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

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.