A TRP channel contributes to insulin secretion by pancreatic β-cells

The release of insulin by pancreatic beta cells involves a complex interplay of conductances that generate oscillations and drive secretion. A recent report identifies a new player in this process, the ion channel TRPM5. TRPM5 was originally identified in taste cells, where it forms a Ca²⁺-activated cation channel that is required for sensory responses to bitter and sweet tastes. New research now shows that TRPM5 is expressed within the pancreatic islets of Langerhans, where it regulates the frequency of Ca²⁺ oscillations and contributes to insulin release by β-cells.     

TRPM2 modulates insulin secretion in pancreatic β-cells

Insulin secretion from pancreatic β-cells is the primary mechanism by which the body lowers blood glucose concentrations. Glucose is the principal stimulator of insulin secretion, and the primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K+ channel voltage-gated Ca2+ channel-mediated pathway. Several TRP channels expressed in pancreatic β-cells have been reported to be involved in insulin secretion. One recent report found that TRPM2 is expressed in pancreatic β-cells and modulates insulin secretion stimulated by glucose and further potentiated by incretin hormones. TRPM2 is a Ca2+-permeable non-selective cation channel activated by adenosine dinucleotides, hydrogen peroxide, and intracellular Ca2+. Glucose tolerance was impaired and insulin secretion was decreased in TRPM2 knockout mice. Insulin secretion via TRPM2 occurs not only through control of intracellular Ca2+ concentrations but also through Ca2+ influx-independent mechanisms. Although further examination is needed to clarify the mechanism of TRPM2-mediated insulin secretion, TRPM2 may be a key player in regulation of insulin secretion and could represent a new target for diabetes therapy.     

Mathematical models for insulin secretion in pancreatic β-cells

Insulin secretion is one of the most characteristic features of β-cell physiology. As it plays a central role in glucose regulation, a number of experimental and theoretical studies have been performed since the discovery of the pancreatic β-cell. This review article aims to give an overview of the mathematical approaches to insulin secretion. Beginning with the bursting electrical activity in pancreatic β-cells, we describe effects of the gap-junction coupling between β-cells on the dynamics of insulin secretion. Then, implications of paracrine interactions among such islet cells as α-, β-, and δ-cells are discussed. Finally, we present mathematical models which incorporate effects of glycolysis and mitochondrial glucose metabolism on the control of insulin secretion.     

Mathematical models for insulin secretion in pancreatic β-cells

Insulin secretion is one of the most characteristic features of β-cell physiology. As it plays a central role in glucose regulation, a number of experimental and theoretical studies have been performed since the discovery of the pancreatic β-cell. This review article aims to give an overview of the mathematical approaches to insulin secretion. Beginning with the bursting electrical activity in pancreatic β-cells, we describe effects of the gap-junction coupling between β-cells on the dynamics of insulin secretion. Then, implications of paracrine interactions among such islet cells as α-, β-, and δ-cells are discussed. Finally, we present mathematical models which incorporate effects of glycolysis and mitochondrial glucose metabolism on the control of insulin secretion.     

Characterization of preptin-induced insulin secretion in pancreatic β-cells

We aimed to characterize the effects of preptin on insulin secretion at the single-cell level, as well as the mechanisms underlying these changes, with respect to regulation by intracellular Ca(2+) [Ca(2+)](i) mobilization. This study assessed the effect of preptin on insulin secretion and investigated the link between preptin and the phospholipase C (PLC)/protein kinase C (PKC) pathway at the cellular level using fura-2 pentakis(acetoxymethyl) ester-loaded insulin-producing cells (Min 6 cells). Our results demonstrate that preptin promotes insulin secretion in a concentration-dependent manner. Using a PLC inhibitor (chelerythrine) or a PKC inhibitor (U73122) resulted in a concentration-dependent decrease in insulin secretion. Also, preptin mixed with IGF2 receptor (IGF2R) antibodies suppressed insulin secretion in a dose-dependent manner, which indicates that activation of IGF2R is mediated probably because preptin is a type of proIGF2. In addition, preptin stimulated insulin secretion to a similar level as did glibenclamide. The activation of PKC/PLC by preptin stimulation is highly relevant to the potential mechanisms for increase in insulin secretion. Our results provide new insight into the insulin secretion of preptin, a secreted proIGF2-derived peptide that can induce greater efficacy of signal transduction resulting from PLC and PKC activation through the IGF2R.     

TRPM2 modulates insulin secretion in pancreatic β-cells

Insulin secretion from pancreatic β-cells is the primary mechanism by which the body lowers blood glucose concentrations. Glucose is the principal stimulator of insulin secretion, and the primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K+ channel voltage-gated Ca2+ channel-mediated pathway. Several TRP channels expressed in pancreatic β-cells have been reported to be involved in insulin secretion. One recent report found that TRPM2 is expressed in pancreatic β-cells and modulates insulin secretion stimulated by glucose and further potentiated by incretin hormones. TRPM2 is a Ca2+-permeable non-selective cation channel activated by adenosine dinucleotides, hydrogen peroxide, and intracellular Ca2+. Glucose tolerance was impaired and insulin secretion was decreased in TRPM2 knockout mice. Insulin secretion via TRPM2 occurs not only through control of intracellular Ca2+ concentrations but also through Ca2+ influx-independent mechanisms. Although further examination is needed to clarify the mechanism of TRPM2-mediated insulin secretion, TRPM2 may be a key player in regulation of insulin secretion and could represent a new target for diabetes therapy.     

Impaired insulin secretion in transgenic mice over-expressing calpastatin in pancreatic β-cells

Calpains are a family of calcium-activated proteases involved in a number of cellular functions including cell death, proliferation and exocytosis. The finding that variation in the calpain-10 gene increases type 2 diabetes risk in some populations has increased interest in determining the potential role of calpains in pancreatic β-cell function. In the present study, transgenic mice (Cast (RIP)) expressing an endogenous calpain inhibitor, calpastatin, in pancreatic β-cells were used to dissect the role of the calpain system in the regulation insulin secretion in vivo and in vitro. Glucose concentrations after the administration of intraperitoneal glucose were significantly increased in Cast (RIP) mice compared with wildtype littermate controls. This was associated with a reduction in glucose-stimulated insulin secretion in vivo. Using pancreas perfusion, static islet incubation and islet perifusion, it was demonstrated that Cast (RIP) islets hypersecreted insulin at low glucose, but exhibited significantly impaired insulin responses to high glucose. Examination of insulin release and calcium signals from isolated islets indicated that distal components of the insulin exocytotic pathway were abnormal in Cast (RIP) mice. Cast (RIP) islets had modestly reduced expression of Rab3a and other critical components in the late steps of insulin exocytosis. These studies provide the first evidence that blocking endogenous calpain activity partially impairs insulin release in vivo and in vitro by targeting distal components of the insulin exocytotic machinery.     

Impaired insulin secretion in transgenic mice over-expressing calpastatin in pancreatic β-cells

Calpains are a family of calcium-activated proteases involved in a number of cellular functions including cell death, proliferation and exocytosis. The finding that variation in the calpain-10 gene increases type 2 diabetes risk in some populations has increased interest in determining the potential role of calpains in pancreatic β-cell function. In the present study, transgenic mice (Cast^RIP) expressing an endogenous calpain inhibitor, calpastatin, in pancreatic β-cells were used to dissect the role of the calpain system in the regulation insulin secretion in vivo and in vitro. Glucose concentrations after the administration of intraperitoneal glucose were significantly increased in Cast^RIP mice compared with wildtype littermate controls. This was associated with a reduction in glucose-stimulated insulin secretion in vivo. Using pancreas perfusion, static islet incubation and islet perifusion, it was demonstrated that Cast^RIP islets hypersecreted insulin at low glucose, but exhibited significantly impaired insulin responses to high glucose. Examination of insulin release and calcium signals from isolated islets indicated that distal components of the insulin exocytotic pathway were abnormal in Cast^RIP mice. Cast^RIP islets had modestly reduced expression of Rab3a and other critical components in the late steps of insulin exocytosis. These studies provide the first evidence that blocking endogenous calpain activity partially impairs insulin release in vivo and in vitro by targeting distal components of the insulin exocytotic machinery.     

Effect of troglitazone on insulin secretion in pancreatic β-cells and its mechanism

The effect of troglitazone on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and its mechanism were investigated.10 μmol/L troglitazone had no effect on basal insulin secretion,but significantly decreased GSIS and stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylations (all P<0.01).These reactions were completely reversed by AMPK inhibitor compound C,suggesting that the troglitazone acutely inhibits insulin secretion via stimulating AMPK activity in beta cells.     

Effect of troglitazone on insulin secretion in pancreatic β-cells and its mechanism

The effect of troglitazone on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells and its mechanism were investigated.10 μmol/L troglitazone had no effect on basal insulin secretion,but significantly decreased GSIS and stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylations (all P<0.01).These reactions were completely reversed by AMPK inhibitor compound C,suggesting that the troglitazone acutely inhibits insulin secretion via stimulating AMPK activity in beta cells.     

α 1-antitrypsin enhances insulin secretion and prevents cytokine-mediated apoptosis in pancreatic β-cells

α1-antitrypsin (AAT) is a serine protease inhibitor, which recently has been shown to prevent type 1 diabetes (T1D) development, to prolong islet allograft survival and to inhibit β-cell apoptosis in vivo. It has also been reported that T1D patients have significantly lower plasma concentrations of AAT suggesting the potential role of AAT in the pathogenesis of T1D. We have investigated whether plasma-purified AAT can affect β-cell function in vitro. INS-1E cells or primary rat pancreatic islets were used to study the effect of AAT on insulin secretion after glucose, glucagon-like peptide-1 (GLP-1) and forskolin stimulation and on cytokine-mediated apoptosis. The secreted insulin and total cyclic AMP (cAMP) were determined using radioimmunoassay and apoptosis was evaluated by propidium iodide staining followed by FACS analysis. We found that AAT increases insulin secretion in a glucose-dependent manner, potentiates the effect of GLP-1 and forskolin and neutralizes the inhibitory effect of clonidine on insulin secretion. The effect of AAT on insulin secretion was accompanied by an increase in cAMP levels. In addition, AAT protected INS-1E cells from cytokine-induced apoptosis. Our findings show that AAT stimulates insulin secretion and protects β-cells against cytokine-induced apoptosis, and these effects of AAT seem to be mediated through the cAMP pathway. In view of these novel findings we suggest that AAT may represent a novel anti-inflammatory compound to protect β-cells under the immunological attack in T1D but also therapeutic strategy to potentiate insulin secretion in type 2 diabetes (T2D).     

T-cadherin (Cdh13) in association with pancreatic β-cell granules contributes to second phase insulin secretion

Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human β-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated β-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on β-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN.     

T-cadherin (Cdh13) in association with pancreatic β-cell granules contributes to second phase insulin secretion

Glucose homeostasis depends on adequate control of insulin secretion. We report the association of the cell-adhesion and adiponectin (APN)-binding glycoprotein T-cadherin (Cdh13) with insulin granules in mouse and human β-cells. Immunohistochemistry and electron microscopy of islets in situ and targeting of RFP-tagged T-cadherin to GFP-labeled insulin granules in isolated β-cells demonstrate this unusual location. Analyses of T-cadherin-deficient (Tcad-KO) mice show normal islet architecture and insulin content. However, T-cadherin is required for sufficient insulin release in vitro and in vivo. Primary islets from Tcad-KO mice were defective in glucose-induced but not KCl-mediated insulin secretion. In vivo, second phase insulin release in T-cad-KO mice during a hyperglycemic clamp was impaired while acute first phase release was unaffected. Tcad-KO mice showed progressive glucose intolerance by 5 mo of age without concomitant changes in peripheral insulin sensitivity. Our analyses detected no association of APN with T-cadherin on β-cell granules although colocalization was observed on the pancreatic vasculature. These data identify T-cadherin as a novel component of insulin granules and suggest that T-cadherin contributes to the regulation of insulin secretion independently of direct interactions with APN.     

Leptin promotes KATP channel trafficking by AMPK signaling in pancreatic β-cells

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (K_ATP) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting K_ATP channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. K_ATP channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce K_ATP channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent K_ATP channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.The K_ATP channel, an inwardly rectifying K⁺ channel that consists of pore-forming Kir6.2 and regulatory sulfonylurea receptor 1 (SUR1) subunits (1), functions as an energy sensor: its gating is regulated mainly by the intracellular concentrations of ATP and ADP. In pancreatic β-cells, K_ATP channels are inhibited or activated in response to the rise or fall in blood glucose levels, leading to changes in membrane excitability and insulin secretion (2, 3). Thus, K_ATP channel gating has been considered an important mechanism in coupling blood glucose levels to insulin secretion. Recently, trafficking of K_ATP channels to the plasma membrane was highlighted as another important mechanism for regulating K_ATP channel activity (4–6).AMP-activated protein kinase (AMPK) is a key enzyme regulating energy homeostasis (7). We recently demonstrated that K_ATP channels are recruited to the plasma membrane in glucose-deprived conditions via AMPK signaling in pancreatic β-cells (6). Inhibition of AMPK signaling significantly reduces K_ATP currents, even after complete wash-out of intracellular ATP (6). Given these results, we proposed a model that recruitment of K_ATP channels to the plasma membrane via AMPK signaling is crucial for K_ATP channel activation in low-glucose conditions. However, the physiological relevance of this model remains unclear because pancreatic β-cells had to be incubated in media containing less than 3 mM glucose to recruit a sufficient number of K_ATP channels to the plasma membrane (6). We thus hypothesized that there should be an endogenous ligand in vivo that promotes AMPK-dependent K_ATP channel trafficking sufficiently to stabilize pancreatic β-cells at physiological fasting glucose levels.Leptin is an adipocyte-derived hormone that regulates food intake, body weight, and glucose homeostasis (8, 9). In addition to its central action, leptin regulates the release of insulin and glucagon, the key hormones regulating glucose homeostasis, by direct actions on β- and α-cells of pancreatic islets, respectively (10–12). It thus was proposed that the adipoinsular axis is crucial for maintaining nutrient balance and that dysregulation of this axis contributes to obesity and diabetes (12). However, intracellular signaling mechanisms underlying leptin effects are largely unknown. Leptin was shown to increase K_ATP currents in pancreatic β-cells (13, 14), but the possibility that K_ATP channel trafficking mediates leptin-induced K_ATP channel activation has not been explored.In the present study, we demonstrate that the surface levels of K_ATP channels increase in pancreatic β-cells under fasting conditions in vivo. Translocation of K_ATP channels to the plasma membrane in fasting was absent in pancreatic β-cells from ob/ob mice, but restored by treatment with leptin, suggesting a role for leptin in K_ATP channel trafficking in vivo. We further show that leptin-induced AMPK activation, which is essential for K_ATP channel trafficking to the plasma membrane, is mediated by activation of canonical transient receptor potential 4 (TRPC4) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). Our results highlight the importance of trafficking regulation in K_ATP channel activation and provide insights into the action of leptin on glucose homeostasis.     

Protein farnesylation is requisite for mitochondrial fuel-induced insulin release: Further evidence to link reactive oxygen species generation to insulin secretion in pancreatic β-cells

Several lines of recent evidence implicate regulatory roles for reactive oxygen species (ROS) in islet function and insulin secretion. The phagocyte-like NADPH oxidase (Nox2) has recently been shown to be one of the sources of ROS in the signaling events leading to glucose stimulated insulin secretion (GSIS). We recently reported inhibition of glucose- or mitochondrial fuel-induced Nox2-derived ROS by a specific inhibitor of protein farnesyl transferse (FTase; FTI-277), suggesting that activation of FTase might represent one of the upstream signaling events to Nox2 activation. Furthermore, FTase inhibitors (FTI-277 and FTI-2628) have also been shown to attenuate GSIS in INS 832/13 cells and normal rodent islets. Herein, we provide further evidence to suggest that inhibition of FTase either by pharmacological (e.g., FTI-277) or gene silencing (siRNA-FTase) approaches markedly attenuates mitochondrial fuel-stimulated insulin secretion (MSIS) in INS 832/13 cells. Together, our findings further establish a link between nutrient-induced Nox2 activation, ROS generation and insulin secretion in the pancreatic β-cell.     

Synapsins I and II are not required for insulin secretion from mouse pancreatic β-cells

Synapsins are a family of phosphoproteins that modulate the release of neurotransmitters from synaptic vesicles. The release of insulin from pancreatic β-cells has also been suggested to be regulated by synapsins. In this study, we have utilized a knock out mouse model with general disruptions of the synapsin I and II genes [synapsin double knockout (DKO)]. Stimulation with 20 mm glucose increased insulin secretion 9-fold in both wild-type (WT) and synapsin DKO islets, whereas secretion in the presence of 70 mm K(+) and 1 mm glucose was significantly enhanced in the synapsin DKO mice compared to WT. Exocytosis in single β-cells was investigated using patch clamp. The exocytotic response, measured by capacitance measurements and elicited by a depolarization protocol designed to visualize exocytosis of vesicles from the readily releasable pool and from the reserve pool, was of the same size in synapsin DKO and WT β-cells. The increase in membrane capacitance corresponding to readily releasable pool was approximately 50fF in both genotypes. We next investigated the voltage-dependent Ca(2+) influx. In both WT and synapsin DKO β-cells the Ca(2+) current peaked at 0 mV and measured peak current (I(p)) and net charge (Q) were of similar magnitude. Finally, ultrastructural data showed no variation in total number of granules (N(v)) or number of docked granules (N(s)) between the β-cells from synapsin DKO mice and WT control. We conclude that neither synapsin I nor synapsin II are directly involved in the regulation of glucose-stimulated insulin secretion and Ca(2)-dependent exocytosis in mouse pancreatic β-cells.     

Protein phosphatase 1 (PP-1)-dependent inhibition of insulin secretion by leptin in INS-1 pancreatic β-cells and human pancreatic islets

Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca2(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.     

Adhesion molecule CADM1 contributes to gap junctional communication among pancreatic islet α-cells and prevents their excessive secretion of glucagon

Cell adhesion molecule-1 (CADM1) is a recently identified adhesion molecule of pancreatic islet α-cells that mediates nerve–α-cell interactions via trans-homophilic binding and serves anatomical units for the autonomic control of glucagon secretion. CADM1 also mediates attachment between adjacent α-cells. Since gap junctional intercellular communication (GJIC) among islet cells is essential for islet hormone secretion, we examined whether CADM1 promotes GJIC among α-cells and subsequently participates in glucagon secretion regulation. Dye transfer assays using αTC6 mouse α-cells, which endogenously express CADM1, supported this possibility; efficient cell-to-cell spread of gap junction-permeable dye was detected in clusters of αTC6 cells transfected with nonspecific, but not with CADM1-targeting, siRNA. Immunocytochemical analysis of connexin 36, a major component of the gap junction among αTC6 cells, revealed that it was localized exclusively to the cell membrane in CADM1-non-targeted αTC6 cells, but diffusely to the cytoplasm in CADM1-targeted cells. Next, we incubated CADM1-targeted and non-targeted αTC6 cells in a medium containing 1 mM glucose and 200 mM arginine for 30 min to induce glucagon secretion, and found that the targeted cells secreted three times more glucagon than did the non-targeted. We conducted similar experiments using pancreatic islets that were freshly isolated from wild-type and CADM1-knockout mice, and expressed glucagon secretion as ratios relative to baseline values. The increase in ratio was larger in CADM1-knockout islets than in wild-type islets. These results suggest that CADM1 may serve as a volume limiter of glucagon secretion by sustaining α-cell attachment necessary for efficient GJIC.     

Small-molecule inducers of insulin expression in pancreatic α-cells

High-content screening for small-molecule inducers of insulin expression identified the compound BRD7389, which caused α-cells to adopt several morphological and gene expression features of a β-cell state. Assay-performance profile analysis suggests kinase inhibition as a mechanism of action, and we show that biochemical and cellular inhibition of the RSK kinase family by BRD7389 is likely related to its ability induce a β-cell-like state. BRD7389 also increases the endocrine cell content and function of donor human pancreatic islets in culture.