Using 13C6 glucose labeling coupled to GC-MS and 2D 1H-13C HSQC NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning steady state insulin release and β cell function. In both mouse and human islets, the contribution of glucose to the TCA cycle is similar. Pyruvate-fueling of the TCA cycle is found to be mediated primarily by the activity of pyruvate dehydrogenase (PDH), with only a limited contribution from pyruvate carboxylase (PC). While conversion

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... of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in both species, lactate accumulation via this route is six-fold higher in human islets. Transcriptomic analysis reveals that human β cells specifically express lactate dehydrogenase B (LDHB) at high levels, in keeping with the phenotype of patients harboring gain-of-function mutations in MCT1/ SLC16A1 (HHF7). Thus, glycolytically-derived acetyl CoA preferentially feeds the TCA cycle in both mouse and human β cells. However, human β cells possess the machinery needed to generate extra-mitochondrial lactate, which might reflect a key mechanism to balance the reducing activity of NADH-producing pathways.