Crocin ameliorates hepatic steatosis through activation of AMPK signaling in db/db mice

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity, type 2 diabetes and other metabolic disorders worldwide. Crocin is a carotenoid compound possessing various pharmacological activities. In the present study, we aimed to investigate the effect on fatty liver under diabetic and obese condition and to examine the possible role of AMP-activated protein kinase (AMPK) signaling.

Methods: db/db mice were administrated with crocin and injected with LV-shAMPK or its negative control lentivirus. Metabolic dysfunction, lipogenesis and fatty acid-oxidation in liver were evaluated.

Results: In db/db mice, we found that oral administration of crocin significantly upregulated the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in liver. Crocin reduced liver weight, serum levels of alanine aminotransferase, alanine aminotransferase, and liver triglyceride content, and attenuated morphological injury of liver in db/db mice. Crocin inhibited the mRNA expression of lipogenesis-associated genes, including sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor γ, fatty acid synthase, stearoyl-CoA desaturase 1, and diacylglycerol acyltransferase 1, and increased the mRNA expression of genes involved in the regulation of β-oxidation of fatty acids, including PPARα, acyl-CoA oxidase 1, carnitine palmitoyltransferase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2. Moreover, treatment of crocin resulted in a amelioration of general metabolic disorder, as evidenced by decreased fasting blood glucose, reduced serum levels of insulin, triglyceride, total cholesterol, and non-esterified fatty acid, and improved glucose intolerance. Crocin-induced protective effects against fatty liver and metabolic disorder were significantly blocked by lentivirus-mediated downregulation of AMPK.

Conclusions: The results suggest that crocin can inhibit lipogenesis and promote β-oxidation of fatty acids through activation of AMPK, leading to improvement of fatty liver and metabolic dysfunction. Therefore, crocin may be a potential promising option for the clinical treatment for NAFLD and associated metabolic diseases.

Keywords: AMPK; NAFLD; antioxidant; crocin; type 2 diabetes.

Fig. 1

Effects of crocin on AMPK signaling in liver in db/db mice. a western blot analysis of AMPK and mTOR phosphorylation in liver tissue. b statistical analysis of AMPK phosphorylation (n = 3). (c) statistical analysis of mTOR phosphorylation (n = 3). The significance of differences among groups was assessed by one-way ANOVA analysis followed by Dunnett’s test. # p < 0.05, indicates statistical significance between the two groups

Fig. 2

Effects of crocin on lipid accumulation and injury of liver in db/db mice. (a) liver weight (n = 10). (b) serum level of AST (n = 10). (c) serum level of ALT (n = 10). (d) liver content of TG (n = 10). (E) HE staining of liver tissue (n = 10). f Quantitative analysis was performed and expressed as folds of volume density (Vv) of steatosis versus control (n = 10). The significance of differences among groups was assessed by one-way ANOVA analysis followed by Dunnett’s test. # p < 0.05, indicates statistical significance between the two groups

Fig. 3

Effects of crocin on lipogenesis and β-oxidation of fatty acids in liver in db/db mice. mRNA expression of Srebp-1c (a), FAS (b), SCD1 (c), PPARγ (d), DGAT1 (e), PPARα (f), Acox1 (g), Cpt1 (h), and Hmgcs2 (i) in liver tissue were determined using real-time qPCR (n = 6). (J) Protein expression of Srebp-1 and PPARα was determined using western blot and statistical analysis was performed (n = 3). The significance of differences among groups was assessed by one-way ANOVA analysis followed by Dunnett’s test. # p < 0.05, indicates statistical significance between the two groups

Fig. 4

Effects of crocin on general glucose and lipid metabolism in db/db mice. (a) body weight (n = 10). (b) fasting blood glucose level (n = 10). (c) serum insulin level (n = 10). (d) OGTT test (n = 6). (e) area under the curve of OGTT (n = 6). (f) IPITT test (n = 6). (g) area under the curve of IPITT (n = 6). (h) serum level of TG (n = 10). (i) serum level of TC (n = 10). (j) serum level of NEFA (n = 10). The significance of differences among groups was assessed by one-way ANOVA analysis followed by Dunnett’s test. # p < 0.05, indicates statistical significance between the two groups