Feasibility of pathways for transfer of acyl groups from mitochondria to the cytosol to form short chain acyl-CoAs in the pancreatic beta cell.

The mitochondria of pancreatic beta cells are believed to convert insulin secretagogues into products that are translocated to the cytosol where they participate in insulin secretion. We studied the hypothesis that short chain acyl-CoA (SC-CoAs) might be some of these products by discerning the pathways of SC-CoA formation in beta cells. Insulin secretagogues acutely stimulated 1.5-5-fold increases in acetoacetyl-CoA, succinyl-CoA, malonyl-CoA, hydroxymethylglutaryl-CoA (HMG-CoA), and acetyl-CoA in INS-1 832/13 cells as judged from liquid chromatography-tandem mass spectrometry measurements. Studies of 12 relevant enzymes in rat and human pancreatic islets and INS-1 832/13 cells showed the feasibility of at least two redundant pathways, one involving acetoacetate and the other citrate, for the synthesis SC-CoAs from secretagogue carbon in mitochondria and the transfer of their acyl groups to the cytosol where the acyl groups are converted to SC-CoAs. Knockdown of two key cytosolic enzymes in INS-1 832/13 cells with short hairpin RNA supported the proposed scheme. Lowering ATP citrate lyase 88% did not inhibit glucose-induced insulin release indicating citrate is not the only carrier of acyl groups to the cytosol. However, lowering acetoacetyl-CoA synthetase 80% partially inhibited glucose-induced insulin release indicating formation of SC-CoAs from acetoacetate in the cytosol is important for insulin secretion. The results indicate beta cells possess enzyme pathways that can incorporate carbon from glucose into acetyl-CoA, acetoacetyl-CoA, and succinyl-CoA and carbon from leucine into these three SC-CoAs plus HMG-CoA in their mitochondria and enzymes that can form acetyl-CoA, acetoacetyl-CoA, malonyl-CoA, and HMG-CoA in their cytosol.

Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion.

The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells.

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.

The succinate mechanism of insulin release.

Nutrient secretagogues can increase the production of succinyl-CoA in rat pancreatic islets. When succinate esters are the secretagogue, succinyl-CoA can be generated via the succinate thiokinase reaction. Other secretagogues can increase production of succinyl-CoA secondary to increasing alpha-ketoglutarate production by glutamate dehydrogenase or mitochondrial aspartate aminotransferase followed by the alpha-ketoglutarate dehydrogenase reaction. Although secretagogues can increase the production of succinyl-CoA, they do not increase the level of this metabolite until after they decrease the level of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This suggests that the generated succinyl-CoA initially reacts with acetoacetate to yield acetoacetyl-CoA plus succinate in the succinyl-CoA-acetoacetate transferase reaction. This would be followed by acetoacetyl-CoA reacting with acetyl-CoA to generate HMG-CoA in the HMG-CoA synthetase reaction. HMG-CoA will then be reduced by NADPH to mevalonate in the HMG-CoA reductase reaction and/or cleaved to acetoacetate plus acetyl-CoA by HMG cleavage enzyme. Succinate derived from either exogenous succinate esters or generated by succinyl-CoA-acetoacetate transferase is metabolized to malate followed by the malic enzyme reaction. Increased production of NADPH by the latter reaction then increases reduction of HMG-CoA and accounts for the decrease in the level of HMG-CoA produced by secretagogues. Pyruvate carboxylation catalyzed by pyruvate carboxylase will supply oxaloacetate to mitochondrial aspartate aminotransferase. This would enable this aminotransferase to supply alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex and would, in part, account for secretagogues increasing the islet level of succinyl-CoA after they decrease the level of HMG-CoA. Mevalonate could be a trigger of insulin release as a result of its ability to alter membrane proteins and/or cytosolic Ca(2+). This is consistent with the fact that insulin secretagogues decrease the level of the mevalonate precursor HMG-CoA. In addition, inhibitors of HMG-CoA reductase interfere with insulin release and this inhibition can be reversed by mevalonate.

Normal thyroid thermogenesis but reduced viability and adiposity in mice lacking the mitochondrial glycerol phosphate dehydrogenase.

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is important for metabolism of glycerol phosphate for gluconeogenesis or energy production and has been implicated in thermogenesis induced by cold and thyroid hormone treatment. mGPD in combination with the cytosolic glycerol phosphate dehydrogenase (cGPD) is proposed to form the glycerol phosphate shuttle, catalyzing the interconversion of dihydroxyacetone phosphate and glycerol phosphate with net oxidation of cytosolic NADH. We made a targeted deletion in Gdm1 and produced mice lacking mGPD. On a C57BL/6J background these mice showed a 50% reduction in viability compared with wild-type littermates. Uncoupling protein-1 mRNA levels in brown adipose tissue did not differ between mGPD knockout and control pups, suggesting normal thermogenesis. Pups lacking mGPD had decreased liver ATP and slightly increased liver glycerol phosphate. In contrast, liver and muscle metabolites were normal in adult animals. Adult mGPD knockout animals had a normal cold tolerance, normal circadian rhythm in body temperature, and demonstrated a normal temperature increase in response to thyroid hormone. However, they were found to have a lower body mass index, a 40% reduction in the weight of white adipose tissue, and a slightly lower fasting blood glucose than controls. The phenotype may be secondary to consequences of the obligatory production of cytosolic NADH from glycerol metabolism in the mGPD knockout animal. We conclude that, although mGPD is not essential for thyroid thermogenesis, variations in its function affect viability and adiposity in mice.