13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells.
Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.
Cline
Gary W
GW
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA. gary.cline@yale.edu
Lepine
Rebecca L
RL
Papas
Klearchos K
KK
Kibbey
Richard G
RG
Shulman
Gerald I
GI
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes / Q000378:metabolism; D002460:Cell Line; D002952:Citric Acid Cycle; D003470:Culture Media / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism; D010477:Perfusion; D011522:Protons; D011766:Pyruvate Carboxylase / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D002247:Carbon Isotopes; D003470:Culture Media; D007328:Insulin; D011522:Protons; D005973:Glutamine; D018698:Glutamic Acid; D002244:Carbon; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D011766:Pyruvate Carboxylase; D007930:Leucine; D005947:Glucose; D010100:Oxygen