Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

Integrating glucose and fat Consuming too much glucose makes you fat, but it is unclear how this conversion is mediated by the body. Glycolysis links to gene transcription via the essential coenzyme nicotinamide adenine dinucleotide in its oxidized state (NAD+). Ryu et al. found that compartmentalized NAD+ synthesis and consumption integrate glucose metabolism and adipogenic (fat-promoting) transcription during adipocyte differentiation (see the Perspective by Trefely and Wellen). Competition between the NAD+ precursors—nuclear NMNAT-1 and cytosolic NMNAT-2—for their common substrate, nicotinamide mononucleotide, regulates the balance between nuclear NAD+ synthesis for adipogenic gene regulation and cytosolic NAD+ synthesis used in metabolism. Science, this issue p. eaan5780; see also p. 603 Compartmentalized NAD+ synthesis integrates cellular metabolism and signal-dependent transcriptional programs. 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. Compartmentalized NAD+ biosynthesis by NMNATs regulates adipogenesis through PARP-1. NMNATs synthesize NAD+ from nicotinamide mononucleotide (NMN) and adenosine triphosphate. Nuclear NMNAT-1 provides NAD+ for nuclear ADP-ribosylation and gene regulation by PARP-1, whereas cytoplasmic NMNAT-2 provides NAD+ for cytosolic ADP-ribosylation and cellular metabolism. Competition between NMNAT-1 and NMNAT-2 for their common substrate, NMN, promotes compartmentalized regulation of NAD+ levels, allowing for discrete nuclear and cytoplasmic events. The fluorescent images of NAD+ in the bottom panel were generated using a NAD+ sensor localized to the nucleus (left) or the cytoplasm (right). NAD+ (nicotinamide adenine dinucleotide in its oxidized state) is an essential molecule for a variety of physiological processes. It is synthesized in distinct subcellular compartments by three different synthases (NMNAT-1, -2, and -3). We found that compartmentalized NAD+ synthesis by NMNATs integrates glucose metabolism and adipogenic transcription during adipocyte differentiation. Adipogenic signaling rapidly induces cytoplasmic NMNAT-2, which competes with nuclear NMNAT-1 for the common substrate, nicotinamide mononucleotide, leading to a precipitous reduction in nuclear NAD+ levels. This inhibits the catalytic activity of poly[adenosine diphosphate (ADP)–ribose] polymerase–1 (PARP-1), a NAD+-dependent enzyme that represses adipogenic transcription by ADP-ribosylating the adipogenic transcription factor C/EBPβ. Reversal of PARP-1–mediated repression by NMNAT-2–mediated nuclear NAD+ depletion in response to adipogenic signals drives adipogenesis. Thus, compartmentalized NAD+ synthesis functions as an integrator of cellular metabolism and signal-dependent transcriptional programs.