Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress

Abstract

The effects of high (H)-fructose (FR) diet (D) (HFRD) on hepatic lipid homeostasis, oxidative stress, inflammation and hepatocyte apoptosis were investigated in 6-week old female C57BL/6J mice fed a regular chow (ContD) or HFRD (35% fructose-derived calories) for 3 weeks. HFRD-fed mice exhibited increased levels of hepatic steatosis with a significant elevation of serum levels of triglyceride, cholesterol and TNFα compared to ContD-fed mice (P<0.05). HFRD-fed mice exhibited ∼2.7- fold higher levels FAS along with significantly decreased protein levels of adiponection-R2 (∼30%), P-AMPK (∼60%), P-ACC (∼70%) and RXR-α (∼55%), suggesting decreased hepatic fat oxidation compared to controls. Interestingly, hepatic fatty acid uptake into hepatocytes and lipolysis were significantly increased in HFRD-fed mice, as shown by decreased CD36 and fatty acid transporter protein-2, and increased adipose triglyceride lipase, respectively (P<0.05). Increased hepatic levels of iNOS and GSSG/GSH suggest elevated oxidative stress with a higher number of macrophages in the adipose tissue in HFRD-fed mice (P<0.05). Significantly elevated rates of hepatocyte apoptosis (∼2.4-fold), as determined by TUNEL analysis with increased Bax/Bcl2 ratio and PARP-1 levels (∼2- and 1.5-fold, respectively), were observed in HFRD-fed mice. Thus, HFRD exposure increased hepatic steatosis accompanied by oxidative stress and inflammation, leading to hepatocyte apoptosis.

Figure 1

Effects of solid HFRD on hepatic steatosis and metabolic parameters in mice. (A) Representative liver histology with H&E staining, (B) steatosis score, (C) inflammation score, (D) concentration of hepatic TG, (E) serum TG, (F) serum cholesterol, (G) serum adiponectin and (H) serum ALT of experimental mice are shown. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 2

Effects of HFRD on hepatic levels of key proteins involved in lipid metabolism. (A) Representative images of the immunoblot analysis and the densitometric levels for SCD1 and FAS and (B) PPARα, PPARγ and RXRα in the livers of the indicated mouse groups are presented. Immunoblot results of the target proteins were normalized to β-actin (for SCD1 and FAS) or Histone H3 (for PPARα, PPARγ and RXRα), used as a loading control. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 3

Effects of HFRD on hepatic levels of genes involved in adiponectin signaling. Representative images of the immunoblot analysis and densitometric levels for (A) Adiponectin R2, (B) P-AMPK and AMPK, (C) P-ACC and ACC are shown. Immunoblot results of the target protein was normalized to β-actin, used as a loading control. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 4

Effects of dietary HFRD on hepatic levels of the key proteins involved in fatty acid uptake and lipolysis. Representative images of the immunoblot analysis and densitometric levels for (A) CD36 and FATP2 and (B) HSL and ATGL are provided. Immunoblot results of the target protein was normalized to β-actin, used as a loading control. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 5

Effects of HFRD on oxidative stress markers. (A) Representative images of the immunoblot analysis and the densitometric levels (B) for CYP2E1, iNOS and HO-1 in the livers of experimental mice are presented. Immunoblot results for the target protein was normalized to β-actin, used as a loading control. The levels of (C) GSH, (D) GSSG, and (E) GSSG/GSH as well as the activities of (F) SOD and (G) Catalase in HFRD-fed mice compared to control mice are shown. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 6

Effects of HFRD on adipose tissue inflammation. (A and B) Representative F4/80 immunohistochemistry showing macrophage infiltration and (C) visceral fat-pad weights. (D) Relative mRNA levels of inflammatory markers and (E) serum TNFα concentration are shown. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; ** P < 0.01.

Figure 7

Effects of HFRD on hepatic apoptosis. (A) Representative images of TUNEL- positive apoptotic hepatocytes (marked with black arrows) in the livers of indicated groups are presented. (B) Number of TUNEL-positive hepatocyte in 10 high-powered fields (×200) was calculated. (C) Representative images and densitometric levels of the immunoblot analysis for Bax, Bcl2, and PARP-1 in the livers of the experimental groups. All results are presented as mean ± S.E.M. (n=6~7/group). Significant differences between the two groups are indicated by asterisks: *P < 0.05; **P < 0.01.

Figure 8

Proposed mechanisms for the induction of hepatic apoptosis and steatosis by high-fructose diet (HFRD).