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.

Experiments do not support a recent claim that glutamate formed from the amination of citric acid cyclederived ␣-ketoglutarate is a messenger in glucose-induced insulin secretion (Maechler, P., and Wollheim, C. (1999) Nature 402, 685-689). Glucose, leucine, succinic acid methyl ester, and ␣-ketoisocaproic acid all markedly stimulate insulin release but do not increase glutamate levels in pancreatic islets. Increasing the intracellular glutamate levels to 10-fold higher than basal levels by adding glutamine to islets does not stimulate insulin release. When leucine, in addition to glutamine, is applied to islets, insulin release is almost as high as with glucose alone. This is consistent with the known ability of leucine to allosterically activate glutamate deamination by glutamate dehydrogenase, which can supply ␣-ketoglutarate to the citric acid cycle. Experiments with mitochondria from pancreatic islets suggest that flux through the glutamate dehydrogenase reaction is quiescent during glucose-induced insulin secretion. These experiments support the traditional idea that when insulin release is associated with flux through glutamate dehydrogenase, the flux is in the direction of ␣-ketoglutarate.Recently Maechler and Wollheim (1), on the basis of intricate and broad-based experiments, proposed that glutamate generated from citric acid cycle-derived ␣-ketoglutarate is a messenger in glucose-induced insulin secretion. The glutamate effect was not robust, and its demonstration seemed to require rather narrowly defined conditions. For example, dimethylglutamate, a glutamate precursor that is permeable to the plasma membrane, caused a leftward shift in the concentration dependence of glucose-stimulated insulin release in INS-1 insulinoma cells. Dimethylglutamate did not stimulate insulin release at a basal concentration of glucose (2.5 mM) or augment insulin release at concentrations of glucose (16.7 to 25 mM) optimal for insulin release. Insulin release by dimethylglutamate was potentiated only at intermediate glucose concentrations. It was reported that when rat insulinoma INS-1 cells were incubated in the presence of a concentration of glucose (12.8 mM) that stimulates insulin release, cellular glutamate levels increased 4.8-fold to a stimulated level of 0.22 mM within 30 min. It was also observed that glucose (16.7 mM) augmented glutamate levels in human pancreatic islets from a basal level of 0.78 to a stimulated level of 3.93 nmol of glutamate per mg of islet protein. However, even these stimulated levels of glutamate are quite low. Our calculations indicate that the basal (0.04 to 0.08 mM) and stimulated glutamate levels (0.2 to 0.4 mM) reported by Maechler and Wollheim (1) are far lower than the basal concentration of glutamate of 1 to 7 mM found in many tissues (2) including the pancreatic islets used in our current study (see below) and in islets studied by others (3, 4).1 In addition, others have observed previously that glucose does not increase glutamate in islets (3). Thus, even though sophisticated app...

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.

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 importance of oscillations to biological organisms can be judged from the fact that almost all cells exhibit some kind of oscillations. 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 (1-5). However, oscillations in citric acid cycle intermediates have never been studied in any system. The beta cell is a unique fuel sensing organ in which mitochondria transduce a metabolic stimulus into multiple pharmacologic stimuli that activate the movement of insulin granules to the plasma membrane and granule extrusion into the circulation (6 -9). Glucose, the most potent insulin secretagogue, is metabolized via aerobic glycolysis. Anaplerosis, the synthesis of cycle intermediates, and cataplerosis, the export of cycle intermediates from mitochondria (10), are most likely very important for insulin secretion. In the beta cell about one-half of pyruvate, the terminal product of aerobic glycolysis in the cytosol, enters mitochondrial metabolism via carboxylation through the reaction catalyzed by pyruvate carboxylase, and the other half enters mitochondrial metabolism via decarboxylation catalyzed by pyru...

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.The glycerol phosphate shuttle, composed of the FAD-dependent mitochondrial glycerol phosphate dehydrogenase (mGPD, 1 EC 1.1.99.5) and the NAD(H)-dependent cytosolic glycerol phosphate dehydrogenase (cGPD, EC 1.1.1.8), is generally considered to play a role in the oxidation of cytosolic NADH formed during glycolysis. In mice the mitochondrial enzyme is encoded by a single gene, Gdm1, on chromosome 2 (1). The cytosolic enzyme has both an adult form, encoded by Gdc1 on chromosome 15 (2) and an embryonic form, encoded by Gdc2 on chromosome 9 (3). The embryonic form has not been found in liver or kidney during gestation but persists in brain for several weeks following birth (4) and in the epididymal white adipose tissue until at least 5 days of age (5). It has recently been reported that mice lacking the adult form of cGPD have elevated dihydroxyacetone phosphate and decreased glycerol phosphate and ATP levels in muscle following exercise (6), although these mice otherwise appear normal, having normal weights and litter sizes (7). Surprisingly, these cGPD-deficient mice grow normally even on a diet essentially free of glycerol (6), presumably by using the dihydroxyacetone phosphate acyl transferase pathway for lipid synthesis. The findings suggest that a lack of cGPD alters the cellular redox status in muscle, whereas pancreatic islet function is relatively normal and liver is only mildly affected, confirming the ability of alter...