C2-ceramide, a cell permeable analogue of ceramide [CER] markedly reduced mitochondrial membrane potential [MMP] in insulin-secreting INS cells, which was followed by a significant accumulation of cytochrome c [Cyt c] into the cytosolic compartment. In a manner akin to CER, exposure of these cells to interleukin-1beta [IL-1beta] also resulted in reduction in MMP and cytosolic accumulation of Cyt c. Further, long-term exposure of these cells to either CER [but not its inactive analogue] or IL-1beta caused a marked reduction in their metabolic viability. However, unlike IL-1beta, which increased nitric oxide [NO] release, CER-treatment of INS cells had no effects of CER on NO release were demonstrable. Together, these findings suggest that CER-induced mitochondrial effects may not be mediated via iNOS gene expression and NO production. CER also activated an okadaic acid -sensitive protein phosphatase [CAPP] in the purified mitochondrial fraction, suggesting that CAPP might represent one of the target proteins for CER in the beta cell mitochondria. Together, our findings suggest direct detrimental effects of CER on mitochondrial function in beta cells leading to their dysfunction and demise via apoptosis. Moreover, our findings provide evidence for a potential difference in the mechanisms underlying CER- and IL-1beta-induced mitochondrial defects and apoptotic demise of the effete beta cell.
Palanivel, Rengasamy, Rajakrishnan Veluthakal, and Anjaneyulu Kowluru. Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells. Am J Physiol Endocrinol Metab 286: E1032-E1041, 2004. First published February 17, 2004 10.1152/ajpendo.00587.2003.-Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrientinduced insulin secretion from isolated -cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated -cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phosphoenolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion. glucose metabolism; insulin-secreting cells THE PHOSPHORYLATION STATUS of proteins is regulated by the balance of activities of protein kinases and phosphatases, which induce the addition and removal of phosphoprotein phosphate, respectively (8, 42). Although several previous studies were focused on the identification and characterization of protein kinases in islets (4, 11, 16), little information is available on the localization and regulation of phosphoprotein phosphatases in the pancreatic -cell. With use of relatively specific inhibitors of protein phosphatase function, recent studies (1,2,23,31,36,40) have suggested important roles for various phosphoprotein phosphatases, such as protein phosphatase 2A (PP2A), in the pancreatic -cell function. For example, okadaic acid (OKA), a selective inhibitor of protein phosphatases, stimulates basal and cAMP-stimulated insulin secretion from electrically permeabilized islets (31). There is also evidence to indicate inhi...
We previously identified and characterized a glutamate- and magnesium-sensitive PP2A-like phosphatase (GAPP), which dephosphorylated and activated acetyl-CoA carboxylase (ACC) in the islet beta cell. Herein, we studied potential regulatory mechanisms by which GAPP is activated by glutamate and magnesium, and also quantitated the degree of activation, by glutamate- and magnesium, of ACC in normal rat islets and islets derived from the diabetic Goto-Kakizaki (GK) rat, a model for type 2 diabetes in humans. Our findings indicate that magnesium, but not glutamate, specifically activates the post-translational carboxylmethylation (CML) of the 36 kDa catalytic subunit of GAPP. Okadaic acid (OKA), which inhibits GAPP-mediated activation of ACC, also reduced the magnesium-stimulated CML of the catalytic subunit of GAPP in all the beta cell preparations studied. These data suggest that the CML step may be necessary for magnesium- and glutamate-mediated activation of ACC. We also observed a marked attenuation in magnesium- and glutamate-facilitated activation of ACC activity in islets derived from the GK rat. Together, our findings raise an interesting possibility that inhibition of GAPP-catalyzed inactivation of ACC (and subsequent reduction in the generation of long-chain fatty acids) could contribute toward the abnormalities in insulin secretion demonstrable in this animal model for type 2 diabetes.
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