Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP ؉ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.The mechanism of glucose-stimulated insulin secretion (GSIS) 2 from pancreatic islet ␤-cells is not completely understood. One component of the signaling pathway involves glucose-induced increases in cytosolic ATP/ADP ratio, leading to closure of K ATP channels at the plasma membrane. K ATP channel closure results in membrane depolarization and activation of voltage-dependent Ca 2ϩ channels, increasing the concentration of cytosolic Ca 2ϩ (1-4). Elevation of cytosolic Ca 2ϩ promotes exocytosis of insulin-containing secretory granules (5). However, fluctuations in cytosolic Ca 2ϩ are not the only signal, because under conditions of clamped cytosolic Ca 2ϩ concentrations, glucose can still cause significant insulin secretion (6). This suggests that glucose generates signals/second messengers that are distinct from ATP and membrane depolarization for regulation of insulin secretion (7,8). Some of the suggested mitochondrial factors include glutamate, malonyl-CoA, longchain acyl-CoAs (LC-CoA), and/or NADPH (7-17).The production of malonyl-CoA, LC-CoA, and NADPH in the cytosol depends on the export of mitochondrial metabolites. NADPH can be produced via one of three pyruvate cycling pathways, the pyruvate/malate pathway, the pyruvate/citrate pathway, or the pyruvate/isocitrate pathway, via cytosolic NADP ϩ -dependent isoforms of malic enzyme (used in the pyruvate/malate and pyruvate/citrate pathways) or cytosolic, NADP ϩ -dependent isocitrate dehydrogenase (ICDc) (used in the pyruvate/isocitrate cycle) (18,19). Citrate emanating from mitochondrial metabolism can also be cleaved by ATP-citrate lyase to produce malonyl-CoA and LC-CoA (19). We and others have previously established that anap...

Pancreatic β-cells normally produce adequate insulin to control glucose homeostasis, but in obesity-related diabetes, there is a presumed deficit in insulin production and secretory capacity. In this study, insulin production was assessed directly in obese diabetic mouse models, and proinsulin biosynthesis was found to be contrastingly increased, coupled with a significant expansion of the rough endoplasmic reticulum (without endoplasmic reticulum stress) and Golgi apparatus, increased vesicular trafficking, and a depletion of mature β-granules. As such, β-cells have a remarkable capacity to produce substantial quantities of insulin in obesity, which are then made available for immediate secretion to meet increased metabolic demand, but this comes at the price of insulin secretory dysfunction. Notwithstanding, it can be restored. Upon exposing isolated pancreatic islets of obese mice to normal glucose concentrations, β-cells revert back to their typical morphology with restoration of regulated insulin secretion. These data demonstrate an unrealized dynamic adaptive plasticity of pancreatic β-cells and underscore the rationale for transient β-cell rest as a treatment strategy for obesity-linked diabetes.

The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonylCoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/ 13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucoseinduced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all ␤-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli. Diabetes 53:

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (<2 h) this is due to increases in the translation of pre-existing mRNA. However, prolonging glucose stimulation (24 h) also increases preproinsulin mRNA levels. It has been proposed that secreted insulin from the pancreatic ␤-cell regulates its own synthesis through a positive autocrine feedback mechanism. Here the comparative contributions of translation and mRNA levels on the levels of proinsulin biosynthesis were examined in isolated pancreatic islets. Also, the autocrine role of insulin upon four ␤-cell functions (insulin secretion, proinsulin translation, preproinsulin mRNA levels, and total protein synthesis) was investigated in parallel. The results showed that proinsulin biosynthesis is regulated, in the short term (1 h), solely at the level of translation, through an ϳ6-fold increase in response to glucose (2.8 mM versus 16.7 mM glucose). In the longer term, when preproinsulin mRNA levels have increased ϳ2-fold, a corresponding increase was observed in the fold response of proinsulin translation to a stimulatory glucose concentration (>10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the ␤-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.