Sudachitin, a polymethoxylated flavone, improves glucose and lipid metabolism by increasing mitochondrial biogenesis in skeletal muscle

Abstract

Background: Obesity is a major risk factor for insulin resistance, type 2 diabetes, and stroke. Flavonoids are effective antioxidants that protect against these chronic diseases. In this study, we evaluated the effects of sudachitin, a polymethoxylated flavonoid found in the skin of the Citrus sudachi fruit, on glucose, lipid, and energy metabolism in mice with high-fat diet-induced obesity and db/db diabetic mice. In our current study, we show that sudachitin improves metabolism and stimulates mitochondrial biogenesis, thereby increasing energy expenditure and reducing weight gain.

Methods: C57BL/6 J mice fed a high-fat diet (40% fat) and db/db mice fed a normal diet were treated orally with 5 mg/kg sudachitin or vehicle for 12 weeks. Following treatment, oxygen expenditure was assessed using indirect calorimetry, while glucose tolerance, insulin sensitivity, and indices of dyslipidemia were assessed by serum biochemistry. Quantitative polymerase chain reaction was used to determine the effect of sudachitin on the transcription of key metabolism-regulating genes in the skeletal muscle, liver, and white and brown adipose tissues. Primary myocytes were also prepared to examine the signaling mechanisms targeted by sudachitin in vitro.

Results: Sudachitin improved dyslipidemia, as evidenced by reduction in triglyceride and free fatty acid levels, and improved glucose tolerance and insulin resistance. It also enhanced energy expenditure and fatty acid β-oxidation by increasing mitochondrial biogenesis and function. The in vitro assay results suggest that sudachitin increased Sirt1 and PGC-1α expression in the skeletal muscle.

Conclusions: Sudachitin may improve dyslipidemia and metabolic syndrome by improving energy metabolism. Furthermore, it also induces mitochondrial biogenesis to protect against metabolic disorders.

Keywords: Glucose metabolism; Lipid metabolism; Mitochondria; Sudachitin.

Figure 1

Sudachitin inhibits weight gain in high-fat diet-fed mice. Mice were fed a low-fat control diet or a high-fat diet and were treated with 5 mg/kg sudachitin or vehicle for 12 weeks (n = 10 per group). Mice were fasted for 16 h at the end of the study. Body weight was measured weekly (A) and food intake was measured every 2 days (B) for 12 weeks. Values are mean ± standard error of the mean (A) or mean ± standard deviation (B). *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high-fat diet-fed vs. control diet-fed animals. Cont + Veh: vehicle-treated, control diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group (open squares); HFD + Veh: vehicle-treated, high-fat diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles).

Figure 2

Sudachitin reduces fat in high-fat diet-fed mice. (A) Body composition was assessed by computed tomography, with the relative body fat content, subcutaneous fat content (g/body weight; B) and visceral fat content (g/body weight; C) quantified. Adipocyte size was measured after 12 weeks of treatment with sudachitin or vehicle (D). Hematoxylin/eosin staining of WAT after 12 weeks of feeding in vehicle- (upper panel) and sudachitin-treated (lower panel) mice (E). Values are mean ± standard deviation (A-C). *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high fat diet vs control diet. Cont + Veh: vehicle-treated, control diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group (open squares); HFD + Veh: vehicle-treated, high-fat diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles).

Figure 3

Sudachitin reduces serum triglyceride and non-esterified fatty acid. Serum triglyceride (A), non-esterified fatty acid (B), and total cholesterol (C levels were evaluated at the end of the 12-week treatment. Values are means ± standard deviation (A-C). *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high-fat diet-fed vs control diet-fed animals. Cont + Veh: vehicle-treated, control diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group (open squares); HFD + Veh: vehicle-treated, high-fat diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles); NEFA: non-esterified fatty acid; T-CHO: total cholesterol.

Figure 4

Sudachitin improves glucose and insulin sensitivity in mice fed a high-fat diet. Fasting blood glucose (A) and plasma insulin concentrations (B) after 12 weeks of sudachitin treatment. Glucose (C) and insulin (D) tolerance tests were performed in high-fat diet-fed mice after an overnight (16 h) (C) or 6 h (D) fast. Mice received an oral dose of 1 g/kg glucose (C) or 0.75 mU/kg insulin by intraperitoneal injection (D). Blood glucose was measured at the indicated times. Glucose utilization and insulin sensitivity were determined from the area under the curve (AUC; inset). Open symbols: sudachitin-treated group; closed symbols: vehicle-treated groups. Plasma leptin levels were measured after 12 weeks of treatment (E). Plasma adiponectin levels were measured after 5, 8, and 12 weeks of starting treatment (F). Values are means ± standard deviation. Closed bars: vehicle-treated, control diet group; dark gray bars: sudachitin-treated, control diet group; light gray bars: vehicle-treated, high-fat diet group; open bars: sudachitin-treated, high-fat diet group; *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high fat diet vs control diet-fed animals.

Figure 5

Effects of sudachitin on mRNA levels in white adipose tissue and liver. Gene transcription was normalized for 36B4 in subcutaneous white adipose tissue (A-B) or 18S in liver (C). Data are means ± standard deviation. *P < 0.05 vs the indicated groups. Closed bars: vehicle-treated, high-fat diet group; open bars: sudachitin-treated, high-fat diet group.

Figure 6

Sudachitin improves fasting glucose, insulin sensitivity, and lipid metabolism in db/db mice. While sudachitin treatment did not affect weight gain over 9 weeks of treatment (A), it significantly decreased fasting blood glucose levels (B). Insulin tolerance tests were performed after a 6 h fast (C). Mice received an intraperitoneal injection of 3 mU/kg insulin, and blood glucose levels were measured at indicated times. Serum triglyceride (D), non-esterified fatty acids (E), and total cholesterol levels (F) were measured in db/db mice. Data are presented as means ± standard deviation. *P < 0.05 vs the indicated groups. closed circles: vehicle-treated group, open circles: sudachitin-treated group.

Figure 7

Sudachitin increases energy expenditure. Ten-week-old mice treated with vehicle or sudachitin (5 mg/kg) for 4 weeks were placed into an Oxymax open-circuit indirect calorimetry system for 4 days. Measurements of O₂ consumption in control C57/Bl6 mice (A) or db/db mice (C), and energy expenditure were obtained in control C57/Bl6 mice (B) or db/db mice (D).

Figure 8

Sudachitin increases energy metabolism-related gene expressions by activating mitochondrial biogenesis. Relative mRNA expression of energy metabolism-related genes in the gastrocnemius muscle (A, B) and brown adipose tissue (A). Gene expression was normalized for GAPDH in both tissues. Phosphorylation of AMPK in sudachitin- or vehicle-treated mice with or without insulin (C). Protein expression of GLUT4 in mice administrated vehicle or sudachitin (D). Skeletal muscle ATP content in mice fed a high-fat diet and treated with sudachitin or vehicle (E). Citrate synthase activity was measured in mitochondria isolated from high-fat diet-fed mice treated with 5 mg/kg sudachitin or vehicle (F). Data are presented as mean ± standard deviation. *P < 0.05 vs. the indicated group. Closed bars; vehicle-treated group; open bars: sudachitin-treated group.

Figure 9

Effects of sudachitin on gene expression in mouse primary skeletal muscle myocytes in vitro. Gene transcription in myocytes treated with 30 μmol/L sudachitin or vehicle for 48 h, normalized for β-actin (A). Data are means ± standard deviation. *P < 0.05 vs the indicated group. Closed bars: untreated cells; open bars: sudachitin-treated cells. The number of mitochondria in myocytes was increased by sudachitin (B, C). Myocytes were incubated with the fluorophore Mito-Tracker Green, which specifically labels mitochondria. Upper panel: control cells; lower panel: cells treated with 30 μmol/L sudachitin for 48 h. Arrows indicate stained mitochondria (B). Quantitative analysis of mitochondrial staining. Fluorescence intensity was measured by ImageJ software using the analyze particle function. *P < 0.05.