RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis*

Background: Hepatic steatosis is an increasing health concern associated with metabolic syndrome. Results: RGC-32 deficiency protects mice from HFD- and ethanol-induced hepatic steatosis by decreasing hepatic lipogenic gene expression. Conclusion: RGC-32 is a novel regulator for hepatic lipogenesis. Significance: RGC-32 may be a novel target for treating hepatic steatosis. Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32−/−) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32−/− mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.

Hepatic steatosis, also known as fatty liver, is characterized by the accumulation of triglycerides in the liver (1) and is a uniform response of the liver to excessive energy intake or alcohol consumption (2)(3)(4). It is the most common liver disease in the United States, with a high prevalence in the obese and type 2 diabetic population, but it is probably underestimated as a cause for cirrhosis (5). Therefore, there is an increasing interest in understanding the pathogenesis of the fatty liver so that effective therapies may be developed by targeting factors that sustain steatosis.
Although the molecular mechanism regulating the development of hepatic steatosis is complex, recent studies have shown that targeting enzymes important for lipogenesis in the liver may be an effective approach to treat hepatic steatosis (6). It is well established that sterol regulatory element (SRE) 3 binding protein (SREBP) 1c (SREBP-1c), a transcriptional factor primarily expressed in the liver, is a critical mediator for the transcription of lipogenic enzymes (7). Stearoyl-CoA desaturase 1 (SCD1), a lipogenic gene regulated by SREBP-1c, plays a central role in the de novo lipogenic pathway (8). SCD1 catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids from saturated fatty acids. It introduces a single double bond into its substrates, palmitic acid (16:0) and stearic acid (18:0), to generate palmitoleic (16:1n-7) and oleic acid (18:1n-9). These products are the most abundant fatty acids found in triglycerides, cholesterol esters, and phospholipids. Mice with a global or liver-specific knockout of SCD1 are protected from diet-induced obesity, hepatic steatosis, and insulin resistance (9 -11).

Experimental Procedures
Animals and Diets-RGC32 Ϫ/Ϫ mice on the C57BL/6 background were generated and genotyped as described previously (21). Parallel line WT C57BL/6 mice were purchased from The Jackson Laboratory. Age-matched WT and RGC32 Ϫ/Ϫ male mice were maintained on a regular chow for 8 weeks, after which they were fed with either normal chow (13% calories from fat, 3.07 kcal/g; catalog no. 5053, LabDiet) or an HFD (40% calories from fat, 4.5 kcal/g; catalog no. D12108C, Research Diets) for an additional 12 weeks. Mice were fasted overnight and anesthetized (2.0% isoflurane), and blood was collected by direct cardiac puncture. The liver was removed carefully and weighed. For generating ethanol-induced fatty liver, 8-weekold WT and RGC32 Ϫ/Ϫ male mice received three acute doses of ethanol (4.7 g/kg bodyweight, 40% diluted in water, v/v) with a 12-h interval by oral gavage. Mice were sacrificed 12 h after the final administration, and whole livers were collected. A portion of the liver was fixed in 4% paraformaldehyde for histological analysis, whereas the other portions were stored at Ϫ80°C for other assays. All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by Institutional Animal Care and Use Committee of the University of Georgia.
Isolation and Culture of Primary Hepatocytes-Hepatocytes were isolated from 8-to 12-week-old male C57BL/6 mice as described previously (22). Briefly, mice were anesthetized with 2.0% isoflurane, portal veins were cannulated, and livers were perfused with buffer A (10 mmol/liter HEPES, 143 mmol/liter NaCl, 7 mmol/liter KCl, and 0.2 mmol/liter EDTA (pH 7.4)), followed by buffer B (50 mmol/liter HEPES, 100 mmol/liter NaCl, 7 mmol/liter KCl, and 5 mmol/liter CaCl 2 (pH 7.6)) containing type IV collagenase (0.5 g/liter, Sigma) at 37°C for 10 min. After perfusion, the livers were excised, transferred to DMEM, and cut into small pieces. The cell suspension was passed through 70-m nylon mesh filters, centrifuged at 50 ϫ g, and resuspended in DMEM supplemented with 10% FBS to inhibit residual collagenase activity. The hepatocytes were then plated onto gelatin-coated plates at a density of 10 5 cells/cm 2 and cultured in DMEM supplemented with 10% FBS. The viability of the cells was tested with 0.4% trypan blue.
RNA Extraction and Real-time Quantitative RT-PCR (qPCR) Analysis-qPCR was performed as described previously (23). Briefly, total RNA from tissues or cultured cells was extracted using TRIzol reagent (Invitrogen). 1 g of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad). qPCR was performed in a Mx3005P qPCR machine using SYBR Green Master Mix (Agilent Technologies). Each sample was amplified in triplicate. The expression of each gene was normalized with cyclophilin.
Histological Analysis-Liver sections (5 m) were deparaffinized and stained with H&E. Oil Red O staining of the frozen liver sections (12 m) and quantitative analysis were performed as described previously (25). Images were captured with a Nikon microscope.
Measurement of Desaturase Indices-Total lipids were extracted from serum or liver samples, methylated, and analyzed by gas chromatography as described previously (26). Briefly, total fatty acid composition was determined using a Shimadzu GC-2010 analyzer. The column was a DB-23 (catalog no. 123-2332, J&W Scientific): length, 30 m; inner diameter, 0.32-mm-wide bore; film thickness, 0.25 m. The carrier gas is helium with a column flow rate of 2.5 ml/min. The analysis of fatty acid methyl esters was made on the basis of the area under the curve, calculated with Shimadzu Class VP 7.4 software. Fatty acid identification was made on the basis of retention times of authenticated fatty acid methyl ester standards (Matreya LLC Inc.). Our primary analysis focused on the principle substrates (16:0, 18:0) and products of SCD1 (16:1n-7, 18:1n-9). The desaturase indices were calculated as the ratios of 16:1/16:0 and 18:1/18:0.
SCD1 Promoter Luciferase Reporter Constructs-The mouse SCD1 promoter DNA fragments from Ϫ500 or Ϫ250 to ϩ300 bp relative to the mapped transcription start sites were obtained by PCR amplification from mouse genomic DNA using primers containing restriction enzyme sites of KpnI or XhoI. The PCR fragments were digested with restriction enzymes and cloned into the pGL4 vector. The SRE mutation was generated by replacing AGAG to CGCG as described previously (27, 28) using a site-directed mutagenesis kit (Agilent). The promoter regions of PCR products were verified by sequencing.
Transient Transfection and Luciferase Assay-Transient transfection and the luciferase assay were performed as described previously (29). Briefly, mouse primary hepatocytes were cultured for 24 h and then transduced with an adenovirus expressing GFP (Ad-GFP), RGC-32 shRNA (Ad-shRGC32), or RGC-32 (Ad-RGC32). 24 h later, different SCD1 promoter constructs were transfected into the cells with Lipofectamine LTX (Invitrogen) for 48 h, followed by a luciferase assay using the Dual-Luciferase reporter assay system (Promega). For each sample, a Renilla luciferase vector was cotransfected as an internal control, and promoter activities were normalized accordingly.
Statistical Analysis-Data are presented as means Ϯ S.D., and the number of independent experiments is indicated for each data set. For statistical analysis, two groups were compared using two-tailed Student's t tests, and three groups were evaluated by one-way analysis of variance followed by Tukey's multiple comparisons. Four groups were evaluated by two-way analysis of variance followed by Bonferroni post hoc tests for multiple comparisons using GraphPad Prism 5.0 software. p Ͻ 0.05 was considered statistically significant.

RGC-32 Deficiency Prevented HFD-induced Hepatic Steatosis-
Our previous studies have shown that RGC-32 expression in adipose tissue was induced by HFD challenge, and RGC-32 deficiency prevented HFD-induced obesity and insulin resistance (20). It is well known that obesity is often accompanied by hepatic steatosis (30). To test whether RGC-32 affects liver during HFD-induced obesity, we first detected RGC-32 expression in liver tissues from WT mice fed normal chow or a 12-week HFD. We found that both mRNA and protein expression of RGC-32 was dramatically up-regulated by the HFD challenge (Fig. 1, A and B). Then we examined whether RGC-32 plays a role in the regulation of HFD-induced hepatic steatosis. Both H&E and Oil Red O staining showed that HFD-fed WT mice had severe hepatic steatosis with numerous lipid droplets ( Fig.  1, C-E). However, RGC32 ؊/؊ dramatically alleviated HFD-induced hepatic steatosis, as evidenced by significantly fewer lipid droplets (Fig. 1, C-E). The hepatic triglyceride content of RGC32 ؊/؊ mice was also significantly lower than that of WT mice fed both normal chow and an HFD (Fig. 1F). Consistently, an HFD significantly increased the liver weight of WT but not RGC32 ؊/؊ mice (Fig. 1G). These data indicate that RGC32 ؊/؊ protects mice from developing HFD-induced hepatic steatosis.

RGC-32 Deficiency Reduced de Novo Hepatic Lipogenesis-
Hepatic steatosis can occur as a result of increased lipogenesis and/or reduced ␤-oxidation (6). To determine how RGC-32 affects HFD-induced hepatic steatosis, we detected a variety of genes involved in lipogenesis and ␤-oxidation of the liver. Interestingly, SREBP-1c, FAS, and SCD1 mRNA expression was decreased dramatically in livers of HFD-fed RGC32 ؊/؊ mice compared with HFD-fed WT control mice ( Fig. 2A), whereas no significant change of ␤-oxidation-related genes was observed ( Fig. 2A). Decreased expression of SREBP-1c, FAS, and SCD1 proteins was also evident in RGC32 ؊/؊ mice fed both normal chow and an HFD (Fig. 2, B-E), consistent with RGC32 ؊/؊ mice having a lower hepatic triglyceride content when fed both normal chow and an HFD (Fig. 1, C-E). SCD1 is the rate-limiting enzyme in lipogenesis. It catalyzes the desaturation of long-chain saturated fatty acids into monounsaturated fatty acids (11), the major components of membrane phospholipids, triglycerides, and cholesterol esters. The desaturase indices were calculated as the product-to-substrate ratio of the fatty acids metabolized by SCD1 (16:1n-7/16:0 and 18:1n-9/18:0) and were used as a proxy for the activity of the enzyme (31,32). Consistent with the decreased expression of SCD1, significantly reduced desaturase indices of C16 and C18 fatty acids were observed in both the liver and serum of RGC32 ؊/؊ mice fed both normal chow and an HFD (Fig. 3,  A-D). These data suggest that RGC-32 deficiency may ameliorate hepatic steatosis through inhibiting de novo hepatic lipogenesis.
RGC-32 Deficiency Protected Mice from Ethanol-induced Hepatic Steatosis-In addition to HFD-induced hepatic steatosis, increased lipogenesis was also observed in ethanol-induced fatty liver (4,33,34). To determine whether RGC-32 plays a broader role in de novo hepatic lipogenesis, we examined the effect of RGC-32 on the development of hepatic steatosis in response to ethanol administration. Ethanol administration dramatically increased RGC-32 expression at both mRNA and protein levels in the liver of WT mice (Fig. 4, A-C). Although H&E staining did not show an alteration in morphology (Fig.  4D), Oil Red O staining revealed that RGC32 ؊/؊ mouse liver had significantly fewer lipid droplets compared with the WT control both with and without ethanol administration (Fig. 4, E  and F). Importantly, ethanol significantly induced the expression of SREBP-1c, FAS, and SCD1 in the livers of WT mice both at the mRNA and protein levels, whereas this effect was dramatically inhibited in RGC32 ؊/؊ mice (Fig. 5, A-C). These results demonstrate that RGC-32 deficiency also protects mice from ethanol-induced hepatic steatosis through inhibiting de novo lipogenesis.
Insulin and Ethanol Increased Hepatic RGC-32 Expression through the NF-B Signaling Pathway-To determine the mechanism by which an HFD induced hepatic RGC-32 expression, mouse primary hepatocytes were isolated and treated with glucose and insulin, and then RGC-32 expression was detected. As shown in Fig. 6A, glucose slightly increased RGC-32 expression, whereas insulin dramatically induced RGC-32 expression, suggesting that insulin is the major contributor to increased RGC-32 expression during HFD challenge. To further determine how insulin regulated RGC-32 expression, we pretreated mouse primary hepatocytes with LY294002 or PDTC to block the PI3K-Akt and NF-B signaling pathways, respectively, before insulin treatment. PDTC, but not LY294002, blocked insulin-induced RGC-32 expression (Fig. 6, B-E), suggesting that insulin increased RGC-32 expression through the NF-B signaling pathway. To determine whether ethanol induces RGC-32 expression in vitro and whether it shares a common mechanism with insulin, we treated mouse primary hepatocytes with ethanol with or without PDTC pretreatment. As shown in Fig. 6F, ethanol increased RGC-32 expression, and PDTC blocked this effect. These data suggest that both insulin and ethanol induce hepatic RGC-32 expression through the NF-B signaling pathway.

RGC-32 Regulated Hepatic SCD1 Expression in a SREBP-1cdependent Manner-Previous
studies have shown that SCD1 is regulated at the transcriptional level via an SREBP-1c-dependent mechanism (28,35). Our results showed that RGC-32 deficiency prevented HFD-and ethanol-induced SREBP-1c and SCD1 expression in the liver (Figs. 2 and 5). To determine the mechanism by which RGC-32 regulates hepatic SCD1 gene expression, mouse primary hepatocytes were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32. Consistent with the in vivo findings, RGC-32 knockdown down-regulated, whereas RGC-32 overexpression up-regulated, the expression of SREBP-1c and SCD1 at both the mRNA and protein levels (Fig.  7, A-C), suggesting that RGC-32 directly regulates SREBP-1c and SCD1 expression in hepatocytes. Because RGC-32 does not have a DNA binding domain, we hypothesized that RGC-32 may regulate SCD1 expression through SREBP-1c. An SCD1 promoter analysis showed that RGC-32 knockdown decreased,
RGC-32 Regulated Hepatic SREBP-1c Expression through Activating LXR-Previous studies have shown that the binding of LXR to the functional LXR element in the SREBP-1c promoter is critical for SREBP-1c transcription (36 -38). Neither RGC-32 knockdown nor RGC-32 overexpression had any effect on LXR protein expression (Fig. 9, A and B). However, arachidonic acid, an inhibitor of LXR activation, blocked RGC-32induced SREBP-1c expression (Fig. 9, C and D), suggesting that RGC-32 may be involved in LXR activation. Therefore, we tested LXR binding to the SREBP-1c promoter using a ChIP assay. As shown in Fig. 9, E and F, RGC-32 knockdown decreased, whereas RGC-32 overexpression increased, LXR binding to the SREBP-1c promoter, suggesting that RGC-32 regulates hepatic SREBP-1c expression through activating LXR. Taken together, these data demonstrate that RGC-32 increases hepatic SREBP-1c expression through LXR activation, followed by enhancing SREBP-1c binding to the SCD1 promoter, leading to activation of SCD1 gene expression and lipogenesis.

Discussion
Nonalcoholic fatty liver disease is defined as a fatty liver (liver fat Ͼ 5-10% of liver weight) not due to excess alcohol consumption or other causes of steatosis (39). It is the most common cause of liver dysfunction and associated with obesity, metabolic syndrome, dyslipidemia, insulin resistance, and type 2 diabetes (6, 39). Our study demonstrates that RGC-32 plays a   6). B and C, SREBP-1c, FAS, and SCD1 protein expression in liver tissues of WT and RGC32 ؊/؊ mice with or without ethanol administration was detected by Western blot analysis and normalized to ␣-tubulin (n ϭ 6). *, p Ͻ 0.05 and **, p Ͻ 0.01 compared with WT groups; #, p Ͻ 0.05 and ##, p Ͻ 0.01 compared with WT ethanol groups.
critical role in the development of hepatic steatosis. RGC-32 is strongly induced in the liver by HFD challenge, which is consistent with the finding of Guo et al. (40). RGC-32 deficiency prevents HFD-induced hepatic steatosis, which may explain the improved insulin sensitivity in HFD-fed RGC32 ؊/؊ mice. More importantly, the liver triglyceride content of RGC32 ؊/؊ mice is much lower than that of WT mice, even under normal chow conditions, suggesting that RGC-32 is essential for regulating hepatic triglyceride content under both physiological and pathological conditions. Excessive accumulation of hepatic triglycerides can occur as a result of increased fat synthesis or reduced fat oxidation (41,42). Studies in humans and rodents have demonstrated that it is mainly linked to enhanced de novo lipid synthesis via the lipogenic pathway in the liver, whereas lipid disposal via ␤-oxidation is less important (6,43,44). It is now well accepted that the transcription factor SREBP-1c, induced by insulin through LXR activation (38), mediates the transcriptional effect on the enzymes involved in fatty acid synthesis and triglyceride synthesis, including FAS and SCD1 (8,45,46). Although lack of FAS reduces the rate of lipogenesis, liver-specific FAS knockout fails to protect mice against the development of fatty liver but rather exacerbates it because of a reduction in ␤-oxidation (47). Mice with a global or liver-specific knockout of SCD1 show decreased lipogenic gene expression and increased ␤-oxidation and are protected from diet-induced obesity and hepatic steatosis (9 -11). Our results show that RGC-32 affects hepatic lipid  accumulation by regulating the expression of lipogenic genes because SREBP-1c, FAS, and SCD1 are decreased significantly in the livers of RGC32 Ϫ/Ϫ mice. However, RGC-32 appears not to be involved in the regulation of fatty acid ␤-oxidation because the expression of peroxisome proliferator-activated receptor ␣ and its downstream target carnitine palmitoyltrans-ferase 1␣ are not affected in HFD-fed RGC32 Ϫ/Ϫ mice compared with the WT controls. Peroxisome proliferator-activated receptor ␣ is a key transcription factor regulating several genes important for fatty acid ␤-oxidation. These observations are consistent with previous findings showing that SCD1 function in hepatic steatosis is independent of peroxisome proliferator-FIGURE 8. RGC-32 regulated hepatic SCD1 expression in an SREBP-1c-dependent manner. A, mouse primary hepatocytes were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32 for 24 , followed by transfection of different SCD1 promoter constructs, as indicated, for 48 h. A luciferase assay was performed. Luciferase activity was normalized to Renilla activity. B and C, mouse primary hepatocytes were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32 for 48 h, and a ChIP assay was performed. SREBP-1c binding enrichment was detected by RT-PCR (B) and qPCR (C). D and E, mouse primary hepatocytes were transfected with scramble (siCtrl) or SREBP-1c siRNA (siSREBP-1c) for 24 h and then transduced with Ad-GFP or Ad-RGC32 for 48 h as indicated. SCD1, RGC-32, and SREBP-1c were detected by Western blot analysis and normalized to ␣-tubulin. All results are representative of at least three independent experiments. *, p Ͻ 0.05 and **, p Ͻ 0.01 compared with Ad-GFP groups; ##, p Ͻ 0.01 compared with siCtrl ϩ AdRGC32 groups. . RGC-32 regulated hepatic SREBP-1c expression through activating LXR. A and B, mouse primary hepatocytes were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32 for 48 h. LXR-␣ and RGC-32 were detected by Western blot analysis and normalized to ␣-tubulin. C and D, mouse primary hepatocytes were preincubated with 100 M arachidonic acid (AA) or vehicle for 1 h and then transduced with Ad-GFP or Ad-RGC32 for 48 h. SREBP-1c and RGC-32 were detected by Western blot analysis and normalized to ␣-tubulin. E and F, mouse primary hepatocytes were transduced with Ad-GFP, Ad-shRGC32, or Ad-RGC32 for 48 h, and a ChIP assay was performed. LXR-␣ binding enrichment was detected by RT-PCR (E) and qPCR (F). All results are representative of at least three independent experiments. *, p Ͻ 0.05 and **, p Ͻ 0.01 compared with Ad-GFP groups; ##, p Ͻ 0.01 compared with Ad-RGC32 groups. activated receptor ␣ (48). Remarkably, RGC-32 regulates SCD1 gene transcription, although RGC-32 does not directly bind to the SCD1 promoter. It appears that RGC-32 increases SREBP-1c expression through LXR activation and increases SREBP-1c binding to the SRE in the SCD1 promoter, which is critical for SCD1 transcription. Therefore, the beneficial effects of RGC-32 deficiency are at least partially due to the suppression of hepatic de novo lipogenesis.
In addition to nonalcoholic fatty liver disease, alcoholic liver disease is also a serious health problem. Alcoholic fatty liver is the earliest and most common response of the liver to alcohol and may be a precursor of more severe forms of liver injury, like steatohepatitis, progressive fibrosis, cirrhosis, and hepatocellular carcinoma (4,49). Although the pathogenesis and diagnosis are different between nonalcoholic and alcoholic fatty liver disease, excessive triglyceride accumulation in the liver induced by activation of lipogenesis is their common feature. Our data demonstrate that RGC-32 is a common regulator for the hepatic lipogenesis observed in both alcoholic and nonalcoholic hepatic steatosis. RGC-32 deficiency prevents both HFDand ethanol-induced hepatic steatosis through suppressing hepatic de novo lipogenesis by reducing the lipogenic genes SREBP-1c, FAS, and SCD1.
Our results also demonstrate that insulin instead of glucose is the major contributor to increased hepatic RGC-32 expression under HFD challenge. The study of Guo et al. (40) shows that glucose increases RGC-32 expression more dramatically than insulin in vascular endothelial cells. This discrepancy is likely due to cell type-specific effects. In vascular endothelial cells, RGC-32 is involved in glucose homeostasis (40), whereas, in hepatocytes, RGC-32 participates in lipogenesis. Taken together, our study identifies RGC-32 as a novel regulator for both nonalcoholic and alcoholic hepatic steatosis, which makes RGC-32 a potential drug target in preventing or reversing the process of hepatic steatosis.
Author Contributions-X. B. C. and S. Y. C. conceived the study, designed and analyzed the experiments, and wrote the paper. X. B. C. performed the majority of experiments. J. N. L. performed the Western blot analysis. All authors reviewed the results and approved the final version of the manuscript.