The physiological role of soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in insulin exocytosis has been reported in pancreatic beta-cells. To determine whether the beta-cells of GK rats, a nonobese rodent model of type 2 diabetes, exhibit abnormalities in their SNARE proteins, we studied the expression and function of target (t)-SNAREs, syntaxin 1A, and synaptosomal-associated protein of 25 kDa (SNAP-25) in GK rat islets. Although insulin release and insulin content of islets isolated from 12-week-old GK rats were reduced, the proinsulin biosynthetic rate was about twofold higher than that in control rat islets, and no change in the preproinsulin mRNA level was observed. Pulse-chase experiments suggested the increased degradation of insulin in GK rat islets. Immunoblot analysis revealed that protein levels of syntaxin 1A and SNAP-25 in GK rat islets decreased to approximately 60% of the levels in control rat islets. We then examined whether the restoration of the decreased expression of t-SNAREs to the normal level in GK rat islets affected insulin secretion. Restoration was achieved by the overexpression of syntaxin 1A and SNAP-25 via the recombinant adenovirus-mediated gene transduction system, which recovered levels of these proteins to almost control levels. Glucose-stimulated insulin release from AdexlCA syntaxin 1A and Adex1CA SNAP-25-infected GK rat islets increased up to approximately 135 and 200%, respectively, of those from uninfected GK rat islets, although no difference in basal (2.2 mmol/l glucose) insulin release was evident between them. We conclude that decreased expression of t-SNAREs in GK rat islets is in part the defect responsible for impaired insulin secretion.

No abstract

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P Ͻ 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin-and AMPKactivated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P Ͻ 0.05). AMPK-and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P Ͻ 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P Ͻ 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.glucose transporter 4; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; enhanced green fluorescent protein IT HAS BEEN ESTABLISHED that insulin-stimulated glucose uptake into adipocytes and skeletal myocytes involves the translocation of GLUT4 from an intracellular pool to the plasma membrane. The intracellular mechanism for the recruitment of GLUT4-containing vesicle into plasma membrane has been investigated, and it has been revealed that phosphatidylinositol 3-kinase (PI3K) plays a crucial role in insulin-stimulated GLUT4 translocation (7,14,24). However, little has been elucidated concerning other mechanisms to enhance the GLUT4 translocation than the insulin signaling system. AMP-activated protein kinases (AMPKs) have been known to act as a metabolic sensor in mammalian cells (9,30). The kinase activity is enhanced by a relative increase in cellular AMP level (increase in AMP-to-ATP ratio) through a metabolic uncoupler, dinitrophenol (DNP), to decrease ATP concentration and by 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR), an adenosine analog. After entering into cells, AICAR is phosphorylated, and ...

No abstract

We investigated whether adenovirus-mediated preproinsulin gene transfer into insulin target tissues (adipocytes) ameliorates hyperglycemia in diabetic mice. KKA y 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 KKA y 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. ß