Poster Topical Area: Dietary Bioactive Components
Location: Hall D
Poster Board Number: 385
Purified enzyme and cell-based assay development to screen 5' AMP-activated protein kinase (AMPK) activators that upregulate AMPK activity from natural products.
Natural products Momordica grosvenori (Luo Han Guo, LHG) extract, Sweet tea extract and EGCG were selected as model samples to establish the screening method of AMPK activity assay. Natural product samples treated purified AMPK enzyme or HepG2 cell lysates were added to the well of CycLex AMPK kinase assay kit (CycLex Co., Ltd. Japan) to test AMPK activity according to User's Manual, and then Western Blotting with antibodies against phospho-ACC (acetyl-CoA carboxylase).
Natural products LHG extract (A), sweet tea extract (B) and EGCG (C) demonstrated activation effect on purified AMPK enzyme. (Fig. 1) Since the commercial purified AMPK enzyme is not stable and readily to be inactive, we tried to set up the AMPK activity assay in cell system. HepG2 cell were treated with or without LHG extract (A), sweet tea extract (B) and EGCG (C), these natural products were verified to activate AMPK in the cultured cells. AICAR was referred to as positive control compound. (Fig.2) Thus, the AMPK activity assay was set up based on HepG2 cell line.
Snow Lotus tissue culture material (D) and Pepper extract (E) were identified as AMPK activator with this established AMPK activity assay model, and the AMPK activation effect by Pepper and Snow Lotus samples were decreased in the present of AMPK inhibitor, compound C, in cultured cells. (Fig. 3) Then, the Pepper and Snow Lotus samples were investigated on acetyl-CoA carboxylase activity (ACC) in liver cells. ACC is downstream substrate of AMPK, the rate-limiting enzyme of fatty acid synthesis. The phosphorylation of ACC reduced fatty acid synthesis pathway. Pepper and Snow Lotus samples show inhibitory effect on ACC activity by stimulating phosphorylation of ACC. (Fig. 4)
We established a quick method to screen AMPK activators from natural products, and identified Pepper extract and Snow Lotus tissue culture material as AMPK activator.They directly activate the enzyme AMPK in vitro, stimulate AMPK and inhibit key enzyme-ACC of fatty acid synthesis in cell system, which provides the plausibility to the preventive effect of these two natural products on AMPK relevant metabolism regulation.
Scientist SR. Associate
Nu Skin (China) Daily-Use & Health Products Co.,Ltd, Center for Anti-Aging Research
Shanghai, Shanghai, China (People's Republic)