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- Author: Michael B Wheeler x
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Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
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Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
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Departments of Physiology and Medicine, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
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We hypothesized that the loss of glucose homeostasis in ob/ob mice is associated with upregulation of islet uncoupling protein-2 (UCP2) expression, leading to impaired glucose-stimulated insulin secretion (GSIS). Changes in glucose homeostasis in lean and ob/ob mice from 5 to 16 weeks were assessed by fasting blood glucose, plasma insulin, oral glucose tolerance, and tissue insulin sensitivity. In vitro GSIS and ATP content were assayed in isolated islets, while UCP2 expression was determined by quantitative real-time PCR and immunoblotting. Short-term reduction of UCP2 expression was achieved through transfection of islets with specific small interfering RNA. Insulin resistance was detected in 5-week-old ob/ob mice, but GSIS and blood glucose levels remained normal. By 8 weeks of age, ob/ob mice displayed fasting hyperglycemia, hyperinsulinemia and glucose intolerance, and also had elevated non-esterified fatty acid concentration in plasma. In vitro, GSIS and ATP generation were impaired in ob/ob islets. Islet UCP2 expression was elevated at 5 and 8 weeks of age. Short-term knockdown of islet UCP2 increased GSIS in islets of lean mice, but had no effect in islets from ob/ob mice. Loss of glucose homeostasis and impairment of insulin secretion from isolated islets at 8 weeks in ob/ob mice is preceded by an increase in UCP2 expression in islets. Moreover, the glucolipotoxic conditions observed are predicted to increase UCP2 activity, contributing to lower islet ATP and GSIS.
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Currently, the physiological function of uncoupling protein-2 (UCP2) in pancreatic islets and its role in the development of diabetes is a matter of great debate. To further investigate the impact of UCP2 on diabetes development, we used streptozotocin (STZ) to experimentally generate diabetes in both wild-type (WT) and UCP2-knockout (UCP2KO) mice. While multiple low-dose STZ injections led to hyperglycemia development over a 14-day period in both WT and UCP2KO mice, we found the development of hyperglycemia to be significantly less severe in the UCP2KO mice. Measurement of insulin and glucagon secretion (in vitro), as well as their plasma concentrations (in vivo), indicated that UCP2-deficiency showed enhanced insulin secretion but impaired α-cell function. Glucagon secretion was attenuated, despite reduced insulin secretion after exposure to STZ, which together contributed to less severe hyperglycemia development in UCP2KO mice. Further experimentation revealed that UCP2-deficient α- and β-cells had chronically higher cellular reactive oxygen species (ROS) levels than the WT prior to STZ application, which correlated with increased basal β- and α-cell mass. Overall, we suggest that increased chronic ROS signaling as a result of UCP2-deficiency contributes to enhanced β-cell function and impairment of α-cell function, leading to an attenuation of STZ-induced hyperglycemia development.
Medicine and
Obstetrics and Gynaecology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8
Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8
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Medicine and
Obstetrics and Gynaecology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8
Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8
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Medicine and
Obstetrics and Gynaecology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8
Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8
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Medicine and
Obstetrics and Gynaecology, University of Toronto, Medical Sciences Building 3247A, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8
Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8
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The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.
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Departments of Physiology and Medicine, Department of Pharmacology, Division of Endocrinology and Metabolism, Neurology and GI Centre of Excellence for Drug Discovery, University of Toronto, Room 7310, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8
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Departments of Physiology and Medicine, Department of Pharmacology, Division of Endocrinology and Metabolism, Neurology and GI Centre of Excellence for Drug Discovery, University of Toronto, Room 7310, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8
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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic β-cells. We then examined the functional characterization of these channels in β-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat β-cells significantly increased HCN current (I h), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous I h. Compared to control β-cells, over-expression of I h increased insulin secretion at 2.8 mmol/l glucose. However, suppression of I h did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced β-cell membrane input resistance (R in), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of R in, which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K+), suppression of I h resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that I h in β-cells possess the potential to modulate β-cell membrane potential and insulin secretion under hypokalemic conditions.
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The aim of this study was to assess the effects of chronic free fatty acid (FFA) exposure on gene expression and the functional state of human pancreatic islets. Chronic exposure of islets to oleate (OA) resulted in a significant reduction in glucose-stimulated insulin secretion (GSIS) compared with control (466±82 vs 234±57 ng/μg DNA, P<0.05). OA treatment also led to reduction in total insulin content of the islets (17 609±3816 vs 10 599±3876 ng insulin/μg DNA) and to an increase in the rate of reactive oxygen species (ROS) generation. Interestingly, the suppressive effects of OA on biosynthesis and secretion of insulin were accompanied by alteration in the expression of 40 genes, as determined by microarray analysis and subsequent qPCR validation. The majority of genes regulated by OA encoded metabolic enzymes. The expression of enzymes involved in oxidative defense was elevated, indicating a link between ROS generation and antioxidant defense activation. Additionally, pretreatment of human islets with OA led to a significant increase (30%) in the rate of oxidation of this fatty acid and to a significant decrease (75%) in glucose oxidation. Importantly, individual analysis of gene clusters from the islets of all donors revealed the induction of genes involved in inflammation and immunity, which provides further evidence that FFA are risk factors for the development of type 2 diabetes. In summary, our data indicate that chronic exposure of human islets to FFA activates inflammatory and metabolic pathways that lead to oxidative stress, reduced β-cell insulin content, and inhibition of GSIS.
