. Despite a wealth of evidence showing astrocytes in the NTS/DVC are involved in synaptic integration of vagal signals and control of autonomic physiology, the potential role of these cells in feeding control has not been investigated.
We hypothesised that NTS astrocytes, and those in the wider DVC, would be
responsive to increases in food intake and, in turn, their activation would act in concert with NTS neurons to drive a corresponding suppression of food intake.
To investigate this prospect we used dietary manipulation,
immunohistochemistry, selective chemogenetic manipulation of DVC astrocytes, behavioural assays and electrophysiology in mice. The key findings of these studies show that in response to acute nutrient excess
and gastric distention NTS astrocytes increase their expression of the
cytoskeletal glial fibrillary acidic protein and adopt a more ramified morphology, indicative of activation. We also show that selective activation of Gq-proteincoupled receptor signalling in DVC astrocytes suppresses nocturnal food intake and refeeding after a fast. These studies provide evidence that astrocytes may be integrators and effectors of satiety signals and appropriate feeding responses
in the DVC.
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Cruz AM, Beall C (2020). Exogenous ATP promotes glucose uptake and utilisation in skeletal muscle cells but does not alter glucose clearance in vivo.
Author URL.
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 Full text.
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.
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MacDonald AJ, Holmes FE, Beall C, Pickering AE, Ellacott KLJ (2020). Regulation of food intake by astrocytes in the brainstem dorsal vagal complex.
Glia,
68(6), 1241-1254.
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Cruz AM (2020). The integrated physiology of glucose. homeostasis: regulation by extracellular. and intracellular nucleotide sensors.
Abstract:
The integrated physiology of glucose. homeostasis: regulation by extracellular. and intracellular nucleotide sensors
Physiological glucose levels are maintained by the complex integration of neuroendocrine, hormonal and nutritional signals controlled by multiple tissues in the body. A dysregulation in these mechanisms leads to increasingly prevalent conditions characterised by an inability to regulate blood glucose levels, such as diabetes. Maintaining glycaemia within a target range remains a daily challenge for individuals with both Type 1 and Type 2 diabetes and a better understanding of the pathophysiology of impaired glucose homeostasis in these conditions is still required to identify more effective and targeted therapeutic approaches.
Work in this thesis focused on elucidating the mechanisms by which lipid overflow, be it from increasingly sedentary behaviour or overfeeding, leads to the development of insulin and anabolic resistance in skeletal muscle. Loss of insulin-stimulated glucose clearance by skeletal muscle is a main driver for impaired glucose disposal in Type 2 diabetes and a role for excessive lipid availability in this pathology is well established. Here, muscle cells were treated with high concentrations of a saturated fatty acid and data demonstrated that lipid overflow led to impaired anabolic sensitivity, inflammatory cytokine release and mitochondrial dysfunction. Furthermore, these experiments elucidated a novel role for adenosine tri-phosphate, acting as a signalling molecule, in the regulation of muscle glucose metabolism, identifying insulin and exercise mimetic roles of the nucleotide that could be therapeutically targetable.
This work was translated into humans, where the effect of lipid overflow by high-fat overfeeding was assessed in an experimental model of inactivity-induced insulin and anabolic resistance. Data suggested that two days of disuse (by forearm immobilisation) were sufficient to cause substantial muscle insulin resistance. After 7 days, muscle strength was significantly reduced and anabolic resistance was evident due to decreased forearm balance of potent anabolic amino acids such as leucine. Contrary to the hypothesis, high-fat overfeeding did not accelerate or exacerbate these impairments, suggesting that removal of contraction represents a potent stimulus for loss of substrate demand by muscle, irrespective of energy balance.
Insulin replacement therapy has been the cornerstone of treatment for Type 1 and advanced Type 2 diabetes for over 8 decades. A serious and inadvertent consequence of prolonged insulin therapy is the increased risk of hypoglycaemia. Hypoglycaemia can lead to impaired physiological defences against a decrease in blood glucose and loss of awareness of these changes. AMP-activated protein kinase activators, which are widely used (to target peripheral tissues) as anti-hyperglycaemic agents in Type 2 diabetes have demonstrated central effects that amplify the first defence against hypoglycaemia, or counterregulatory response. Data presented here demonstrated that peripheral administration of a brain permeable AMP-activated protein kinase activator amplified the counterregulatory response to hypoglycaemia by enhancing glucagon levels in healthy rats, without altering fasting blood glucose. This demonstrates important clinical implications for the pharmaceutical use of AMP-activated protein kinase activators as the central roles that regulate blood glucose may supersede the peripheral effects of these compounds, during hypoglycaemia.
Work presented here highlights the complexity of the regulation of glycaemia and discusses the contribution of extracellular and intracellular nucleotides/nucleotide sensors to glucose homeostasis. It can be concluded from this work that strategies to manage or treat diabetes in future should consider the importance of tissue-specific or metabolic status specific actions of the targets of interest.
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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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2019
Robb JL, Hammad NA, Beall C, Ellacott KL (2019). A role for translocator protein 18kDa (TSPO) in immunometabolic regulation in astrocytes.
Author URL.
Cruz AM, Malekizadeh Y, Walker JMV, Shaw S, Ellacott KLJ, Beall C (2019). AMP-activated protein kinase (AMPK) activator R481 improves the counterregulatory response to hypoglycaemia by amplifying glucagon release in healthy rats.
Author URL.
Cruz AML, Malekizadeh Y, Vlachaki Walker J, Ellacott K, Shaw S, Beall C (2019). Amplified Glucagon Response to Hypoglycemia following AMP-Activated Protein Kinase (AMPK) Activator R481 Treatment in Healthy Rats. American Diabetes Association. 7th - 11th Jun 2019.
Abstract:
Amplified Glucagon Response to Hypoglycemia following AMP-Activated Protein Kinase (AMPK) Activator R481 Treatment in Healthy Rats
Abstract.
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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Weightman Potter P (2019). The impact of glucose variation on human astrocytes.
Abstract:
The impact of glucose variation on human astrocytes
Diabetes is a metabolic disorder dysregulating glucose homeostasis. The role of astrocytes in central glucose sensing is poorly understood. But it is recognised they take part in whole-body energy homeostasis, specifically as glucose sensors necessary for the counterregulatory response (CRR) to hypoglycaemia. Iatrogenic hypoglycaemia is the limiting factor to glycaemic control in people with type 1 or type 2 diabetes. Severe hypoglycaemia occurs approximately once per year, whereas, the incidence of minor hypoglycaemia is much greater. Hypoglycaemia impairs awareness of future hypoglycaemia and blunts the CRR, eventually causing hypoglycaemia-associated autonomic failure. The mechanisms of this process are poorly understood.
This thesis utilised isolated human astrocytes exposed to acute or recurrent low glucose (RLG) in vitro to mimic glucose variation in diabetes. Cellular responses were characterised of three key astrocyte functions. Firstly, is astrocyte metabolism altered by acute and RLG treatment? Secondly, do isolated human astrocytes become activated by low glucose treatment, and is this affected by RLG? Thirdly, are astrocytic inflammatory pathways altered by acute or RLG?
The key findings from this thesis shows for the first time that astrocytic mitochondrial oxidation is increased following RLG, with a concurrent increase in fatty acid dependency but decreased coupling efficiency; glycolytic function is also enhanced. Together, this indicates that astrocytes successfully adapt to low glucose to sustain intracellular nucleotide ratios. Contrary to previous work, these human astrocytes do not respond to low glucose by Ca2+-dependent activation. However, the astrocytes do increase inflammatory cytokine release following acute and RLG. Lastly, for the first time an RNA-sequencing approach has been used to identify low glucose-induced differential gene expression. Together these findings support the argument that astrocytes are sensitive to low glucose and may be important in glucose sensation and the CRR.
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2018
Hall BC, Beall C, Randall AD (2018). Alzheimer's disease drug Memantine inhibits Adenosine triphosphate (ATP)-sensitive potassium (KATP) channel activation in hypothalamic GT1-7 cells.
Author URL.
Logie L, Beall C, Rena G (2018). Effect of metformin but not AICAR on total adenosine triphosphate (ATP) levels in primary hepatocytes.
Author URL.
Potter PGW, Walker JMV, Robb JL, Chilton J, Williamson R, Randall A, Ellacott KLJ, Beall C (2018). Recurrent hypoglycaemia increases human primary astrocyte oxygen consumption, fatty acid dependency and pentose phosphate pathway activity.
Author URL.
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) Full text.
Robb J, Hammad N, Beall C, Ellacott K (2018). The 18-kDa translocator protein (TSPO) regulates cellular metabolism in astrocytes.
Author URL.
2017
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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Walker JMV, Potter PGW, Somes A, Beall C (2017). Altered adenosine triphosphate (ATP)-induced calcium responses in glucosensing GT1-7 neurons during hypoglycaemia and in astrocytes following recurrent hypoglycaemia.
Author URL.
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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Walker JMV, Potter PGW, Robb J, Ellacott KLJ, Beall C (2017). Exposure of Astrocytes to Recurrent Hypoglycemia in Vitro Alters Gliotransmission and Purinergic Signaling.
Author URL.
Potter PGW, Cruz JMV, Cruz AM, Williamson R, Randall A, Beall C (2017). Human astrocytes are altered following chronic glucose variation: differential regulation of cytokine release and increased basal metabolism.
Author URL.
Beall C, Dadak S, Walker JMV, Soutar MPM, McCrimmon RJ, Ashford MLJ (2017). Oleate induces ATP-sensitive channel (K-ATP)-dependent hyperpolarisation of mouse hypothalamic glucose-excited neurons without altering cellular energy charge.
Author URL.
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.
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Logie L, Beall C, Rena G (2017). Repression of hepatic glucose production by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) is inhibited by 8-CPT-cAMP mediated blockade of equilibrative nucleotide transporter 1 (ENT1).
Author URL.
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.
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2016
Walker JMV, Malhi A, Hardy H, Chilton J, Beall C (2016). A769662 regulates intracellular calcium and extracellular ATP independent of AMPK.
Author URL.
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.
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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.
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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.
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2015
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.
Author URL.
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.
2014
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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2013
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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2012
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.
Abstract.
Author URL.
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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2010
Piipari K, Beall C, Al-Qassab H, Smith MA, Carling D, Viollet B, Ashford MLJ, Withers DJ (2010). Key Role of AMP-Activated Protein Kinase in Pancreatic Beta Cell Glucose-Sensing and Whole Body Glucose Homeostasis.
Author URL.
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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