WDR76 mediates obesity and hepatic steatosis via HRas destabilization

Ras/MAPK (mitogen active protein kinase) signaling plays contradictory roles in adipocyte differentiation and is tightly regulated during adipogenesis. However, mechanisms regulating adipocyte differentiation involving Ras protein stability regulation are unknown. Here, we show that WD40 repeat protein 76 (WDR76), a novel Ras regulating E3 linker protein, controls 3T3-L1 adipocyte differentiation through HRas stability regulation. The roles of WDR76 in obesity and metabolic regulation were characterized using a high-fat diet (HFD)-induced obesity model using Wdr76−/− mice and liver-specific Wdr76 transgenic mice (Wdr76Li−TG). Wdr76−/− mice are resistant to HFD-induced obesity, insulin resistance and hyperlipidemia with an increment of HRas levels. In contrast, Wdr76Li-TG mice showed increased HFD-induced obesity, insulin resistance with reduced HRas levels. Our findings suggest that WDR76 controls HFD-induced obesity and hepatic steatosis via HRas destabilization. These data provide insights into the links between WDR76, HRas, and obesity.


WDR76 mediated adipocyte differentiation of 3T3-L1 cells via destabilization of HRas.
To elucidate the role of WDR76 on HRas protein stability during adipogenesis, we examined the effect of the knockdown or overexpression of WDR76 in 3T3-L1 cells. 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). The role of WDR76 in inhibition of adipocyte differentiation was confirmed by decreases in both mRNA and protein levels of PPARγ and C/EBPα as well as reduction of Oil Red O (ORO) staining of 3T3-L1 cells (Fig. 1d) after WDR76 knockdown ( Fig. 1a-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). ORO staining showed that lipid accumulation was increased by WDR76 overexpression (Fig. 1h). Degradation rates of HRas were also accelerated by WDR76 overexpression, as shown by measurements in the presence of the de novo protein synthesis inhibitor, cycloheximide (CHX) (Fig. 1i). The proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN), reversed the WDR76-mediated HRas reduction (Fig. 1j). WDR76 overexpression increased HRas polyubiquitination ( Fig. 1k and Supplementary Fig. 1). These results indicated that HRas degradation occurred through the polyubiquitination-dependent proteasomal machinery. The role of HRas stability regulation in adipocyte differentiation by WDR76 was confirmed by co-expression of WDR76 and HRas (Fig. 1l,m). HRas overexpression reduced the levels of both PPARγ and C/EBPα; whilst WDR76 overexpression increased PPARγ and C/EBPα expression with a decrease of HRas protein levels and ERKs phosphorylation (Fig. 1l).
HRas overexpression consistently blocked adipogenesis, but the effect was reversed by overexpression of WDR76 as shown by ORO staining (Fig. 1m). To examine the role of HRas in the WDR76-dependent adipocyte differentiation, we checked the effects of HRas knockdown on the WDR76-induced differentiation of 3T3-L1 cells ( Supplementary Fig. 2a,b). The adipogenesis of 3T3-L1 cells monitored by ORO staining was increased by HRas knockdown or WDR76 overexpression together with induction of both PPARγ and C/EBPα ( Supplementary  Fig. 2a,b). The WDR76-induced adipogenesis was further increased by HRas knockdown (Supplement Fig. 2a,b).
Because WDR76 revealed a regulatory effect on preadipocyte differentiation, we examined expression levels of WDR76 and HRas during the preadipocyte differentiation of 3T3-L1 cells. WDR76 expression was increased up to day 2, and decreased thereafter. Contrarily, HRas level was gradually decreased up to day 8 after initiation of the differentiation (Supplementary Fig. 3a). In addition, we examined the levels of WDR76 and HRas in WAT and liver tissue of mice fed either normal chow diet (Chow) or HFD. WDR76 and HRas expression were increased and decreased, respectively, in both WAT and liver tissue of HFD-induced obese mice compared with the control mice fed a Chow diet ( Supplementary Fig. 3b). Overall, WDR76 plays a role in adipocyte differentiation through destabilization of HRas in 3T3-L1 preadipocyte cells.
WDR76 deficiency ameliorated HFD-induced obesity in mice. To assess whether there were overt phenotypic effects in the context of a HFD, we fed Wdr76 +/+ and Wdr76 −/− mice with a HFD (60% kcal from fat) for 9 weeks, and monitored the body weights. The male Wdr76 −/− mice showed reduced body weights (41.2 ± 2.02 g vs 30.3 ± 1.54 g) (Fig. 2a,b) and the body weight reduction of the Wdr76 −/− mice was not attributed to differences in their food intake (Fig. 2c) or body length ( Supplementary Fig. 4). 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). To identify the effects of 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).
Obesity is correlated with glucose intolerance and insulin resistance. Therefore, we next assessed the effects of WDR76 deficiency on glucose homeostasis and insulin sensitivity. Wdr76 −/− mice had improved glucose tolerance and insulin sensitivity (Fig. 2i,j), suggesting that WDR76 deficiency could reduce HFD-induced insulin resistance.
The thickness and surface area of subcutaneous fat in HFD-fed Wdr76 −/− mice was smaller than that of HFD-fed Wdr76 +/+ mice as determined by hematoxylin and eosin (H&E) staining (Fig. 3a-c). The diameters of epididymal white fat adipocytes were also reduced (Fig. 3d). Immunohistochemical (IHC) and western blotting analyses of epididymal WAT showed that protein levels of PPARγ and C/EBPα were lowered with an increase of HRas protein levels in Wdr76 −/− mice (Fig. 3e,f). The reduction of mRNA levels of the adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) in Wdr76 −/− mice were further confirmed by real-time quantitative polymerase chain reaction (qRT-PCR) (Fig. 3g). The ubiquitin-conjugated proteins were detected by IB analyses. Lower panels show graphs for quantification of IB analyses. (l,m) The 3T3-L1 cells were infected with control or WDR76 with or without HRas lentivirus and grown under MDI-induced differentiation condition. WCLs were subjected to IB analyses using PPARγ, C/EBPα, WDR76, HRas, pERKs, ERKs or β-actin antibody. Lower panels show graphs for quantification of IB analyses (l). Lipid droplets were stained using ORO staining. Lower panels show graphs depicting the relative area of ORO staining as determined by Image J. Scale bars, 100 µm (m). All data are presented as the mean ± SD and representative results of at least three experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.005, Student's t-test.
www.nature.com/scientificreports www.nature.com/scientificreports/ Wdr76 −/− mice had decreased hepatic steatosis. Obesity is closely related with hepatic steatosis in humans as well as in rodents. To further characterize the role of WDR76 in the hyperlipidemia of liver, histological characteristics of liver tissues of Wdr76 −/− mice were analyzed with ORO staining to characterize the possibility of fatty livers (Fig. 4a). Consistent with the lean phenotype in Wdr76 −/− mice, smaller lipid vesicles and decreased numbers of hepatocytes with lipid droplets were observed in the livers of Wdr76 −/− mice (Fig. 4a). Because hepatic PPARγ and C/EBPα proteins are known to play a role in the development and maintenance of hepatic steatosis, we assessed the expression levels of these in the liver tissues of HFD-fed Wdr76 +/+ and Wdr76 −/− mice [43][44][45] . IHC analyses and western blotting of liver tissues revealed that expression levels of C/EBPα and PPARγ decreased with an increase of HRas protein in Wdr76 −/ − mice (Fig. 4b,c). To further investigate the role of WDR76 in hepatic steatosis, we analyzed the expression of key lipogenic and metabolic genes in the livers of HFD-fed Wdr76 +/+ and Wdr76 −/− mice. The mRNA expression levels for both liver lipogenesis and gluconeogenesis markers were reduced in Wdr76 −/− mice (Fig. 4d). Taken together, the results showed that Wdr76 −/− mice had reduced hepatic steatosis.
The liver-specific overexpression of WDR76 increased obesity and insulin resistance. The liver plays a vital role in central metabolism by regulating key aspects of glucose and fatty acid regulation and storage 46 . Because Wdr76 −/− mice exhibited decreased hepatic steatosis, we examined the impact of WDR76 in the liver by using liver-specific Wdr76 transgenic mice (Wdr76 Li−TG ). Wdr76 Li−TG mice were generated by crossing Wdr76 conditional transgenic mice with albumin-Cre transgenic mice as previously described 34 . Based on the knockout mouse experiments, we hypothesized that Wdr76 Li−TG mice will be more sensitive to the HFD, resulting in a severe obesity phenotype compared with Wdr76 +/+ . Therefore, we fed Wdr76 +/+ and Wdr76 Li−TG mice with a HFD (45% kcal from fat) in order to accurately quantify the effect of WDR76 overexpression 47 . Although WDR76 was specifically overexpressed in the liver, Wdr76 Li−TG mice showed a severe obesity phenotype after HFD feeding when compared with Wdr76 +/+ mice (Fig. 5a). Wdr76 Li-TG mice showed more glucose intolerant and insulin resistant phenotypes (Fig. 5b,c). Both sizes and weights of epididymal, perirenal WATs and liver were increased (Fig. 5d). Histological analyses of subcutaneous, epididymal adipose tissues and liver were performed to show that Wdr76 Li-TG mice had elevated fat depots with thicker subcutaneous fat, increased sizes of epididymal adipocytes and hepatic steatosis ( Fig. 5e-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. 5h-j). www.nature.com/scientificreports www.nature.com/scientificreports/ We observed that WDR76 expression was increased in both WAT and liver tissue of HFD-fed Wdr76 Li-TG mice compared with Wdr76 +/+ mice (Fig. 6a). As shown in Supplementary Fig. 3b, the level of WDR76 was increased in the WAT of HFD-fed mice. These data suggest that the increased level of WDR76 in WAT of HFD-fed Wdr76 Li-TG mice was affected by the severe obesity phenotype of Wdr76 Li-TG mice (Fig. 6a). The level of adipogenic markers, PPARγ and C/EBPα, was higher with decreased HRas levels in the liver and WAT of Wdr76 Li-TG mice (Fig. 6a). The mRNA expressions of PPARγ, C/EBPα, and SREBP1 were increased in WAT of Wdr76 Li-TG mice (Fig. 6b). Consistently, hepatic gene expression profiles of lipogenesis and gluconeogenesis in the HFD-fed Wdr76 Li-TG mice were increased, suggesting that WDR76 promotes HFD-induced hepatic steatosis (Fig. 6c).

Discussion
The Ras/ERK pathway has been known as an important signaling cascade in adipogenesis 14,48 . ERK has been reported to promote differentiation in the early stages of adipocyte differentiation and then ERK needs to be downregulated preceding adipocyte maturation 49,50 . HRas has been shown to block adipocyte differentiation, probably due to sustained activation of the ERK pathway 15,51 . These studies suggested that HRas protein might play roles in blocking adipocyte differentiation and should be down-regulated during adipogenesis. However, the regulatory mechanism of HRas during adipogenesis has been poorly explored.
Using a proteomics approach with tumor and non-tumor tissues from a human patient with hepatocellular carcinoma (HCC), the E3 linker protein, WDR76, was identified as an HRas binding protein, which promoted HRas degradation, thus functioning as a tumor suppressor in liver cancer 34 . However, the physiological roles of WDR76 regarding the stability regulation of Ras are unknown.
In this study, we found that WDR76-mediated Ras destabilization was directly related to adipocyte differentiation in 3T3-L1 cells. The effects of decreases and increases of HRas protein levels and ERK activity by overexpression and knockdown of WDR76, respectively, were verified in the regulation of 3T3-L1 preadipocyte cell differentiation. WDR76-mediated HRas degradation was regulated via polyubiquitination-dependent proteasomal degradation in 3T3-L1 cells. The role of HRas in blocking the adipogenesis was confirmed. HRas overexpression inhibited MDI-induced adipocyte differentiation of 3T3-L1 cells, which was consistent with the previous www.nature.com/scientificreports www.nature.com/scientificreports/ reports 51 . Overexpression of WDR76 reversed this effect through HRas degradation with the decrease of ERK activation. Consistently, HRas knockdown increased adipocyte differentiation of 3T3-L1 cells and these effects were further promoted by WDR76 overexpression. These results suggest that WDR76 mediates adipocyte differentiation of 3T3-L1 preadipocyte cells via HRas destabilization.
The role of WDR76 in adipocyte hypertrophy was confirmed by in vivo experiments using a HFD-induced obesity model. As we previously reported, whole-body Wdr76 knockout mice had similar body weight and size, and no obvious developmental abnormalities 34 . When mice were fed with a HFD (60% kcal from fat), the Wdr76 −/− mice improved the risk factors of metabolic disorders induced by HFD, such as obesity, insulin resistance, size increments of the epididymal and perirenal fat tissues, hepatic steatosis as well as the increment of expressions of the lipogenic and gluconeogenic genes. These results suggest that WDR76 plays multiple pathophysiological roles related to metabolism and energy homeostasis. The impaired lipid storage in adipose tissue could lead to fat deposition in non-adipose tissues such as liver, heart, and muscle 52,53 . However, HFD-fed Wdr76 −/− mice showed decreased lipid storage in adipose tissue as results of size reduction of WATs but do not exhibit increased circulating TG levels or hepatic steatosis compared with Wdr76 +/+ mice. The reduced accumulation of fat in the liver of the HFD-fed Wdr76 −/− mice is consistent with reduced expression of lipogenic genes, such as PPARγ, C/EBPα, and SREBP1 in the liver which play roles in the development of hepatic steatosis [54][55][56][57] . Because food intake or body length did not significantly alter between Wdr76 −/− and Wdr76 +/+ , it is possible that decreased body weight and hepatic steatosis phenotypes in HFD-fed Wdr76 −/− mice could be attributed to improved insulin sensitivity, altered glucose and lipid metabolism, or enhanced energy consumption etc. In addition, we also consider the possibility that deficiency of WDR76 in liver tissue could prevent fatty liver and improve lipid metabolism due to whole-body Wdr76 knockout mice. Further investigations for lipid accumulation in muscle or brown adipose tissue, and the difference in energy expenditure compared with Wdr76 +/+ mice will help to assess fat distribution of HFD-fed Wdr76 −/− mice.
The liver has a vital role in fatty acid synthesis and glucose and lipid metabolism. Fatty acid synthesized by the liver is converted to TG and transported to the blood 58 . Lipid accumulation in the liver is related to insulin www.nature.com/scientificreports www.nature.com/scientificreports/ resistance 59 . In addition, liver-specific gene regulation affects whole-body composition through lipid metabolism regulation 60,61 . To determine the effects of WDR76 in the liver, we introduced Wdr76 Li-TG mice with a HFD (45% kcal from fat) model. The differences in the H&E staining patterns of liver tissues and levels of TC, TG, and FFAs may be attributed to the differences in body weights of the control groups for Wdr76 Li-TG and Wdr76 −/− mice, respectively. A difference between the experimental conditions for the two mice groups is their dietary conditions; Wdr76 +/+ and Wdr76 −/− mice fed a HFD (60% calories from fat) for 9 weeks, whereas Wdr76 +/+ and Wdr76 Li-TG mice fed a HFD (45% calories from fat) for 8 weeks. Therefore, the observed phenotypic differences could likely be attributed to their differences in dietary conditions. The decrease in HRas protein levels by WDR76 overexpression in vivo was verified. HFD-fed Wdr76 Li-TG mice had increased HFD-related metabolic defects such as obesity, insulin resistance, and hyperlipidemia, with whole-body composition changes. These results supported the concept that the liver is one of the main systemic organs that regulate whole body glucose and lipid metabolism, and HRas stability regulation by WDR76 plays a role in metabolism regulation.
Consistent with our study, several studies reported the role of HRas in energy homeostasis. For example, adipose specific HRas transgenic mice showed reduced gonadal fat pad weight and reduced adipocyte size 62 . In addition, mice with oncogenic HRas mutation (HRas G12S/+ ) are resistant to the HFD-induced obesity 63 . Taken together, these results imply that the stability regulation of HRas by WDR76 is important in the maintenance of liver homeostasis.
There are multiple factors involved in the etiology of HCC 64,65 . Hepatic steatosis is one of the factors that can progress to cirrhosis and, subsequently to HCC 64 . We recently verified the tumor-suppressive role of WDR76, which functions via Ras degradation, by monitoring effects the diethylnitrosamine (DEN)-induced heptocarcinogenesis in Wdr76 −/− mice and the HRas G12V -driven liver carcinogenesis in Wdr76 Li-TG mice 34 . In the current study, (g) Distribution of adipocyte size in epididymal WAT was measured (n = 5 per group). The diameters of epididymal white fat adipocytes were determined using the NIS element AR image program, and more than 300 adipocytes were examined for each group. (h-j) Serum levels of total cholesterol (h), triglyceride (i), and free fatty acids (j) in HFD-fed Wdr76 +/+ and Wdr76 Li-TG mice were measured (n = 5 per group). All data are presented as the mean ± SD. Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.005. www.nature.com/scientificreports www.nature.com/scientificreports/ however, we demonstrated that HFD-fed Wdr76 Li-TG mice had a severe obesity phenotype with increased hepatic steatosis which is a potential risk factor for HCC. Therefore, these results implicate that the stability regulation of HRas by WDR76 should be considered depending on pathophysiological conditions. In summary, our data indicated that WDR76 plays a role as a positive regulator of diet-induced obesity and hepatic steatosis in mice through HRas destabilization. Considering a single E3 ligase may target multiple substrates, identifying the other potential targets for WDR76 will help to identify additional molecular mechanisms of WDR76. Moreover, further studies using adipose or liver tissue-specific Wdr76 knockout mice and validating the role of WDR76 in adipogenesis in vivo will help to clarify the roles of WDR76 in metabolism and as a potential therapeutic target to control obesity-associated metabolic diseases.

Animals and dietary treatments. All animal experiments were performed in accordance with the Korean
Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University (IACUC-201711-652-01). The generation of Wdr76 −/− and Wdr76 Li-TG mice were previously described 34 . The 4-5-week-old Wdr76 +/+ and Wdr76 −/− littermates (weightmatched) 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. All mice were in the C57BL/6 background.
Histological analysis and immunohistochemistry. For Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), dissected tissues were fixed in 4% paraformaldehyde in PBS and embedded in paraffin and sectioned into 4 μm slices. Paraformaldehyde-fixed paraffin sections were deparaffinized using xylene and rehydrated in serially diluted in ethanol, and stained with H&E or used for IHC. Images of the H&E staining were recorded with a TE-2000U bright-field optical microscope (Nikon, Tokyo, Japan). The diameters of epididymal WAT were determined using the NIS element AR image program, and more than 300 adipocytes were examined for each group. For IHC analyses, the slides were autoclaved in retrieval buffer (10 mM sodium citrate buffer, pH 6.0; Sigma-Aldrich, St. Louis, MO, USA) for antigen retrieval. The sections were blocked in PBS containing 5% bovine serum albumin (BSA) and 1% normal goat serum (Vector Laboratories, CA) at room temperature for 1 h. The sections were incubated overnight at 4 °C with the following dilution of primary antibodies: PPARγ (Santa Cruz Biotechnology, sc-271392, 1:300) and C/EBPα (Cell Signaling Technology, #2295, 1:100). The slides were washed with PBS, incubated with Alexa Fluor 488-(Molecular Probes, A32731, 1:300) or Alexa Fluor 555-conjugated IgG secondary antibodies (Molecular Probes, A32732, 1:300) at room temperature for 1 h, www.nature.com/scientificreports www.nature.com/scientificreports/ and counterstained with DAPI (Roche Life Science, 10236276001, 1:5,000) for 10 min. The images were captured using a LSM700 META confocal microscope (Carl Zeiss).
Cell culture and adipocyte differentiation. The 3T3-L1 preadipocytes were cultured as previously described 12,13 . The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% (v/v) calf serum (BCS; Gibco), 100 µg/mL penicillin, and 100 µg/mL streptomycin in a 5% CO 2 incubator at 37 °C. The 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.
Oil red O staining. For ORO staining, liver tissue samples were fixed using 4% paraformaldehyde in PBS of the liver, specimens were submerged in 15%, 20% and 30% sucrose solution per day. The samples were embedded in OCT compound (Tissue tek; Sakura Finetek USA) and sectioned into 20 μm slices. 3T3-L1 cells and liver tissues were washed with PBS and 70% isopropanol (Duksan Pure Chemicals), and stained with ORO solution (Sigma-Aldrich) at room temperature overnight. Samples were washed thoroughly with distilled water. Tissues were counterstained with Gill's hematoxylin (Sigma-Aldrich). Images of the ORO staining were visualized with a bright field microscope (Nikon TE-2000U). To quantify the ORO staining intensities, image files were analyzed using ImageJ 66 . First, original images of staining were converted into RGB images, and the red monochromatic image was used to calculate the area of the staining. "Threshold" tool in the "Adjust" box in the "Image" menu was used to adjust threshold manually. And then staining positive area was obtained by clicking "Measure" button under the "Analyze" menu.
Ubiquitination assay. For the ubiquitination assay, cells were washed in ice-cold PBS and lysed with RIPA buffer. Ten millimolar N-ethylmaleimide (NEM; Sigma-Aldrich) was subsequently added to the RIPA buffer for the ubiquitination assays. The lysates were incubated with the indicated antibodies and protein A/G agarose at 4 °C for 12 h, and the immunoprecipitated beads were washed three times in RIPA buffer. The ubiquitin-conjugated proteins were resolved by SDS-PAGE, and detected by IB analyses.
Virus production and viral packaging. Lentiviral plasmid was co-transfected with the packaging plasmids, psPAX2 and pMD2.G into HEK 293 T cells using a ratio of 2:1.2:0.8, respectively. Lentivirus-containing media were collected twice every 24 h and filtered through 0.45 µm pore filters (EMB Millipore). During ultraconcentration of the virus in two steps, FBS was removed; samples were filtered through Centricon filters (EMB Millipore) with a 100 K cut-off, and then subjected to ultracentrifugation at 100,000 × g for 2 h at 4 °C. Concentrated lentivirus was reconstituted in PBS, prepared as 10 µL aliquots, and stored at −80 °C.