Chronic Reduction of the Cytosolic or Mitochondrial NAD(P)-malic Enzyme Does Not Affect Insulin Secretion in a Rat Insulinoma Cell Line

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2009; 284(51):35359-67. DOI: 10.1074/jbc.M109.040394
Source: PubMed


The cytosolic malic enzyme (ME1) has been suggested to augment insulin secretion via the malate-pyruvate and/or citrate-pyruvate shuttles, through the production of NADPH or other metabolites. We used selectable vectors expressing short hairpin RNA (shRNA) to stably decrease Me1 mRNA levels by 80-86% and ME1 enzyme activity by 78-86% with either of two shRNAs in the INS-1 832/13 insulinoma cell line. Contrary to published short term ME1 knockdown experiments, our long term targeted cells showed normal insulin secretion in response to glucose or to glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. We found no increase in the mRNAs and enzyme activities of the cytosolic isocitrate dehydrogenase or glucose-6-phosphate dehydrogenase, which also produce cytosolic NADPH. There was no compensatory induction of the mRNAs for the mitochondrial malic enzymes Me2 or Me3. Interferon pathway genes induced in preliminary small interfering RNA experiments were not induced in the long term shRNA experiments. We repeated our study with an improved vector containing Tol2 transposition sequences to produce a higher rate of stable transferents and shortened time to testing, but this did not alter the results. We similarly used stably expressed shRNA to reduce mitochondrial NAD(P)-malic enzyme (Me2) mRNA by up to 95%, with severely decreased ME2 protein and a 90% decrease in enzyme activity. Insulin release to glucose or glutamine plus 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid remained normal. The maintenance of robust insulin secretion after lowering expression of either one of these malic enzymes is consistent with the redundancy of pathways of pyruvate cycling and/or cytosolic NADPH production in insulinoma cells.

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    • "Several studies, but not all [31], have shown that reduction of MEc expression in INS 832/13 cells, MIN6 cells and in mouse islets impaired GIIS and correlated with a decrease in NADPH level [24], [25], [26], [32]. In rat islets, siRNA knockdown of MEc mRNA did not affect GIIS. "
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    ABSTRACT: Cytosolic NADPH may act as one of the signals that couple glucose metabolism to insulin secretion in the pancreatic ß-cell. NADPH levels in the cytoplasm are largely controlled by the cytosolic isoforms of malic enzyme and isocitrate dehydrogenase (IDHc). Some studies have provided evidence for a role of malic enzyme in glucose-induced insulin secretion (GIIS) via pyruvate cycling, but the role of IDHc in ß-cell signaling is unsettled. IDHc is an established component of the isocitrate/α-ketoglutarate shuttle that transfers reducing equivalents (NADPH) from the mitochondrion to the cytosol. This shuttle is energy consuming since it is coupled to nicotinamide nucleotide transhydrogenase that uses the mitochondrial proton gradient to produce mitochondrial NADPH and NAD(+) from NADP(+) and NADH. To determine whether flux through IDHc is positively or negatively linked to GIIS, we performed RNAi knockdown experiments in ß-cells. Reduced IDHc expression in INS 832/13 cells and isolated rat islet ß-cells resulted in enhanced GIIS. This effect was mediated at least in part via the KATP-independent amplification arm of GIIS. IDHc knockdown in INS 832/13 cells did not alter glucose oxidation but it reduced fatty acid oxidation and increased lipogenesis from glucose. Metabolome profiling in INS 832/13 cells showed that IDHc knockdown increased isocitrate and NADP(+) levels. It also increased the cellular contents of several metabolites linked to GIIS, in particular some Krebs cycle intermediates, acetyl-CoA, glutamate, cAMP and ATP. The results identify IDHc as a component of the emerging pathways that negatively regulate GIIS.
    PLoS ONE 10/2013; 8(10):e77097. DOI:10.1371/journal.pone.0077097 · 3.23 Impact Factor
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    • "The fact that ME1-repressed cells could not survive in a glucose-free medium supplied with pyruvate revealed the vital role of ME1 in NADPH production in CNE-2 cells. A study conducted in rat insulinoma cells has shown that there were no differences in the activity of G6PD and IDH1 between ME1-repressed and control cells[33]. However, this result could be due to differences between species or various tissues. "
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    ABSTRACT: A large amount of nicotinamide adenine dinucleotide phosphate (NADPH) is required for fatty acid synthesis and maintenance of the redox state in cancer cells. Malic enzyme 1 (ME1)-dependent NADPH production is one of the three pathways that contribute to the formation of the cytosolic NADPH pool. ME1 is generally considered to be overexpressed in cancer cells to meet the high demand for increased de novo fatty acid synthesis. In the present study, we found that glucose induced higher ME1 activity and that repressing ME1 had a profound impact on glucose metabolism of nasopharyngeal carcinoma cells. High incorporation of glucose and an enhancement of the pentose phosphate pathway were observed in ME1-repressed cells. However, there were no obvious changes in the other two pathways for glucose metabolism: glycolysis and oxidative phosphorylation. Interestingly, NADPH was decreased under low-glucose conditions in ME1-repressed cells relative to wild-type cells, whereas there was no significant difference under high-glucose conditions. ME1-repressed cells had significantly decreased tolerance to low glucose conditions. Moreover, NADPH produced by ME1 was not only important for fatty acid synthesis but also essential for maintenance of the intracellular redox state and the protection of cells from oxidative stress. Furthermore, diminished migration and invasion were observed in ME1-repressed cells due to a reduced level of Snail protein. Collectively, these results suggest an essential role for ME1 in the production of cytosolic NADPH and maintenance of cellular migratory and invasive abilities in nasopharyngeal carcinoma cells.
    Chinese journal of cancer 11/2012; 31(11). DOI:10.5732/cjc.012.10088 · 2.16 Impact Factor
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    • "Another possible, and likely explanation, is that there is a redundancy of these mitochondrial-cytosolic substrate cycle, and that malate-pyruvate cycle and the citrate-malate-pyrvate cycle work concurrently. The loss of any one cycle with low degree of metabolic control [27], as was done in the shRNA knock-down studies in islets [28], would lead to the conclusion that that particular cycle was unimportant for regulating glucose stimulated insulin secretion. A redundancy of cycles would share responsibility for the transfer of reducing equivalents to the cytosol, and could result in no single cycle having a high degree of metabolic control [27,28]. "
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    ABSTRACT: The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet β-cells.
    Diabetes & metabolism journal 10/2011; 35(5):458-65. DOI:10.4093/dmj.2011.35.5.458
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