During cell differentiation, intracellular energetic pathways are drastically reorganized. Although this alteration had been considered as a passive process to adapt to environmental change during differentiation, recent studies suggested that the metabolic alteration rather actively regulates the differentiation process. It is known that the dynamic metabolic reprogramming occurs during differentiation of preadipocytes, however its implication in adipogenesis is unknown. In this study, we employed mass spectrometry-based metabolomics and investigated the metabolic change during differentiation of 3T3-L1 preadipocytes. We found that the level of nicotinamide adenine dinucleotide (NAD), a cofactor mediating redox reaction and protein modification, was significantly increased during the differentiation. Consistently Nampt, a rate-limiting enzyme of NAD synthesis, was also upregulated in the early phase of adipogenesis. Further, we found that pharmacological or genetic inhibition of Nampt reduced NAD synthesis and repressed the metabolic reprogramming during adipogenesis. Notably, inhibition of Nampt blocked the gene expression of Pparg and impeded the preadipocyte differentiation. The effect of Nampt inhibition was cancelled by the supplementation of nicotinamide mononucleotide, a precursor of NAD. Our metabolomic analysis also revealed that TCA cycle intermediate, α-ketoglutarate (αKG) was upregulated during the differentiation and Nampt inhibition disturbed the rise of αKG. Interestingly, αKG is known as a cofactor of the demethylation of histones. The ChIP assay revealed that αKG mediated the demethylation of H3K9me3 on Pparg promoter region and promote the gene expression of Pparg during adipogenesis. This process was inhibited by Nampt inhibitor treatment, and was reversed by the supplementation of αKG. Altogether, our data indicated that Nampt-mediated NAD synthesis is necessary for differentiation of preadipocyte.
K. Okabe: None. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. T. Kado: None. Y. Igarashi: None. K. Yagi: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura & Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. T. Nakagawa: None.
