Anti-adipogenic effect of the flavonoids through the activation of AMPK in palmitate (PA)-treated HepG2 cells

Abstract

Background Flavonoids are natural polyphenols found widely in citrus fruit and peel that possess anti-adipogenic effects. On the other hand, the detailed mechanisms for the anti-adipogenic effects of flavonoids are unclear. Objectives The present study observed the anti-adipogenic effects of five major citrus flavonoids, including hesperidin (HES), narirutin (NAR), nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), on AMP-activated protein kinase (AMPK) activation in palmitate (PA)-treated HepG2 cells. Methods The intracellular lipid accumulation and triglyceride (TG) contents were quantified by Oil-red O staining and TG assay, respectively. The glucose uptake was assessed using 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) assay. The levels of AMPK, acetyl-CoA carboxylase (ACC), and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and levels of sterol regulatory element-binding protein 2 (SREBP-2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression were analyzed by Western blot analysis. The potential interaction between the flavonoids and the γ-subunit of AMPK was investigated by molecular docking analysis. Results The flavonoid treatment reduced both intracellular lipid accumulation and TG content in PA-treated HepG2 cells significantly. In addition, the flavonoids showed increased 2-NBDG uptake in an insulin-independent manner in PA-treated HepG2 cells. The flavonoids increased the AMPK, ACC, and GSK3β phosphorylation levels and decreased the SREBP-2 and HMGCR expression levels in PA-treated HepG2 cells. Molecular docking analysis showed that the flavonoids bind to the CBS domains in the regulatory γ-subunit of AMPK with high binding affinities and could serve as potential AMPK activators. Conclusion The overall results suggest that the anti-adipogenic effect of flavonoids on PA-treated HepG2 cells results from the activation of AMPK by flavonoids.

Effect of Citrus bergamia extract on lipid profile: A combined in vitro and human study

“…Preliminary evidence has been also documented for C. bergamia , although most data are focused on single active bio‐components. Citrus bergamia bioflavonoids impair adipogenesis in mature 3T3‐L1 adipocytes in vitro, by acting at the HMGCR level (Ballistreri et al, 2021; Richard et al, 2013) and drive AMPK phosphorylation in both high glucose‐treated and palmitate‐challenged HepG2 cells (Dayarathne et al, 2021; Rajan et al, 2022), thus paving the way to the identification of such enzyme as a master regulator of the functional properties of bergamot. Thus, with the aim to characterize the possible lipid‐related mechanism(s) underlying the function of Brumex™, we performed an in vitro characterisation in HepG2 liver cells.…”

“…Preliminary evidence has been also documented for C. bergamia, although most data are focused on single active bio-components. Citrus bergamia bioflavonoids impair adipogenesis in mature 3T3-L1 adipocytes in vitro, by acting at the HMGCR level (Ballistreri et al, 2021;Richard et al, 2013) and drive AMPK phosphorylation in both high glucosetreated and palmitate-challenged HepG2 cells (Dayarathne et al, 2021;Rajan et al, 2022), thus paving the way to the identification of such enzyme as a master regulator of the functional properties (characterised over a concentration window that is non-cytotoxic to liver cells in vitro, Figure 1) specifically stimulates the Thr 175 phosphorylation of AMPK (Figure 2, Figure S2), a post-translational event specifically tuned by drugs that are routinely used in the treatment of obesity and type 2 diabetes mellitus, like AICAR and metformin (Luo et al, 2016;Sun et al, 2007).…”

Antiadipogenic Effect of Citrus Flavonoids: Evidence from RNA Sequencing Analysis and Activation of AMPK in 3T3-L1 Adipocytes

“…The optimal model for the AMPK flavonoids docked complex was chosen based on its binding energy and subsequently examined with PyMOL (The PyMOL Molecular Graphics System, Ver.2.5.0; Schrodinger, LLC, NY). Interactions involving hydrogen bonds and hydrophobic forces between the receptor and ligand were assessed using Ligplot software. , …”

“…Adipogenesis refers to the process of differentiation, in which preadipocytes develop into a mature state characterized by the accumulation of lipid droplets . AMPK serves as a key energy regulator and is instrumental in modulating lipid metabolic processes in adipocytes. , AMPK can directly modulate the function of acetyl-CoA carboxylase (ACC) via phosphorylation and inhibit the sterol regulatory element-binding protein-1 (SREBP-1), hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and fatty acid synthase (FASN) in adipocytes. , Therefore, flavonoid treatments that inhibit energy metabolism by modulating these metabolic elements might offer a viable solution for obesity prevention or treatment.…”

“…Earlier studies have reported that citrus flavanones glycosides, such as hesperidin (HES) and narirutin (NAR), might play a pivotal role in modulating cellular processes, particularly those related to lipid metabolism and adipocyte development . Furthermore, specific flavonoids known as polymethoxylated flavones (PMFs), particularly nobiletin (NOB), sinensetin (SIN), and tangeretin (TAN), have garnered attention for their possible health benefits. , Given the escalating global obesity epidemic, understanding the mechanisms through which these citrus flavanone glycosides exert their effects can pave the way for the development of novel therapeutic strategies. , Recently, flavonoid supplements have been introduced as a beverage on the global market, and several studies have elucidated their beneficial effects on metabolic diseases. − …”