C-C chemokine receptor 2 inhibitor ameliorates hepatic steatosis by improving ER stress and inflammation in a type 2 diabetic mouse model

Abstract

Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study, db/db and db/m (n = 9) mice were fed CCR2 inhibitor (2 mg/kg/day) for 9 weeks. In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers (e.g., BiP, ATF4, CHOP, and XBP-1) and inflammatory cytokines (e.g., TNFα, IL-6, and MCP-1) while increasing markers of mitochondrial biogenesis (e.g., PGC-1α, Tfam, and COX1) in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.

Fig 1. Changes in body weight, liver weight, TG content, and lipid synthesis genes in experimental animals.

CCR2 inhibitor-treated and control db/db mice were fed an NCD for 9 weeks. (A) Body weight changes of mice and (B) liver weights of treated (n = 9) and control (n = 8) mice. (C) Histological analysis and (D) triglyceride (TG) content of liver. Sections were stained with hematoxylin and eosin. Results shown are means ± SEM. Original magnification is x200 (scale bar = 100 μm). (E) Levels of lipid synthesis gene (SREBP1c, FasN, HSL, L-FABP and SCD1) mRNAs in the livers of treated db/db mice. mRNA levels were estimated by real-time reverse transcriptase-PCR. Data shown are means ± SEM of 9 animals for each group. *P < 0.05 compared with the db/m mice. #P < 0.05 compared with control db/db mice.

Fig 2. Changes of hepatic macrophages in experimental animals.

(A) CD68 staining in the liver tissue sections by immunohistochemistry. Original magnification is x200 (scale bar = 100 um). (B) Western blots of (C) macrophage (CD68), and (D) M1 macrophage (CD16) in the liver of CCR2 inhibitor-treated (n = 9) and control (n = 8) mice. The band intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with db/m mice. #P < 0.05 compared with control db/db mice.

Fig 3. Hepatic inflammatory responses in experimental animals.

(A-D) Concentrations of inflammatory proteins such as TNF-α, IL-6, MCP-1 and anti-inflammatory cytokine (IL-10) in the livers of CCR2 inhibitor-treated (n = 9) and control (n = 8) db/db mice. (A-C) Cytokine levels in homogenates were measured by enzyme-linked immunosorbent assay and normalized for protein content. (D) Western blots of anti-inflammatory cytokine (IL-10) in the livers of treated (n = 9) and control mice (n = 8). (E-G) Inflammatory signaling pathway markers IKKβ and JNK in the livers of treated (n = 9) and control mice (n = 8). The band intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with db/m mice. #P < 0.05 compared with control db/db mice.

Fig 4. ER stress, oxidative stress proteins, and mitochondrial biogenesis gene levels in the livers of experimental animals.

Liver tissue (300 μg) was homogenized with 500 μL of lysis buffer supplemented with protease and phosphatase inhibitors. Western blots showing levels of (A and B) ER stress proteins (BiP, ATF4, CHOP and XBP-1s) in livers of CCR2 inhibitor-treated (n = 9) and control (n = 8) db/db mice. (C) XBP-1 staining of liver sections by immunohistochemistry. Original magnification is x200 (scale bar = 100 um). (D) Levels of mitochondrial biogenesis genes (PGC1a, Tfam, and COX1) in liver from treated (n = 9) and control (n = 8) db/db mice. (E) Western blots showing levels of antioxidation (SOD2) and oxidative stress (4HNE) proteins in liver from treated (n = 9) and control (n = 9) mice. Band intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with db/m mice. #P < 0.05 compared with control db/db mice.

Fig 5. CCR2 inhibitor ameliorates insulin resistance in experimental mice.

(A, B and C) Western blots of insulin signaling (pIRS-1 Ser³⁰⁷, IRS-1, pAkt and Akt) in liver from CCR2 inhibitor-treated (n = 9) and control (n = 8) db/db mice. (D) Western blots of mTOR in liver from CCR2 inhibitor-treated (n = 9) and control (n = 8) db/db mice. (E) GLUT4 mRNA in the livers of treated db/db mice. mRNA levels were evaluated by real-time reverse transcriptase-PCR. Bands intensities were measured with an Image J Analyzer. Results shown are means ± SEM (n = 8–9 mice per group). *P < 0.05 compared with db/m mice. #P < 0.05 compared with control db/db mice.

Fig 6. Upregulation of lipid synthesis on hepatocytes by palmitate.

Hepatocytes (AML12) were treated with a fatty liver-related factor (palmitate 250 μM). Palmitate was prepared in ethanol containing bovine serum albumin (BSA, 50 μM). (A) AML12 cells were stained with Oil red O. Original magnification is x200 (scale bar = 100 μm). (B) Western blots of SREBP-1 and PGC-1a in AML12 cells. (C) mRNA levels of lipid synthesis genes (SREBP1c, FasN, HSL, L-FABP, and SCD1) in AML12 cells. (D) Western blots of inflammatory signaling pathway markers (IKKβ and JNK) in AML12 cells. Bands intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with control. #P < 0.05 compared with palmitate-treated control.

Fig 7. Hepatic inflammatory and ER stress responses in AML12 cells.

Western blots of (A) oxidative stress markers (SOD2 and 4-HNE) and (B) ER stress proteins (BiP, ATF4, CHOP, and XBP-1s) in AML12 cells. Band intensities were measured with an Image J Analyzer. Results shown are means ± SEM. *P < 0.05 compared with control. #P < 0.05 compared with palmitate-treated control. (C) Schematic presentation for the effect of CCR2 inhibitor in hepatocyte.