PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic ß-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic ß-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

Endogenous hydrogen sulfide protects pancreatic beta-cells from a high-fat diet-induced glucotoxicity and prevents the development of type 2 diabetes.

Chronic exposure to high glucose induces the expression of cystathionine gamma-lyase (CSE), a hydrogen sulfide-producing enzyme, in pancreatic beta-cells, thereby suppressing apoptosis. The aim of this study was to examine the effects of hydrogen sulfide on the onset and development of type 2 diabetes. Middle-aged (6-month-old) wild-type (WT) and CSE knockout (CSE-KO) mice were fed a high-fat diet (HFD) for 8weeks. We determined the effects of CSE knockout on beta-cell function and mass in islets from these mice. We also analyzed changes in gene expression in the islets. After 8weeks of HFD, blood glucose levels were markedly increased in middle-aged CSE-KO mice, insulin responses were significantly reduced, and DNA fragmentation of the islet cells was increased. Moreover, expression of thioredoxin binding protein-2 (TBP-2, also known as Txnip) was increased. Administration of NaHS, a hydrogen sulfide donor, reduced TBP-2 gene levels in isolated islets from CSE-KO mice. Gene levels were elevated when islets were treated with the CSE inhibitor dl-propargylglycine (PPG). These results provide evidence that CSE-produced hydrogen sulfide protects beta-cells from glucotoxicity via regulation of TBP-2 expression levels and thus prevents the onset/development of type 2 diabetes.

Activated Cdc42-bound IQGAP1 determines the cellular endocytic site.

Recruitment of specific molecules to a specific membrane site is essential for communication between specialized membranous organelles. In the present study, we identified IQGAP1 as a novel GDP-bound-Rab27a-interacting protein. We found that IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42. We also found that IQGAP1 regulates the endocytosis of insulin secretory membranes. Silencing of IQGAP1 inhibits both endocytosis and the glucose-induced redistribution of endocytic machinery, including Rab27a and its binding protein coronin 3. These processes can also be inhibited by disruption of the trimeric complex with dominant negative IQGAP1 and Cdc42. These results indicate that activation of Cdc42 in response to the insulin secretagogue glucose recruits endocytic machinery to IQGAP1 at the cell periphery and regulates endocytosis at this membrane site.

Distribution of hydrogen sulfide (H2S)-producing enzymes and the roles of the H2S donor sodium hydrosulfide in diabetic nephropathy.

BACKGROUND: Hydrogen sulfide (H(2)S) has recently been found to play beneficial roles in ameliorating several diseases, including hypertension, atherosclerosis and cardiac/renal ischemia-reperfusion injuries. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), the main enzymes in the transsulfuration pathway, catalyze H(2)S production in mammalian tissues. However, the distributions and precise roles of these enzymes in the kidney have not yet been identified. METHODS: The present study examined the localization of both enzymes in the normal kidney and the effect of the H(2)S donor sodium hydrosulfide (NaHS) in the renal peritubular capillary (PTC) under conditions of diabetic nephropathy, using pancreatic ß-cell-specific calmodulin-overexpressing transgenic mice as a model of diabetes. RESULTS: In the normal kidney, we detected expression of both CBS and CSE in the brush border and cytoplasm of the proximal tubules, but not in the glomeruli, distal tubules and vascular endothelial cells of renal PTCs. Administration of NaHS increased PTC diameter and blood flow. We further evaluated whether biosynthesis of H(2)S was altered in a spontaneous diabetic model that developed renal lesions similar to human diabetic nephropathy. CSE expression was markedly reduced under diabetic conditions, whereas CBS expression was unaffected. Progressive diabetic nephropathy showed vasoconstriction and a loss of blood flow in PTCs that was ameliorated by NaHS treatment. CONCLUSION: These findings suggest that CSE expression in the proximal tubules may also regulate tubulointerstitial microcirculation via H(2)S production. H(2)S may represent a target of treatment to prevent progression of ischemic injury in diabetic nephropathy.

Protein phosphorylation involved in the gene expression of the hydrogen sulphide producing enzyme cystathionine γ-lyase in the pancreatic ß-cell.

Cystathionine γ-lyase (CSE) is one of the major enzymes for the production of hydrogen sulphide (H(2)S), a multifunctional gasotransmitter in the pancreatic ß-cell. We examined the mechanisms by which glucose induces CSE expression in mouse pancreatic islets and the insulin-secreting cell line MIN6. CSE expression was increased by anti-diabetic sulphonylureas, and decreased by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+) channel blocker nitrendipine. Application of the synthetic inhibitors of protein kinases revealed the involvement of Ca(2+)/calmodulin-dependent protein kinase (CaMK) II and extracellular signal-regulated protein kinase (ERK) in glucose- and thapsigargin-induced CSE expression. The CaMK IIδ knockdown also suppressed CSE expression. Knockdown of the transcription factors Sp1 and Elk1, both of which can be phosphorylated by ERK, blunted CSE expression. By a reporter assay, we found Sp1 may directly and Elk1 may indirectly regulate CSE expression. These findings suggest Ca(2+)-dependent CSE expression may be mediated via protein phosphorylation of Sp1 and Elk1 in pancreatic ß-cells.

Rab27a in pancreatic beta-cells, a busy protein in membrane trafficking.

The small GTPases have the 'active' GTP-bound and 'inactive' GDP-bound states, and thereby act as a molecular switch in cells. Rab27a is a member of this family and exists in T-lymphocytes, melanocytes and pancreatic beta-cells. Rab27a regulates secretion of cytolytic granules from cytotoxic T-lymphocytes and intracellular transport of melanosomes in melanocytes. In pancreatic beta-cells, Rab27a controls pre-exocytotic stages of insulin secretion. A few GTP-dependent Rab27a effectors are known to mediate these cellular functions. We recently found that Rab27a also possesses the GDP-dependent effector coronin 3. Coronin 3 regulates endocytosis in pancreatic beta-cells through its interaction with GDP-Rab27a. These results imply that GTP- and GDP-Rab27a actively regulate distinct stages in the insulin secretory pathway. In this review, we provide an overview of the roles of both GTP- and GDP-Rab27a in pancreatic beta-cells.

Significance of hydrogen sulfide production in the pancreatic ß-cell.

Hydrogen sulfide (H(2)S) is an important signaling molecule in various mammalian cells and tissues. H(2)S is synthesized from L-cysteine and regulates several cellular and physiological phenomena (vasorelaxation, hormone secretion, and apoptosis) and multicellular events (neuromodulation and inflammatory responses). H(2)S can be produced in pancreatic ß-cells by cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE). H(2)S inhibits insulin release and regulates ß-cell survival. We found that glucose stimulation increased CSE expression at transcript and protein levels in mouse pancreatic islets. We also found that H(2)S protects ß-cells that were chronically exposed to high glucose from apoptotic cell death. Loss of ß-cell mass and failures of ß-cell function are important in the pathogenesis and/or progression of diabetes mellitus; therefore, molecular analyses of the mechanisms of H(2)S production and its protective effects on ß-cells may lead to new insights into diabetes mellitus.

Rab27a, actin and beta-cell endocytosis.

The output and time-course of insulin release from pancreatic beta-cells are elegantly controlled. The secretory process comprises pre-exocytotic stages, exocytosis and post-exocytotic stages. The small GTPase Rab27a is known to regulate pre-exocytotic stages that determine the size of the readily-releasable pool of insulin granules. GTP-Rab27a and its specific effectors are responsible for this process like other GTPases. Recently, we searched for Rab27a-interacting proteins and identified coronin 3. Unexpectedly, coronin 3 only bound GDP-Rab27a and this interaction regulated post-exocytotic stages via reorganization of the actin cytoskeleton. Since glucose converts Rab27a from the GTP- to GDP-bound form, we suggested that Rab27a plays a crucial role in stimulus-endocytosis coupling in pancreatic beta-cells, and that this is the key molecule for membrane recycling of insulin granules. In this review, we provide an overview of the roles of Rab27a and its GTP- and GDP-dependent effectors in the insulin secretory pathway of pancreatic beta-cells.

Glucose-induced translocation of coronin 3 regulates the retrograde transport of the secretory membrane in the pancreatic beta-cells.

GTP-Rab27a is known to regulate insulin exocytosis. We have recently reported that coronin 3, which paradoxically binds GDP-Rab27a, participates in endocytosis of the insulin secretory membrane. Here, we demonstrate that glucose stimulation caused redistribution of coronin 3 in the vicinity of the plasma membrane, which was mimicked by overexpression of the GDP-Rab27a mutant or the Rab27a GAP. Glucose-induced translocation of coronin 3 was inhibited by Rab27a knock-down. The internalized phogrin, an insulin granule associated protein, located near the plasma membrane by the dominant-negative coronin 3, but the protein at the outer surface of the plasma membrane was decreased. These results indicate that glucose recruits coronin 3 near the plasma membrane, and that it regulates the retrograde transport of the secretory membrane in pancreatic beta-cells.

Actin assembly controlled by GDP-Rab27a is essential for endocytosis of the insulin secretory membrane.

We have recently reported that GDP-bound Rab27a regulates endocytosis of the insulin secretory membrane via its binding to coronin 3, an actin-binding protein. The aim of this study was to examine the participation of actin assembly in the Rab27a-dependent regulation of endocytosis using a pancreatic beta cell line, MIN6. Coronin 3 promoted F-actin bundling only in the presence of GDP-Rab27a. This effect was independent of coronin-3-binding to the actin-related proteins 2 and 3 (Arp2/3). Uptake of anti-phogrin-lumen antibody into MIN6 was inhibited by anti-coronin-3-C antibody which recognizes the actin-binding site. This inhibition was also observed with coronin-3-R28D, which lacks in actin binding. These results suggest that coronin 3 is a genuine GDP-Rab27a effector, and that controls endocytosis of the secretory membrane via modulating actin assembly in pancreatic beta-cells.

Establishment and characterization of a novel method for evaluating gluconeogenesis using hepatic cell lines, H4IIE and HepG2.

The liver gluconeogenic pathway is recognized as a target for treating diabetes mellitus. In this study, we attempted to establish a new method to evaluate gluconeogenesis using rat H4IIE hepatoma cells. High-density preculture and exposure to hypertonic solutions, which are known to upregulate the expression of gluconeogenic genes, enhanced glucose release (GR) promoted by gluconeogenic substrates (GS: 1mM pyruvate and 10mM lactate). Our method was also applicable to the human hepatoma HepG2 cells. Measurement of glycogen content in HepG2 cells revealed that GR was compensated by glycogenolysis in the basal state and was generated by gluconeogenesis in the presence of GS. The optimized conditions increased the expression of gluconeogenic genes in HepG2 cells. Insulin and metformin dose-dependently inhibited GR and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) increased it. These results suggest that the present method is useful to evaluate the effects of nutrients, hormones and hypoglycemic agents on hepatic gluconeogenesis.

Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose.

We examined the expression of the major H(2)S-producing enzymes, cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). CBS was ubiquitously distributed in the mouse pancreas, but CSE was found only in the exocrine. Freshly isolated islets expressed CBS, while CSE was faint. However, high glucose increased the CSE expression in the beta-cells. L-Cysteine or NaHS suppressed islet cell apoptosis with high glucose, and increased glutathione content in MIN6 beta-cells. Pretreatment with L-cysteine improved the secretory responsiveness following stimulation with glucose. The CSE inhibitor DL-propargylglycine antagonized these L-cysteine effects. We suggest H(2)S may function as an 'intrinsic brake' which protects beta-cells from glucotoxicity.

The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines.

Rab27a is involved in the control of membrane traffic, a crucial step in the regulated secretion. Typically, the guanosine triphosphate (GTP)-bound form has been considered to be active and, therefore, searching for proteins binding to the GTP-form has been attempted to look for their effectors. Here, we have identified the actin-bundling protein coronin 3 as a novel Rab27a effector that paradoxically bound guanosine diphosphate (GDP)-Rab27a in the pancreatic beta-cell line MIN6. Coronin 3 directly bound GDP-Rab27a through its beta-propeller structure. The most important insulin secretagogue glucose promptly shifted Rab27a from the GTP- to GDP-bound form. Knockdown of coronin 3 by RNAi resulted in the inhibition of phogrin (an insulin-granule-associated protein) internalization and the uptake of FM4-64 (a marker of endocytosis). Similar results were reproduced by disruption of the coronin-3-GDP-Rab27a interaction with the dominant-negative coronin 3, and coexpression of the GDP-Rab27a mutant rescued these changes. Taken together, our results indicate that interaction of GDP-Rab27a and coronin 3 is important in stimulus-endocytosis coupling, and that GTP- and GDP-Rab27a regulates insulin membrane recycling at the distinct stages.

Radioimmunoscintigraphy of pancreatic cancer in tumor-bearing athymic nude mice using (99m)technetium-labeled anti-KL-6/MUC1 antibody.

PURPOSE: KL-6 is an extracellular epitope of MUC1, a membrane-penetrating glycoprotein, and its overexpression has been reported in pancreatic cancer. The aim of this study was to examine whether radiolabeled anti-KL-6/MUC1 antibody could be used for molecular imaging of pancreatic cancer in vivo. MATERIALS AND METHODS: Anti-KL-6/MUC1 antibody was labeled with 99mTC by the stannous reduction method. Immunoreactivity of the 99mTc-labeled anti-KL-6/MUC1 antibody was evaluated by a whole-cell binding study. In vivo experiments were performed by injecting the 99mTc-labeled anti-KL-6/MUC1 antibody into athymic nude mice bearing the KP-1NL pancreatic cancer cell line. RESULTS: A whole-cell binding study showed that the radiolabeled antibody retained its immunoreactivity. On scintigrams, the density of the tumors remained unchanged during the 16-32 h after injection, whereas that of the kidneys decreased time-dependently. The radioactivity levels of the kidneys and tumors were measured densitometrically, and we found that the intensity in the tumors relative to that in the kidneys increased time-dependently. Radioactivity levels were the highest in the blood 32 h after injection, and those in the liver, kidney, lung, and tumor were also rather high. CONCLUSION: 99mTc-labeled anti-KL-6/MUC1 antibody appears to be a promising agent as a tumor-specific radiotracer for pancreatic cancer.

Overexpression of calmodulin in pancreatic beta cells induces diabetic nephropathy.

Recently, endothelial dysfunction induced by an uncoupling of vascular endothelial growth factor (VEGF) and nitric oxide has been implicated in the pathogenesis of diabetic nephropathy (DN). Investigating the pathogenesis of DN has been limited, however, because of the lack of animal models that mimic the human disease. In this report, pancreatic beta cell-specific calmodulin-overexpressing transgenic (CaMTg) mice, a potential new model of DN, are characterized with particular emphasis on VEGF and related molecules. CaMTg mice developed hyperglycemia at 3 wk and persistent proteinuria by 3 mo. Morphometric analysis showed considerable increases in the glomerular and mesangial areas with deposition of type IV collagen. Moreover, the pathologic hallmarks of human DN (mesangiolysis, Kimmelstiel-Wilson-like nodular lesions, exudative lesions, and hyalinosis of afferent and efferent arteries with neovascularization) were observed. In addition, increased VEGF expression was associated with an increased number of peritubular capillaries. Expression of endothelial nitric oxidase synthase was reduced and that of VEGF was markedly elevated in CaMTg mice kidney compared with nontransgenic mice. No differences in VEGF receptor-1 or VEGF receptor-2 expression were observed between CaMTg mice and nontransgenic kidneys. In summary, CaMTg mice develop most of the distinguishing lesions of human DN, and the elevated VEGF expression in the setting of diminished endothelial nitric oxide synthase expression may lead to endothelial proliferation and dysfunction. This model may prove useful in the study of the pathogenesis and treatment of DN.

Protection of pancreatic beta-cells by exendin-4 may involve the reduction of endoplasmic reticulum stress; in vivo and in vitro studies.

The aim of this study was to investigate the in vivo and in vitro effects of exendin-4, a potent glucagon-like peptide 1 agonist, on the protection of the pancreatic beta-cells against their cell death. In in vivo experiments, we used beta-cell-specific calmodulin-overexpressing mice where massive apoptosis takes place in their beta-cells, and we examined the effects of chronic treatment with exendin-4. Chronic and s.c. administration of exendin-4 reduced hyperglycemia. The treatment caused significant increases of the insulin contents of the pancreas and islets, and retained the insulin-positive area. Dispersed transgenic islet cells lived only shortly, and several endoplasmic reticulum (ER) stress-related molecules such as immunoglobulin-binding protein (Bip), inositol-requiring enzyme-1alpha, X-box-binding protein-1 (XBP-1), RNA-activated protein kinase-like endoplasmic reticulum kinase, activating transcription factor-4, and C/EBP-homologous protein (CHOP) were more expressed in the transgenic islets. We also found that the spliced form of XBP-1, a marker of ER stress, was also increased in beta-cell-specific calmodulin-overexpressing transgenic islets. In the quantitative real-time PCR analyses, the expression levels of Bip and CHOP were reduced in the islets from the transgenic mice treated with exendin-4. These findings suggest that excess of ER stress occurs in the transgenic beta-cells, and the suppression of ER stress and resultant protection against cell death may be involved in the anti-diabetic effects of exendin-4.

L-cysteine inhibits insulin release from the pancreatic beta-cell: possible involvement of metabolic production of hydrogen sulfide, a novel gasotransmitter.

Hydrogen sulfide (H(2)S) was historically recognized as a toxic gas generated by natural resources. However, its enzymatic production from L-cysteine has recently been demonstrated in mammals. Cystathionine beta-synthase and cystathionine gamma-lyase, both of which can produce H(2)S, were expressed in mouse pancreatic islet cells and the beta-cell line, MIN6. L-cysteine and the H(2)S donor NaHS inhibited glucose-induced insulin release from islets and MIN6 cells. These inhibitory effects were reproduced when insulin release was stimulated by alpha-ketoisocaproate, tolbutamide, or high K+. L-cysteine and NaHS inhibited glucose-potentiated insulin release in the copresence of diazoxide and high K+. Real-time imaging of intracellular Ca2+ concentration ([Ca2+](i)) demonstrated that both L-cysteine and NaHS reversibly suppressed glucose-induced [Ca2+](i) oscillation in a single beta-cell without obvious changes in the mean value. These substances inhibited Ca2+ - or guanosine 5'-0-3-thiotriphosphate-induced insulin release from islets permeabilized with streptolysin-O. L-cysteine and NaHS reduced ATP production and attenuated glucose-induced hyperpolarization of the mitochondrial membrane potential. Finally, L-cysteine increased H(2)S content in MIN6 cells. We suggest here that L-cysteine inhibits insulin release via multiple actions on the insulin secretory process through H(2)S production. Because the activities of H(2)S-producing enzymes and the tissue H(2)S contents are known to increase under diabetic conditions, the inhibition may participate in the deterioration of insulin release in this disease.

The HND mouse, a nonobese model of type 2 diabetes mellitus with impaired insulin secretion.

OBJECTIVES: This study aimed to develop a novel type 2 diabetes model designated the HND (Horio-Niki diabetic) mouse, by transferring diabetogenic genes from wild castaneus mice (Mus musculus castaneus) captured in the Philippines into laboratory mice (C57BL/6J:B6). METHODS: Offspring from the cross between a wild male and a B6 female were backcrossed to the sire. One male backcross which exhibited fasting hyperglycemia was crossed with a B6 female to comprise the fundamental stock (F0). Thereafter, full-sib mating was performed, and mice with impaired glucose tolerance were selected and bred from the F2 generation. Characterization of the phenotype of HND mice and insulin release from their islets was evaluated with F12 generation males. RESULTS: The male HND mice were lean, and spontaneously exhibited impaired glucose tolerance at a high incidence rate at 6 weeks of age. Their serum insulin levels in response to intraperitoneal glucose were markedly attenuated. However, glucose-induced insulin release from isolated HND islets was not affected. Notably, inhibition of glucose-induced insulin release by epinephrine was more pronounced in HND islets than in B6 islets. Moreover, in vivo treatment of HND mice with the alpha2-adrenergic receptor agonist clonidine resulted in marked hypoinsulinemic hyperglycemia. CONCLUSIONS: We suggest the HND mouse may be a distinctive and useful model for type 2 diabetes with impaired neural control of insulin secretion.