Succinate stimulates insulin secretion and proinsulin biosynthesis. We studied the effects of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-modulating pathways on glucose- and succinate-stimulated insulin secretion and proinsulin biosynthesis in the rat and the insulin-resistant Psammomys obesus. Disruption of the anaplerotic pyruvate/malate shuttle by phenylacetic acid inhibited glucose- and succinate-stimulated insulin secretion and succinate-stimulated proinsulin biosynthesis in both species. In contrast, phenylacetic acid failed to inhibit glucose-stimulated proinsulin biosynthesis in P. obesus islets. Inhibition of the NADPH-consuming enzyme neuronal nitric oxide synthase (nNOS) with l-N(G)-nitro-l-arginine methyl ester or with N(G)-monomethyl-l-arginine(G) doubled succinate-stimulated insulin secretion in rat islets, suggesting that succinate- and nNOS-derived signals interact to regulate insulin secretion. In contrast, nNOS inhibition had no effect on succinate-stimulated proinsulin biosynthesis in both species. In P. obesus islets, insulin secretion was not stimulated by succinate in the absence of glucose, whereas proinsulin biosynthesis was increased 5-fold. Conversely, under stimulating glucose levels, succinate doubled insulin secretion, indicating glucose-dependence. Pyruvate ester and inhibition of nNOS partially mimicked the permissive effect of glucose on succinate-stimulated insulin secretion, suggesting that anaplerosis-derived signals render the beta-cells responsive to succinate. We conclude that beta-cell anaplerosis via pyruvate carboxylase is important for glucose- and succinate-stimulated insulin secretion and for succinate-stimulated proinsulin biosynthesis. In P. obesus, pyruvate/malate shuttle dependent and independent pathways that regulate proinsulin biosynthesis coexist; the latter can maintain fuel stimulated biosynthetic activity when the succinate-dependent pathway is inhibited. nNOS signaling is a negative regulator of insulin secretion, but not of proinsulin biosynthesis.