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); HFD, untreated HFD-fed mice (white bars); HFD+PD, HFD-fed mice treated with PD153035 (gray bars); NC, negative control; EGF, Huh-7 cells stimulated with 10 ng/mL EGF; EGF+G, Huh-7 cells stimulated with 10 ng/mL EGF and 10 μM gefitinib; PA, Huh-7 cells incubated with 400 μM palmitic acid; PA+G, Huh-7 cells incubated with 400 μM palmitic acid and 10 μM gefitinib for 24 h. Data are expressed as the mean ± SEM. * P P P P P P < 0.0001 HFD vs. HFD+PD group.</p

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 (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 P P P P P < 0.0001 HFD vs. HFD+PD group.</p

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 P P P P P < 0.0001 HFD vs. HFD+PD group.</p

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 P P P P P < 0.0001 HFD vs. HFD+PD group.</p

Three-dimensional model of mTOR and docking simulation between mTOR and PP242 and MHY1485.

<p>A three-dimensional model of mTOR (NCBI GI number: 4826730) was built using SWISS-MODEL program (A). Homologue structural template used was 1E8X (18% sequence identity and 6.9E-44 e-value). The Z-score of the predicted model was −4.8. We checked the quality of each residue on the mTOR structure model. The internal region, which bound ATP and target compounds, was more reliable (blue) than the external region (red) of mTOR. The estimated residue error was visualized using a color gradient from blue (more reliable regions) to red (potentially unreliable regions, estimated error above 3.5 Å). B shows the docking simulation between mTOR and PP242 and MHY1485. The magenta compound is PP242, which was used as a control compound. The cyan compound is MHY1485, and the yellow compound is ATP. The binding energies of the compounds were −7.28 kcal/mol (PP242) and −7.55 kcal/mol (MHY1485).</p

Adipokine, proinflammatory, and macrophage markers by obesity level.

Adipokine, proinflammatory, and macrophage markers by obesity level.</p

Increase of the LC3II/LC3I ratio and LC3II-positive vacuoles by MHY1485.

<p>Western blot analysis was performed to detect LC3II and LC3I. Ac2F cells were treated with different concentrations of MHY1485 and rapamycin 5 µM as a positive control for 6 h. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated or rapamycin-treated samples with the LC3II/LC3I ratios from untreated samples (A). Control cells treated with same volume of vehicle or MHY1485 (2 µM) for 1, 6 or 12 h were collected. Bars represent the LC3II/LC3I ratio calculated by normalizing the LC3II/LC3I ratio from MHY1485-treated samples with LC3II/LC3I ratio from control samples at 1 hour (B). β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05, **p<0.01, ***p<0.001; n = 3). Live-cell confocal microscopic images of AdGFP-LC3-transfected Ac2F cells treated with 2 µM MHY1485 for 1, 6 or 12 h are shown. The images show the GFP-LC3-positive vacuoles (upper, green), corresponding phase contrast images (middle) and merged images (bottom). Scale bar, 20 µm.</p

Activation of mTOR by MHY1485.

<p>Western blot analysis was performed to detect the change of total protein level and levels of phosphorylated forms of mTOR and 4E-BP1 reflecting the activity of mTOR. Ac2F cells were treated with MHY1485 of different concentrations and rapamycin 5 µM as a positive control for 1 h. Bars represent the phospho-mTOR(Ser2448)/mTOR ratio and the phospho-4E-BP1(Thr37/46)/4E-BP1 ratio normalized with the ratio from untreated samples, respectively. β-Actin blot is shown to verify the same amount of protein loaded. The blots were quantified by densitometry expressed as mean±SD (*p<0.05; n = 3).</p

Effects of gefitinib and statin treatments on the serum lipid profile.

<p>Changes in serum lipids after 6 weeks of gefitinib or statin treatment. (A) Total cholesterol, (B) HDL cholesterol, (C) LDL cholesterol, (D) triglyceride levels (n = 10 each, <i>P</i><0.05). Values represent the means ±SEM. *<i>P</i><0.05 relative to baseline. ^&<i>P</i><0.05 relative to Con, ^#<i>P</i><0.05 relative to G. N-C, normal control; HFD, high-fat diet; Con, control; G, gefitinib; S, statin.</p

The Roles of Adipokines, Proinflammatory Cytokines, and Adipose Tissue Macrophages in Obesity-Associated Insulin Resistance in Modest Obesity and Early Metabolic Dysfunction

<div><p>The roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in obesity-associated insulin resistance have been explored in both animal and human studies. However, our current understanding of obesity-associated insulin resistance relies on studies of artificial metabolic extremes. The purpose of this study was to explore the roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in human patients with modest obesity and early metabolic dysfunction. We obtained omental adipose tissue and fasting blood samples from 51 females undergoing gynecologic surgery. We investigated serum concentrations of proinflammatory cytokines and adipokines as well as the mRNA expression of proinflammatory and macrophage phenotype markers in visceral adipose tissue using ELISA and quantitative RT-PCR. We measured adipose tissue inflammation and macrophage infiltration using immunohistochemical analysis. Serum levels of adiponectin and leptin were significantly correlated with HOMA-IR and body mass index. The levels of expression of MCP-1 and TNF-α in visceral adipose tissue were also higher in the obese group (body mass index ≥ 25). The expression of mRNA MCP-1 in visceral adipose tissue was positively correlated with body mass index (r = 0.428, p = 0.037) but not with HOMA-IR, whereas TNF-α in visceral adipose tissue was correlated with HOMA-IR (r = 0.462, p = 0.035) but not with body mass index. There was no obvious change in macrophage phenotype or macrophage infiltration in patients with modest obesity or early metabolic dysfunction. Expression of mRNA CD163/CD68 was significantly related to mitochondrial-associated genes and serum inflammatory cytokine levels of resistin and leptin. These results suggest that changes in the production of inflammatory biomolecules precede increased immune cell infiltration and induction of a macrophage phenotype switch in visceral adipose tissue. Furthermore, serum resistin and leptin have specific roles in the regulation of adipose tissue macrophages in patients with modest obesity or early metabolic dysfunction.</p></div