Dihydromyricetin Imbues Antiadipogenic Effects on 3T3-L1 Cells via Direct Interactions with 78-kDa Glucose-Regulated Protein

Abstract

Background: Obesity is among the most serious public health problems worldwide, with few safe pharmaceutical interventions. Natural products have become an important source of potential anti-obesity therapeutics. Dihydromyricetin (DHM) exerts antidiabetic effects. The biochemical target of DHM, however, has been unknown. It is crucial to identify the biochemical target of DHM for elucidating its physiological function and therapeutic value.

Objectives: The objective of this study was to identify the biochemical target of DHM.

Methods: An abundant antiadipogenic flavanonol was extracted from the herbal plant Ampelopsis grossedentata through bioassay-guided fractionation and characterized with high-resolution LC-MS and 1H and 13C nuclear magnetic resonance. Antiadipogenic experiments were done with mouse 3T3-L1 preadipocytes. A biochemical target of the chemical of interest was identified with drug affinity responsive target stability assay. Direct interactions between the chemical of interest and the protein target in vitro were predicted with molecular docking and subsequently confirmed with surface plasmon resonance. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ), which is associated with 78-kDa glucose-regulated protein (GRP78), were measured with real-time qPCR.

Results: DHM was isolated, purified, and structurally characterized. Cellular studies showed that DHM notably reduced intracellular oil droplet formation in 3T3-L1 cells with a median effective concentration of 294 μM (i.e., 94 μg/mL). DHM targeted the ATP binding site of GRP78, which is associated with adipogenesis. An equilibrium dissociation constant between DHM and GRP78 was 21.8 μM. In 3T3-L1 cells upon treatment with DHM at 50 μM (i.e., 16 μg/mL), the expression level of PPARγ was downregulated to 53.9% of the solvent vehicle control's level.

Conclusions: DHM targets GRP78 in vitro. DHM is able to reduce lipid droplet formation in 3T3-L1 cells through a mode of action that is plausibly associated with direct interactions between GRP78 and DHM, which is a step forward in determining potential applications of DHM as an anti-obesity agent.

Keywords: 78-kDa glucose-regulated protein; GRP78; adipocyte; antiadipogenesis; drug affinity responsive target stability (DARTS); lipid droplet; obesity; proteomics; surface plasmon resonance.