13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells

Gary W. Cline, Rebecca L. LePine, Klearchos K Papas, Richard G. Kibbey, Gerald I. Shulman

Research output: Contribution to journalArticle

98 Citations (Scopus)

Abstract

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic β-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 ∼40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mM stimulated insulin secretion ∼4-fold, and was associated with a ∼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 ∼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 β-cells and that anaplerosis through GDH does not play a major role in this process.

Original languageEnglish (US)
Pages (from-to)44370-44375
Number of pages6
JournalJournal of Biological Chemistry
Volume279
Issue number43
DOIs
StatePublished - Oct 22 2004
Externally publishedYes

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Nuclear magnetic resonance
Pyruvate Carboxylase
Insulin
Fluxes
Glucose
Glutamate Dehydrogenase
Pyruvic Acid
Glutamine
Carbon-13 Magnetic Resonance Spectroscopy
Perfusion
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells. / Cline, Gary W.; LePine, Rebecca L.; Papas, Klearchos K; Kibbey, Richard G.; Shulman, Gerald I.

In: Journal of Biological Chemistry, Vol. 279, No. 43, 22.10.2004, p. 44370-44375.

Research output: Contribution to journalArticle

Cline, Gary W. ; LePine, Rebecca L. ; Papas, Klearchos K ; Kibbey, Richard G. ; Shulman, Gerald I. / 13C NMR isotopomer analysis of anaplerotic pathways in INS-1 cells. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 43. pp. 44370-44375.
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