Systemic depletion of WWP1 improves insulin sensitivity and lowers triglyceride content in the liver of obese mice

Obesity is a metabolic disorder associated with many diseases. WW domain‐containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT‐type E3 ubiquitin ligase involved in several diseases. Recently, we found that the level of WWP1 is increased in white adipose tissue in a mouse model of obesity and that obese Wwp1 knockout (KO) mice exhibit improved whole‐body glucose metabolism. Here, to determine which insulin‐sensitive tissues contribute to this phenotype, we investigated the levels of several insulin signaling markers in white adipose tissue, liver, and skeletal muscle of Wwp1 KO mice, which were fed a normal or high‐fat diet and transiently treated with insulin. In obese Wwp1 KO mice, phosphorylated Akt levels were increased in the liver but not in white adipose tissue or skeletal muscle. Moreover, the weight and triglyceride content of the liver of obese Wwp1 KO mice were decreased. These results suggest that systemic deletion of WWP1 improves glucose metabolism via enhanced hepatic insulin signaling and suppressed hepatic fat accumulation. In summary, WWP1 participates in obesity‐related metabolic dysfunction and pathologies related to hepatic steatosis via suppressed insulin signaling.

Obesity is a metabolic disorder associated with many diseases. WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a HECT-type E3 ubiquitin ligase involved in several diseases. Recently, we found that the level of WWP1 is increased in white adipose tissue in a mouse model of obesity and that obese Wwp1 knockout (KO) mice exhibit improved whole-body glucose metabolism. Here, to determine which insulin-sensitive tissues contribute to this phenotype, we investigated the levels of several insulin signaling markers in white adipose tissue, liver, and skeletal muscle of Wwp1 KO mice, which were fed a normal or high-fat diet and transiently treated with insulin. In obese Wwp1 KO mice, phosphorylated Akt levels were increased in the liver but not in white adipose tissue or skeletal muscle. Moreover, the weight and triglyceride content of the liver of obese Wwp1 KO mice were decreased. These results suggest that systemic deletion of WWP1 improves glucose metabolism via enhanced hepatic insulin signaling and suppressed hepatic fat accumulation. In summary, WWP1 participates in obesity-related metabolic dysfunction and pathologies related to hepatic steatosis via suppressed insulin signaling.
Obesity is abnormal or excessive fat accumulation that causes metabolic disorders and contributes to the development of insulin resistance and type 2 diabetes through chronic inflammation or oxidative stress [1]. Since obesity-related insulin resistance has become a major worldwide health problem, it is necessary to understand the molecular mechanisms underlying insulin resistance and establish preventive methods.
Insulin has important metabolic effects in insulinsensitive tissues, which include the liver, white adipose tissue (WAT), and skeletal muscle. Insulin stimulation suppresses glucose output in the liver and lipolysis in WAT. In addition, it enhances hepatic glycogen production and glucose uptake in WAT and skeletal muscle. Insulin resistance is characterized by a diminished response to insulin stimulation that results, in part, from disruption of the insulin signaling pathway [2].
In this pathway, the binding of insulin to the insulin receptor induces tyrosine phosphorylation of the insulin receptor substrate via autophosphorylation of the insulin receptor. This phosphorylation activates phosphatidylinositol 3-kinase (PI3K), which converts phosphatidylinositol 3,4-bisphosphate (PIP 2 ) into phosphatidylinositol 3,4,5-triphosphate (PIP 3 ), a phospholipid recognized as the cellular second messenger. Phosphatase and tensin homolog (PTEN) is a negative regulator of PI3K [3,4]. Akt (also known as protein kinase B) is phosphorylated and activated by PIP 3 , leading to induced translocation of glucose transporter 4 on the cell surface and glucose uptake in insulinsensitive tissues [5]. Therefore, the insulin signaling pathway is important for whole-body glucose metabolism.
WW domain-containing E3 ubiquitin protein ligase 1 (WWP1; also known as TIUL1 or AIP5) belongs to the HECT-type E3 ubiquitin protein ligase family. WWP1 has a C2 domain at its N-terminal, four WW domains in its central region and a HECT domain at its C-terminal [6,7]. The C2 domain determines subcellular localization of the molecule, while the WW domains bind to proline-rich sequences (PY motif) of substrate proteins. Previous studies have indicated that WWP1 plays important roles in various pathologies such as cancers, infectious diseases, and neurological diseases [7].
We previously revealed that in a mouse model of obesity induced by a high-fat diet (HFD), the expression level of WWP1 in WAT was elevated in a p53dependent manner [8]. Subsequently, to evaluate the involvement of WWP1 in metabolic regulation, we studied systemic Wwp1 knockout (KO) mice and found that the HFD-induced reduction in phosphorylation levels of Akt in WAT was enhanced in Wwp1 KO mice. This result indicates that deletion of WWP1 exacerbates obesity-related insulin resistance in WAT. However, in the same study, the insulin tolerance test (ITT) and glucose tolerance test (GTT) showed that insulin sensitivity and glucose tolerance were unexpectedly improved in Wwp1 KO mice [9]. This discrepancy between insulin sensitivity in the whole body and insulin signal transduction in WAT implies that WWP1 deletion may also affect insulin signal transduction in other insulin-sensitive tissues, including the liver and skeletal muscle. Thus, in this study, we investigated insulin signal transduction in insulin-sensitive tissues in Wwp1 KO mice transiently stimulated with insulin.

Animals
All animal experiments and protocols were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan and were approved by the Ethics Review Committee for Animal Experimentation at Tokyo University of Science (approval numbers: Y20043 and Y21043). Mice with systemic KO of Wwp1 (Wwp1 À/À mice) and wild-type (WT) Wwp1 +/+ mice were generated as shown in our previous report [10]. Genotyping of offspring was performed by PCR using KOD FX neo (Toyobo, Osaka, Japan) with the following primers: forward, 5 0 -AGA GGC AAG AGA ATG GCG TCA AG-3 0 ; reverse, 5 0 -GGA GGT GAA AGG GTT GGA AGA ATA C-3 0 . Mice were maintained under specific-pathogen-free conditions at 23°C, under a 12-h light/dark cycle in the animal facility at the Faculty of Pharmaceutical Sciences, Tokyo University of Science. They had free access to water and were fed a Charles River Formula-1 diet (21.9% crude protein, 5.4% crude fat and 2.9% crude fiber; Oriental Yeast, Tokyo, Japan). At 5 weeks old, WT and KO mice were divided into two groups: the normal diet (ND; Nosan, Yokohama, Japan) group or HFD group. The Charles River Formula-1 diet and High-Fat Diet 32 (25.5% crude protein, 32.0% crude lipid and 2.9% crude fiber; CREA, Tokyo, Japan) were fed as the ND and HFD, respectively. At 13-15 weeks old, mice were euthanized under isoflurane anesthesia (Mylan, Canonsburg, PA, USA) in a fed state, and their liver was collected for measuring the expression of WWP1 by immunoblot (Fig. 3A). At 23 weeks old, mice were fasted for 24 h and intravenously administrated with insulin (1 U per kg body weight) via the inferior vena cava by abdominal section under isoflurane anesthesia. After 10 min, they were euthanized and their epididymal and subcutaneous WAT depots and liver and quadriceps femoris muscle (skeletal muscle) were collected and weighed for immunoblot and for measuring the contents of triglyceride and liver weight. These tissues were immediately diced, frozen in liquid nitrogen, and stored at À80°C.

GTT and ITT
GTT and ITT were performed in HFD-fed Wwp1 WT and KO mice of 13-15 weeks old. Prior to GTT and ITT, mice were fasted for 24 and 8 h, respectively. D-glucose (1.0 kg per body weight, Wako) or insulin (1.0 U per kg body weight, Wako) were injected intraperitoneally for GTT and ITT, respectively. Next, serial blood sampling from the tail vein was performed at 0, 15, 30, 60, and 120 min after injection. Blood glucose levels were measured using an Accu-ChekÒ Aviva blood glucose meter (Roche, Basel, Switzerland).

Amount of triglyceride in liver
Collected liver pieces were homogenized with Solution I (chloroform : methanol = 1 : 1), followed by mixing with a quarter volume of 1 M NaCl. The homogenates were centrifuged for 10 min at 1100 g at 4°C, and their lower layers were collected and dried up into lipid pellets. The pellets were dissolved in Solution II (tert-butanol : methanol: triton-114 = 3 : 1 : 1) as extracted triglyceride samples. The amount of triglyceride in samples was measured using LabAssay TM triglyceride (Wako).
Supernatants were used for the measurement of tissue GSH content with an Infinite F200 PRO microplate reader (Tecan, M€ annedorf, Switzerland). The rate of 5,5-dithiobis-(2-nitrobenzoic) acid (DTNB) formation was calculated, and the concentrations of tGSH and GSSG in each sample were determined using linear regression, with reference to a standard curve. GSH concentration was calculated by subtracting GSSG concentration from tGSH concentration.

Statistical analysis
Statistical significance was determined using the Student's t-test to compare two groups or the Tukey-Kramer test to compare more than two groups after the assessment of significant differences by two-way or three-way analysis of variance. R software (R project for Statistical Computing) and/ or BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan) was used. Differences with P values < 0.05 were considered statistically significant.

Deletion of WWP1 enhanced the insulin signaling response in liver from HFD-fed mice
We initially performed GTT and ITT and confirmed that insulin sensitivity was improved in HFD-fed Wwp1 KO mice, which is consistent with our published results (Fig. S1B,D) [9]. By contrast, these changes were not observed in ND-fed mice (Fig. S1A,C). To evaluate the insulin signaling response in insulin-sensitive tissues (including epididymal and subcutaneous WAT, skeletal muscles and liver), we analyzed insulin-stimulated changes in levels of total and phosphorylated Akt (Akt and pAkt) and PTEN, which are insulin signaling-related proteins, in each tissue in Wwp1 KO mice. The results showed that in the liver of HFD-fed mice, WWP1 deletion significantly increased the ratio of pAkt/Akt protein levels and, despite no significance, decreased PTEN protein levels (Fig. 1A). In the WAT and skeletal muscle of HFD-fed mice, however, WWP1 deletion did not affect levels of these proteins (Fig. 1B-D). Moreover, in NDfed mice, WWP1 deletion did not significantly affect the ratio of pAkt/Akt levels and PTEN levels in any tissue (Fig. 2). These results suggest that the improvement in insulin sensitivity in Wwp1 KO mice results from an enhanced insulin signaling response mainly in the liver.

Deletion of WWP1 decreased the weight and triglyceride content of the liver in HFD-fed mice
Protein levels of WWP1 in WT mice were not different between ND-fed and HFD-fed mice of 13-15 weeks of age in the liver (Fig. 3A). Hepatic insulin sensitivity is reportedly inversely correlated with triglyceride content in nondiabetic obese patients [13]; therefore, we measured the hepatic triglyceride content in all groups of mice of 23 weeks of age. We showed that HFDinduced increases in hepatic triglyceride content were significantly suppressed in Wwp1 KO mice (Fig. 3B). In addition, liver weight was also decreased in HFD- fed Wwp1 KO mice, which correlates with the decrease in triglyceride content (Fig. 3C). De novo lipogenesis is important for regulation of hepatic triglyceride levels [14]. To assess the contribution of WWP1 to lipogenesis in the liver, we examined the expression levels of ACC, FASN, and phosphorylated ACLY, which are important enzymes in de novo fatty acid synthesis. The results showed that expression of these  proteins was not significantly different in any of the groups (Fig. S2).

Discussion
Our previous report demonstrated that obese systemic Wwp1 KO mice exhibit improved whole-body insulin sensitivity ( Fig. S1 and [9]). In this study, we focused on insulin-sensitive tissues and investigated the insulin signaling pathway to assess the role of WWP1 in whole-body glucose metabolism. We found that WWP1 deletion in HFD-fed mice enhanced insulin signaling (pAkt/Akt rate) in the liver (Fig. 1A) but not in other insulin-sensitive tissues (Fig. 1B-D). This enhanced insulin signaling in the liver by WWP1 deletion probably contributes to improved whole-body glucose metabolism in Wwp1 KO obese mice. In addition, WWP1 deletion in HFD-fed mice slightly reduced PTEN protein levels in the liver, suggesting that WWP1 may positively regulate PTEN (Fig. 1D). By contrast, several studies have shown that WWP1 negatively regulates PTEN in cancers. In studies by Lee et al., WWP1 ubiquitinated PTEN and inhibited its dimerization and translocation to the cellular membrane, resulting in the inactivation of PTEN [15,16]. Furthermore, total protein levels of PTEN were not altered in embryonic fibroblasts of Wwp1 KO mice in one of these studies [15]. Considering that the HFD did not affect WWP1 levels in the liver in our study (Fig. 3A), the discrepancy between our results and these findings implies that WWP1 deletion reduces PTEN protein levels in obese liver in an indirect manner, for example, by the influence of other organs. Taken together, although the mechanism remains unclear, WWP1 plays a downregulatory role in hepatic insulin signal transduction via stabilizing PTEN, at least in obese mice. Although HFD did not alter WWP1 expression levels in the liver (Fig. 3A), we previously reported that WWP1 expression was increased by HFD in WAT in a p53-dependent manner [8]. Considering the evidence that obesity upregulated p53 expression not only in WAT but also in liver [17], it is conceivable that the regulation of HFD-induced WWP1 expression in liver is different from that in WAT, which is dependent on p53. Moreover, it was reported that transforming growth factor b (TGFb) [18] and tumor necrosis factor a (TNFa) [19] stimulate the transcription of WWP1 gene via an unknown mechanism. Several micro-RNAs have been also found to regulate the expression of WWP1 [20]. Despite no direct evidence, these regulators may contribute to the regulation of WWP1 expression in obese liver.
WWP1 deletion reduced increases in hepatic triglyceride content normally associated with a HFD (Fig. 3B). We previously demonstrated that WWP1 plays a defensive role against mitochondrial oxidative stress in adipocytes and WAT [8,9]. Hence, the WAT of Wwp1 KO mice is more vulnerable to mitochondrial oxidative stress than that of WT mice. In contrast, while WWP1 deficiency slightly reduced the total glutathione concentration with no significant in liver, the GSH/GSSG ratio, a marker of antioxidative capacity, did not change, unlike WAT (Fig. S3). Mild-tomoderate mitochondrial dysfunction or stress responses in adipocytes prevent obesity-induced hepatic steatosis [21]. For example, Yang et al. [22] have shown that adipose-specific deletion of fumarate hydrase, an integral Krebs cycle enzyme, provokes mitochondrial stress and suppresses hepatic steatosis. Systemic deletion of caseinolytic mitochondrial matrix peptidase proteolytic subunit (ClpP), a mitochondrial matrix protease responsible for quality control of mitochondrial proteins, also reportedly exerts similar effects [23]. These reports suggest that reductions in the weight and triglyceride content of the liver in obese Wwp1 KO mice may result from moderate mitochondrial oxidative stress in adipocytes. Moreover, the above-mentioned mouse models of mitochondrial oxidative stress exhibited improved glucose tolerance [22,23]. Thus, although activation of insulin signal transduction in the liver is a main mechanism by which insulin sensitivity is enhanced in Wwp1 KO mice, mitochondrial oxidative stress in adipocytes also plays a role.
In addition to the extrahepatic influence, Korenblat et al. [13] raise the possibility that decreased hepatic triglyceride content is associated with an enhanced insulin signaling response in the liver; in this study, hepatic triglyceride content was inversely correlated with hepatic insulin sensitivity in obese nondiabetic patients, despite an unproved cause-and-effect relationship. This report also found that hepatic triglyceride content was directly correlated with basal plasma insulin concentration in obese nondiabetic patients, in agreement with our previous finding of decreased plasma insulin levels in HFD-fed Wwp1 KO mice [8]. Collectively, decreased weight and triglyceride content in the liver of Wwp1 KO mice could be explained by two mechanisms: one is oxidative-stress-induced mitochondrial dysfunction in WAT, and the other is enhanced insulin signal transduction in the liver. Clarification of which mechanism is correct requires analysis of adipose-specific or liver-specific Wwp1 KO mice.
In the present study, obese Wwp1 KO mice displayed an enhanced hepatic insulin signaling response. In obese mice, WWP1 deletion also reduced the weight and triglyceride content of the liver. Dysregulation of hepatic triglyceride content is closely linked to nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). NAFLD/NASH is an obesityassociated risk factor for serious liver diseases, including cirrhosis and hepatocellular carcinoma [24]. Zhang et al. [25] have reported that the expression of WWP1 is upregulated in human hepatocellular carcinoma and is highly correlated with its poor outcome. Therefore, further analysis of WWP1 will improve understanding of not only obesity-related metabolic dysfunction but also hepatic steatosis-related pathologies, such as cirrhosis and hepatocellular carcinoma.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.