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Subcellular NAMPT-mediated NAD⁺ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.

ABSTRACT: NAD⁺ is a cofactor required for glycolysis, tricarboxylic acid cycle, and complex I enzymatic reaction. In mammalian cells, NAD⁺ is predominantly synthesized through the salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme. Previously, we demonstrated that NAMPT exerts a neuroprotective effect in ischemia through the suppression of mitochondrial dysfunction. Mammalian cells maintain distinct NAD⁺ pools in the cytosol, mitochondria, and nuclei. However, it is unknown whether mitochondria have an intact machinery for NAD⁺ salvage, and if so, whether it plays a dominant role in bioenergetics, mitochondrial function, and neuronal protection after ischemia. Here, using mouse primary cortical neuron and cortical tissue preparations, and multiple technologies including cytosolic and mitochondrial subfractionation, viral over-expression of transgenes, molecular biology, and confocal microscopy, we provided compelling evidence that neuronal mitochondria possess an intact machinery of NAMPT-mediated NAD⁺ salvage pathway, and that NAMPT and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) are localized in the mitochondrial matrix. By knocking down NMNAT1-3 and NAMPT with siRNA, we found that NMNAT3 has a larger effect on basal and ATP production-related mitochondrial respiration than NMNAT1-2 in primary cultured neurons, while NMNAT1-2 have a larger effect on glycolytic flux than NMNAT3. Using an oxygen glucose deprivation model, we found that mitochondrial, cytoplasmic, and non-subcellular compartmental over-expressions of NAMPT have a comparable effect on neuronal protection and suppression of apoptosis-inducing factor translocation. The current study provides novel insights into the roles of subcellular compartmental NAD⁺ salvage pathways in NAD⁺ homeostasis, bioenergetics, and neuronal protection in ischemic conditions.

SUBMITTER: Wang X

PROVIDER: S-EPMC6917901 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Subcellular NAMPT-mediated NAD+ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.

Wang Xiaowan X Zhang Zhe Z Zhang Nannan N Li Hailong H Zhang Li L Baines Christopher P CP Ding Shinghua S

Journal of neurochemistry 20191016 6

NAD+ is a cofactor required for glycolysis, tricarboxylic acid cycle, and complex I enzymatic reaction. In mammalian cells, NAD+ is predominantly synthesized through the salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme. Previously, we demonstrated that NAMPT exerts a neuroprotective effect in ischemia through the suppression of mitochondrial dysfunction. Mammalian cells maintain distinct NAD+ pools in the cytosol, ...[more]

PMID: 31553812

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Project description:The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV, would suffice for an NAD+ salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli, the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro, and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD+ biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD+, and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD+ biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD+ for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, nadV and natV, share an evolutionary history in their respective phage-host group.

Project description:ObjectiveThe ability of adipose tissue to expand and contract in response to fluctuations in nutrient availability is essential for the maintenance of whole-body metabolic homeostasis. Given the nutrient scarcity that mammals faced for millions of years, programs involved in this adipose plasticity were likely evolved to be highly efficient in promoting lipid storage. Ironically, this previously advantageous feature may now represent a metabolic liability given the caloric excess of modern society. We speculate that nicotinamide adenine dinucleotide (NAD+) biosynthesis exemplifies this concept. Indeed NAD+/NADH metabolism in fat tissue has been previously linked with obesity, yet whether it plays a causal role in diet-induced adiposity is unknown. Here we investigated how the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) supports adipose plasticity and the pathological progression to obesity.MethodsWe utilized a newly generated Nampt loss-of-function model to investigate the tissue-specific and systemic metabolic consequences of adipose NAD+ deficiency. Energy expenditure, glycemic control, tissue structure, and gene expression were assessed in the contexts of a high dietary fat burden as well as the transition back to normal chow diet.ResultsFat-specific Nampt knockout (FANKO) mice were completely resistant to high fat diet (HFD)-induced obesity. This was driven in part by reduced food intake. Furthermore, HFD-fed FANKO mice were unable to undergo healthy expansion of adipose tissue mass, and adipose depots were rendered fibrotic with markedly reduced mitochondrial respiratory capacity. Yet, surprisingly, HFD-fed FANKO mice exhibited improved glucose tolerance compared to control littermates. Removing the HFD burden largely reversed adipose fibrosis and dysfunction in FANKO animals whereas the improved glucose tolerance persisted.ConclusionsThese findings indicate that adipose NAMPT plays an essential role in handling dietary lipid to modulate fat tissue plasticity, food intake, and systemic glucose homeostasis.

Dataset Information

Subcellular NAMPT-mediated NAD⁺ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.

Publications

Subcellular NAMPT-mediated NAD<sup>+</sup> salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.

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