Berberine Improves Glucose and Lipid Metabolism in HepG2 Cells Through AMPKα1 Activation.

Gang Ren,Jiang-Hong Guo,Wei-Jia Kong,Jian-Dong Jiang,Yu-Zhen Qian

doi:10.3389/fphar.2020.00647

Abstract

AimThis study is designed to investigate whether or not AMP-activated protein kinase α1 (AMPKα1) is required for natural product berberine (BBR) to improve glucose and lipid metabolism in HepG2 cells.MethodsAMPKα1 knocked-out (KO, AMPKα1-/-) cells were obtained by co-transfection of the CRISPR/Cas9 KO and HDR (homology-directed repair) plasmid into HepG2 cells, as well as subsequent screen with puromycin. The expression levels of target proteins or mRNAs were determined by western blot or real-time RT-PCR, respectively. Cellular AMPK activity, glucose consumption, lactate release, glucose production, and lipid accumulation were determined by kits.ResultsThe results showed that the AMPKα1 gene was successfully KO in HepG2 cells. In AMPKα1-/- cells, the protein expression of AMPKα1 and phosphorylated-AMPKα1 (p-AMPKα1) disappeared, the level of total AMPKα declined to about 45–50% of wild type (p < 0.01), while p-AMPKα level and AMPK activity were reduced to less than 10% of wild type (p < 0.001). BBR increased p-AMPKα1, p-AMPKα, AMPK activity, and stimulated glucose consumption, lactate release, inhibited glucose production in wild type HepG2 cells (p < 0.05 or p < 0.01). BBR also reduced intracellular lipid accumulation and suppressed the expression of lipogenic genes in oleic acid (OA) treated wild type HepG2 cells (p < 0.05 or p < 0.01). In AMPKα1-/- HepG2 cells, the stimulating effects of BBR on p-AMPKα1, p-AMPKα, AMPK activity, and its improving effects on glucose and lipid metabolism were completely abolished.ConclusionOur study proves that AMPKα1 plays a critical role for BBR to improve glucose and lipid metabolism in HepG2 cells. Our results will provide new information to further understand the molecular mechanisms of BBR.

Highlights

The cellular AMP-activated protein kinase (AMPK) plays a central role in the modulation of energy balance of metabolic tissues by suppressing anabolism and promoting catabolism (Steinberg and Kemp, 2009)
Our results clearly prove that AMPKa1 is essential for BBR to promote glucose and lipid metabolism in HepG2 cells
In order to clarify whether or not AMPK is indispensable for BBR to stimulate glucose metabolism, AMPKa1-/- HepG2 cells were generated by disruption of the AMPKa1 gene and stable monoclonal transfectants were selected

Summary

Introduction

The cellular AMP-activated protein kinase (AMPK) plays a central role in the modulation of energy balance of metabolic tissues by suppressing anabolism and promoting catabolism (Steinberg and Kemp, 2009). AMPK is considered as an important molecular target for the treatment of metabolic diseases such as diabetes, obesity, or dyslipidemia (Steinberg and Kemp, 2009). BBR improves glucose and lipid metabolism through multiple targets, which include the AMPK (Kim et al, 2009; Kumar et al, 2015; Yao et al, 2015). BBR was demonstrated to activate AMPK in liver cells, skeletal muscle cells, and adipocytes, perhaps through inhibiting mitochondrial respiration and ATP biosynthesis (Turner et al, 2008; Yin et al, 2008; Kim et al, 2009; Kumar et al, 2015; Yao et al, 2015)

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