Glucose Metabolism and Pulsatile Insulin Release From Isolated Islets

Abstract

T he circulating insulin concentration displays regular oscillations in normal human subjects (1). Disturbances of these oscillations are an early sign in the development of type 2 diabetes (2,3). Such alterations may lead to insulin resistance and glucose intolerance by downregulation of insulin receptors (4). The plasma insulin oscillations have been attributed to the pulsatile release of the hormone from the pancreas (5), a process that requires coordinated secretion from the pancreatic islets (6).When the ambient glucose concentration is raised, ␤-cell metabolism is stimulated (7). The resulting increase in the ATP/ADP ratio decreases the permeability of the ATP-sensitive K ϩ (K ATP ) channels and depolarizes the cell (8). Subsequent influx of Ca 2ϩ through voltage-dependent channels triggers the exocytosis of insulin granules (9). ATP produced by metabolism also plays an important role in providing energy for secretion by recruitment and priming of insulin granules for exocytosis (10). Impaired glucose metabolism in pancreatic ␤-cells has been implicated in the development of the disturbed plasma insulin pattern seen in type 2 diabetes (11). Support for this idea has been obtained from altered plasma insulin patterns in patients with mutations in genes coding for glycolytic enzymes, such as phosphofructokinase and glucokinase and mitochondrial tRNA (12)(13)(14)(15)(16)(17).In the present study, we attempted to determine the importance of glycolytic and mitochondrial metabolism for pulsatile insulin release by measuring insulin secretion and [Ca 2ϩ ] i from individual pancreatic islets exposed to metabolic inhibitors at basal and stimulatory glucose concentrations. RESEARCH DESIGN AND METHODS Materials.Reagents of analytical grade and deionized water were used. Collagenase, HEPES, and bovine serum albumin (fraction V) were obtained from Boehringer Mannheim (Mannheim, Germany). Antimycin A, iodoacetamide (IAA), 2,4-dinitrophenol (DNP), tetramethylbenzidine, and insulinperoxidase were obtained from Sigma (St. Louis, MO). The rat insulin standard was obtained from Novo Nordisk (Bagsvaerd, Denmark). IgGcertified microtiter plates were purchased from Nunc (Roskilde, Denmark). The insulin antibodies were raised in guinea pigs. Preparation and culture of islets. Pancreatic islets were collagenaseisolated from ob/ob mice taken from a local colony (18). For the insulin experiments, freshly isolated islets were perifused in a medium supplemented with 1 mg/ml albumin and containing (in mmol/l) 125 NaCl, 5.9 KCl, 1.2 MgCl 2 , 1.3 CaCl 2 , and 25 HEPES, titrated to pH 7.4 with NaOH. For the [Ca 2ϩ ] i experiments, the islets were cultured overnight in the presence of 5.5 mmol/l glucose in RPMI 1640 supplemented with 10% fetal calf serum, and the Ca 2ϩ concentration of the perifusion medium was 2.6 mmol/l. Measurements of insulin release. The kinetics of insulin release were studied essentially as described previously (6). A single islet was placed in a temperature-controlled (37°C) 10-l chamber and perifused with med...

Glycolytic and Mitochondrial Metabolism in Pancreatic Islets from MSG-Treated Obese Rats Subjected to Swimming Training

“…These results indicate for the first time that the glycolytic flux is increased in the islets from MSG-treated obese rats and could be contributing to the hypersecretion observed in these islets. Similar data were also observed in isolated pancreatic islets from Zucker (fa/fa) obese rats and ob/ob obese mice [48]. …”

Insulin granule dynamics in pancreatic beta cells

“…At best, secretion can be monitored in single islets (containing ≈1000 cells) with a sample interval longer than1 s [26]. Although this as such represents a considerable accomplishment, the elucidation of the cell biology of islet-hormone secretion requires an even higher temporal resolution.…”

Insulin Constitutively Secreted by β-Cells Is Necessary for Glucose-Stimulated Insulin Secretion

“…Because [Ca 2ϩ ] c controls insulin secretion, ␤-cells secrete insulin in an oscillatory manner (4,5). Oscillations of insulin secretion, however, are synchronized among ␤-cells, and the number of ␤-cells that participate in insulin secretion increases with increasing glucose concentration (6).…”

“…Depolarized ␤-cell membranes activate their voltage-dependent Ca 2ϩ channels (2), leading to an influx of Ca 2ϩ . Increases in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] c ) direct the fusion of insulin-containing vesicles with plasma membrane and the expulsion of insulin (3,4).…”