Ellagic Acid Alleviates Hepatic Oxidative Stress and Insulin Resistance in Diabetic Female Rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) affects more than 70% of patients with type 2 diabetes mellitus (T2DM) and has become one of the most common metabolic liver diseases worldwide. To date, treatments specifically targeting NAFLD do not exist. Oxidative stress and insulin resistance have been implicated in the pathogenesis of NAFLD in diabetes. Accordingly, the goal of this present study was to determine whether Ellagic acid (EA), a natural antioxidant polyphenol found in berries and nuts, mitigates hepatic oxidative stress and insulin resistance in T2DM rats, and thus alleviates NAFLD. Using adult female Goto Kakizaki (GK) rats, a non-obese and spontaneous model of T2DM, we found that EA treatment significantly lowered fasting blood glucose and reduced insulin resistance, as shown by a 21.8% reduction in the homeostasis model assessment index of insulin resistance (HOMA-IR), while triglyceride and total cholesterol levels remained unchanged. Increased hepatic lipid accumulation and oxidative stress present in diabetic GK rats was markedly reduced with EA treatment. This effect was associated with a downregulation of the NADPH oxidase subunit, p47-phox, and overexpression of NF-E2-related factor-2 (NRF2). Moreover, EA was able to decrease the hepatic expression of hypoxia-inducible factor (HIF-α), a transcription factor linked to hypoxia and hepatic steatosis. We further showed that EA treatment activated an insulin signaling pathway in the liver, as evidenced by increased levels of phosphorylated Akt (Ser 473). In conclusion, our results demonstrate that EA diminishes blood glucose levels and potently suppress NAFLD in diabetic rats via mechanisms that involve reductions in p47-phox and HIF-α, upregulation of NRF2 and enhancement of the Akt signaling pathway in the liver. Together, these results reveal that EA improves hepatic insulin sensitivity and lipid metabolism as a result of its antioxidant effects. This implies an anti-diabetic effect of EA with beneficial effects for the treatment of hepatic complications in T2DM.

Keywords: Ellagic Acid; Goto-kakizaki (GK) rats; HIF-α; NRF2; Type II Diabetes Mellitus (T2DM); antioxidant; hepatic steatosis; insulin resistance; oxidative stress; p47-phox.

Figure 1

Effect of EA treatment on glucose tolerance in diabetic GK rats. (A) oGTT was performed in female diabetic GK rats with or without EA (50 mg/kg) treatment for 45 days. After 10 h fasting, rats received 2 mg/kg glucose solution by oral gavage and blood samples were collected at 15, 30, 45, 60, 75, 90, 105 and 120 min after the administration of glucose solution. Area under the curve of glucose measured during oGTT (B). Data are presented as mean ± SEM. (n = 6 per group); * p < 0.05 vs. GK without EA treatment.

Figure 2

EA treatment enhances insulin sensitivity and reduces insulin resistance in diabetic GK rats. (A) Insulin Tolerance Test (B) Rate constant for the disappearance of glucose in insulin tolerance test (kITT) and (C) insulin resistance index estimation by homeostasis model assessment method (HOMA-IR) were performed in female diabetic GK rats with or without EA (50 mg/kg) treatment for 45 days. Data are presented as mean ± SEM, * p < 0.05, (n = 8).

Figure 3

EA treatment reduces hepatic steatosis in diabetic GK Rats. Oil Red staining for detection of fat droplets was preformed in frozen sections of livers from age matched Wistar control, and diabetic GK rats with or without EA treatment (50 mg/kg) for 45 days. (A) Representative oil red staining showing steatosis (black arrows). Scale bar: 50 μm; (B) Morpho-quantitative analysis of hepatic steatosis area. Data are represented as mean ± SEM; * p < 0.05, (n = 6 per group).

Figure 4

EA treatment reduces hepatic oxidative stress in diabetic GK rats. DHE staining was performed in frozen section of liver from diabetic GK rats with or without treatment with EA (50 mg/kg) for 45 days for detection of superoxide anion. (A) Representative image of DHE staining in liver section. Scale bar: 50 μm; (B) Quantitative analysis of DHE fluorescence. Data are presented as mean ± SEM; * p < 0.05, (n = 6 per group).

Figure 5

EA treatment reduces stress reduces stress machinery in diabetic livers. Expression of p47-phox (A), antioxidant transcription factors, NRF2 (B) and HIF-α (C) and in hepatic homogenate from diabetic GK rats with or without treatment with EA (50 mg/kg) for 45 days. Top panels: representative blots. Bottom panels: densitometry analysis. GAPDH was used as loading control. Data are presented as mean ± SEM; * p < 0.05, (n = 6 per group).

Figure 6

EA treatment ameliorates hepatic insulin signaling. Expression of phosphorylated Akt at serine 473 in hepatic homogenate from diabetic GK rats with or without treatment with EA (50 mg/kg) for 45 days. Top panel: representative blot. Bottom panel: densitometry analysis. Data are presented as mean ± SEM; * p < 0.05, (n = 6 per group).