To investigate whether the beiging process changes the interactive effects of salt and MEK6 gene on inflammatory adipogenesis, the salt treatment (NaCl 50 mM) and MEK6 transfection of Tg(+/+) cells were performed with white adipocytes (WAT) and beige-like-adipocytes (BLA). BLA induced by T3 were confirmed by UCP-1 expression and the MEK6 protein was 3.5 times higher in MEK6 transfected WAT than the control. The adipogenic genes, PPAR-γ and C/EBP-α, were 1.5 times more highly expressed in the salt-treated groups than the non-salt-treated groups, and adipogenesis was greatly increased in Tg(+/+) WAT compared to non-transfected Tg(−/−). The adipogenesis induced by salt treatment and MEK6 transfection was significantly reduced in BLA. The inflammatory adipocytokines, TNF-α, IL-1β, and IL-6, were increased in the salt-treated Tg(+/+) WAT, but an anti-inflammation biomarker, the adiponectin/leptin ratio, was reduced in Tg(+/+), to tenth of that in Tg(−/−). However, the production of adipocytokines in WAT was strongly weakened in BLA, although a combination of salt and MEK6 transfection had the most significant effects on inflammation in both WAT and BLA. Oxygen consumption in mitochondria was maximized in salt-treated and MEK6 transfected WAT, but it was decreased by 50% in BLA. In conclusion, beiging controls the synergistic effects of salt and MEK6 on adipogenesis, inflammation, and energy expenditure.

Objectives Both obesity and obesogenic environments(OE) to induce the fat accumulation are being the risk factors of various metabolic diseases. By 6 year-cohort study, we found that MEK6 gene and salt intake were positively related with inducing obese as part of OE factors such as lifestyle, genes, and eating habits etc. Objectives of study are to find the effects of MEK6 gene and salt treatment on adipogenesis using MEK6 over-expressed 3T3-L1 cells. Methods After transformation for MEK6 over-expressed 3T3-L1 with lipofectamine 3000 (Invitrogen, California, USA) was confirmed by PCR method, salt was treated as 50 mM in the form of NaCl without cytotoxicity (MTT assay). Cell differentiation and TG synthesis (Oil Red O; ORO & DAPI/Nile red staining), and protein expression (western blotting analysis) associated with adipogenesis genes (PPAR-γ, C/EBP-α & aP2) and adipocytokines (adiponectin & leptin) were performed. ELISA kits were used for estimating inflammatory factors such as TNF-α, IL-1β, IL-10, MCP-1, PAI-1 etc. Real-time oxygen consumption in alive cells was measured every 17 seconds for 100 minutes. (Mito-Xpress O2 consumption array kit) All analyses were performed using SAS9.1 software and p-values of <0.05 were interpreted as statistically significant. Results We confirmed the salt induced the protein expression associated with adipogenesis (PPAR-γ, C/EBP-α and aP2) in both control and MEK6 over-expressed cells. The levels of inflammatory cytokine factors (TNF-α, IL-1β, IL-10) were also increased by salt treatment in both groups. We found that obesity-linked metabolism such as lipolysis, insulin resistance, and adipocyte production were significantly higher in MEK6 over-expressed cells than them in the control. However, the leptin level did not seem to be associated with MEK6 gene. Salt also increased O2 consumption in both groups but the time to reach maximum of O2 consumption in MEK6 over-expressed cells was slower than the case of control. MEK6 might be associated with mitochondria function to control O2 consumption. Conclusions The obesity metabolism related adiopgenesis in 3T3-L1 was activated by MEK6 expression and salt treatment. These findings will contribute to a future study for finding MEK6 linked mechanisms involved in adipogenesis and for setting DRI of salt intake in obese Koreans. Funding Sources This work was funded by the Ministry of Health&Welfare, Republic of Korea grant number: HI17C0863.