Since WNK overexpression accelerated expression of adipogeni

Since WNK4 overexpression accelerated motilin receptor agonist of adipogenic genes (Fig. S7), we expected the overexpression of WNK4 in adipocytes would cause the phenotypes opposite to those in WNK4−/− mice. We first checked the WNK4 protein levels in the adipose tissues of WNK4D561A/+ and WNK4-Tg mice, which we have generated previously (Fig. S10). Unexpectedly, however, we found that the increase of WNK4 in adipose tissue of these mice was not so dramatic as we observed previously in the kidney (Figs. S10a and S10b). Indeed, the WNK4-Tg mice showed similar body weights to WT mice at 8weeks of age and even after the introduction of a HFD (Fig. S10). There were no apparent differences in the metabolic phenotypes between the genotypes. The reason(s) for the unexpected minimal increase in the levels of WNK4 protein in the adipose tissue of WNK4-Tg mice is not clear, but some compensatory mechanisms may be working to prevent excessive increases in WNK4 in tissues other than kidney. Thus, it is not clear at present whether WNK4 overexpression in adipose tissue causes diet-induced obesity. WNK4 in adipose tissue of PHAII patients might not be increases as in the PHAII model mice, which may be consistent with the clinical observation that PHAII patients do not necessarily show metabolic disorders. In conclusion, we revealed a role for WNK4 as a regulator of core adipogenic factors. Our results may offer insights into the complex process of obesity. Our data indicated that WNK4 inhibition would be beneficial in management of blood pressure and MetS. Since the recently reported pan-WNK inhibitor (Yamada et al., 2016) specifically targets WNK\'s kinase domain, it may not be effective for inhibiting WNK4’s adipogenic function. The strategy to inhibit WNK4 induction in the early adipogenesis and/or to stimulate WNK4 degradation by the recently identified KLHL3/Cul3 E3 ligase would be possible.
Acknowledgements and Funding Sources We thank C. Iijima, K. Hashimoto, and A. Imono for help in the experiments. This study was supported in part by Grants-in-Aid for Scientific Research (S) from the Japan Society for the Promotion of Science (No. 25221306), a Health Labor Science Research Grant from the Ministry of Health Labour and Welfare (No. 1211001), the Salt Science Research Foundation (No. 1026, 1228), and the Takeda Science Foundation (No. X2365).
Conflicts of Interest
Author Contributions
Introduction Calorie restriction (CR) has been reported to extend the lifespan of many model organisms including rodents and possibly non-human primates (Colman et al., 2009; Guarente, 2000; McCay et al., 1935). It has been proposed that some of the positive health effects of CR are mediated, at least in part, by the Sir2 family of NAD+-dependent protein deacetylases (Guarente, 2000). Although increasing the expression levels of Sirt1, the mammalian orthologue of Sir2, does not extend lifespan, it appears to extend healthspan (Bordone and Guarente, 2005; Herranz et al., 2010), improve insulin sensitivity and protect against hepatic steatosis in transgenic mice (Banks et al., 2008; Bordone et al., 2007; Boutant et al., 2016; Pfluger et al., 2008). Therefore, Sirt1 may be a target for developing therapeutics to treat aging-related diseases. Resveratrol, a polyphenol found in red wine and other plant-based foods, was the first of the Sirt1-activating compounds (STACs) to be reported and has been described as a CR-mimetic (Howitz et al., 2003). Resveratrol increased mitochondrial biogenesis, metabolic rate, insulin sensitivity and glucose tolerance and protected against premature death due to a high fat diet in mice (Baur et al., 2006; Lagouge et al., 2006). However, resveratrol was not Sirt1-specific (Borra et al., 2005; Kaeberlein et al., 2005) so a group of synthetic STACs, which are structurally unrelated to resveratrol, were subsequently developed. Synthetic STACs such as SRT1720 protected mice against obesity; increased mitochondrial biogenesis, energy metabolism and glucose tolerance (Feige et al., 2008; Milne et al., 2007); decreased hepatic lipid accumulation (Walker et al., 2010; Yamazaki et al., 2009) and extended the lifespan in mice (Minor et al., 2011b; Mitchell et al., 2014). Because weight loss can be related to all of these benefits, it was not known whether they are all simply due to the weight loss induced by SRT1720.