The flavonoid, naringenin, decreases adipose tissue mass and attenuates ovariectomy-associated metabolic disturbances in mice

Abstract

Objective: Adverse metabolic changes associated with loss of ovarian function increase the risk of developing metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) in postmenopausal women. Naringenin improves metabolic disturbances in vitro and in vivo. In the present study, we tested the effects of naringenin on metabolic disturbances resulting from estrogen deficiency in ovariectomized mice.

Materials/methods: Ovariectomized C57BL/6 J female mice were fed a control diet (10% calories from fat) for 11 weeks. Mice either continued on the control diet (n = 9) or were switched to the control diet supplemented with 3% naringenin (n = 10) for the next 11 weeks. Energy expenditure was measured by indirect calorimetry and activity was monitored by infrared beam breaks. Intra-abdominal and subcutaneous adiposity was evaluated by magnetic resonance imaging (MRI). Blood biochemical measures of metabolic response included glucose, insulin, adipokines, and lipids. Lipid content in liver and muscle and expression of relevant genes in adipose tissue, liver, and muscle were quantified.

Results: Ovariectomized mice fed naringenin exhibited lower fasting glucose and insulin levels compared to controls, with over 50% reduction of intra-abdominal and subcutaneous adiposity. Plasma leptin and leptin mRNA in adipose depots were also decreased in mice fed a naringenin diet. Monocyte chemoattractant protein-1 (MCP1/Ccl2) and interleukin 6 (IL-6/Il6) mRNA expression levels were significantly lower in perigonadal adipose tissue of naringenin-supplemented mice. We also observed that mice fed a naringenin diet had less hepatic lipid accumulation with corresponding alterations of hepatic gene expression associated with de novo lipogenesis, fatty acid oxidation, and gluconeogenesis.

Conclusion: Dietary naringenin attenuates many of the metabolic disturbances associated with ovariectomy in female mice.

Keywords: Adipose tissue inflammation; Fatty liver; Insulin sensitivity; Menopause; Naringenin; Obesity.

Figure 1

Effects of dietary naringenin on caloric intake, body weight, and metabolic measurements. OVX C57BL/6 J mice were fed the control diet for 11 weeks then randomized to continuation of the control diet (n = 9) or switched to 3% wt/wt naringenin supplementation of the control diet (n = 10) for the weeks 12–22. Average daily caloric intake (A) of two groups (n = 2/group) and weekly body weight (B) of the CON (n = 9) and NAR (n = 10) group were determined. At week 17, CLAMS chambers were used to measure locomotor activity (ambulation) in the horizontal plane by infrared beam breaks (C) and estimated energy expenditure (heat) after controlling for body weight (D) in the dark and light phase for 24-h following a 24-h acclimation (n = 6/group). Fasting glucose levels (E) were measured at week 5 and week 18, (CON, n = 9; NAR, n = 10). Fasting insulin levels (F) were determined after 22 weeks of experimental period (n = 6/group). HOMA-IR values (G) were derived from fasting plasma glucose and insulin (n = 4/group). Values are presented as mean ± SEM. Significance between groups was determined by Student’s t test, except metabolic data of heat was analyzed by ANCOVA with body weight as a covariate. ^#P < 0.05 compared CON with NAR.

Figure 2

Effects of dietary naringenin on adiposity, plasma adipokines, and adipose tissue gene expression. At week 19, MRI analysis was performed in a subset of mice (n = 6/group) to measure percentage of total body fat and percentage of two adipose tissue depots, intra-abdominal and subcutaneous adipose tissues (A). Fasting plasma leptin and adiponectin in the CON (n = 6) and NAR (n = 5-6) groups were measured at the end of study at 22 weeks (B). Panel (C) shows representative coronal MRI views from each group. Effects of dietary naringenin on the mRNA levels of genes in perigonadal adipose tissue (D) and in subcutaneous adipose tissue (E) are shown (CON, n = 6-8; NAR, n = 10). Values are presented as mean ± SEM. Significance between groups was determined by Student’s t test. ^#P < 0.05 compared CON with NAR.

Figure 3

Effects of dietary naringenin on plasma lipids and lipid accumulation in liver and muscle. Plasma cholesterol (A; CON, n = 6; NAR, n = 10), triglyceride (B; n = 4/group) and NEFA (C; n = 6/group) after 5-h fasting obtained prior to necropsy were measured by enzymatic colorimetric method. Total lipids (D; CON, n = 9; NAR n = 9) and triglyceride (E; CON, n = 8; NAR n = 9) per gram of liver section were determined. Panel (F) shows representative H&E staining of livers (10x) from each group. Total lipids (G) and triglyceride (H) per gram of muscle section were also determined (CON, n = 9; NAR n = 9). Values are presented as mean ± SEM. Significance between groups was determined by Student’s t test. ^#P < 0.05 compared CON with NAR.

Figure 4

Effects of dietary naringenin on gene expression in liver and muscle. mRNA expression of hepatic genes related to de novo lipogenesis and hepatic steatosis (A), mRNA expression of hepatic genes related to beta-oxidation and gluconeogenesis (B), and mRNA expression of genes related to de novo lipogenesis and beta-oxidation in muscle (C) were quantified using qRT- PCR (n = 6-10/group). Values are presented as mean ± SEM. Significance between groups was determined by Student’s t test. ^#P < 0.05 compared CON with NAR.