br Contributors br Declaration of Interests br Acknowledgeme

Contributors
Declaration of Interests
Acknowledgements The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project (MAL-ED) is carried out as a collaborative project supported by the Bill & Melinda Gates Foundation (47075), the Foundation for the National Institutes of Health, and the National Institutes of Health, Fogarty International Center. We particularly want to thank the children and caregivers who participated in the study for their invaluable contributions. Tom Brewer and Gretchen Meller are also thanked for their early support of the project. We are truly indebted to the following individuals for their advisory role in the MAL-ED study: Henry Binder, Maureen Black, Kathleen Braden, Robert Breiman, Susan Bull, Katherine Dewey, Christopher Duggan, Christine Grady, Gerry Keusch, Nancy Krebs, Claudio Lanata, James Nataro, Jerome Singh, Peter Smith, Phillip Tarr, Katherine Tucker and Theodore Wachs. Fraser Lewis is recognized and appreciated for his critical review of and guidance on the analytical methods and statistical approaches applied in the MAL-ED study. We thank Leslie Pray and Alicia Livinski for their editorial assistance. Special thanks go to Roger Glass and George Griffin for their continued support and guidance.
Introduction Metabolic syndrome (MetS), characterized by insulin resistance, dyslipidemia, hypertension, and central obesity, is a significant clinical problem associated with cardiovascular disease and diabetes (Alberti et al., 2009). Because white adipose tissue (WAT) is the main buy flumazenil storage organ and secretes several humoral factors (Cristancho and Lazar, 2011), it has important roles in MetS. Core transcriptional factors regulating adipocyte differentiation include the master regulators, peroxisome proliferator-activated receptor gamma (PPARγ), and CCAAT/enhancer-binding protein alpha (C/EBPα) (Lefterova et al., 2008), and moderate reduction of PPARγ activity was shown to prevent obesity and improve insulin sensitivity (Kubota et al., 1999; Yamauchi et al., 2001; Jones et al., 2005). The with-no-lysine kinases (WNKs) are a family of serine/threonine kinases composed of four human genes, WNK1, WNK2, WNK3, and WNK4. WNK1 and WNK4 were identified as being responsible for pseudohypoaldosteronism type II (PHAII; (Wilson et al., 2001), a hereditary hypertensive disease characterized by hyperkalemia, metabolic acidosis, and thiazide sensitivity (Gordon, 1986). We and others have clarified that WNK regulates the NaCl cotransporter (NCC) in the distal convoluted tubules of the kidney through phosphorylation of oxidative stress-responsive 1 (OSR1) and Ste20-like proline/alanine-rich kinase (SPAK) (Yang et al., 2007; Chiga et al., 2011). Recent studies have added further information that insulin phosphorylates WNK1 through protein kinase B (PKB)/Akt (Jiang et al., 2005), and that insulin is a powerful activator of the WNK4–OSR1/SPAK–NCC signaling cascade in vitro and in vivo (Sohara et al., 2011; Nishida et al., 2012; Takahashi et al., 2014). These data suggest that activation of the WNK–OSR1/SPAK–NCC signaling cascade caused by hyperinsulinemia may underlie the pathogenesis of salt-sensitive hypertension in MetS. In extrarenal organs, recent studies have suggested that WNKs regulate cell growth, differentiation, and development. For example, WNK1 is required for mitosis in cultured cells (Tu et al., 2011). In addition, WNK2 is known to suppress cell growth (Hong et al., 2007), while WNK3 increases cell survival (Veríssimo et al., 2006), and WNK4 is required for the anterior formation of Xenopus embryos (Shimizu et al., 2013). However, the functions of WNKs in energy metabolism remain unknown. In the present study, we reported a role of WNK4 as a regulator of adipocyte development. We found that WNK4 was expressed in the mouse adipose tissue. In primary preadipocytes and 3T3-L1 adipocytes, WNK4 was increased dramatically in the early phase of differentiation. The suppression of endogenous WNK4 decreased expression of PPARγ and C/EBPα, resulting in the impaired formation of mature adipocytes in 3T3-L1 cells and human mesenchymal stem cells. We also found that WNK4 was involved in cell cycle progression during mitotic clonal expansion (MCE), which might be the cause of decreased expression of PPARγ by C/EBPβ by WNK4 suppression. Consistent with the reduced expression of PPARγ, adult WNK4−/− (WNK4-KO) mice exhibited reduced adiposity and the decreased expression of adipogenic genes on high-fat diets (HFDs), suggesting the involvement of WNK4 in the development of obesity. These results suggested that the hypertension-causing gene WNK4 not only is involved in the regulation of salt-sensitive hypertension, but also in energy metabolism.