[show abstract][hide abstract] ABSTRACT: OBJECTIVE The inability of pancreatic beta-cells to appropriately respond to glucose and secrete insulin are primary defects associated with beta-cell failure in type 2 diabetes. Mitochondrial dysfunction has been implicated as a key factor in the development of type 2 diabetes; however, a link between mitochondrial dysfunction and defective insulin secretion is unclear. RESEARCH DESIGN AND METHODS We investigated the changes in islet mitochondrial function and morphology during progression from insulin resistance (3 weeks old), immediately before hyperglycemia (5 weeks old), and after diabetes onset (10 weeks old) in transgenic MKR mice compared with controls. The molecular and protein changes at 10 weeks were determined using microarray and iTRAQ proteomic screens. RESULTS At 3 weeks, MKR mice were hyperinsulinemic but normoglycemic and beta-cells showed negligible mitochondrial or morphological changes. At 5 weeks, MKR islets displayed abrogated hyperpolarization of mitochondrial membrane potential (DeltaPsi(m)), reduced mitochondrial Ca(2+) uptake, slightly enlarged mitochondria, and reduced glucose-stimulated insulin secretion. By 10 weeks, MKR mice were hyperglycemic and hyperinsulinemic and beta-cells contained swollen mitochondria with disordered cristae. beta-Cells displayed impaired stimulus-secretion coupling including reduced hyperpolarization of DeltaPsi(m), impaired Ca(2+)-signaling, and reduced glucose-stimulated ATP/ADP and insulin release. Furthermore, decreased cytochrome c oxidase-dependent oxygen consumption and signs of oxidative stress were observed in diabetic islets. Protein profiling of diabetic islets revealed that 36 mitochondrial proteins were differentially expressed, including inner membrane proteins of the electron transport chain. CONCLUSIONS We provide novel evidence for a critical role of defective mitochondrial oxidative phosphorylation and morphology in the pathology of insulin resistance-induced beta-cell failure.
[show abstract][hide abstract] ABSTRACT: Voltage-gated eag-related gene (Erg) K(+) channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in alpha-TC6 and Min6 cells alpha- and beta-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in alpha- and beta-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca(2+)-imaging experiments were performed on isolated alpha- and beta-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca(2+) increase in both alpha- and beta-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of alpha- and beta-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.
Journal of Biological Chemistry 09/2009; 284(44):30441-52. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele.
Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols.
ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay.
ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.
[show abstract][hide abstract] ABSTRACT: The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, discovered initially in cardiac and neuronal cells, mediate the inward pacemaker current (I (f) or I (h)). Recently, we have demonstrated the presence of HCN channels in pancreatic beta cells. Here, we aim to examine the presence and function of HCN channels in glucagon-secreting alpha cells.
RT-PCR and immunocytochemistry were used to examine the presence of HCN channels in alpha cells. Whole-cell patch-clamp, calcium imaging and glucagon secretion experiments were performed to explore the function of HCN channels in alpha cells.
HCN transcripts and proteins were detected in alpha-TC6 cells and dispersed rat alpha cells. Patch-clamp recording showed hyperpolarisation-activated currents in alpha-TC6 cells, which could be blocked by HCN channel inhibitor ZD7288. Glucagon secretion RIA studies demonstrated that at both low and high glucose concentrations (2 and 20 mmol/l), ZD7288 significantly enhanced glucagon secretion in alpha-TC6 and IN-R1-G9 cell lines. Conversely, activation of HCN channels by lamotrigine significantly suppressed glucagon secretion at the low glucose concentration. Calcium imaging studies showed that blockade of HCN channels by ZD7288 significantly increased intracellular calcium in alpha-TC6 cells, while lamotrigine or the Na(+) channel blocker tetrodotoxin suppressed the effect of ZD7288 on intracellular calcium. Furthermore, we found the HCN channel inhibitors ZD7288 and cilobradine both significantly increased glucagon secretion from rat islets.
These results suggest a potential role for HCN channels in regulation of glucagon secretion via modulating Ca(2+) and Na(+) channel activities.
[show abstract][hide abstract] ABSTRACT: During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).
Journal of Biological Chemistry 05/2008; 283(15):10184-97. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Prolonged elevation of glucose can adversely affect beta-cell function. In vitro studies have linked glucose-induced beta-cell dysfunction to oxidative stress; however, whether oxidative stress plays a role in vivo is unclear. Therefore, our objective was to investigate the role of oxidative stress in an in vivo model of glucose-induced beta-cell dysfunction.
Wistar rats were infused intravenously with glucose for 48 h to achieve 20 mmol/l hyperglycemia with/without co-infusion of one of the following antioxidants: taurine (2-amino ethanesulfonic acid) (TAU), an aldehyde scavenger; N-acetylcysteine (NAC), a precursor of glutathione; or tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) (TPO), a superoxide dismutase mimetic. This was followed by islet isolation or hyperglycemic clamp.
A 48-h glucose infusion decreased glucose-stimulated insulin secretion (GSIS) and elevated reactive oxygen species (ROS), total superoxide, and mitochondrial superoxide in freshly isolated islets. TPO prevented the increase in total and mitochondrial superoxide and the beta-cell dysfunction induced by high glucose. However, TAU and NAC, despite completely normalizing H(2)DCF-DA (dihydro-dichlorofluorescein diacetate)-measured ROS, did not prevent the increase in superoxide and the decrease in beta-cell function induced by high glucose. TPO but not TAU also prevented beta-cell dysfunction induced by less extreme hyperglycemia (15 mmol/l) for a longer period of time (96 h). To further investigate whether TPO is effective in vivo, a hyperglycemic clamp was performed. Similar to the findings in isolated islets, prolonged glucose elevation (20 mmol/l for 48 h) decreased beta-cell function as assessed by the disposition index (insulin secretion adjusted for insulin sensitivity), and co-infusion of TPO with glucose completely restored beta-cell function.
These findings implicate superoxide generation in beta-cell dysfunction induced by prolonged hyperglycemia.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Endocrinology 04/2007; 192(3):605-14. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: In contrast to mouse, rat islet beta-cell membrane potential is reported not to oscillate in response to elevated glucose despite demonstrated oscillations in calcium and insulin secretion. We aim to clarify the electrical activity of rat islet beta-cells and characterize and compare the electrical activity of both alpha- and beta-cells in rat and mouse islets. We recorded electrical activity from alpha- and beta-cells within intact islets from both mouse and rat using the perforated whole-cell patch clamp technique. Fifty-six percent of both mouse and rat beta-cells exhibited an oscillatory response to 11.1 mm glucose. Responses to both 11.1 mm and 2.8 mm glucose were identical in the two species. Rat beta-cells exhibited incremental depolarization in a glucose concentration-dependent manner. We also demonstrated electrical activity in human islets recorded under the same conditions. In both mouse and rat alpha-cells 11 mm glucose caused hyperpolarization of the membrane potential, whereas 2.8 mm glucose produced action potential firing. No species differences were observed in the response of alpha-cells to glucose. This paper is the first to demonstrate and characterize oscillatory membrane potential fluctuations in the presence of elevated glucose in rat islet beta-cells in comparison with mouse. The findings promote the use of rat islets in future electrophysiological studies, enabling consistency between electrophysiological and insulin secretion studies. An inverse response of alpha-cell membrane potential to glucose furthers our understanding of the mechanisms underlying glucose sensitive glucagon secretion.
[show abstract][hide abstract] ABSTRACT: We investigated whether an increase in cAMP could normalize glucose-stimulated insulin secretion (GSIS) in uncoupling protein-2 (UCP2) overexpressing (ucp2-OE) beta-cells. Indices of beta-cell (beta-TC-6f7 cells and rodent islets) function were measured after induction of ucp2, in the presence or absence of cAMP-stimulating agents, analogs, or inhibitors. Islets of ob/ob mice had improved glucose-responsiveness in the presence of forskolin. Rat islets overexpressing ucp2 had significantly lower GSIS than controls. Acutely, the protein kinase A (PKA) and epac pathway stimulant forskolin normalized insulin secretion in ucp2-OE rat islets and beta-TC-6f7 beta-cells, an effect blocked by specific PKA inhibitors but not mimicked by epac agonists. However, there was no effect of ucp2-OE on cAMP concentrations or PKA activity. In ucp2-OE islets, forskolin inhibited ATP-dependent potassium (K(ATP)) channel currents and (86)Rb(+) efflux, indicative of K(ATP) block. Likewise, forskolin application increased intracellular Ca(2+), which could account for its stimulatory effects on insulin secretion. Chronic exposure to forskolin increased ucp2 mRNA and exaggerated basal secretion but not GSIS. In mice deficient in UCP2, there was no augmentation of either cAMP content or cAMP-dependent insulin secretion. Thus, elevating cellular cAMP can reverse the deficiency in GSIS invoked by ucp2-OE, at least partly through PKA-mediated effects on the K(ATP) channel.
Journal of Endocrinology 10/2006; 190(3):669-80. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The antioxidant compound alpha-lipoic acid (alpha-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, alpha-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of alpha-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of alpha-LA on pancreatic beta cell function.
Isolated rat islets and MIN6 beta cells were treated acutely (15-90 min) or chronically (18-24 h) with alpha-LA or the known AMPK-activating compounds 5'-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed.
Acute or chronic treatment of islets and MIN6 cells with alpha-LA led to dose-dependent rises in phosphorylation of the AMPK alpha-subunit and acetyl CoA carboxylase. Chronic exposure to alpha-LA, AICAR or metformin caused a reduction in insulin secretion. alpha-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, alpha-LA also acutely inhibited insulin secretion. Examination of the effect of alpha-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca(2+).
This study is the first to demonstrate that alpha-LA directly affects beta cell function. The chronic effects of alpha-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, alpha-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function.
[show abstract][hide abstract] ABSTRACT: In pancreatic beta-cells Zn(2+) is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn(2+) transport into beta-cells or the regulation and compartmentalization of Zn(2+) within this cell type. Evidence suggests that Zn(2+) in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn(2+)-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC(4)(3) we applied fluorescent microscopy techniques for analysis of Zn(2+)-accumulating pathways in mouse islets, dispersed islet cells, and beta-cell lines (MIN6 and beta-TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn(2+) (5-10 mum) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K(+)) conditions. Under both basal and depolarized states, beta-cells were capable of Zn(2+) uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn(2+) accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca(2+) channels, Gd(3+) and La(3+), inhibited Zn(2+) uptake in beta-cells under stimulatory conditions with little or no change in Zn(2+) accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn(2+) uptake the expression of a number of ZIP family Zn(2+) transporter mRNAs in islets and beta-cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn(2+) transport into beta-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn(2+) accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn(2+) transport across the plasma membrane is dependent upon the metabolic status of the cell.
Journal of Biological Chemistry 05/2006; 281(14):9361-72. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: In pancreatic β-cells Zn2+ is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn2+ transport into β-cells or the regulation and compartmentalization of Zn2+ within this cell type. Evidence suggests that Zn2+ in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn2+-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC4(3) we applied fluorescent microscopy techniques for analysis of Zn2+-accumulating pathways in mouse islets, dispersed islet cells, and β-cell lines (MIN6 and β-TC6f7 cells). Because the stimulation
of insulin secretion is associated with cell depolarization, Zn2+ (5-10 μm) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K+) conditions. Under both basal and depolarized states, β-cells were capable of Zn2+ uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn2+ accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific
inhibitors of Ca2+ channels, Gd3+ and La3+, inhibited Zn2+ uptake in β-cells under stimulatory conditions with little or no change in Zn2+ accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn2+ uptake the expression of a number of ZIP family Zn2+ transporter mRNAs in islets and β-cells was investigated. In conclusion, we demonstrate for the first time that, in part,
Zn2+ transport into β-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn2+ accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn2+ transport across the plasma membrane is dependent upon the metabolic status of the cell.
Journal of Biological Chemistry 04/2006; 281(14):9361-9372. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA(A)Rs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA(A)Rs in the pancreatic islets. However, the reported effects of GABA(A)R activation on insulin secretion from islet beta cells have been controversial.
This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration.
Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 micromol/l, induced a transmembrane current (I(GABA)) which was sensitive to the GABA(A)R antagonist bicuculline. The current-voltage relationship revealed that I(GABA) reversed at -42+/-2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V (M)), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V (M) of INS-1 cells was below the I(GABA) reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V (M) of INS-1 cells was above the I(GABA) reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5).
These findings indicate that activation of GABA(A)Rs in beta cells regulates insulin secretion in concert with changes in glucose levels.