Bee Venom Suppresses the Differentiation of Preadipocytes and High Fat Diet-Induced Obesity by Inhibiting Adipogenesis

Abstract

Bee venom (BV) has been widely used in the treatment of certain immune-related diseases. It has been used for pain relief and in the treatment of chronic inflammatory diseases. Despite its extensive use, there is little documented evidence to demonstrate its medicinal utility against obesity. In this study, we demonstrated the inhibitory effects of BV on adipocyte differentiation in 3T3-L1 cells and on a high fat diet (HFD)-induced obesity mouse model through the inhibition of adipogenesis. BV inhibited lipid accumulation, visualized by Oil Red O staining, without cytotoxicity in the 3T3-L1 cells. Male C57BL/6 mice were fed either a HFD or a control diet for 8 weeks, and BV (0.1 mg/kg or 1 mg/kg) or saline was injected during the last 4 weeks. BV-treated mice showed a reduced body weight gain. BV was shown to inhibit adipogenesis by downregulating the expression of the transcription factors CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptor gamma (PPARγ), using RT-qPCR and Western blotting. BV induced the phosphorylation of AMP-activated kinase (AMPK) and acetyl-CoA carboxylase (ACC) in the cell line and in obese mice. These findings demonstrate that BV mediates anti-obesity/differentiation effects by suppressing obesity-related transcription factors.

Keywords: AMPK; Bee venom; MAPK; PPARγ; adipogenesis.

Figure 1

Bee venom (BV) suppressed lipid accumulation in 3T3-L1 preadipocytes. (A) Cells were cultured in the growth medium or the differentiation medium containing concentrations ranging from 1.3 to 40 μg/mL of BV for 3 days. (B) Preadipocytes were differentiated with and without BV (2.5, 5, and 10 μg/mL) for 9 days. Differentiated cells were stained with Oil red O and images were taken with a Leica DM IL LED microscope (100× and 200× magnifications). (C) Oil red O was extracted from lipid droplets using isopropanol and was measured at 510 nm. ^### p < 0.001 vs. non-differentiation cells. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. differentiation cells.

Figure 2

The effects of BV on the expression levels of lipid metabolism and adipogenesis in 3T3-L1 cells. (A) The mRNA levels of C/EBPβ, C/EBPδ, and C/EBPα were measured by qRT-PCR. (B) The protein and the mRNA expression levels of PPARγ were determined by Western blot analysis or by qRT-PCR. β-actin was used as an internal control. ^### p < 0.001 vs. non-differentiation cells, *** p < 0.001 vs. differentiation cells.

Figure 3

The effects of BV on the mitogen-activated protein kinase (MAPK) and the adenosine monophosphate-activated protein kinase (AMPK) pathways in 3T3-L1 preadipocytes. 3T3-L1 cells were treated with BV (2.5, 5, and 10 μg/mL for 3 days). The total cell lysates were analyzed by Western blotting to determine the expression of (A) MAPKs (B) AMPK and acetyl coenzyme A carboxylase (ACC). β-actin was used as an internal control. ^### p < 0.001 vs. non-differentiation cells. ** p < 0.01, *** p < 0.001 vs. differentiation cells.

Figure 4

Effects of BV injection on (A) total body weight, (B) fat weight, (C) a decrease in body weight, (D) body weight gain, and (E) Epididymis adipose tissue (100× magnifications) from mice. The duration of the experimental window was 14 weeks during which mice were fed on normal diet or a high-fat diet. The representative photographic images of mice were from different treatment/feeding groups at the time of sacrifice. Values are expressed as the mean ± SEM of 10 mice per group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. the normal diet (ND) group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the high fat diet (HFD) group.

Figure 5

BV regulated adipogenic markers and the AMPK pathway in HFD-induced mice. (A) Important adipogenic transcription factors, PPAR γ and C/EBPα, determined using Western blot analysis. (B) AMPK and (C) ACC determined using Western blot analysis. β-actin was used as an internal control. ^### p < 0.001 vs. ND group. * p < 0.05, *** p < 0.001 vs. HFD group.