K_ATPchannel-deficient pancreatic β-cells are streptozotocin resistant because of lower GLUT2 activity

Abstract

Ϫ/Ϫ isolated pancreatic islets also transported less glucose than wild-type ones. Quantification of glucose transporter 2 (GLUT2) protein content by Western blot using an antibody with an epitope in the extracellular loop showed no significant difference in GLUT2 content between wild-type and Kir6.2 Ϫ/Ϫ pancreatic islets. However, visualization by immunofluorescence with the same antibody gave rise to 32% less fluorescence in Kir6.2 Ϫ/Ϫ pancreatic islets. The fluorescence intensity using another antibody, with an epitope in the COOH terminus, was 5.6 times less in Kir6.2 Ϫ/Ϫ than in wild-type pancreatic islets. We conclude that 1) Kir6.2 Ϫ/Ϫ mice are STZ resistant because of a decrease in STZ transport by GLUT2 in pancreatic ␤-cells and 2) the decreased transport is due to a downregulation of GLUT2 activity involving an effect at the COOH terminus. knockout; insulin; liver; glucose transporter; glucose tolerance; diabetic model GLUCOSE-STIMULATED INSULIN secretion in pancreatic ␤-cells comprises an ATP-sensitive K ϩ channel (K ATP channel)-dependent and -independent pathway (14, 21). For the K ATP channel-dependent mechanism, glucose-stimulated insulin secretion first involves the transport of glucose into the cytosol by glucose transporter 2 (GLUT2). As glucose is metabolized, the intracellular ATP concentration increases, and that of ADP decreases. The changes in ATP and ADP cause the closing of the K ATP channels, which results in the depolarization of the cell membrane. Voltage-dependent calcium channels are then activated, allowing for a calcium influx. Finally, calcium triggers the exocytosis of insulin-containing granules (20, 21).In pancreatic ␤-cells, the K ATP channel is made up of four Kir6.2 subunits, which make up the pore of the channel, and four SUR1 subunits, which are regulatory subunits (6, 21). Homozygous null mice for the Kir6.2 gene (Kir6.2 Ϫ/Ϫ mice) have no K ATP channel activity in several tissues, including pancreatic ␤-cells (14). These mice have normal plasma glucose and insulin levels during prolonged fasting and secrete insulin following a mixed meal (21) but not during hyperglycemia induced with parenteral glucose administration (14). Despite the latter deficiency, Kir6.2 Ϫ/Ϫ mice only have a mild glucose intolerance: they are only slightly more hyperglycemic than wild-type mice following an intraperitoneal glucose injection (14). It therefore appears that Kir6.2 Ϫ/Ϫ mice develop compensatory mechanisms allowing them to be less dependent on insulin to prevent hyperglycemia. One of these mechanisms may involve greater basal and insulin-induced glucose uptake by adipose tissues and skeletal muscles (13).Streptozotocin (STZ) is a glucosamine-nitrosourea compound that shows selective cytotoxicity to pancreatic ␤-cells and is widely used to generate diabetic animal models (2, 7, 10). STZ is believed to cause DNA alkylation (4), leading to the activation of poly(ADP-ribose) polymerase for DNA repair (1,3,16). This process consumes an excessive amount of NAD ϩ (1, 3). It has been suggested ...

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

“…STZ is reported to increase oxidative stress (39,40), and the upregulation of antioxidant enzymes could account for the observed resistance. Reduced sensitivity to STZ has been reported for β cells from Kir6.2 -/-mice, which lack K ATP channels (62). The effect is attributed to reduced Glut2 activity, expected to limit the rate of STZ uptake into β cells.…”

“…The effect is attributed to reduced Glut2 activity, expected to limit the rate of STZ uptake into β cells. However, maximal plasma STZ concentration was significantly lower in Kir6.2 -/-mice than in WT mice (62). Therefore, reduced tissue accumulation could also be explained by the differential STZ plasma concentrations of the 2 genotypes rather than the lower Glut2 activity of the knockout.…”

Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus

“…Based on several lines of evidence, streptozotocin toxicity toward the ␤-cells depends on the expression of GLUT2. Human islet cells, vs. those of rat or mouse, were resistant to streptozotocin toxicity due to the differential profiles of glucose transporter expression (7); insulinoma cells expressing GLUT2 had increased levels of streptozotocin uptake and susceptibility to streptozotocin-induced destruction in vivo (45); complete GLUT2 deficiency caused the rare Fanconi-Bickel syndrome of kidney syndrome, hepatomegaly, and impaired glucose homeostasis; the GLUT2 Ϫ/Ϫ mice displayed diabetic hyperglycemia and a lack of the first-phase glucose-stimulated insulin secretion, and died at ϳ2-3 wk (54); transgenic expression of glucose transporter genes demonstrated that GLUT2 (but not GLUT1)-reexpressing islets became susceptible to streptozotocin (22); moreover, K ATP channel-deficient (Kir6.2 Ϫ/Ϫ ) pancreatic ␤-cells were insensitive to streptozotocin because of significantly decreased GLUT2 expression on the cell membrane (not necessarily total protein amount) (59). We thus propose that RIP-I/Reg3␤ mice became resistant to streptozotocin due at least in part to diminished level and/or membrane localization of GLUT2, thereby reducing streptozotocin uptake by the ␤-cells.…”

“…We thus propose that RIP-I/Reg3␤ mice became resistant to streptozotocin due at least in part to diminished level and/or membrane localization of GLUT2, thereby reducing streptozotocin uptake by the ␤-cells. Although the mechanism of Reg3␤ action within the islet cells is still unclear, decreased GLUT2 expression might be the result of increased p8 expression (see below) or caused by mild hyperglycemia, as chronic exposure to high glucose concentration led to decreased GLUT2 activity (59). Under basal conditions, the decreased GLUT2 level in transgenic islets might contribute to the subtle changes in glycemia, glucose tolerance, and even insulin immunohistochemistry, which require further evaluation using more detailed and dynamic approaches.…”

Commonalities of genetic resistance to spontaneous autoimmune and free radical-mediated diabetes

“…While inbred strains differ in their susceptibility to chemically mediated diabetes [35], the majority of the studies designed to better understand the mechanisms of streptozotocin or alloxan-induced diabetes have done so using single gene knockouts [36–53]. Missing from these knockout studies, demonstrating either increased sensitivity or resistance to chemically-mediated diabetes, have been direct comparisons to spontaneous T1D.…”