Cell suspension culture of Eriobotrya japonica regulates the diabetic and hyperlipidemic signs of high-fat-fed mice

Abstract

The present study investigates the anti-hyperlipidemic and antihyperglycemic effects and mechanism in high-fat (HF)-fed mice of cell suspension culture of Eriobotrya japonica (TA), which contains a great number of pentacyclic terpenoids. Firstly, C57BL/6J mice were randomly divided into two groups: the control (CON) group was fed with a low-fat diet (n = 9), whereas the experimental group was fed a 45% HF diet for 8 weeks. Afterwards, the CON group was treated with vehicle, whereas the HF group was subdivided into five groups and was orally given TA or rosiglitazone or not for 4 weeks. Blood and visceral adipose tissue, liver tissue and skeletal muscle were examined. Treatment with TA reduced body weight gain, weights of white adipose tissue (WAT) (including epididymal, perirenal, mesenteric WAT and visceral fat), and hepatic triacylglycerol content significantly without affecting food intake in diet-induced diabetic mice. TA effectively prevented HF diet-induced increases in the levels of blood glucose, insulin, leptin and HOMA-IR index (p < 0.001, p < 0.05, p < 0.05, p < 0.01, respectively) and attenuated insulin resistance. Treatment with TA, adipocytes in the visceral depots showed a reduction in size. TA effectively significantly increased the protein contents of phosphorylation of AMPK-α (Thr172) both in liver and adipose tissue. It is shown that TA exhibits hypolipidemic effect in HF-fed mice by decreasing gene expressions of fatty acid synthesis, including acyl-coenzyme A: diacylglycerol acyltransferase (DGAT) 2, which catalyzes the final step in the synthesis of triglycerides, and antidiabetic properties occurred as a result of decreased hepatic glucose production via phosphenolpyruvate carboxykinase (PEPCK) down- regulation, improved insulin sensitization and TA (at 1.0 g/kg dose) decreased expression of hepatic and adipose 11-β-hydroxysteroid dehydroxygenase (11β-HSD1) gene, which contributed in attenuating diabetic state. Futhermore, TA at doses of 0.5 and 1.0 g/kg had serum lipid-lowering action characterized by the inhibition of DGAT 1 expression. Thus, amelioration of diabetic and dyslipidemic state by TA in HF-fed mice occurred by regulation of PEPCK, DGAT2 and AMPK phosphorylation.

Figure 1

The chemical structures of five triterpenes.

Figure 2

Effect of cell suspension culture of Eriobotrya japonica (TA) on (A) Body weight gain over 4-week treatment and (B) blood glucose levels at week 12. Blood samples were collected from the retro-orbital sinus of fasting mice and the level of glucose was measured by the glucose oxidase method. Mice were fed with 45% high-fat diet (HF) or low-fat diet (CON) for 12 weeks. After 8 weeks, the HF mice were treated with vehicle (water; p.o.), or TA, or rosiglitazone (p.o.) accompanied with HF diet for 4 weeks. All values are means ± S.E. (n=9). ^## p < 0.01, ^### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the high-fat + vehicle (distilled water) (HF) group by ANOVA. T1, T2 and T3: cell suspension culture of Eriobotrya japonica (T1: 0.2, T2: 0.5 and T3: 1.0 g/kg bodyweight); Rosi: rosiglitazone (0.01 g/kg body weight).

Figure 3

Histology of the epididymal white adipose tissue (WAT) of mice in the (A) Low-fat (LF); (B) High-fat (HF); (C) HF+T1; (D) HF+T2; (E) HF+T3; or (F) HF+Rosi groups. Each presented is typical and representative of nine mice. Magnification: 10 (ocular) × 40 (object lens). T1: 0.2, T2: 0.5 and T3: 1.0 g/kg bodyweight cell suspension culture of Eriobotrya japonica; Rosi: rosiglitazone (0.01 g/kg body weight).

Figure 4

Semiquantative RT-PCR analysis for (A) apo C-III, (B) DGAT1, (C) DGAT2, (D) PEPCK, (E) 11β-HSD1 and (F) ATGL mRNA expression in liver tissue; (G) leptin, and (H) 11β-HSD1 mRNA expression in adipose tissue of the mice by oral gavage cell suspension culture of Eriobotrya japonica for 4 weeks. All values are means ± S.E. (n = 9). ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the high-fat + vehicle (distilled water) (HF) group. Total RNA (1μg) isolated from tissue was reverse transcripted by MMLV-RT, 10μL of RT products was used as templates for PCR. Signals were quantitated by image analysis; each value was normalized by GAPDH. T1, T2, T3, cell suspension culture of Eriobotrya japonica.

Figure 5

The phospho-AMPK (Thr172) protein contents in liver and white adipose tissue of the mice by oral gavage cell suspension culture of Eriobotrya japonica for 4 weeks. Protein was separated by 12% SDS-PAGE detected by Western blot. All values are means ± S.E. (n = 9). ^# p < 0.05 compared with the control (CON) group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the high-fat (HF) + vehicle (distilled water) group by ANOVA. T1, T2, T3, cell suspension culture of Eriobotrya japonica.

Figure 6

Effects of cell suspension culture of Eriobotrya japonica on oral glucose tolerance in normal mice. Animals in all groups received oral glucose 30 min after the extract administration. Blood samples were collected and centrifuged at 3,000 rpm for 10 min. Each point is the mean ± S.E. of 5 separate mice. * p < 0.05, ** p < 0.01, *** p < 0.001 significantly different compared with the control group in the same time by ANOVA.