Hypoglycemic effects of brassinosteroid in diet-induced obese mice

Abstract

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.

Fig. 1.

Chemical structure of 28-homobrassinolide (HB; A) compared with other synthetic and natural brassinosteroid analogs (B).

Fig. 2.

Acute lowering effect of HB on plasma glucose in C57BL/6J mice 3 h after administration of 50, 100, or 300 mg/kg HB (n = 8). Metformin at 300 mg/kg was used as a positive control. Values are means ± SE. *P < 0.05 and **P < 0.01 when compared with control by 1-way ANOVA followed by Dunnett's post hoc test.

Fig. 3.

Chronic insulin-sensitizing effect of HB on body weight gain (A), fasting blood glucose (B), and oral glucose tolerance test (C) in the C57BL/6J mice. Six-week-old male mice were fed a high-fat diet for 6 wk and kept on the same diet for additional 8 wk combined with daily gavage with vehicle or 50 mg/kg HB. B: fasting blood glucose levels in animals fed high-fat diet (n = 8) or high-fat diet treated with 50 mg/kg HB (n = 8) at 6 wk. C: oral glucose tolerance test curves of groups fed high-fat diet or high-fat diet animals receiving the HB treatment. Values are means ± SE. *P < 0.05 and ***P < 0.001 when compared with control by 1-way ANOVA followed by Dunnett's post hoc test. **P > 0.01.

Fig. 4.

Hepatic expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) (A) and activation of liver AMP-acticated protein kinase phosphorylation (AMPK-P; B) in C57BL/6J mice treated with 50 mg/kg HB. PEPCK and G-6-Pase mRNAs were normalized to β-actin mRNA. Liver tissue lysates were analyzed by immunoblotting with phospho- and non-phospho-specific AMPK antibodies. Values are means ± SE. *P < 0.05, 2-tailed t-test vs. control.

Fig. 5.

Effect of HB administration on insulin signaling in liver (A) and skeletal muscle (B). Tissue lysates were analyzed by immunoblotting with phospho- and non-phospho-specific antibodies for insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), Akt1, Akt2, and β-actin as loading controls. Four animals were tested from each group, and a representative Western blot is shown. Ctr, control.

Fig. 6.

A: production of glucose in the H4IIE rat hepatoma cell culture in response to HB treatment following inducement with the dexamethasone (Dex)-cAMP mixture. B and C: hepatic expression of the gluconeogenic enzymes PEPCK and G-6-Pase in cell culture induced with the Dex-cAMP mixture (B) or cAMP alone (C). PEPCK and G-6-Pase mRNAs were normalized to β-actin mRNA. Values are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 when compared with control by 1-way ANOVA followed by Dunnett's post hoc test.

Fig. 7.

Production of glucose in the H4IIE rat hepatoma cell culture in response to treatment of HB (1), homocastasterone (2), and other synthetic and natural brassinosteroid analogs (3–9). Cells were incubated with 10 μM brassinosteroids for 8 h at 37°C. Values are means ± SE. *P < 0.05 and **P < 0.01 when compared with control by 1-way ANOVA followed by Dunnett's post hoc test.