Impaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase

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dc.contributor.authorHasan, N.
dc.contributor.authorLongacre, M.
dc.contributor.authorStoker, S.
dc.contributor.authorBoonsaen, T.
dc.contributor.authorJitrapakdee, S.
dc.contributor.authorKendrick, M.
dc.contributor.authorWallace, J.
dc.contributor.authorMacDonald, M.
dc.date.issued2008
dc.description.abstractAnaplerosis, 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 β-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 14C into lipid from [U-14C]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.
dc.description.statementofresponsibilityNoaman M. Hasan, Melissa J. Longacre, Scott W. Stoker, Thirajit Boonsaen, Sarawut Jitrapakdee, Mindy A. Kendrick, John C. Wallace and Michael J. MacDonald
dc.identifier.citationJournal of Biological Chemistry, 2008; 283(42):28048-28059
dc.identifier.doi10.1074/jbc.M804170200
dc.identifier.issn0021-9258
dc.identifier.issn1083-351X
dc.identifier.urihttp://hdl.handle.net/2440/52371
dc.language.isoen
dc.publisherAmer Soc Biochemistry Molecular Biology Inc
dc.relation.granthttp://purl.org/au-research/grants/arc/DP0346807
dc.relation.granthttp://purl.org/au-research/grants/arc/DP0346807
dc.source.urihttps://doi.org/10.1074/jbc.m804170200
dc.subjectCell Line
dc.subjectAnimals
dc.subjectHumans
dc.subjectMice
dc.subjectRats
dc.subjectInsulinoma
dc.subjectInsulin
dc.subjectPyruvate Carboxylase
dc.subjectLipids
dc.subjectRNA, Small Interfering
dc.subjectRNA, Messenger
dc.subjectTransfection
dc.subjectPhosphorylation
dc.subjectModels, Biological
dc.subjectInsulin-Secreting Cells
dc.subjectInsulin Secretion
dc.titleImpaired anaplerosis and insulin secretion in insulinoma cells caused by small interfering RNA-mediated suppression of pyruvate carboxylase
dc.typeJournal article
pubs.publication-statusPublished

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