Effects of balloon injury on neointimal hyperplasia in steptozotocin-induced diabetes and in hyperinsulinemic nondiabetic pancreatic islet-transplanted rats.

BACKGROUND: The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. METHODS AND RESULTS: Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras-negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. CONCLUSIONS: In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals

Insulin-secreting pituitary GH3 cells : a potential beta-cell surrogate for diabetes cell therapy

In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 7 106 InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic \u3b2TC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced \u3b2TC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential \u3b2-cell surrogate because they are more resistant than pancreatic \u3b2 cells to different apoptotic insults and might therefore be particularly suitable for encapsulation

Energy Expenditure Evaluation in Humans and Non-Human Primates by SenseWear Armband : Validation of Energy Expenditure Evaluation by SenseWear Armband by Direct Comparison with Indirect Calorimetry

Introduction:The purpose of this study was to compare and validate the use of SenseWear Armband (SWA) placed on the arm (SWA ARM) and on the back (SWA BACK) in healthy humans during resting and a cycle-ergometer exercise and to evaluate the SWA to estimate Resting Energy Expenditure (REE) and Total Energy Expenditure (TEE) in healthy baboons.Methods:We studied 26 (15F/11M) human subjects wearing SWA in two different anatomical sites (arm and back) during resting and a cycle-ergometer test and directly compared these results with indirect calorimetry evaluation (IC), performed at the same time. We then inserted the SWA in a metabolic jacket for baboons and evaluated the TEE and REE in free living condition for 6 days in 21 (8F/13M) non-human primates.Results:In humans we found a good correlation between SWA place on the ARM and on the BACK with IC during the resting experiment (1.1\ub10.3 SWAs, 1\ub10.2 IC kcal/min) and a slight underestimation in the SWAs data compared with IC during the cycle-ergometer exercise (5\ub11.9 SWA ARM, 4.5\ub11.5 SWA BACK and 5.4\ub12.1 IC kcal/min). In the non-human primate (baboons) experiment SWA estimated a TEE of 0.54\ub10.009 kcal/min during free living and a REE of 0.82\ub10.06 kcal/min.Conclusion:SWA, an extremely simple and inexpensive apparatus, provides quite accurate measurements of energy expenditure in humans and in baboons. Energy expenditure data obtained with SWA are highly correlated with the data obtained with "gold standard", IC, in humans. \ua9 2013 Casiraghi et al

Exenatide regulates pancreatic islet integrity and insulin sensitivity in the nonhuman primate baboon Papio hamadryas.

The glucagon-like peptide-1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β cell and α cell function and relative volumes, and islet cell apoptosis and replication in nondiabetic nonhuman primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an l-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated, and the remnant pancreas (head-body) was harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by about 2-fold. β, α, and δ cell relative volumes in exenatide-treated baboons were significantly increased compared with saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β, α, and δ cells and produces a robust increase in insulin sensitivity in nonhuman primates

Disproportionate Hyperproinsulinemia, \u3b2-Cell Restricted Prohormone Convertase 2 Deficiency, and Cell Cycle Inhibitors Expression by Human Islets Transplanted Into Athymic Nude Mice: Insights Into Nonimmune-Mediated Mechanisms of Delayed Islet Graft Failure

To learn more about nonimmune-mediated islet graft failure, we transplanted different preparations (preps) of isolated human islets under the kidney capsule of streptozotocin (STZ)-diabetic nude mice. One month after the implantation of 1,000 or 2,000 islets, grafts were harvested for morphological, immunohistochemical, and ultrastructural analysis. Only a single islet prep cured the diabetes out of all the recipients, while the remaining preps showed only partial function after the implantation of 2,000 islets. Transplanted mice showed high circulating proinsulin levels but, with the exclusion of those bearing curative grafts, relatively low mature insulin levels. Engrafted beta-cells showed positive carboxypeptidase E (CPE) and prohormone convertase 1 (PC1) staining, while prohormone convertase 2 (PC2) was undetectable. In contrast, PC2 was abundantly expressed by engrafted alpha-cells. Moreover, engrafted beta-cells did not show evidence of replication, and preapoptotic beta-cells, with intra- and extracellular amyloid deposition, were detected with electron microscopy. Cell cycle inhibitors p16(INK4), p21(WAF1), and p27(Kip1) were abundantly expressed in the islet grafts and showed a predominant nuclear localization. In conclusion, diabetic nude mice transplanted with human islets showed disproportionate hyperproinsulinemia and graft evidence of beta-cell restricted PC2 depletion, amyloid deposition and beta-cell death, and lack of beta-cell replication with nuclear translocation of p27(Kip1) and p21(WAF1) that together may contribute to delayed graft failur

The potential role of glutamate in the current diabetes epidemic

In the present article, we propose the perspective that abnormal glutamate homeostasis might contribute to diabetes pathogenesis. Previous reports and our recent data indicate that chronically high extracellular glutamate levels exert direct and indirect effects that might participate in the progressive loss of \u3b2-cells occurring in both T1D and T2D. In addition, abnormal glutamate homeostasis may impact all the three accelerators of the "accelerator hypothesis" and could partially explain the rising frequency of T1D and T2D

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

The glial glutanmate transporter 1 (GLT1) controls glutamate homeostasis and preserves beta-cell integrity in islet of Langerhans

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained under physiological concentrations by high affinity glutamate transporters of the SoLute Carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans, where it is secreted by the alpha-cells together with glucagon and acts as signalling molecule to modulate hormone secretion. Nevertheless, our knowledge regarding the effects of glutamate on islet cells\u2019 physiology is still incomplete. The aim of this work was to investigate whether glutamate also affect islet cells viability and hormone secretion. We demonstrated that chronic exposure to glutamate exerted a cytotoxic effect in clonal beta-cell lines and human islet beta-cells but not in alpha-cells. In human islets, glutamate-induced beta-cell cytotoxicity was associated to increased oxidative stress and led to apoptosis and autophagy. We also provided evidence that the key regulator of extracellular islet glutamate concentration was the glial glutamate transporter 1 (GLT1). GLT1 localized to the plasma membrane of beta-cells, modulated hormone secretion and prevented glutamate-induced cytotoxicity, as shown by the fact that its down-regulation induced beta-cell death while GLT1 up-regulation promoted beta-cell survival. In conclusion, glutamate homeostasis is critical to preserve islet cell signalling and beta-cell integrity and GLT1, by controlling extracellular glutamate levels, may play an important role in the pathogenesis of beta-cell and islet remodelling in diabetes

Modulation of glutamate signalling in human islets of Langerhans under hyperglycaemia

Background and aims: Increasing evidence suggests that the excitatory neurotransmitter L-glutamate functions as a paracrine/autocrine signal in human islet of Langerhans. L-glutamate is released by \u3b1-cells together with glucagon and modulates hormone secretion by acting on specific glutamate receptors. When present at elevated concentrations, it may induce beta-cell cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its interstitial concentration is regulated by glutamate transporters of the SLC1A family which are expressed on the plasma membrane of endocrine cells. Their functional activity is essential for islet function as shown by the fact that their pharmacological inhibition increases glutamate concentration in the islets and causes beta-cell death. Aim of this study was to verify whether chronic hyperglycaemia may modulate the glutamate signalling system in human islets of Langerhans. Materials and methods: Human islets were incubated under chronic (3 days) hyperglycaemia (16.7 mmol/l glucose) or normoglycemia (5.5 mmoll glucose), and the expression and function of plasma membrane glutamate transporters and intracellular signalling proteins were studied by quantitative PCR analysis, western blotting, immunofluorescence, [3H]-Glutamate uptake and Ca2+-imaging experiments. Results: Quantitative PCR analysis revealed a 40\ub13% reduction in the total ASCT2/SLC1A5 expression after incubation in chronic hyperglycaemia. No changes in the total GLT1/SLC1A2 mRNA and protein expression in human islets were detected. Immunofluorescence experiments performed on human islets exposed to hyperglycaemia revealed GLT1 relocalization into intracellular vesicular compartments. Because of this relocalization, the GLT1-mediated surface activity measured by [3H]D-glutamate uptake was inhibited by 31\ub15% relative to normoglycemic conditions (p<0.05; n=4 in triplicate). Chronic hyperglycaemia induced a downregulation of the PI3K/Akt pathway in human beta-cells, suggesting a possible involvement of this pathway in the modulation of GLT-1 trafficking (35\ub13 % downregulation of P-Akt expression, n=5 islet preparations). In line with this possibility, PI3K inhibition with 100 \uf06dM LY293 caused the GLT1 relocalization in intracellular compartments, and a 75\ub18% downregulation of its activity, relative to control conditions (p<0.001; n=2 different islet isolations, in quadruplicate). Chronic treatments with 10 nM ceftriaxone a drug capable to up-regulate GLT1 significantly prevented hyperglycaemia-induced apoptosis in human islets (65\ub112% reduction of apoptosis. P<0.001; n=2 preparations, in quadruplicate). Conclusions: Our data indicate that glutamate signalling in human islet is altered in hyperglycaemia and this may further contribute to beta-cell death. Targeting glutamate signalling system components may be a promising approach to prevent beta-cell death and to control glucose homeostasis in diabetes

Secretory defects induced by immunosuppressive agents on human pancreatic &#946;-cells

Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human beta-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (delta-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human beta-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic beta-cell point of view