Metformin is considered to be one of the most effective therapeutics for the treatment of type 2 diabetes (T2D) since it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain, or posing a risk of hypoglycemia1,2. For over half a century, this agent has been prescribed to T2D patients worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase (mGPD), resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production (EGP), while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide (ASO) knockdown of hepatic mGPD in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decrease in plasma glucose concentrations and inhibition of EGP. These findings were replicated in whole-body mGPD knockout mice. These results have significant implications for understanding the mechanism of metformin’s blood glucose lowering effects and provide a novel therapeutic target for T2D.

OBJECTIVE-Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. RESEARCH DESIGN AND METHODS-The INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed Ͻ45 years). None had a molecular genetic diagnosis of monogenic diabetes. RESULTS-We identified heterozygous INS mutations in 33 of141 probands diagnosed at Ͻ6 months, 2 of 86 between 6 and 12 months, and none of 58 between 12 and 24 months of age. Three known mutations (A24D, F48C, and R89C) account for 46% of cases. There were six novel mutations: H29D, L35P, G84R, C96S, S101C, and Y103C. INS mutation carriers were all insulin treated from diagnosis and were diagnosed later than ATP-sensitive K ϩ channel mutation carriers (11 vs. 8 weeks, P Ͻ 0.01). In 279 patients with PND, the frequency of KCNJ11, ABCC8, and INS gene mutations was 31, 10, and 12%, respectively. A heterozygous R6C mutation cosegregated with diabetes in a MODY family and is probably pathogenic, but the L68M substitution identified in a patient with young-onset type 2 diabetes may be a rare nonfunctional variant.CONCLUSIONS-We conclude that INS mutations are the second most common cause of PND and a rare cause of MODY. Insulin gene mutation screening is recommended for all diabetic patients diagnosed before 1 year of age.

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.

Pancreatic beta cells are specialised endocrine cells that continuously sense the levels of blood sugar and other fuels and, in response, secrete insulin to maintain normal fuel homeostasis. During postprandial periods an elevated level of plasma glucose rapidly stimulates insulin secretion to decrease hepatic glucose output and promote glucose uptake into other tissues, principally muscle and adipose tissues. Beta cell mitochondria play a key role in this process, not only by providing energy in the form of ATP to support insulin secretion, but also by synthesising metabolites (anaplerosis) that can act, both intra-and extramitochondrially, as factors that couple glucose sensing to insulin granule exocytosis. ATP on its own, and possibly modulated by these coupling factors, triggers closure of the ATP-sensitive potassium channel, resulting in membrane depolarisation that increases intracellular calcium to cause insulin secretion. The metabolic imbalance caused by chronic hyperglycaemia and hyperlipidaemia severely affects mitochondrial metabolism, leading to the development of impaired glucose-induced insulin secretion in type 2 diabetes. It appears that the anaplerotic enzyme pyruvate carboxylase participates directly or indirectly in several metabolic pathways which are important for glucoseinduced insulin secretion, including: the pyruvate/malate cycle, the pyruvate/citrate cycle, the pyruvate/isocitrate cycle and glutamate-dehydrogenase-catalysed α-ketoglutarate production. These four pathways enable 'shuttling' or 'recycling' of these intermediate(s) into and out of mitochondrion, allowing continuous production of intracellular messenger(s). The purpose of this review is to present an account of recent progress in this area of central importance in the realm of diabetes and obesity research.

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...

A new clinical entity that is prevalent in young type I (insulin-dependent) diabetic patients, postexercise late-onset (PEL) hypoglycemia, is described. A prospective case-finding study suggested that PEL hypoglycemia occurred in 48 of approximately 300 diabetic type I patients who were diagnosed as diabetic before age 20 yr and who were monitored for up to 2 yr. Typically, hypoglycemia was nocturnal and occurred 6-15 h after the completion of unusually strenuous exercise or play. In more than half the cases the hypoglycemia resulted in loss of consciousness or seizures and necessitated treatment with subcutaneous glucagon or intravenous glucose and/or attendance by a health professional. The hypoglycemia was not limited to patients in good or excellent metabolic control and often occurred after a single bout of exercise in patients unaccustomed to exercise or in athletic patients who were making the transition from an untrained to a trained state. Surprisingly, 12 of the patients who experienced nocturnal PEL hypoglycemia were not using significant amounts of insulin that peaked at night. Type I diabetic patients should be made aware of the possibility of PEL hypoglycemia to enable them to make adjustments in their management plans in anticipation of unusually strenuous exercise, so that they may attempt to minimize or avoid late-onset hypoglycemia.

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.

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 ...