Amarogentin, a secoiridoid glycoside, activates AMP- activated protein kinase (AMPK) to exert beneficial vasculo-metabolic effects

Abstract

Introduction: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined.

Methods: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α₂β₁γ₁ trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α₂ subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining.

Results: AG interacts with the α₂ subunit of AMPK and activates the trimeric kinase with an EC₅₀ value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice.

Conclusion: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK.

General significance: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.