Role of NADH Shuttles in Glucose-Induced Insulin Secretion From Fetal β-Cells

Cynthia Tan 0 Bernard E. Tuch 0 Jian Tu 0 Shane A. Brown 0 0 From the Diabetes Transplant Unit, Department of Endocrinology, Prince of Wales Hospital and the University of New South Wales , Sydney, New South Wales , Australia. Diabetes Transplant Unit, Prince of Wales Hospital , High Street, Randwick, New South Wales 2031 , Australia The NADH shuttle system, which transports the substrate for oxidative metabolism directly from the cytosol to the mitochondrial electron transport chain, has been shown to be essential for glucose-induced activation of mitochondrial metabolism and insulin secretion in adult -cells. We examined the role of these shuttles in the fetal -cell, which is immature in being unable to secrete insulin in response to glucose. The activity and concentration of the two key enzymes of the NADH shuttles, mitochondrial glycerol phosphate dehydrogenase (mGPDH) and mitochondrial malate dehydrogenase (mMDH), were eight- and threefold lower, respectively, in fetal compared with adult rat islets. Likewise, mGPDH and mMDH activity was fivefold lower in isletlike cell clusters (ICCs) and sevenfold lower in purified -cells compared with adult islets in the pig. The low level of enzyme activity was a result of low gene expression of the mitochondrial enzymes in the fetal -cells. Increasing NADH shuttle activity by transduction of fetal rat islets with mGPDH cDNA enabled the fetal islets to secrete insulin when stimulated with glucose. We concluded that the immaturity of the NADH shuttles contributes to the inability of fetal -cells to secrete insulin in response to glucose. Diabetes 51:2989 -2996, 2002 - Gunable to cause a similar response in fetal lucose is the major physiological stimulus for insulin secretion in adult -cells (13) but is -cells (4,5). The secretion of insulin from an adult -cell involves the transport of glucose into the -cell via the GLUT2 transporter, followed by the production of ATP, as a result of glucose metabolism in the glycolytic pathway, the tricarboxylic acid (TCA) cycle, and the mitochondrial electron transport chain. The resulting increase in ATP-to-ADP ratio leads to the closure of the ATP-dependent K channels on the cell surface, which results in the depolarization of the cell membrane and the opening of the voltage-activated Ca2 channels on the cell surface to allow the influx of extracellular Ca2 . The increase in intracellular Ca2 triggers insulin secretion. It is hypothesized that the failure of fetal -cells to respond to glucose is due to an immaturity in one or more of the above steps (4). Glucose transport into the fetal -cell does not appear to be as defective as GLUT2 and has been identified in human fetal -cells at 13 weeks gestation (6) and in fetal rat by 19 days gestation (7). The glycolytic pathway in the fetal -cell appears to be intact (5). This is shown by normal glucose utilization and normal activity of the rate-limiting enzyme, glucokinase (5). A reduction of glucose oxidation has been observed more distally (8). Several factors may be responsible for this reduction: 1) immaturity in the TCA cycle; 2) the lack of substrate for the TCA cycle, e.g., the accumulation of lactate due to high lactate dehydrogenase activity, which results in the unavailability of pyruvate for glucose oxidation; or 3) reduced NADH shuttle activity. Immaturity of the TCA cycle in fetal -cells is suggested because this is the pathway in which most ATP required for glucose-stimulated insulin secretion is produced. Against this hypothesis is the fact that leucine is able to cause insulin secretion from fetal rat -cells (9). Leucine acts via two different intramitochodrial pathways, one involving the degradation of ketoisocaproic acid to acetyl Co-A, which enter the TCA cycle and the other by acting as an allosteric activator of glutamate dehydrogenase, resulting in the formation of 2-ketoglutarate from endogenous glutamate, which is further oxidized in the TCA cycle (10). It is unlikely that lack of substrate for the TCA cycle occurs in fetal -cells, as lactate dehydrogenase is low in adult -cells (11). Furthermore, the amount of pyruvate entering the TCA cycle appears to not be pivotal to glucose-stimulated insulin secretion, since pyruvate alone is unable to stimulate insulin secretion, even though it is readily metabolized by -cells (2,12). The final steps required for insulin secretion, i.e., the closure of the ATP-dependent K channels and the opening of the Ca2 channels have been shown to be normal in fetal -cells (9). The NADH shuttles, which transport NADH, the substrate for oxidative metabolism and ATP production, from the cytosol to the mitochondria are crucial to glucoseinduced activation of mitochondrial metabolism and insulin secretion in the adult -cell (13). Two NADH shuttles have been identified: the glycerol phosphate shuttle and the malate-aspartate shuttle. The glycerol phosphate shuttle enables the transfer of electrons from NADH generated from glycolysis to coenzyme Q (complex II) in the electron transport chain. The malate-aspartate shuttle results in the regeneration of NADH utilized in the mitochondria and its subsequent transfer to complex I in the electron transport chain, contributing to the production of ATP in the mitochondria (14,15). The activity of mitochondrial glycerol phosphate dehydrogenase (mGPDH), the key enzyme of the glycerolphosphate shuttle is 40- to 70-fold higher in islets relative to other tissues, indicating that mitochondrial oxidation of metabolized glucose is favored in -cells (1). A reduction in the activity of this enzyme theoretically should result in diminished ATP production in the mitochondria and hence a reduction in glucose-induced insulin secretion. Indeed this is true for both animals models (16,17) and some humans with type 2 diabetes (18). In contrast, insulin secretion is normal in transgenic mice with no functional mGPDH (13). When the second NADH shuttle, the malate aspartate shuttle, was inhibited in these transgenic mice, insulin secretion in response to glucose was adversely affected (13). Such studies show the importance of the NADH shuttles in glucose-stimulated insulin secretion. The current study was undertaken to investigate the role of the NADH shuttles, in particular the role of the mitochondrial enzymes that are rate limiting, in glucose-stimulated insulin secretion from fetal -cells. RESEARCH DESIGN AND METHODS Unless indicated otherwise, all reagents were analytical grade and were purchased from Sigma Chemical (St Louis, MO). Islets, islet-like cell clusters, and -cell preparation Rat islets. Fetal rat pancreata were obtained from Wistar pregnant rats at gestational age 20 21 days (crown-rump length 45 1 mm). Approval to conduct the experiments was obtained from the Animal Care and Ethics Committee of the University of New South Wales, Australia. Pancreata from within a litter were pooled, and islets were isolated by digestion with Collagenase A (Roche D (...truncated)