title: Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion.
abstract: BACKGROUND
  There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.
  
  
  METHODS
  With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%-90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.
  
  
  RESULTS
  With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.
  
  
  CONCLUSIONS
  The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.
  
  
  CONCLUSIONS
  As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.
authors.affiliation: Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA. Electronic address: mjmacdon@wisc.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacDonald
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Brown
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Longacre
authors.forename: Scott W
authors.initials: SW
authors.lastname: Stoker
authors.forename: Mindy A
authors.initials: MA
authors.lastname: Kendrick
authors.forename: Noaman M
authors.initials: NM
authors.lastname: Hasan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D019343:Citric Acid / Q000378:metabolism; D002952:Citric Acid Cycle / Q000502:physiology; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000201:enzymology* / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
chemical_list: D007328:Insulin; D007656:Ketoglutaric Acids; D008293:Malates; D012333:RNA, Messenger; D005973:Glutamine; D019343:Citric Acid; C030298:malic acid; D000255:Adenosine Triphosphate; D007521:Isocitrate Dehydrogenase; D005969:Glutamate Dehydrogenase; D005947:Glucose
keywords: Cytosolic isocitrate dehydrogenase; INS-1 832/13 cell line; Insulin secretion; Mitochondrial isocitrate dehydrogenase; Stable knockdown of isocitrate dehydrogenase; shRNA