2-heptyl-formononetin increases cholesterol and induces hepatic steatosis in mice

Abstract

Consumption of isoflavones may prevent adiposity, hepatic steatosis, and dyslipidaemia. However, studies in the area are few and primarily with genistein. This study investigated the effects of formononetin and its synthetic analogue, 2-heptyl-formononetin (C7F), on lipid and cholesterol metabolism in C57BL/6J mice. The mice were fed a cholesterol-enriched diet for five weeks to induce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplemented formononetin or C7F for three weeks. Body weight and composition, glucose homeostasis, and plasma lipids were compared. In another experiment, mice were fed the above diets for five weeks, and hepatic triglyceride accumulation and gene expression and histology of adipose tissue and liver were examined. Supplementation with C7F increased plasma HDL-cholesterol thereby increasing the plasma level of total cholesterol. Supplementation with formononetin did not affect plasma cholesterol but increased plasma triglycerides levels. Supplementation with formononetin and C7F induced hepatic steatosis. However, formononetin decreased markers of inflammation and liver injury. The development of hepatic steatosis was associated with deregulated expression of hepatic genes involved in lipid and lipoprotein metabolism. In conclusion, supplementation with formononetin and C7F to a cholesterol-enriched diet adversely affected lipid and lipoprotein metabolism in C57BL/6J mice.

Figure 1

Body weight of cholesterol fed C57BL/6 mice (n = 23–25) supplemented with either formononetin or 2-heptyl-formonetin (C7F) for three weeks (Experiment  1). Graphs show mean ± SEM. *P ≤ 0.05.

Figure 2

Glucose homeostasis of cholesterol fed C57BL/6 mice (n = 23–25) supplemented with either formononetin or 2-heptyl-formononetin (C7F) for three weeks (Experiment  1). (a) Glucose clearance assessed by oral glucose tolerance test (2 g/kg glucose). (b) Fasting plasma glucose concentration. Graphs show mean ± SEM.

Figure 3

Development of hepatic steatosis in C57BL/6 mice fed chow, cholesterol, or cholesterol supplemented with formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment  2). (A) Weight of liver (n = 8). (B) Triglyceride content in liver (n = 6). Total lipids were extracted from liver using a modified version of the Bligh and Dyer protocol, and the content of triglyceride were analysed with a commercial kit. (C) Liver sections stained with hematoxylin and eosin. (D) Hepatic gene expression of Tnf (tumour necrosis factor α) measured by RT-PCR. Data is normalised to 18S ribosomal RNA and presented relatively to the expression in chow (n = 6). (E) Plasma level of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (n = 6). Graphs show mean ± SEM. Different letters (a, b) denote significant difference (P ≤ 0.05) between the groups.

Figure 4

Hepatic gene expression measured by RT-PCR in C57BL/6 mice fed chow, cholesterol, or cholesterol supplemented formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment  2). (A) Genes involved in lipogenesis (Srebf1 (sterol regulatory element-binding protein-1c), Mlxipl (carbohydrate response element binding protein), Acaca (acyl-CoA carboxylase 1), Fasn (fatty acid synthase), and Scd1 (stearoyl-CoA desaturase 1)) and synthesis of triglycerides (Gpam (glycerol phosphate acyltransferase) and Dgat2 (diglyceride acyltransferase 2)). (B) Genes involved in hydrolysis and beta-oxidation of fatty acids, Atgl (adipose triglyceride lipase), Ppara (peroxisome proliferator-activated receptor α), Cpt1a (carnitine palmitoyltransferase 1a), and Acox1 (acyl CoA oxidase). (C) Genes involved in lipoprotein metabolism, Acat2 (acetyl-CoA acetyltransferase), Mttp (microsomal triglyceride transfer protein), and Ldlr (low-density lipoprotein receptor). (D) Genes involved in cholesterol metabolism, Hmgcr (3-hydroxy-3-methyl-glutaryl-CoA reductase), Cyp7a1 (cholesterol 7 alpha-hydroxylase), Nr1 h3 (liver X receptor), and Nr1 h3 (farnesoid X receptor). Data is normalised to 18S ribosomal RNA and presented relative to the expression in chow (n = 6). Graphs show mean ± SEM. Different letters (a, b, c) denote significant difference (P ≤ 0.5) between the groups.

Figure 5

Adipocyte gene expression in C57BL/6 mice fed chow, cholesterol, or cholesterol supplemented formononetin or 2-heptyl-formonetin (C7F) for five weeks (Experiment  2). Gene expression measured by RT-PCR of Srebf1 (sterol regulatory element-binding protein-1c), Pparg (peroxisome proliferator-activated receptor γ), Cebpa (CCAAT/enhancer-binding protein α), Acaca (acyl-CoA carboxylase 1), Fasn (fatty acid synthase), Scd1 (stearoyl-CoA desaturase 1), Atgl (adipose triglyceride lipase), Ucp1 (uncoupling protein 1), and Emr1 (F4/80) in (A) eWAT, (B) iWAT, and (C) iBAT measured by RT-PCR. Data is normalised to 18S ribosomal RNA and presented relative to the expression in chow (n = 6). Graphs show mean ± SEM. Different letters (a, b) denote significant difference (P ≤ 0.5) between the groups.