Allomyrina dichotoma Larva Extract Ameliorates the Hepatic Insulin Resistance of High-Fat Diet-Induced Diabetic Mice

Abstract

Allomyrina dichotoma larva is a nutritional-worthy future food resource and it contributes to multiple pharmacological functions. However, its antidiabetic effect and molecular mechanisms are not yet fully understood. Therefore, we investigated the hypolipidemic effect of A. dichotoma larva extract (ADLE) in a high-fat diet (HFD)-induced C57BL/6J mice model. Glucose tolerance and insulin sensitivity in HFD-induced diabetic mice significantly improved after ADLE administration for six weeks. The levels of serum triglyceride (TG), aspartate aminotransferase (AST), alanine transferase (ALT) activity, and lipid accumulation were increased in the liver of HFD-fed mice, but the levels were significantly reduced by the ADLE treatment. Moreover, hepatic fibrosis and inflammatory gene expression in the liver from HFD-treated mice were ameliorated by the ADLE treatment. Dephosphorylation of AMP-activated protein kinase (AMPK) by palmitate was inhibited in the ADLE treated HepG2 cells, and subsequently reduced expression of lipogenic genes, such as SREPBP-1c, ACC, and FAS were observed. The reduced expression of lipogenic genes and an increased phosphorylation of AMPK was also observed in the liver from diabetic mice treated with ADLE. In conclusion, ADLE ameliorates hyperlipidemia through inhibition of hepatic lipogenesis via activating the AMPK signaling pathway. These findings suggest that ADLE and its constituent bioactive compounds are valuable to prevent or treat hepatic insulin resistance in type 2 diabetes.

Keywords: Allomyrina dichotoma larva; hepatic insulin resistance; lipogenesis.

Figure 1

Treatment of ADLE reduced body weight and blood glucose level of HFD-induced diabetic mice. High-fat diet-induced diabetic mice were treated with vehicle, Allomyrina dichotoma larva extract (ADLE, 100 mg/kg) or metformin (Met, 100 mg/kg) for 6 weeks. (A) Body weight for 6 weeks, (B) changes in body weight for 6 weeks, (C) food intake (g/day), (D) FER over 6 weeks, and (E) fasting blood glucose levels for 6 weeks. Values are presented as means ± SD (n = 6–8). *** p < 0.001 vs. NFD group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. HFD group. NFD, normal-fat diet; HFD, high-fat diet; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.

Figure 2

Treatment of ADLE improved glucose and insulin tolerance of HFD-induced diabetic mice. Mice were treated as described in Figure 1. (A) Intraperitoneal glucose tolerance test (GTT), (B) area under the curve (AUC) of GTT, (C) intraperitoneal insulin tolerance test (ITT), and (D) area under the curve (AUC) of ITT. Values are presented as mean ± SD (n = 6–8). *** p < 0.001 vs. NFD group. ^# p < 0.05 vs. HFD group. NFD, normal-fat diet; HFD, high-fat diet; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.

Figure 3

ADLE altered lipid profiles, AST, and ALT levels in serum of HFD-induced diabetic mice. Mice were treated as described in Figure 1. After 6 weeks of ADLE treatment, serum was collected, and (A) lipid profiles: total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) and (B) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by specific assay kits. The results are expressed as means ± SD (n = 6–8). ** p < 0.01, *** p < 0.001 vs. NFD group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. HFD group. NFD, normal-fat diet; HFD, high-fat diet; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.

Figure 4

ADLE reduced hepatic fibrosis and inflammation in the liver of HFD-induced diabetic mice. (A) Liver sections were stained with Masson’s trichrome (scale bar = 200 μm) and (B,C) the mRNA expression of Col1a2, TNF-α, and MCP-1 in the liver was determined by RT-PCR. The results are expressed as means ± SD (n = 6–8). ** p < 0.01, *** p < 0.001 vs. NFD group. ^# p < 0.05 and ^## p < 0.01 vs. HFD group. NFD, normal-fat diet; HFD, high-fat diet; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.

Figure 5

ADLE reduced lipid accumulation in the liver of HFD-induced diabetic mice. (A) Mice were treated as described in Figure 1. After 6 weeks of ADLE treatment, sections of liver tissue were stained with Oil red O staining (scale bar = 200 μm). (B) Hepatic triglyceride (TG, mg/g protein) and total cholesterol (TC, mg/g protein) levels from liver lysates were measured by assay kits. The results are expressed as means ± SD (n = 6–8). * p < 0.05 and ** p < 0.01 vs. NFD group. ^# p < 0.05 and ^## p < 0.01 vs. HFD group. NFD, normal-fat diet; HFD, high-fat die; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.

Figure 6

ADLE reduced lipogenic gene expression in palmitate-treated HepG2 cells. (A) HepG2 cells were cultured with various concentrations of ADLE for 24 h and cell viability was determined by MTT assay. (B) HepG2 cells were treated with 0.5 mM palmitate (PAL) with or without ADLE (0.5 mg/ml) for 24 h and the intracellular TG levels were measured by triglyceride assay kit. (C) The cells were treated as described in Figure 6B, and mRNA levels (SREBP-1c, FAS, and ACC) were determined by qRT-PCR analysis. The mRNA levels were normalized with those of cyclophilin. (D) Protein expression levels of SREBP-1, FAS, and ACC were measured by western blot analysis. (E) Protein expression levels of AMPK and phospho-AMPK were measured by western blot analysis. The intensity of each band was measured with Quantity one software, and the relative quantity was calculated over β-actin. The results are expressed as the mean ± SD (n = 3–5). * p < 0.05, ** p < 0.001, and *** p < 0.001 vs. untreated control (CON). ^# p < 0.05, ^## p < 0.001, and ^### p < 0.001 vs. 0.5 mM PAL only.

Figure 7

ADLE reduced the expression of lipogenic genes and dephosphorylation of AMPK in the liver of HFD-induced diabetic mice. Mice were treated as described in Figure 1. After 6 weeks of ADLE treatment, total RNA and protein were extracted from the liver as described in the Materials and Methods section. (A) The qRT-PCR analysis of gene expression for SREBP-1c, FAS, and ACC was normalized with those of cyclophilin. (B) Protein expression levels of SREBP-1, FAS, and ACC were measured by western blot analysis. The intensity of the bands was quantified by densitometric analysis and normalized with corresponding β-actin. (C) Western blots of phosphorylated AMPK (pAMPK) in the liver were performed, and the intensity of the bands was quantified by densitometric analysis and normalized with AMPK. Results are calculated as the means ± SD (n = 6–8). ** p < 0.01 and *** p < 0.001 vs. NFD group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. HFD group. NFD, normal-fat diet; HFD, high-fat diet; HFD + ADLE, high-fat diet + ADLE; HFD + Met, high-fat diet + Met.