Allopurinol ameliorates high fructose diet induced hepatic steatosis in diabetic rats through modulation of lipid metabolism, inflammation, and ER stress pathway

Abstract

Excess fructose consumption contributes to development obesity, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD). Uric acid (UA), a metabolite of fructose metabolism, may have a direct role in development of NAFLD, with unclear mechanism. This study aimed to evaluate role of fructose and UA in NAFLD and explore mechanisms of allopurinol (Allo, a UA lowering medication) on NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats fed a high fructose diet (HFrD), with Long-Evans Tokushima Otsuka (LETO) rats used as a control. There were six groups: LETO, LETO-Allo, OLETF, OLETF-Allo, OLETF-HFrD, and OLETF-HFrD-Allo. HFrD significantly increased body weight, epididymal fat weight, and serum concentrations of UA, cholesterol, triglyceride, HbA1c, hepatic enzymes, HOMA-IR, fasting insulin, and two hour-glucose after intraperitoneal glucose tolerance tests, as well as NAFLD activity score of liver, compared to the OLETF group. Allopurinol treatment significantly reduced hepatic steatosis, epididymal fat, serum UA, HOMA-IR, hepatic enzyme levels, and cholesterol in the OLETF-HFrD-Allo group. Additionally, allopurinol significantly downregulated expression of lipogenic genes, upregulated lipid oxidation genes, downregulated hepatic pro-inflammatory cytokine genes, and decreased ER-stress induced protein expression, in comparison with the OLETF-HFrD group. In conclusion, allopurinol ameliorates HFrD-induced hepatic steatosis through modulation of hepatic lipid metabolism, inflammation, and ER stress pathway. UA may have a direct role in development of fructose-induced hepatic steatosis, and allopurinol could be a candidate for prevention or treatment of NAFLD.

Figure 1

Effects of HFrD and allopurinol treatment on glucose tolerance test. (A) Glucose concentrations and (B) insulin concentrations in LETO, LETO-Allo, OLETF, OLETF-Allo, OLETF-HFrD, and OLETF-HFrD-Allo group during intraperitoneal glucose tolerance test (IPGTT). IPGTT were conducted. 2 g/kg glucose was intraperitoneally administered to rats after overnight fasting. And blood samples were collected for the measurement of biochemical parameters at 0 min, 30 min, 60 min, 90 min, and 120 min after glucose injection. Data are expressed as mean ± SE. †P < 0.05, versus the LETO group. *P < 0.05, versus OLETF group. LETO, Long-Evans Tohushima Otsuka; LETO-Allo, LETO rats treated with allopurinol; OLETF, Otsuka Long-Evans Tokushima Fatty rats; OLETF-Allo, OLETF rats treated with allopurinol; OLETF-HFrD, OLETF rats fed a high fructose diet; OLETF-HFrD-Allo rats, OLETF rats fed a high fructose diet treated with allopurinol.

Figure 2

Liver pathology and NAFLD activity score after 16 weeks of LETO, OLETF, and HFrD-fed OLETF rats with or without allopurinol treatment. Liver morphology was visualized and evaluated using light microscopy (400 × magnification) on tissue sections subjected to hematoxylin and eosin (H&E) and Masson’s trichrome stains. (A). H&E-stained sections from rats in the OLETF and OLETF-HFrD group showed significant hepatic lipid accumulation compared to LETO group. Hepatic steatosis was decreased in the OLETF-HFrD-Allo group. (B). Significant fibrotic changes were not observed in all experimental groups. (C). NAFLD activity score was significantly elevated in the OLETF and OLETF-HFrD group compared to LETO group. Also, it was significantly decreased in OLETF-HFrD-Allo group, compared to OLETF-HFrD group. † P < 0.05 versus LETO group. *P < 0.05 versus OLETF-HFrD group. LETO, Long-Evans Tohushima Otsuka; LETO-Allo, LETO rats treated with allopurinol; OLETF, Otsuka Long-Evans Tokushima Fatty rats; OLETF-Allo, OLETF rats treated with allopurinol; OLETF-HFrD, OLETF rats fed with high fructose diet; OLETF-HFrD-Allo rats, OLETF rats fed with high fructose diet which were treated with allopurinol.

Figure 3

Hepatic mRNA expression of lipid metabolism genes and pro-inflammatory cytokine genes. Hepatic expression of genes was evaluated using real-time PCR. (A and B): Lipogenic genes expressions (SREBP-1c and SCD-1) were significantly upregulated in the OLETF and OLETF-HFrD groups compared to LETO group and significantly downregulated in the OLETF-HFrD-Allo group compared to the OLETF-HFrD group. (C and D): The expressions of lipid oxidation genes (PPARα and CPT-1) were significantly suppressed in the OLETF and OLETF-HFrD groups compared to the LETO group. However, allopurinol-treatment (OLETF-HFrD-Allo group) ameliorated the downregulation of lipid oxidation genes observed in OLETF-HFrD group. (E and F): Proinflammatory cytokine genes (TNF-α and PAI-1) significantly increased in the OLETF and OLETF-HFrD groups, and this increase was abolished in the OLETF-HFrD-Allo group. Data are expressed as mean ± SE. †P < 0.05 versus LETO group, *P < 0.05 versus OLETF group, #P < 0.05 versus OLETF-HFrD group LETO, Long-Evans Tohushima Otsuka; LETO-Allo, LETO rats treated with allopurinol; OLETF, Otsuka Long-Evans Tokushima Fatty rats; OLETF-Allo, OLETF rats treated with allopurinol; OLETF-HFrD, OLETF rats fed with high fructose diet; OLETF-HFrD-Allo rats, OLETF rats fed with high fructose diet which were treated with allopurinol; SREBP-1c, sterol regulatory element-binding protein 1c; SCD-1, stearoyl-CoA desaturase 1; PPARα, peroxisome proliferator-activated receptor-alpha; CPT-1, carnitine palmitoyltransferase 1; TNF-α, tumor necrosis factor-alpha; PAI-1, plasminogen activator inhibitor 1.

Figure 4

Hepatic ER stress pathway. BiP, p-IRE1α, and t-IRE1α protein expression levels were determined by Western blot analysis. Β-actin was used as a loading control. (A) OLETF and OLETF-HFrD groups significantly increased BiP over-expression and phosphorylation of IRE1 compared to LETO group. allopurinol-treatment significantly attenuated the HFrD-induced increased expression of BiP, and p-IRE1 in the OLETF-HFrD-Allo group compared to OLETF-HFrD group. (B,C). Relative protein expression was quantified using densitometry. *P < 0.05 versus NC group; #P < 0.05 versus HFrD group. ‘Full-length blots/gels’ are presented in Supplementary Fig. 1. LETO, Long-Evans Tohushima Otsuka; LETO-Allo, LETO rats treated with allopurinol; OLETF, Otsuka Long-Evans Tokushima Fatty rats; OLETF-Allo, OLETF rats treated with allopurinol; OLETF-HFrD, OLETF rats fed with high fructose diet; OLETF-HFrD-Allo rats, OLETF rats fed with high fructose diet which were treated with allopurinol; BiP, immunoglobulin heavy chain-binding protein; p- and t-IRE1, phosphorylated, and total inositol-requiring enzyme 1.