Jonctions communicantes et sécrétion [Gap junctions and secretion]

The emergence of multicellular organisms has necessitated the development of mechanisms for interactions between adjacent and distant cells. A consistent feature of this network is the expression of gap junction channels between the secretory cells of all glands so far investigated in vertebrates. Here, we reviewed the distribution of the gap junctions proteins, named connexins, in a few mammalian glands, and discussed the recent evidence pointing to the participation of these proteins in the functioning of endocrine and exocrine cells. Specifically, available data indicate the importance of gap junctions for the proper control of glucose-induced insulin secretion. Understanding the functions of beta-cell connexins are crucial for the engineering of surrogate cells, which is necessary for implementation of a replacement cell therapy in diabetic patients.

The role of microRNAs in the development of type 2 diabets

Pancreatic ß cells are highly specialized endocrine cells located within the islets of Langerhans in the pancreas. Their main role is to produce and secrete insulin, the hormone essential for the regulation of glucose homeostasis and body's metabolism. Diabetes mellitus develops when the amount of insulin released by ß cells is not sufficient to cover the metabolic demand. In type 1 diabetes (5-10% of diagnoses) insulin deficiency is caused by the autoimmune destruction of pancreatic ß cells. Type 2 diabetes (90% of diagnoses) results from a genetic predisposition and from the presence of adverse environmental conditions. The combination of these factors reduces insulin sensitivity of peripheral target tissues, causes impairment in ß-cell function and can lead to partial loss of ß cells. The development of novel therapeutic strategies for the treatment of diabetes necessitates the comprehension of the cellular processes involved in dysfunction and loss of ß cells. My thesis was focused on the involvement in the physiopathological processes leading to the development of diabetes of a class of small regulatory RNA molecules, called microRNAs (miRNAs) that post- transcriptionally regulate gene expression. Global miRNA profiling in pancreatic islets of two animal models of diabetes, the db/db mice and mice that were fed a high fat diet (HFD), characterized by obesity and insulin resistance, led us to identify two groups of miRNAs displaying expression changes under pre-diabetic and diabetic conditions. Among the miRNAs already upregulated in pre-diabetic db/db mice and HFD mice, miR- 132 was found to have beneficial effects on pancreatic ß cell function and survival. Indeed, mimicking the upregulation of miR-132 in primary pancreatic islet cells and ß-cell lines improved glucose- induced insulin secretion and favored survival of the cells upon exposure to pro-apoptotic stimuli such as palmitate and cytokines. MiR-132 was found to exert its action by enhancing the expression of MafA, a transcription factor essential for ß-cell function, survival and identity. On the other hand, up-regulation of miR-199a-5p and miR-199a-3p was detectable only in the islets of diabetic db/db mice and resulted in impaired insulin secretion and sensitization of the cells to apoptosis. MiR-199a- 5p was found to decrease insulin secretion by inducing the expression of granuphilin, a potent inhibitor of ß cell exocytosis. In contrast, miR-199a-3p was demonstrated to directly target and reduce the expression of two key ß-cell genes, mTOR and cMET, resulting in impaired ß-cell adaptation to metabolic demands and loss by apoptosis. Our findings suggest that miRNAs are important players in the onset of type 2 diabetes. MiRNA expression is adjusted in pancreatic ß cells exposed to a diabetogenic environment. These changes initially concern miRNAs responsible for adaptive processes aimed at compensating the onset of insulin resistance, but later such changes can be overlapped by modifications in the level of several additional miRNAs that favor ß-cell failure and the onset of type 2 diabetes.

Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted beta-cells.

Either 200 or 400 syngeneic islets were transplanted under the kidney capsule of normal or streptozocin-induced diabetic B6/AF1 mice. The diabetic mice with 400 islets became normoglycemic, but those with 200 islets, an insufficient number, were still diabetic after the transplantation (Tx). Two weeks after Tx, GLUT2 expression in the islet grafts was evaluated by immunofluorescence and Western blots, and graft function was examined by perfusion of the graft-bearing kidney. Immunofluorescence for GLUT2 was dramatically reduced in the beta-cells of grafts with 200 islets exposed to hyperglycemia. However, it was plentiful in grafts with 400 islets in a normoglycemic environment. Densitometric analysis of Western blots on graft homogenates demonstrated that GLUT2 protein levels in the islets, when exposed to chronic hyperglycemia for 2 weeks, were decreased to 16% of those of normal recipients. Moreover, these grafts had defective glucose-induced insulin secretion, while the effects of arginine were preserved. We conclude that GLUT2 expression in normal beta-cells is promptly down-regulated during exposure to hyperglycemia and may contribute to the loss of glucose-induced secretion of diabetes.

Engineering tolerance into transplanted beta cell lines.

In this paper we explore the possibility of improving, by genetic engineering, the resistance of insulin-secreting cells to the metabolic and inflammatory stresses that are anticipated to limit their function and survival when encapsulated and transplanted in a type 1 diabetic environment. We show that transfer of the Bcl-2 antiapoptotic gene, and of genes specifically interfering with cytokine intracellular signaling pathways, greatly improves resistance of the cells to metabolic limitations and inflammatory stresses.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Inhibition of glucose-induced insulin secretion by long-term preexposure of pancreatic islets to leptin.

Here we evaluated the effect of leptin on glucose-induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of beta-cells. However, following preexposure to leptin a pronounced time- and dose-dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long-term preexposure to the hormone and which may involve alteration in beta-cell gene expression.

Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice.

Insulin controls glucose homeostasis by regulating glucose use in peripheral tissues, and its own production and secretion in pancreatic beta cells. These responses are largely mediated downstream of the insulin receptor substrates, IRS-1 and IRS-2 (refs 4-8), through distinct signalling pathways. Although a number of effectors of these pathways have been identified, their roles in mediating glucose homeostasis are poorly defined. Here we show that mice deficient for S6 kinase 1, an effector of the phosphatidylinositide-3-OH kinase signalling pathway, are hypoinsulinaemic and glucose intolerant. Whereas insulin resistance is not observed in isolated muscle, such mice exhibit a sharp reduction in glucose-induced insulin secretion and in pancreatic insulin content. This is not due to a lesion in glucose sensing or insulin production, but to a reduction in pancreatic endocrine mass, which is accounted for by a selective decrease in beta-cell size. The observed phenotype closely parallels those of preclinical type 2 diabetes mellitus, in which malnutrition-induced hypoinsulinaemia predisposes individuals to glucose intolerance.

Pancreatic islet adaptation to fasting is dependent on peroxisome proliferator-activated receptor alpha transcriptional up-regulation of fatty acid oxidation.

The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor-alpha (PPARalpha)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPARalpha null (PPARalphaKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPARalpha expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPARalpha expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARalphaKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPARalpha null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPARalpha, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPARalpha, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.

ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis.

The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic beta-cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP-raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA-dependent manner by chronic hyperglycemia and cAMP-raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of beta-cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMP-raising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic beta-cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes.

Glucose uptake, utilization, and signaling in GLUT2-null islets.

We previously reported that pancreatic islet beta-cells from GLUT2-null mice lost the first phase but preserved the second phase of glucose-stimulated insulin secretion (GSIS). Furthermore, we showed that the remaining secretory activity required glucose uptake and metabolism because it can be blocked by inhibition of oxidative phosphorylation. Here, we extend these previous studies by analyzing, in GLUT2-null islets, glucose transporter isoforms and glucokinase expression and by measuring glucose usage, GSIS, and glucose-stimulated insulin mRNA biosynthesis. We show that in the absence of GLUT2, no compensatory expression of either GLUT1 or GLUT3 is observed and that glucokinase is expressed at normal levels. Glucose usage by isolated islets was increased between 1 and 6 mmol/l glucose but was not further increased between 6 and 20 mmol/l glucose. Parallel GSIS measurements showed that insulin secretion was not stimulated between 2.8 and 6 mmol/l glucose but was increased by >4-fold between 6 and 20 mmol/l glucose. Stimulation by glucose of total protein and insulin biosynthesis was also markedly impaired in the absence of GLUT2. Finally, we re-expressed GLUT2 in GLUT2-null beta-cells using recombinant lentiviruses and demonstrated a restoration of normal GSIS. Together, these data show that in the absence of GLUT2, glucose can still be taken up by beta-cells, albeit at a low rate, and that this transport activity is unlikely to be attributed to GLUT1 or GLUT3. This uptake activity, however, is limiting for normal glucose utilization and signaling to secretion and translation. These data further demonstrate the key role of GLUT2 in murine beta-cells for glucose signaling to insulin secretion and biosynthesis.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.

cDNA cloning and mapping of a novel islet-brain/JNK-interacting protein.

IB1/JIP-1 is a scaffold protein that regulates the c-Jun NH(2)-terminal kinase (JNK) signaling pathway, which is activated by environmental stresses and/or by treatment with proinflammatory cytokines including IL-1beta and TNF-alpha. The JNKs play an essential role in many biological processes, including the maturation and differentiation of immune cells and the apoptosis of cell targets of the immune system. IB1 is expressed predominantly in brain and pancreatic beta-cells where it protects cells from proapoptotic programs. Recently, a mutation in the amino-terminus of IB1 was associated with diabetes. A novel isoform, IB2, was cloned and characterized. Overall, both IB1 and IB2 proteins share a very similar organization, with a JNK-binding domain, a Src homology 3 domain, a phosphotyrosine-interacting domain, and polyacidic and polyproline stretches located at similar positions. The IB2 gene (HGMW-approved symbol MAPK8IP2) maps to human chromosome 22q13 and contains 10 coding exons. Northern and RT-PCR analyses indicate that IB2 is expressed in brain and in pancreatic cells, including insulin-secreting cells. IB2 interacts with both JNK and the JNK-kinase MKK7. In addition, ectopic expression of the JNK-binding domain of IB2 decreases IL-1beta-induced pancreatic beta-cell death. These data establish IB2 as a novel scaffold protein that regulates the JNK signaling pathway in brain and pancreatic beta-cells and indicate that IB2 represents a novel candidate gene for diabetes.

Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

Cloning and functional expression of the human islet GLP-1 receptor. Demonstration that exendin-4 is an agonist and exendin-(9-39) an antagonist of the receptor.

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatic islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9-39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9-39) was an antagonist of the receptor, inhibiting GLP-1-induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.