Polyphenol Stilbenes from Fenugreek (Trigonella foenum-graecum L.) Seeds Improve Insulin Sensitivity and Mitochondrial Function in 3T3-L1 Adipocytes

Abstract

Fenugreek (Trigonella foenum-graecum L.) is a well-known annual plant that is widely distributed worldwide and has possessed obvious hypoglycemic and hypercholesterolemia characteristics. In our previous study, three polyphenol stilbenes were separated from fenugreek seeds. Here, we investigated the effect of polyphenol stilbenes on adipogenesis and insulin resistance in 3T3-L1 adipocytes. Oil Red O staining and triglyceride assays showed that polyphenol stilbenes differently reduced lipid accumulation by suppressing the expression of adipocyte-specific proteins. In addition, polyphenol stilbenes improved the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by promoting the phosphorylation of protein kinase B (AKT) and AMP-activated protein kinase (AMPK). In present studies, it was found that polyphenol stilbenes had the ability to scavenge reactive oxygen species (ROS). Results from adenosine triphosphate (ATP) production and mitochondrial membrane potentials suggested that mitochondria play a critical role in insulin resistance and related signaling activation, such as AKT and AMPK. Rhaponticin, one of the stilbenes from fenugreek, had the strongest activity among the three compounds in vitro. Future studies will focus on mitochondrial biogenesis and function.

Figure 1

Chemical structure of polyphenol stilbenes from fenugreek.

Figure 2

Timeline of 3T3-L1 differentiation and induction of insulin resistance. CS/DMEM: 10% calf serum/Dulbecco's Modified Eagle's Medium; IMI (induction media I): 10% fetal bovine serum (FBS)/DMEM + 0.5 mmol/L isobutylmethylxanthine (IBMX) + 1 μmol/L dexamethasone (Dex) + 10 μg/mL insulin; IMII (induction media II): 10% FBS/DMEM + 1 μg/mL insulin; FBS/DMEM: 10% FBS/DMEM; IMIII (induction media III): 10% FBS/DMEM + 1 μmol/L Dex.

Figure 3

Effects of polyphenol stilbenes on viability and differentiation of 3T3-L1 adipocytes. (a) 3T3-L1 preadipocytes were incubated with different concentrations of polyphenol stilbenes for 48 hr after which cell viability was determined by extracellular LDH assay in the media. Data are expressed as the mean ± SD of three independent experiments. (b) 3T3-L1 preadipocytes were induced to full differentiation for 8 days as described in Figure 2. Lipid droplets were stained by Oil Red O. Con: undifferentiated 3T3-L1 preadipocytes; Mod: fully differentiated 3T3-L1 adipocytes. (c) On day 8, the cellular triglyceride (TG) content was measured and normalized against total protein. Data are presented as the mean ± SD of three independent experiments (n = 3). ^##P < 0.01 mature adipocytes versus preadipocytes; ^∗P < 0.05, ^∗∗P < 0.01 compound-treated adipocytes versus mature adipocytes. (d) Adipocyte-specific protein expression on day 8 was determined by Western blot analysis.

Figure 4

Effects of polyphenol stilbenes on 2-NBDG uptake and p-AKT and p-AMPK expression in 3T3-L1 adipocytes. (a) Results are presented as the means ± SD of three independent experiments (n = 3). ^##p < 0.01 Insulin-resistant (IR) adipocytes versus mature adipocytes; ^∗P < 0.05, ^∗∗P < 0.01 compound-treated adipocytes versus IR adipocytes. (b) p-AKT and p-AMPK expression in 3T3-L1 adipocytes was determined by Western blot analysis.

Figure 5

Effects of polyphenol stilbenes on ROS production in 3T3-L1 adipocytes. 1 μmol/L of Rosi and 10 μmol/L of the three polyphenol stilbenes were used in the experiments. Results are shown as the mean ± SD of three independent experiments (n = 3). ^##P < 0.01, IR adipocytes versus mature adipocytes; ^∗P < 0.05, ^∗∗P < 0.01 compound-treated adipocytes versus IR adipocytes.

Figure 6

Effects of polyphenol stilbenes on ATP production (a) and mitochondrial membrane potential (b) in 3T3-L1 adipocytes. 1 μmol/L of Rosi and 10 μmol/L of the three polyphenol stilbenes were used in the experiments. Results are presented as the mean ± SD of three independent experiments (n = 3). ^##P < 0.01, IR adipocytes versus mature adipocytes; ^∗P < 0.05, ^∗∗P < 0.01, compound-treated adipocytes versus IR adipocytes.