Hitoshi Ishida

Adenovirus-mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKAy mice

We investigated whether adenovirus‐mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKAy mice, a genetically obese type 2 diabetic animal model, were treated with a single subcutaneous injection of recombinant adenovirus, Adex1CA‐human preproinsulin (Adex1CA‐pchi), into the epididymal fat pads. pchi mRNA was expressed only in adipose tissue in which mature insulin was produced. Three days after virus injection these mice showed a marked decrease of blood glucose levels (from about 400 to 200 mg/dl), and an intraperitoneal glucose tolerance test revealed the markedly improved glucose tolerance. There was no significant difference in serum insulin levels between control and recombinant adenovirus‐treated KKAy mice. The normalized glucose levels in diabetic mice were maintained for at least 2 weeks after the virus injection. This strategy could provide a novel and, most importantly, a simple and convenient gene therapy for obese type 2 diabetes patients.

PPAR-γ overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions; however, its physiological role in pancreatic beta cell functions remains to be determined. In the present study, we investigated the synergistic effect of PPAR-gamma and its agonist, pioglitazone, on proinsulin biosynthesis and insulin release in a glucose-responsible insulinoma cell line, MIN6 cells. Expression of PPAR-gamma in MIN6 cells was not detectable by RT-PCR and immunoblot analysis. When PPAR-gamma-1 was overexpressed adenovirally in MIN6 cells, glucose-stimulated proinsulin biosynthesis and insulin release were inhibited. Pioglitazone treatment alone had no effects on these parameters of beta cell function in control MIN6 cells, although pioglitazone synergistically augmented the inhibitory effect of PPAR-gamma on proinsulin biosynthesis and insulin release under the condition of PPAR-gamma overexpression. Our results demonstrate that PPAR-gamma plays a negative role in pancreatic beta cells.

Pancreatic Endocrine Function and Glucose Transporter (GLUT)-2 Expression in Rat Acute Pancreatitis

Introduction Impairment in pancreatic endocrine function is believed to play an important role in the development of glucose intolerance in acute pancreatitis. Aim To investigate the functional aspects of endocrine cells in acute pancreatitis and the expression of glucose transporter (GLUT) 2 in the pancreatitis islet. Methodology A mild form of acute pancreatitis was induced in rats by an injection of a sodium taurocholate solution via a cannulated biliopancreatic duct. Isolated islets were stimulated by glucose, and insulin secretion was analyzed by radioimmunoassay. Immunohistochemical detection of GLUT2 with use of a specific antibody was attempted to determine GLUT2 expression in pancreatic islets. Results A marked elevation of glucose levels observed in the current rat pancreatitis model confirmed that glucose intolerance can occur even in a mild form of pancreatitis. The architecture of the islets, however, remained intact despite marked inflammatory changes in the neighboring exocrine region. Insulin secretion studies revealed that the ability of islets to secrete insulin in response to glucose was markedly reduced in pancreatitis islets. GLUT2 immunoreactivity in endocrine cells was found to be intact in pancreatitis islets. Conclusion The amount of insulin released from isolated islets following glucose stimulation is reduced in acute edematous pancreatitis, although pancreatic islets remain histologically intact. On the basis of the present findings, it appears that although the mechanisms responsible for this functional deficiency remain to be determined, the decrease in insulin secretion is possibly caused by impairment of some pancreatic B-cell functions rather than GLUT2-mediated glucose transportation.

An insulin-related peptide expressed in 3T3L1 adipocytes is localized in GLUT4 vesicles and secreted in response to exogenous insulin, which augments the insulin-stimulated glucose uptake

If an adipocyte is programmed to secrete insulin, then the insulin released may amplify the insulin action by an autocrine manner. To examine this hypothesis in vitro, we investigated the effects of expressing the preproinsulin gene in 3T3L1 adipocytes on (pro)insulin release and glucose uptake. The human preproinsulin gene was transferred into 3T3L1 adipocytes by infecting the cells with recombinant adenovirus Adex1CA human preproinsulin. Immunocytochemical studies showed that (pro)insulin is associated with vesicular structures that colocalize with GLUT4 vesicles but not with GLUT1 vesicles. We then examined insulin-induced proinsulin release from 3T3L1 adipocytes expressing the insulin gene. The exogenously administered insulin stimulated proinsulin release from these cells in a dose-dependent manner. HPLC determination revealed the existence of mature human insulin in these cells, which suggested the release of mature insulin into the medium. Further we monitored the (pro)insulin release from these cells with confocal laser microscopy using the expression of a fusion protein between insulin and green fluorescent protein (GFP). Time-lapse confocal laser-scanning microscopy revealed that the total number of vesicles containing insulin-GFP was decreased by the addition of 10-7 M insulin within 1 minute. Finally, we examined the insulin-stimulated glucose uptake by these cells. The data showed that insulin-stimulated glucose uptake increased to about 150% of that of control cells in response to exogenously administered insulin, indicating that the insulin released augmented the insulin-stimulated glucose uptake in an autocrine manner. Thus, the data support our hypothesis, indicating that we could construct the insulin-regulated insulin release system in adipocytes by introducing the preproinsulin gene.