Culturing rat islets in high glucose (HG) increased 1-(14)C-alpha-ketoisocaproate (KIC) oxidation compared with culturing them in low glucose. Leucine caused insulin secretion (IS) in low glucose but not in HG rat islets, whereas KIC did so in both. Pretreatment with HG for 40 min abolished leucine stimulation of IS by mouse islets and prevented the cytosolic Ca(2+) rise without inhibiting IS and Ca(2+) increments caused by KIC. When islets were pretreated without glucose and glutamine, aminooxyacetic acid (AOA) markedly decreased KIC effects. When islets were pretreated without glucose and with glutamine, AOA potentiated leucine effects but attenuated KIC effects. AOA stimulated glutamine oxidation in the presence but not the absence of +/-2-amino-2-norbornane-carboxylic acid, a nonmetabolized leucine analog. Pretreatment with HG and glutamine partially reversed AOA inhibition of KIC effects. Glucose increased intracellular ATP and GTP, whereas it decreased ADP and GDP in beta HC9 cells. Glutamate dehydrogenase activity of beta HC9 cell extracts was increased by leucine and attenuated by GTP, but it was potentiated by ADP. In conclusion, leucine and KIC stimulated beta-cells via distinct mechanisms. Glutamate dehydrogenase is the sensor of leucine, whereas transamination plays an important role in KIC stimulation of pancreatic beta-cells.

Our goal was to investigate whether leucine culture affects ␤-cell glucose sensing. One-day culture of rat islets with 10 mM leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mM glucose. Glucose-induced cytosolic free Ca 2؉ was increased at 1 week but not at 1 day of leucine culture. Glucose is the main secretagogue of insulin secretion from pancreatic ␤-cells. The mechanisms of glucose-induced insulin secretion have been studied extensively. Through glycolysis and oxidation, glucose increases pancreatic ␤-cell ATP/ADP ratio, which closes ATP-sensitive potassium (K ATP ) channels and depolarizes the cell membrane. This results in an influx of extracellular Ca 2ϩ and increase of free cytosolic [Ca 2ϩ ] that stimulates exocytosis of insulin granules (1-5). Another mechanism is K ATP channel-independent and involves increased effectiveness of [Ca 2ϩ ] (6, 7). In pancreatic ␤-cells, most of the intracellular ATP comes from the oxidation of glucose-derived pyruvate and oxidation of NADH in the mitochondria via the electron transport chain. Damage or inhibition of ATP synthesis results in ␤-cell dysfunction and impairs glucose-stimulated insulin secretion (8, 9). Some recent publications indicate that superoxide produced by hyperglycemia activates uncouplingprotein-2 (UCP2) and destroys the proton gradient between inner and outer mitochondrial membranes. This negatively affects the activity of ATP synthase, decreases ATP production, and impairs glucose-stimulated insulin secretion of pancreatic ␤-cells resulting in diabetes (8, 9). More recently, it is shown that mitochondrial metabolism sets the maximal limit of fuel stimulated-insulin secretion in ␤-cells (10). These findings imply that mitochondrial ATP synthesis may play a vital role in fuel-stimulated insulin secretion of pancreatic ␤-cells.Some amino acids, particularly leucine and its non-metabolizable analogue 2-amino-2-norbornanecarboxylic acid, have been known to stimulate insulin secretion from pancreatic ␤-cells by activation of glutamate dehydrogenase (11-15). More recently, the branched-chain amino acids, including leucine, isoleucine, and valine, have been reported to activate the mammalian target of rapamycin (mTOR) 1 signaling pathway (16 -19) in ␤-cells. Leucine stimulates protein synthesis and pancreatic ␤-cell proliferation via the mTOR signaling pathway at physiological concentrations (16). These studies indicate a new role of branched-chain amino acids in pancreatic ␤-cell biology in addition to serving as fuels or residues for protein synthesis.As the rate-limiting enzyme of glucose metabolism, it is believed that glucokinase sets a strict control on glucose metabolism in pancreatic ␤-cells. However, overexpression of glucokinase or hexokinase I fails to increase the maximal insulin output induced by glucose, although it decreases the threshold for glucose-induced insulin secretion in pancreatic ␤-cel...

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318-332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(-) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(-) splice variants of the type-I IP3R.

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