Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

Abstract

Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit.

Keywords: AMPK; Cordycepin; LKB1; Molecular docking.

Fig 1

Cordycepin inhibits intracellular lipid accumulation in HepG2 cells. Cells were incubated with oleic acid (OA, 100 μM) for 12 hrs, then treated with various agents [5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) (1 mM), cordycepin (0.1–10 μM) or cordycepin + compound C (10 μM + 40 μM)] for additional 6 hrs. Neutral lipids were determined by spectrophotometry at 358 nm after oil-red O staining (A); total lipids (B), total cholesterol (C) and triglycerides (D) were measured by kits according to the manufacturer's instructions. Bars depict the means ± SEM of at least three experiments. Asterisks represent statistically significant differences from the OA-overloaded control group (^†P < 0.05, ^‡P < 0.01). cpn: cordycepin; C.C; compound C.

Fig 2

Cordycepin decreases LD size in HepG2 cells. Cells were transfected with the LD-specific GFP-Perilipin A expression vector followed by treatment with OA (OA, 100 μM) either with or without cordycepin (1 μM) for 16 hrs. The LDs were visualized by oil-red O staining (bar = 50 μm; A) and fluorescence microscopy (bar = 5 μm; B). Over 100 fluorescence photos were taken for each group, and the LD number (C) and the diameter of the largest LD (D) in each cell were recorded and plotted. Asterisks represent statistically significant differences from the OA-treated control group (*P < 0.05). OA: oleic acid; LD: lipid droplet.

Fig 3

Cordycepin inhibits de novo lipogenesis (A) and promotes fatty acid oxidation (B). HepG2 cells were incubated with either [1-¹⁴C]acetate for 2 hrs or [1-¹⁴C]palmitate for 24 hrs co-treated with AICAR (1 mM), cordycepin (0.1–10 μM) or cordycepin + compound C (10 μM + 40 μM), and the radioactivity in the saponifiable fatty acid fractions was measured. The data depict the means ± SEM of at least three experiments. Asterisks represent statistically significant differences from the oleic acid–treated control group (^†P < 0.05, ^‡P < 0.01). cpn: cordycepin; C.C; compound C.

Fig 4

Cordycepin increases AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation (A) and AMPK activity (B). Cells were treated with AICAR (1 mM), cordycepin (0.1–10 μM) or cordycepin + compound C (10 μM + 40 μM) for 1 hr. Phosphorylation of AMPK (pThr172-AMPK) and its substrate ACC (pSer79-ACC) were detected by western blot analysis. The mean grey values of each lane in the western blot photos were quantified by ImageJ 4.1 software and the ratios of p-AMPK/t-AMPK and p-ACC/t-ACC were calculated. The AMPK activity was determined by the SAMS peptide assay. The data depict the means ± SEM of at least three experiments. Asterisks represent statistically significant differences from the oleic acid–treated control group (*P < 0.05, **P < 0.01, **^*P < 0.001). cpn: cordycepin; C.C; compound C.

Fig 5

Cordycepin activates AMP-activated protein kinase (AMPK) without changing intracellular AMP level but is inhibited by LKB1 dysfunction. (A) Effect of cordycepin on intracellular AMP, ADP and ATP levels and the AMP/ATP ratio. Cells were treated with AICAR (1 mM), cordycepin (0.1–10 μM) for 1 hr. AMP, ADP and ATP levels were determined by high-performance liquid chromatography (HPLC). (B) Treatment with the CaMKK inhibitor STO-609 (10 μg/ml) did not suppress cordycepin-mediated AMPK and acetyl-CoA carboxylase (ACC) phosphorylation. (C) LKB1 knockdown impaired cordycepin-induced AMPK and ACC phosphorylation. Before western blot analysis, cells were treated with respective agents for 1 hr. The mean grey values of each lane in the western blot photos were quantified by ImageJ 4.1 software and the ratios of p-AMPK/t-AMPK and p-ACC/t-ACC were calculated. The data depict the means ± SEM of at least three experiments. cpn: cordycepin; C.C; compound C; ST: STO609.

Fig 6

Molecular docking study of the binding affinity of cordycepin to the AMPKγ1 subunit. Hydrogen bonds are represented as dotted lines.

Fig 7

Cordycepin interacts with AMPKγ1 in vitro. (A) The fluorescence quenching spectra of AMPKγ1 with different concentrations of cordycepin at 298 K. The concentration of AMPKγ1 was fixed at 2.0 μM, and the ratios of protein versus cordycepin ranged from 1:1 to 1:4. (B) The CD spectra of the AMPKγ1-cordycepin system obtained in 10 μM PBS buffer at pH 7.4 at room temperature. The AMPKγ1 concentration was fixed at 2.7 μM, and the molar ratios of protein to cordycepin ranged from 1:1 to 1:8.

Fig 8

Cordycepin-induced AMP-activated protein kinase (AMPK) phosphorylation was suppressed in HepG2 cells in which AMPKγ1 was stably knocked down. Real-time quantitative PCR (A) and Western blot (B) showed that the expression of AMPKγ1 was significantly decreased, whereas AMPKα1 was not inhibited. Cordycepin-mediated AMPK-and acetyl-CoA carboxylase (ACC)-phosphorylation (C) and TC-and TG-decrease (D) were substantially reduced by knockdown of AMPKγ1. Cells were co-treated with oleic acid (OA, 100 μM) and either cordycepin alone (0.1–10 μM) or cordycepin + compound C (10 μM + 40 μM) for 6 hrs. Total cholesterol (TC) and triglycerides (TG) were measured by kits according to the manufacturers’ protocols. Bars depict the means ± SEM of at least three experiments. cpn: cordycepin; C.C; compound C.

Fig 9

The regulatory effects of cordycepin on the expression of key lipid metabolic genes were reversed in AMPKγ-deficient cells. Cells were treated with oleic acid (OA, 100 μM) and cordycepin (10 μM) for 6 hrs. Real-time PCR was conducted with specific oligonucleotide primers, with the amplification of beta-actin serving as the internal control. The data depict the means ± SEM of at least three experiments. *P < 0.05, **P < 0.01, cordycepin-treated cells versus control cells in HepG2 cells transfected with scrambled RNA, ^#P < 0.05, ^##P < 0.01, cordycepin-treated cells versus control cells in HepG2 cells transfected with specific AMPKγ1-targeting RNA. ABCA1, ATP-binding cassette transporter A1; ACC, acetyl-CoA carboxylase; ApoA1, apolipoprotein A1; CPT1, carnitine palmitoyltransferase 1; CYP7A1, cytochrome P450 7A1; DGAT, diacylglycerol acyltransferase; FAS, fatty acid synthase; HMGR, HMG-CoA reductase; LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; LXRα, liver X receptor α; PPARs, peroxisome proliferator–activated receptors; SREBPs, sterol regulatory element–binding proteins; UCPs, uncoupling proteins.