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

Fig 1. Effect of epidermal growth factor receptor (EGFR) inhibition on metabolic phenotypes in high fat diet (HFD)-fed mice. (A) Body weight. (B) Body weight gain. (C) Subcutaneous and epididymal fat mass (g/100 g body weight). (D) Food intake (g/day/mouse). (E) Glucose tolerance test (GTT); glucose (1 g/kg body weight) was injected into overnight-fasted mice. (F) Area under the curve (AUC) for GTT. (G) Fasting blood glucose levels. (H) Fasting blood insulin levels. (I) Homeostasis model of assessment for insulin resistance index (HOMA-IR) assessed as fasting insulin (μU/mL) × fasting glucose (mg/dL)/22.5. NCD, normal chow diet (NCD)-fed mice (NCD, black bars or white circles); HFD, untreated HFD-fed mice (white bars or white squares); HFD+PD, HFD-fed mice treated with PD153035 (gray bars or black triangles). Data are expressed as the mean ± standard error of the mean (SEM). * P < 0.05, ** P < 0.001, *** P < 0.0001 NCD vs. HFD group. # P < 0.05, ## P < 0.001, ### P < 0.0001 HFD vs. HFD+PD group.

Fig 2. Effects of EGFR inhibition on serum lipid levels in HFD-fed mice. Serum concentrations were measured after 12 weeks of NCD, HFD, and HFD+PD treamtment. (A) Serum total cholesterol (TC) concentration (mg/dL). (B) HDL cholesterol concentration (mg/dL). (C) LDL cholesterol concentration (mg/dL). (D) Triglyceride (TG) concentration (mg/dL). (E) Non-esterified fatty acid (NEFA) levels (mg/dL). NCD, NCD-fed mice (NCD, black bars); HFD, untreated HFD-fed mice (white bars); HFD+PD, HFD-fed mice treated with PD153035 (gray bars). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.001, *** P < 0.0001 NCD vs. HFD group. # P < 0.05, ## P < 0.001, ### P < 0.0001 HFD vs. HFD+PD group.

Fig 3. Effect of EGFR inhibition on hepatic steatosis in HFD-fed mice. (A) The weight of liver tissue (g/100 g body weight). (B) Hepatic triglyceride(TG) level (μmol/g tissue). (C) Alanine transaminase (ALT) (U/L) levels. (D) Aspartate transaminase (AST) (U/L) levels. (E) The m RNA levels of selected inflammatory genes were showed by real time PCR for liver tissue. (F) Steatosis score. Pathology scores were as follows: 0, no significant lesions (0%); 1, minimal (<10%); 2, mild (10–25%); 3, moderate (25–40%); 4, marked (40–50%); and 5, severe (>50%) (G) Representative images of H&E staining of liver sections. Scale bars = 10 μm. NCD, NCD-fed mice (NCD, black bars); HFD, untreated HFD-fed mice (white bars); HFD+PD, HFD-fed mice treated with PD153035 (gray bars). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.001, *** P < 0.0001 NCD vs. HFD group. # P < 0.05, ## P < 0.001, ### P < 0.0001 HFD vs. HFD+PD group.

Fig 4. Effects of EGFR inhibition on hepatic lipid metabolism in HFD-fed mice. (A) Analysis of proteins in the EGFR signaling pathway in mouse liver tissues. The figure shows the total and phosphorylated forms of EGFR and Akt. (B) Analysis of proteins associated with de novo lipogenesis in mouse liver tissues. (C–E) Real time quantitative PCR showing the transcriptional levels of genes related to lipid and cholesterol metabolism. (C) Transcriptional levels of cholesterol biosynthesis-related genes in liver tissues. (D) Transcriptional levels of de novo lipogenesis-related genes in liver tissues. (E) Transcriptional levels of fatty acid β-oxidation-related genes in liver tissues. (F) Huh-7 cells were treated with EGF and gefitinib. The expression of proteins in the EGFR signaling pathway were measured in Huh-7 cells. Real-time quantitative PCR assay showing the transcriptional levels of lipid metabolism-related genes. (G) Huh-7 cells were treated with palmitic acid (PA) and gefitinib. Representative images of oil Red O staining of Huh-7 cells and the transcriptional levels of lipid metabolism-related genes, as examined by real-time quantitative PCR. NCD, NCD-fed mice (NCD, black bars);…