Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis.

INTRODUCTION Nicotinamide adenine dinucleotide (NAD) is an essential small molecule that is involved in a variety of physiological and pathological processes. The oxidized form, NAD + , serves as a cofactor in metabolic pathways, as well as a substrate for various enzymes that consume it, such as the poly[adenosine diphosphate (ADP)–ribose] polymerases (PARPs) and sirtuins (SIRTs). PARPs and SIRTs cleave NAD + into nicotinamide and ADP-ribose, resulting in the irreversible breakdown of NAD + . Therefore, the resynthesis of NAD + is necessary for maintaining normal cellular functions. Increasing evidence has revealed that (i) reduced NAD + levels result in altered metabolism and increased disease susceptibility and (ii) restoration of NAD + levels can prevent disease progression. Thus, understanding NAD + synthesis and catabolism is important for understanding physiological and pathological processes. RATIONALE NAD + is synthesized by a family of enzymes known as nicotinamide mononucleotide adenylyl transferases (NMNATs). In mammalian cells, NMNATs exhibit distinct subcellular localizations (NMNAT-1 in the nucleus, NMNAT-2 in the cytoplasm and Golgi, and NMNAT-3 in the mitochondria), suggesting that NAD + biosynthesis is compartmentalized within the cell. Despite the biological importance of NAD + , the physiological role of compartmentalized NAD + biosynthesis in cells is largely unexplored. Given the dual role of NAD + as a metabolic cofactor and a substrate for enzymes involved in gene regulation, we hypothesized that compartmentalized synthesis of NAD + might connect cellular metabolism and gene regulation. RESULTS Here we show that compartment-specific NAD + biosynthesis acts as a key mediator of PARP-1–regulated transcription during adipocyte differentiation, integrating cellular metabolism and the adipogenic transcription program. During adipogenesis, nuclear NAD + levels drop concomitantly with a rapid induction of NMNAT-2, the cytoplasmic NAD + synthase. Increased NMNAT-2 levels limit the availability of nuclear NMN, a common substrate of NMNATs, thereby leading to a precipitous reduction in nuclear NAD + synthesis by NMNAT-1. This reduction of nuclear NAD + results in decreased PARP-1 catalytic activity, which in turn reduces inhibitory ADP-ribosylation of the adipogenic transcription factor C/EBPβ. Reduced ADP-ribosylation of C/EBPβ allows it to bind its target genes and drive a proadipogenic transcriptional program that promotes the differentiation of preadipocytes into adipocytes. Experimentally, we found that decreasing nuclear NAD + synthesis by NMNAT-1 depletion significantly reduced PARP-1 enzymatic activity and enhanced adipogenesis, whereas NMNAT-2 depletion inhibited the drop in nuclear NAD + levels and significantly reduced adipocyte differentiation. Moreover, providing exogenous NMN to preadipocytes in culture “short-circuited” the competition between NMNAT-1 and NMNAT-2 for NMN, leading to increased nuclear NAD + synthesis during differentiation. This, in turn, increased PARP-1 activity and inhibited adipocyte differentiation. Adipogenic signaling pathways and increased glucose metabolism were required for the rapid induction of NMNAT-2, and inhibition of glucose metabolism completely abolished the induction of NMNAT-2 during adipogenesis. Preventing NMNAT-2 induction by glucose deprivation restored PARP-1 activity and inhibited C/EBPβ-dependent gene expression. Collectively, these results suggest that NMNAT-1 and NMNAT-2 function as sensors to integrate cellular metabolism and the adipogenic transcription program. CONCLUSION We have elucidated a pathway leading from glucose uptake and metabolism, to competition between nuclear and cytoplasmic NMNATs for the NAD + biosynthesis precursor NMN, and ultimately to alterations in the activity of PARP-1 and its catalytic target C/EBPβ, a transcription factor that promotes adipogenic gene expression and initiates the process of adipocyte differentiation. Such mechanisms are also likely to play a key role in other biological systems that exhibit dramatic changes in nuclear PARylation as differentiation proceeds or have a high metabolic load.