Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic ␤-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 13 C-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 ϳ40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mM stimulated insulin secretion ϳ4-fold, and was associated with a ϳ4-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 ϳ6-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 ␤-cells and that anaplerosis through GDH does not play a major role in this process.Glucose homeostasis depends on the appropriate release of insulin from the pancreatic ␤-cell in response to increases in blood glucose concentrations. Although, it is well established that glucose-stimulated insulin secretion requires mitochondrial carbohydrate oxidation to drive ATP production (1), a number of studies have suggested that intermediates (e.g. malonyl-CoA (2, 3), succinate (4), and glutamate (5)) generated, directly or indirectly, from anaplerosis may act as second messengers linking glucose oxidation with insulin secretion.Previous studies of anaplerotic flux have typically examined only a single anaplerotic pathway (pyruvate carboxylase (PC), 1 or glutamate dehydrogenase (GDH)) with a single substrate. Given the complexity and interdependence of these fluxes, a comprehensive and simultaneous evaluation of the relative contribution and regulation of alternate anaplerotic pathways and substrates into the Kreb's cycle is essential to understand the relative role of these individual fluxes in the regulation of glucose-stimulated insulin secretion. The experimental design of most previous studies have either inhibited competing anaplerotic pathways or used methodology that preclude any analysis of the coordination of flux through competing anaplerotic pathways. To address these limitations, we examined the relationship between insulin secretion and the pathways of anaplerosis in clonal INS-1 832/13 cells under conditions of low (2.5 mM) and high (15 mM) glucose. In addition, we also examined the effec...