Epidermal growth factor receptor inhibition attenuates non-alcoholic fatty liver disease in diet-induced obese mice

Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of chronic liver disease. NAFLD begins with excessive lipid accumulation in the liver and progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is closely linked to dysregulated hepatic lipid metabolism. Although recent studies have reported that epidermal growth factor receptor (EGFR) signaling regulates lipid metabolism, the roles of EGFR and EGFR inhibitors as modulators of lipid metabolism are largely unknown. Here, we investigated whether inhibiting EGFR using the EGFR tyrosine kinase inhibitor (TKI) PD153035 improves NAFLD. Our results demonstrate that EGFR was activated in liver tissues from high fat diet (HFD)-induced NAFLD mice. Inhibiting EGFR using PD153035 significantly reduced phosphatidylinositol-3-kinase/protein kinase B signaling and sterol responsive elementary binding protein 1 and 2 expression, which prevented HFD-induced hepatic steatosis and hypercholesterolemia by reducing de novo lipogenesis and cholesterol synthesis and enhancing fatty acid oxidation. Additionally, inhibiting EGFR improved HFD-induced glucose intolerance. In conclusion, these results indicate that EGFR plays an important role in NAFLD and is a potential therapeutic target.


Introduction
The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly worldwide and it is now the most common liver disease, with an estimated global prevalence of~25% [1]. NAFLD is characterized by excess fat accumulation in the liver, including simple fatty liver and nonalcoholic steatohepatitis (NASH). NAFLD can progress to liver cirrhosis and hepatocellular carcinoma [2], and is closely related to obesity and metabolic syndrome. Several previous studies reported that insulin resistance is also strongly associated with NAFLD and that NAFLD and type 2 diabetes mellitus frequently coexist [3,4]. Although NAFLD has become a public health concern worldwide, there is still no approved drug for the treatment of NAFLD. The epidermal growth factor receptor (EGFR) signaling pathway has been implicated in many human diseases, especially in cancer, as it plays a central role in regulating the survival, proliferation, migration, and differentiation of various tissues [5]. Several studies have also suggested that EGFR is associated with metabolic disorders [6,7]. Our previous study showed that serum cholesterol and intrahepatic lipid levels were increased in mice with conditional ablation of mitogen-inducible gene 6 (Mig-6), an EGFR negative feedback inhibitor, in the liver [8], suggesting that EGFR is a possible target for the treatment of dyslipidemia and NAFLD. However, the effectiveness of EGFR-targeted treatments in NAFLD is unknown. Therefore, we aimed to investigate whether inhibiting EGFR using the EGFR tyrosine kinase inhibitor (TKI) PD153035 improves NAFLD.

Animals
Male C57BL/6J mice were purchased from Harlan (Indianapolis, IN, USA). A high-fat diet (HFD) composed of 60% fat was purchased from Research Diets Inc. (D12492; New Brunswick, NJ, USA). The animals were maintained in a controlled environment (12 h light/12 h dark cycle; 50-60% humidity; ambient temperature 22 ± 2˚C). Eight-week-old male mice were fed a normal chow diet (NCD) or HFD for 8 consecutive weeks and then divided randomly into three groups: the NCD group were fed an NCD without treatment, the HFD group were fed a HFD without treatment, and the HFD+PD group were fed a HFD and treated with PD153035 (30 mg/kg/day O.G.; Selleck Chemicals, Houston, TX, USA) for the final 4 weeks. All animals received humane care according to institutional guidelines, and all experimental procedures were approved by the Institutional Review Board of Chungnam National University School of Medicine (Daejeon, South Korea).

Cell culture
The Huh-7 hepatocellular carcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured according to the manufacturer's instructions. Cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin. Huh-7 cells were stimulated with 10 ng/mL EGF (Sigma, St. Louis, MO, USA) and treated with 10 μM gefitinib (Sigma). Huh-7 cells were incubated with 400 μM palmitic acid and 10 μM gefitinib for 24 h.

Histological analysis
Tissue samples were obtained from 18-week-old mice. Samples for light microscopy were fixed in 4% paraformaldehyde (PFA) for 1 h. Paraffin embedding, sectioning, and hematoxylin and eosin (H&E) and oil red O staining were performed according to standard protocols.

Serum biochemical measurements
Blood was collected from the heart under general anesthesia. Samples were centrifuged at 5,000 rpm for 5 min and the supernatants were collected. Serum insulin levels were measured by enzyme-linked immunosorbent assay (ELISA; ALPCO Diagnostics, Salem, NH, USA) using kits obtained from the indicated supplier. Biochemical analyses, including measuring free fatty acid and total cholesterol levels, were performed using a Hitachi 7180 auto analyzer and Wako reagents (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

RNA isolation and real-time PCR
Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was prepared from total RNA using M-MLV reverse transcription and oligo-dT primers (Invitrogen). The resulting cDNA was amplified on a Rotor-Gene 6000 real-time rotary analyzer with software version 1.7 (Corbett Life Science, Sydney, Australia). Real-time polymerase chain reaction (PCR) was performed using cDNA and QuantiTect SYBRGreen PCR Master Mix (Qiagen, Hilden, Germany). All quantitative calculations were performed using the ΔΔCT method. The comparative Ct method was used to quantify transcripts, which were normalized to 18sRNA expression. Values were expressed as fold changes vs. control groups.

Statistical analysis
Statistical analyses were performed using Stat Graph Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± standard error of the mean (SEM). To compare values obtained from two or more groups, data from animal studies were analyzed using two-way repeated-measures analysis of variance followed by Bonferroni's correction for multiple comparisons, one-way analysis of variance followed by Tukey's post hoc test, or two-tailed Student's t-tests. P < 0.05 was considered statistically significant.

Inhibiting EGFR reverses HFD-induced weight gain
Male C57BL/6J mice were maintained on an NCD or HFD for 8 weeks. A significant increase in body weight was observed in the HFD group compared with the NCD group, as expected ( Fig 1A and 1B). At the end of week 8, HFD-fed mice were treated with the specific EGFR inhibitor PD153035. PD153035 is a highly potent and specific inhibitor of EGFR tyrosine kinase; it suppresses the tyrosine phosphorylation and activation of EGFR by competitively binding to the receptor tyrosine kinase domain [9]. Treatment with PD153035 reversed HFDinduced weight gain throughout the 4-week treatment period (Fig 1A and 1B). In addition, the weights of subcutaneous and epididymal fat were reduced significantly in the HFD+PD group compared with the HFD group ( Fig 1C). During the 4-week treatment period, daily food intake was similar between the HFD and HFD+PD groups (data not shown).Inhibiting

EGFR improves glucose tolerance and insulin sensitivity in HFD-induced obese mice
To compare glucose homeostasis parameters among groups, glucose tolerance tests were performed. Treatment with PD153035 for 4 weeks improved glucose tolerance ( Fig 1D) and reduced fasting plasma glucose levels ( Fig 1E) in HFD-induced obese mice. In addition, plasma insulin levels were significantly lower in the HFD+PD group compared with the HFD group ( Fig 1F). Treatment with PD153035 also improved the homeostasis model of assessment for insulin resistance index (HOMA-IR) (Fig 1G). These findings indicate that EGFR inhibition reduces HFD-induced weight gain, leading to improved glucose tolerance and insulin sensitivity.

Inhibiting EGFR reduces serum lipid levels in HFD-induced obese mice
Because NAFLD was strongly linked with dyslipidemia, we next investigate serum lipid levels in each group. HFD-fed mice exhibited significantly increased serum total cholesterol (TC), high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, triglyceride (TG), and non-esterified fatty acid (NEFA) levels compared with those of the NCD group (Fig 2A-2E). Treatment with PD153035 significantly reduced serum TC, HDLC, and LDLC levels in HFD-fed mice to levels close to those observed in NCD-fed mice (Fig 2A-2C). Serum TG and NEFA levels were also significantly lower in the HFD+PD group than in the HFD group (Fig 2D and 2E).

Inhibiting EGFR prevents hepatic steatosis in HFD-induced obese mice
The most common cause of hepatomegaly is hepatic steatosis, which is also the hallmark feature of NAFLD [10]. Furthermore, NAFLD is now the most common cause of elevated liver enzymes, indicating liver damage [11]. Therefore, we next compared liver weights and liver function tests, including measuring serum aspartate transaminase (AST) and alanine transaminase (ALT) levels, between groups. HFD-fed mice had a higher mean liver weight and elevated liver enzymes compared with NCD-fed mice (Fig 3A-3C). As expected, treatment with PD153035 significantly reduced liver weight (Fig 3A) in HFD-fed mice. Treatment with PD153035 also significantly reduced AST and ALT levels in HFD-fed mice (Fig 3B and 3C).
To evaluate the intrahepatic lipid contents and the degree of liver damage, we performed H&E staining of liver tissues. Histopathological analysis of the livers in the HFD group revealed markedly increased hepatic steatosis (Fig 3D), whereas treatment with PD153035 noticeably reduced hepatic lipid contents. Hepatocellular vacuolation, which indicates hepatocellular damage, was also reduced by treatment with PD153035 ( Fig 3D). These results suggest that inhibiting EGFR may improve HFD-induced hepatic steatosis.

Inhibiting EGFR regulates hepatic lipid metabolism in HFD-induced obese mice
To understand the role of EGFR in regulating hepatic lipid metabolism, we investigated whether inhibiting EGFR alters the mRNA and protein expression of proteins associated with hepatic lipid metabolism pathways such as hepatic de novo lipogenesis (DNL) and cholesterol synthesis. First, we examined whether EGFR signaling is activated in the livers of HFD-fed mice. The phosphorylation of EGFR and its downstream protein Akt was increased in the livers of HFD-fed mice. Treatment with PD153035 effectively reduced the phosphorylation of both proteins (Fig 4A).
https://doi.org/10.1371/journal.pone.0210828.g001 are closely linked to the development of NAFDL [13]. The protein expression of SREBP-1 and its lipogenic targets such as fatty acid synthase (FASN) was increased in HFD-induced NAFLD, whereas inhibiting EGFR decreased SREBP-1 and FASN expression. The expression of SREBP-2, a transcription factor that preferentially activates genes involved in cholesterol synthesis, was also decreased after EGFR inhibition (Fig 4B). These findings indicate that inhibiting EGFR reduces protein expression related to lipogenesis and cholesterol synthesis.

Inhibiting EGFR prevents intracellular lipid accumulation in hepatocytes
To reconfirm our results in vitro, we used the human hepatocellular carcinoma cell line, Huh-7. Cells were treated with EGF to stimulate the EGFR signaling pathway, and the activation was reversed by the EGFR TKI, gefitinib (Fig 4F). Consistent with the in vivo data, EGFR inhibition decreased the transcriptional level of genes involved in DNL and cholesterol biosynthesis, such as Srebp1, Srebp2, and Hmgcr ( Fig 4F). Then, Huh-7 cells were treated with palmitic acid to simulate excessive free fatty acid influx into hepatocytes [14]. After 24 h, cells were stained with oil red O to examine intracellular lipid accumulation. Significantly increased intracellular lipid accumulation was observed in cells exposed to palmitic acid. However, inhibiting EGFR using gefitinib attenuated palmitic acid-induced lipid accumulation and DNL (Fig 4G).

Discussion
The mechanisms of NAFLD progression have been studied in recent years. Although several mechanisms have been revealed [15,16], the pathogenesis of NAFDL is not fully understood. In this study, we confirmed that EGFR signaling is associated with the development of NAFLD. Our results demonstrated that EGFR signaling is increased in HFD-induced NAFLD. Moreover, EGFR inhibition using PD153035 induced a significant improvement in hepatic steatosis, which was accompanied by improved intrahepatic lipid levels. In addition, PD153035 also prevented HFD-induced dyslipidemia and insulin resistance. These results suggest that inhibiting the EGFR signaling pathway may be a potential therapeutic target for NAFLD and related dyslipidemia and insulin resistance.
NAFLD is classified into four stages as follows: stage 1, simple fatty liver (steatosis); stage 2, non-alcoholic steatohepatitis (NASH); stage 3, fibrosis; and stage 4, cirrhosis. In the early stages of NAFLD, only hepatic TG accumulation, without abnormal liver function, is found. NASH is characterized by hepatic steatosis with inflammation, and persistent inflammation can ultimately lead to irreversible fibrotic change [17]. Therefore, early diagnosis and intervention for hepatic steatosis are essential to prevent progression to fibrosis. According to the current results, EGFR signaling may be a novel target for the treatment of hepatic steatosis and steatohepatitis, which are the early stages of NAFLD.
The current study also confirmed that inhibiting EGFR improves glucose tolerance. Several studies have suggested that EGFR affects glycolysis and glucose transport through the downstream RAS/RAF/MAPK and PI3K/Akt pathways [21]. In addition, previous reports indicated that EGFR TKIs may exert antidiabetic effects in type 1 and type 2 diabetes [28]. Although several mechanisms have been proposed to explain how TKIs can regulate glucose metabolism, the underlying mechanism is not fully understood. However, reduced insulin resistance, caused by reducing the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 and inhibited M1 pro-inflammatory macrophage infiltration in adipose tissue, was suggested as a potential mechanism [29]. Because insulin resistance is closely associated with NAFLD, reducing cytokine levels and M1 macrophage infiltration may be additive mechanisms through which EGFR inhibition improves NAFLD. However, we did not examine the expression of TNF-α and IL-6 or the degree of macrophage infiltration, which is a limitation of our study.

Conclusions
In conclusion, we demonstrated that inhibiting EGFR significantly improved HFD-induced NAFLD. To our knowledge, this is the first time that the role of EGFR as a therapeutic target of NAFLD has been experimentally investigated. Furthermore, we confirmed that inhibiting EGFR reduces liver fat accumulation and hepatocellular damage by reducing DNL and cholesterol synthesis and by enhancing fatty acid oxidation. However, further studies are needed to address the clinical efficacy and safety of EGFR TKIs such as PD153035.
Supporting information S1