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.

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.

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Anaplerosis, the synthesis of citric acid cycle intermediates, by pancreatic beta cell mitochondria has been proposed to be as important for insulin secretion as mitochondrial energy production. However, studies designed to lower the rate of anaplerosis in the beta cell have been inconclusive. To test the hypothesis that anaplerosis is important for insulin secretion, we lowered the activity of pyruvate carboxylase (PC), the major enzyme of anaplerosis in the beta cell. Stable transfection of short hairpin RNA was used to generate a number of INS-1 832/13-derived cell lines with various levels of PC enzyme activity that retained normal levels of control enzymes, insulin content, and glucose oxidation. Glucose-induced insulin release was decreased in proportion to the decrease in PC activity. Insulin release in response to pyruvate alone, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) plus glutamine, or methyl succinate plus ␤-hydroxybutyrate was also decreased in the PC knockdown cells. Consistent with a block at PC, the most PC-deficient cells showed a metabolic crossover point at PC with increased basal and/or glucose-stimulated pyruvate plus lactate and decreased malate and citrate. In addition, in BCH plus glutamine-stimulated PC knockdown cells, pyruvate plus lactate was increased, whereas citrate was severely decreased, and malate and aspartate were slightly decreased. The incorporation of 14 C into lipid from [U-14 C]glucose was decreased in the PC knockdown cells. The results confirm the central importance of PC and anaplerosis to generate metabolites from glucose that support insulin secretion and even suggest PC is important for insulin secretion stimulated by noncarbohydrate insulin secretagogues.Glucose is the most potent physiological insulin secretagogue in the pancreatic beta cell, and it stimulates insulin secretion via its metabolism by aerobic glycolysis. Pyruvate, the terminal product of glycolysis, is metabolized in mitochondria to make ATP to power intracellular processes. However, somewhat surprisingly, in the beta cell, a large amount of glucosederived pyruvate, equal to one-half the total pyruvate entering mitochondrial metabolism, is carboxylated in the pyruvate carboxylase reaction to form oxaloacetate (1-5). The oxaloacetate can combine with pyruvate-derived acetyl-CoA to form citrate enabling the beta cell mitochondrion to increase the rate of synthesis of any citric acid cycle intermediate (anaplerosis) (6 -9). The rate of pyruvate carboxylation correlates with the glucose concentration applied to islets and is thus correlated with the rate of insulin secretion (2). The level of pyruvate carboxylase in the pancreatic islet beta cell is higher than in most body tissues (4, 10 -13) and is equal to the levels in gluconeogenic tissues, such as liver and kidney (4), which possess very high levels of the enzyme. However, the beta cell does not possess the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (11, 14, 15) or fructose-1,6-bisphosphatase (8), and this explains why it ...

Brown–Vialetto–Van Laere syndrome (BVVLS) is a genetic condition caused by a mutation in the C20orf54 gene, which also codes for an intestinal riboflavin transporter. We report the case of a female who presented at 22 months with acute‐onset stridor and generalized muscle weakness, in whom a genetic diagnosis of BVVLS was made, and whose symptoms improved on therapy with high‐dose riboflavin. She had previously been developing normally and was able to walk at 11 months, then developed progressive muscle weakness at 22 months, and within 2 weeks was unable to sit without support. She also demonstrated stridor and paradoxical breathing indicating diaphragmatic weakness, and required continuous non‐invasive ventilation (NIV) through a tracheostomy. After treatment with riboflavin she was able to walk unaided, and her Gross Motor Functional Classification level improved from level IV to level I, having fully regained the motor function she showed before symptom onset. There were no longer signs of diaphragmatic paralysis while on NIV, and she was able to tolerate 10‐minute periods off NIV before paradoxical breathing again became apparent. We therefore recommend that in all cases suspected to be in the BVVLS or Fazio–Londe spectrum, early treatment with high‐dose riboflavin must be considered.

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...

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Anaplerosis, the net synthesis in mitochondria of citric acid cycle intermediates, and cataplerosis, their export to the cytosol, have been shown to be important for insulin secretion in rodent beta cells. However, human islets may be different. We observed that the enzyme activity, protein level, and relative mRNA level of the key anaplerotic enzyme pyruvate carboxylase (PC) were 80 -90% lower in human pancreatic islets compared with islets of rats and mice and the rat insulinoma cell line INS-1 832/13. Activity and protein of ATP citrate lyase, which uses anaplerotic products in the cytosol, were 60 -75% lower in human islets than in rodent islets or the cell line. In line with the lower PC, the percentage of glucose-derived pyruvate that entered mitochondrial metabolism via carboxylation in human islets was only 20 -30% that in rat islets. This suggests human islets depend less on pyruvate carboxylation than rodent models that were used to establish the role of PC in insulin secretion. Human islets possessed high levels of succinyl-CoA:3-ketoacid-CoA transferase, an enzyme that forms acetoacetate in the mitochondria, and acetoacetyl-CoA synthetase, which uses acetoacetate to form acyl-CoAs in the cytosol. Glucose-stimulated human islets released insulin similarly to rat islets but formed much more acetoacetate. ␤-Hydroxybutyrate augmented insulin secretion in human islets. This information supports previous data that indicate beta cells can use a pathway involving succinyl-CoA:3-ketoacid-CoA transferase and acetoacetyl-CoA synthetase to synthesize and use acetoacetate and suggests human islets may use this pathway more than PC and citrate to form cytosolic acyl-CoAs.Understanding the enzymatic makeup of human pancreatic islets is fundamental to developing strategies for designing artificial beta cells and beta cells differentiated from stem cells as treatments for type 1 diabetes, as well as modulating beta cell metabolism for the treatment of type 2 diabetes. Until recently, most of the information about normal insulin secretion came from studies of rodent islets or clonal cell lines. Although a recent study showed human pancreatic islets respond to insulin secretagogues similarly to rodent islets (1), what is still unknown is whether the use of intracellular pathways of secretagogue metabolism is the same in human islets as in rodent islets and cell lines. During the last few years, human islet preparations from human donors have become more readily available to researchers. By studying the levels of enzymes, the functional units of metabolism, the recent abundant supply of human islets has enabled our laboratory to discover clues suggesting differences in metabolic pathways between pancreatic islets of humans and rodents that have implications for better understanding normal human beta cell physiology.Anaplerosis, the biosynthesis of citric acid cycle intermediates (2), is widely believed to be important for insulin secretion (3). Pyruvate carboxylase (PC) 2 is the key anaplerotic enzyme in this process ...