Claes B. Wollheim

Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis

The hormone insulin is stored in secretory granules and released from the pancreatic β-cells by exocytosis. In the consensus model of glucose-stimulated insulin secretion, ATP is generated by mitochondrial metabolism, promoting closure of ATP-sensitive potassium (KATP) channels, which depolarizes the plasma membrane. Subsequently, opening of voltage-sensitive Ca2+ channels increases the cytosolic Ca2+ concentration ([Ca2+]c) which constitutes the main trigger initiating insulin exocytosis. Nevertheless, the Ca2+ signal alone is not sufficient for sustained secretion. Furthermore, glucose elicits a secretory response under conditions of clamped, elevated [Ca2+]c (refs 5, 6). A mitochondrial messenger must therefore exist which is distinct from ATP. We have now identified this as glutamate. We show that glucose generates glutamate from β-cell mitochondria. A membrane-permeant glutamate analogue sensitizes the glucose-evoked secretory response, acting downstream of mitochondrial metabolism. In permeabilized cells, under conditions of fixed [Ca2+]c, added glutamate directly stimulates insulin exocytosis, independently of mitochondrial function. Glutamate uptake by the secretory granules is likely to be involved, as inhibitors of vesicular glutamate transport suppress the glutamate-evoked exocytosis. These results demonstrate that glutamate acts as an intracellular messenger that couples glucose metabolism to insulin secretion.

α-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains

Glucagon, a hormone secreted from the alpha-cells of the endocrine pancreas, is critical for blood glucose homeostasis. It is the major counterpart to insulin and is released during hypoglycemia to induce hepatic glucose output. The control of glucagon secretion is multifactorial and involves direct effects of nutrients on alpha-cell stimulus-secretion coupling as well as paracrine regulation by insulin and zinc and other factors secreted from neighboring beta- and delta-cells within the islet of Langerhans. Glucagon secretion is also regulated by circulating hormones and the autonomic nervous system. In this review, we describe the components of the alpha-cell stimulus secretion coupling and how nutrient metabolism in the alpha-cell leads to changes in glucagon secretion. The islet cell composition and organization are described in different species and serve as a basis for understanding how the numerous paracrine, hormonal, and nervous signals fine-tune glucagon secretion under different physiological conditions. We also highlight the pathophysiology of the alpha-cell and how hyperglucagonemia represents an important component of the metabolic abnormalities associated with diabetes mellitus. Therapeutic inhibition of glucagon action in patients with type 2 diabetes remains an exciting prospect.

Mitochondrial function in normal and diabetic |[beta]|-cells

The aetiology of type 2, or non-insulin-dependent, diabetes mellitus has been characterized in only a limited number of cases. Among these, mitochondrial diabetes, a rare subform of the disease, is the consequence of pancreatic β-cell dysfunction caused by mutations in mitochondrial DNA, which is distinct from the nuclear genome. The impact of such mutations on β-cell function reflects the importance of mitochondria in the control of insulin secretion. The β-cell mitochondria serve as fuel sensors, generating factors that couple nutrient metabolism to the exocytosis of insulin-containing vesicles. The latter process requires an increase in cytosolic Ca2+, which depends on ATP synthesized by the mitochondria. This organelle also generates other factors, of which glutamate has been proposed as a potential intracellular messenger.

Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from β-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from α-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet α- and β-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although α-cells, like β-cells, possess the inherent capacity to respond to nutrients, secretion from α-cells is normally suppressed by the simultaneous activation of β-cells. Zinc released from β-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.

Hydrogen Peroxide Alters Mitochondrial Activation and Insulin Secretion in Pancreatic Beta Cells

The effects of a transient exposure to hydrogen peroxide (10 min at 200 μmH2O2) on pancreatic beta cell signal transduction and insulin secretion have been evaluated. In rat islets, insulin secretion evoked by glucose (16.7 mm) or by the mitochondrial substrate methyl succinate (5 mm) was markedly blunted following exposure to H2O2. In contrast, the secretory response induced by plasma membrane depolarization (20 mm KCl) was not significantly affected. Similar results were obtained in insulinoma INS-1 cells using glucose (12.8 mm) as secretagogue. After H2O2 treatment, glucose no longer depolarized the membrane potential (ΔΨ) of INS-1 cells or increased cytosolic Ca2+. Both ΔΨ and Ca2+ responses were still observed with 30 mm KCl despite an elevated baseline of cytosolic Ca2+ appearing ∼10 min after exposure to H2O2. The mitochondrial ΔΨ of INS-1 cells was depolarized by H2O2 abolishing the hyperpolarizing action of glucose. These ΔΨ changes correlated with altered mitochondrial morphology; the latter was not preserved by the overexpression of the antiapoptotic protein Bcl-2. Mitochondrial Ca2+ was increased following exposure to H2O2 up to the micromolar range. No further augmentation occurred after glucose addition, which normally raises this parameter. Nevertheless, KCl was still efficient in enhancing mitochondrial Ca2+. Cytosolic ATP was markedly reduced by H2O2 treatment, probably explaining the decreased endoplasmic reticulum Ca2+. Taken together, these data point to the mitochondria as primary targets for H2O2 damage, which will eventually interrupt the transduction of signals normally coupling glucose metabolism to insulin secretion.

ER stress and SREBP-1 activation are implicated in β-cell glucolipotoxicity

The reduction in insulin secretory capacity and β-cell mass observed in type 2 diabetes is thought to be caused by glucolipotoxicity secondary to hyperglycemia and hyperlipidemia. Our aim in this study was to elucidate the underlying molecular mechanisms. We found a strong correlation between chronic high-glucose treatment and SREBP-1c activation in INS-1 cells and rat islets. Both high-glucose treatment and SREBP-1c activation in INS-1 cells resulted in lipid accumulation, impaired glucose-stimulated insulin secretion, apoptosis, and strikingly similar gene expression patterns, including upregulation of lipogenic and pro-apoptotic genes and downregulation of IRS2, Bclxl and Pdx1. These lipotoxic effects of high glucose were largely prevented by induction of a dominant-negative mutant of SREBP-1c, suggesting SREBP-1c is a major factor responsible for β cell glucolipotoxicity. Moreover, overexpression of another lipogenic transcription factor, ChREBP, in INS-1 cells did not cause lipotoxicity. Intriguingly, chronic high glucose treatment in INS-1 cells led to pronounced induction of the ER stress marker genes, BIP and Chop10. Treatment of rat islets with both chronic high glucose and two ER stress inducers, thapsigargin and tunicamycin, enhanced SREBP-1 binding to the human IRS2 promoter. These results suggest that SREBP-1 activation caused by ER stress is implicated in β-cell glucolipotoxicity.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta‐cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP‐2 and cellubrevin, but not VAMP‐1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta‐cells contain secretory granules that store and secrete insulin as well as synaptic‐like microvesicles carrying gamma‐aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP‐2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP‐2 with insulin‐containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta‐cells. Pretreatment of streptolysin‐O permeabilized insulin‐secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP‐2 and cellubrevin and abolished Ca(2+)‐induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S‐stimulated insulin secretion. Taken together, our results show that pancreatic beta‐cells express VAMP‐2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)‐mediated insulin secretion.

Single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic Ca2+ and secretion.

It is generally believed that the initiation of insulin secretion by nutrient stimuli necessitates the generation of metabolic coupling factors, leading to membrane depolarization and the gating of voltage‐sensitive Ca2+ channels. To establish this sequence of events, the kinetics of endogenous fluorescence of reduced pyridine nucleotides [NAD(P)H], reflecting nutrient metabolism, were compared to those of cytosolic calcium ([Ca2+]i) rises in single cultured rat islet beta‐cells. In preliminary experiments, the loss of quinacrine fluorescence from prelabelled cells was used as an indicator of secretion. This dye is concentrated in the acidic insulin‐containing secretory granules. Both glucose and 2‐ketoisocaproate (KIC) raised [Ca2+]i in a dose‐dependent manner. There was marked cellular heterogeneity in the [Ca2+]i response patterns. The two nutrient stimuli also increased NAD(P)H fluorescence, again showing cell‐to‐cell variations. In combined experiments, where the two parameters were measured in the same cell, the elevation of the NAD(P)H fluorescence preceded the rise in [Ca2+]i, confirming the statistical evaluation performed on separate cells. The application of two consecutive glucose challenges revealed coordinated changes in [Ca2+]i and NAD(P)H fluorescence. Finally, quinacrine secretion was stimulated by two nutrients with onset times similar to those recorded for [Ca2+]i elevations. These results clearly demonstrate that increased metabolism occurs during the lag period preceding Ca2+ influx via voltage‐sensitive Ca2+ channels, a prerequisite for the triggering of insulin secretion by nutrient stimuli.

Vasopressin-induced von Willebrand factor secretion from endothelial cells involves V2 receptors and cAMP.

Vasopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrands disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.