Glucose homeostasis depends upon the appropriate release of insulin from pancreatic islet beta-cells. Postpandrial changes in circulating nutrient concentrations are coupled with graded release of stored insulin pools by the proportional changes in mitochondrial metabolism. The corresponding increased synthesis rates of both ATP and of anaplerotic metabolites have been shown to be mediators for nutrient-stimulated insulin secretion. Anaplerosis leads to the export of malate or citrate from the mitochondria, both of which can be recycled through metabolic pathways to reenter the Kreb's cycle. These metabolic cycles have the net effect of either transferring mitochondrial reducing equivalents to the cytosol, or of efficiently providing pyruvate to facilitate responsive changes in the Kreb's cycle flux in proportion to increased availability of glutamate and anaplerotic flux through glutamate dehydrogenase. Here, we describe siRNA knock-down and isotopic labeling strategies to evaluate the role of cytosolic and mitochondrial isoforms of malic enzyme in facilitating malate-pyruvate cycling in the context of fuel-stimulated insulin secretion.