Rate-limiting Enzyme In Salvage Pathway Research Articles

1987-P: Nampt Deletion Changes Metabolism in Adipose Tissue and Prevents Diet-Induced Obesity

Nampt is the rate-limiting enzyme in salvage pathway in which NAD is mainly synthesized in mammalian cells. NAD plays very important role in various biological processes, such as energy production, energy consumption, aging, cell death, and DNA damage repair as a coenzyme of redox reactions or as a substrate of Sirtuins or Poly (ADP-ribose) polymerases. Besides, Nampt is highly expressed in mature adipocyte and secreted into extracellular. The extracellular Nampt is known to regulate systemic metabolic state. In short, Nampt works as a metabolic sensor and NAD integrates systemic metabolism. Recently, metabolism is thought to be closely related to cell differentiation. However, how NAD metabolism affects to adipogenesis remains unknown. In this study, we generated the adipose tissue specific Nampt knock out mice, Adiponectin-Cre Nampt flox/flox (KO) and conducted targeted-metabolomic analysis to investigate the metabolic effect, employing liquid-chromatograph mass spectrometry and gas-chromatograph mass spectrometry. Interestingly, KO showed resistant against diet-induced obesity (DIO), fed high fat high sucrose diet. Body weight and both inguinal and epididymal fat mass were significantly reduced in KO compared to wild type control mice. On the other hand, we revealed that Nampt-mediated NAD synthesis was up-regulated during differentiation of 3T3-L1 preadipocytes and the inhibition of Nampt significantly suppressed the differentiation. Our metabolomic analysis and ChIP-qPCR assay showed NAD contributed to the differentiation through the metabolic reprogramming and the subsequent epigenetic alteration to activate the transcription of adipogenic genes. This result indicated Nampt-mediated NAD synthesis regulated differentiation of preadipocytes to mature adipocytes. Together, our data suggested that adipose tissue specific Nampt deletion resulted in the resistance against DIO and it could be partly attributed to the inhibited differentiation of preadipocytes. Disclosure K. Okabe: None. A. Nawaz: Research Support; Spouse/Partner; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company. Research Support; Self; Japan Society for the Promotion of Science. Research Support; Spouse/Partner; Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Shionogi & Co., Ltd., Takeda Pharmaceutical Company Limited. S. Fujisaka: None. K. Yagi: None. T. Kado: None. I. Usui: None. T. Nakagawa: None. K. Tobe: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Japan Diabetes Society. Research Support; Self; Bristol-Myers Squibb Company. Funding Japan Society for the Promotion of Science

Schedule-dependent synergistic effects of 5-fluorouracil and selumetinib in KRAS or BRAF mutant colon cancer models

Combination of MEK inhibitor and 5-FU had showed limited efficacy in clinical trials. We previously reported that acquired resistance to 5-FU was related with continued activation of salvage pathway. Here we investigated whether combination of 5-FU and a MEK inhibitor had treatment sequence-dependent synergistic effects in KRAS or BRAF mutant colon cancer models. Treatment with 5-FU followed by selumetinib (FS) induced highest cell death and synergy compared with reverse (SF) and concomitant (cFS) treatment in six cell lines. SF or cFS combination induced synergy in 1 or 2 cell lines, respectively, in which the synergy was less than that by FS combination. FS enhanced apoptosis and decreased anchorage-independent growth. Induction of thymidine kinase 1, a rate-limiting enzyme in salvage pathway, by 5-FU was abrogated by subsequent treatment with selumetinib, and ERK reactivation after selumetinib was prohibited by pretreatment with 5-FU. FS altered mRNA expression in groups of genes distinct from SF. Administration of 5-FU (10 or 30 mg/kg/day) for 7 days, followed by selumetinib (10 or 25 mg/kg/day) for another 7 days, in colo205 and HCT8 xenograft models significantly decreased tumor growth compared with a single agent. However, co-administration in the reverse sequence did not show the difference in tumor size compared with the treatment of single agent. Decreased expression of Ki67 was observed in tumors from mice treated with FS. Our results suggest that sequential administration of 5-FU plus selumetinib would be a promising strategy for patients having KRAS or BRAF mutant colon cancers.