Page 49 - N. Metabolism and metabolic diseases

WDR76 mediates obesity and hepatic steatosis via Hras destabilization
Seol Hwa Seo¹,², Jong-Chan Park¹,², Woo-Jeong Jeong¹,², Kang-Yell Choi¹,²,³
¹Translational Research Center for Protein Function Control, Yonsei University, Seoul 03722, Korea, ²Department of
Biotechnology, Yonsei University, Seoul 03722, Korea, ³CK Biotechnology Inc., CK Biotechnology Inc., Seoul 03722, Korea

BACKGROUND AIM

 The Ras/ERK pathway plays important roles in adipogenesis via many  To identified the role of WDR76 in Hras destabilization related to
functions from early to late adipocyte differentiation (1).
adipocyte differentiation of the 3T3-L1 preadipocytes.
 The Ras/ERK pathway activation is required for mitotic clonal expansion in
early adipocyte differentiation (1).  To characterize the role of WDR76 in HFD-induced obesity and hepatic
 WDR76, a component of E3 ubiquitin ligase complex, as one of the Hras steatosis.
binding proteins that mediates Ras degradation (2,3).  To suggest WDR76 as a potential target for the treatment of obesity and
 The role Hras protein stability regulation by WDR76 in HFD-induced obesity metabolic diseases related HFD.
and hepatic steatosis are unknown.
METHODS
Cell culture and adipocyte differentiation. The 3T3-L1 cells were infected with lentivirus and adipocyte differentiation was induced after 48 h. For adipocyte
differentiation, confluent cells were induced to differentiate in DMEM containing 10% fetal bovine serum (FBS; Gibco) and MDI (520 μM methylisobutylxanthine (IBMX;
Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich) and 167 nM insulin(Gibco)). After 2 days, the medium was changed with DMEM containing 10% FBS and
insulin. On day 4, the medium was replaced with DMEM containing 10% FBS and changed with fresh identical medium every 2 days for up to day 8. On day 8, cells
were harvested for further analyses.
Animals and dietary treatments. The 4–5-week-old Wdr76 +/+ and Wdr76 −/− littermates (weight matched) were fed with a HFD (60% calories from fat; Research Diet,
D12492) for 9 weeks. The mice were weighed once a week for 9 weeks. The 4–5-week-old Wdr76 +/+ and Wdr76 Li-TG littermates (weight-matched) were fed with a HFD
(45% calories from fat; Research Diet, D12451) for 8 weeks. The mice were weighed once a week for 8 weeks.
Blood chemistry. Total blood samples from mice were collected by cardiac puncture. The blood was clotted for 30 min and then centrifuged for 10 min at 1,000 × g
to obtain the supernatant. The supernatant was measured for metabolic parameters. Plasma free fatty acid (FFA) concentrations in plasma were measured with an
ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). Serum total cholesterol and triglycerides were measured using FUJI DRI-CHEM slides (Fuji, Japan).
RESULTS

Figure 1. Effects of WDR76 on adipogenesis in 3T3- Figure 2. Wdr76 knockout mice are resistant to Figure 3. Liver-specific Wdr76 overexpression mice
L1 cells. HFD-induced obesity. were susceptible to HFD-induced obesity.

Figure 1. WDR76 mediated adipocyte differentiation of 3T3-L1 cells via destabilization of HRas. In 3T3-L1 cells, knockdown of WDR76 increased HRas protein
levels without changing HRas mRNA levels, resulting in the activation of ERKs (Fig. 1a–c). Knockdown of WDR76 decreases in both mRNA and protein levels of
PPARγ and C/EBPα as well as reduction of adipogenesis (Fig. 1c, d). In contrast, WDR76 overexpression increased the PPARγ and C/EBPα mRNAs and protein
levels with reduction of HRas protein levels and ERKs activation (Fig. 1e–g). Lipid accumulation was increased by WDR76 overexpression (Fig. 1h).
Figure 2. WDR76 deficiency ameliorated HFD-induced obesity in mice. The Wdr76 −/− mice showed reduced body weights (Fig. 2a,b) and the body weight
reduction of the Wdr76 −/− mice was not attributed to differences in their food intake (Fig. 2c). The sizes and weights of epididymal, and perirenal fat tissues of
Wdr76 −/− mice were all reduced compared to those of Wdr76 +/+ mice (Fig. 2d,e). WDR76 deficiency on metabolic disorders, we measured triglyceride (TG), total
cholesterol (TC), and free fatty acid (FFA) levels in the serum of the HFD-fed Wdr76 +/+ and Wdr76 −/− mice. Compared with Wdr76 +/+ mice, Wdr76 −/− mice showed a
decrease in the levels of TG, TC, and FFA in the serum (Fig. 2f–h). Wdr76 −/− mice had improved glucose tolerance and insulin sensitivity (Fig. 2i,j)
Figure 3. The liver-specific overexpression of WDR76 increased obesity and insulin resistance. Wdr76 Li−TG mice showed a severe obesity phenotype after
HFD feeding when compared with Wdr76 +/+ mice (Fig. 3a). Wdr76 Li-TG mice showed more glucose intolerant and insulin resistant phenotypes (Fig. 3b,c). Both sizes
and weights of epididymal, perirenal WATs and liver were increased (Fig. 3d). Wdr76 Li-TG mice had elevated fat depots with thicker subcutaneous fat, increased sizes
of epididymal adipocytes and hepatic steatosis (Fig. 3e–g). Consistent with the increased fat mass by HFD feeding, TG, TC, and FFA levels in serum were increased
in HFD-fed Wdr76 Li-TG mice when compared with those of Wdr76 +/+ mice (Fig. 3h-j).
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

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