SummaryIncretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is nonselectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy-dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2-type ribonuclease, generating 3 0 -NMPs that were immediately converted to nucleosides by the vacuolar non-specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re-assimilated, but excreted from the cell. Bulk non-selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.

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Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADPribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro 35S :AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro 35S :AtNUDX7 plants but increased in the KOnudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro 35S :AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro 35S :AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD + and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro 35S :AtNUDX7 plants. Accumulation of NAD + and ATP was observed in the KO-nudx7-and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD + by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD + levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.