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 C-13-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 sub-stimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was similar to40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mM stimulated insulin secretion similar to4-fold, and was associated with a similar to4-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 similar to6-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.