Fenvalerate exposure induces AKT/AMPK-dependent alterations in glucose metabolism in hepatoma cells

Abstract

Background: Fenvalerate (Fen) is a synthetic pyrethroid insecticide significantly associated with an increased risk of type 2 diabetes. Tumor cells exhibit a shift in glucose metabolism, known as the Warburg effect. Accordingly, we aimed to elucidate whether Fen interferes with insulin signaling and affects hepatoma cell metabolism.

Methods: The cells were subjected to Fen to assess glucose uptake, acidification, oxygen consumption, and ATP production. ROS generation, mitochondrial membrane potentials, and protein expression were evaluated by flow cytometry, immunofluorescence microscopy, and western blot analyses.

Results: Our results demonstrated that Fen promotes glucose uptake, lactate production, and ATP generation in various cancer cells. Moreover, Fen enhanced insulin receptor phosphorylation and upregulated p-AKT/p-AMPK expression. Fen enhanced insulin receptor sensitivity and endocytosis via reactive oxygen species generation rather than the PP2B pathway. Additionally, the antioxidants N-acetyl-L-cysteine and ascorbic acid reversed the Fen-induced increase in glycolysis. Finally, chronic Fen exposure protected hepatoma cells against metformin-induced cell death via the AKT/AMPK pathway.

Conclusion: These findings raise concerns regarding the safety of Fen and its potential role in altering cancer cell metabolism, affecting insulin signaling and treating drug resistance, thereby necessitating further research.

Keywords: cancer cell metabolism; fenvalerate; insulin receptor; reactive oxygen species; warburg-like effect.

FIGURE 1

Fen enhances glucose uptake, lactate production, and ATP generation in several cell lines. (A) In HepG2 cells, extracellular glucose uptake was measured with the glucose (GO) assay kit. (B) HepG2 cells were treated with Fen for 1 h, and glycolysis was measured using a glycolysis assay kit. (C) HepG2 cell glucose uptake was measured by 2-NBDG uptake and flow cytometry. (D) HepG2 cells were incubated with the indicated Fen concentrations for 1 h, and extracellular oxygen consumption was measured using an extracellular oxygen consumption assay kit. (E) HepG2 cells were incubated with various Fen concentrations for 1 h. Cellular ATP levels were measured using a cell ATP assay reagent. (F) Cell culture medium was sampled, and cytotoxicity was determined using an LDH assay. In A549 cells (G), MCF-7 cells (H), and HaCaT cells (I), extracellular glucose uptake was measured with glucose (GO) assay kit (i); glycolysis was determined with a glycolysis assay kit (ii); cellular ATP levels were measured with a cell ATP assay reagent (iii); viability was evaluated with CCK-8 reagent (iv). Statistical analysis was performed using one-way ANOVA (Dunnett’s test). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group.

FIGURE 2

Fen enhanced insulin receptor phosphorylation and upregulated p-AKT/p-AMPK expression. (A) After 24 h of Fen pretreatment, followed by stimulation with 100 nM insulin for 10 min, phosphorylated and total IR, AKT, GSK-3-β, and AMPK levels were analyzed using Western blotting. (C) Expression of phosphorylated and total forms of ERK and JNK were analyzed using Western blotting. (E) HepG2 cells were pretreated with each inhibitor for 1 h before incubation with 20 µM Fen and stimulated with 100 nM insulin for 10 min. The expression levels of p-IR, p-PTEN, PTEN, p-AKT, p-ERK, and p-JNK were analyzed using Western blotting; β-actin was used as the loading control. Average values of the ratio of phosphorylated to total proteins (B, D) or beta-actin (F) were quantified in each group. Statistical analysis was performed using a two-way ANOVA. *P < 0.05 and **P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 reagent pretreatment group vs. the 20 μM Fen combined group.

FIGURE 3

Fen decreased IRS1 expression and promoted insulin receptor endocytosis. HepG2 cells were pretreated with Fen for 24 h and stimulated with 100 nM insulin for 10 min. (A) Expression levels of IRS1 and p-mTOR were analyzed using western blotting. (C) Lyso-Tracker level was measured using flow cytometry with Lyso-Tracker staining. (D) Co-expressed IR-β and Lyso-tracker were stained with anti-IR-β (red), Lyso-Tracker staining (green), and DAPI (blue). Scale bar = 10 µm. Control or pretreatment with each inhibitor for 1 h before incubation with 20 μM Fen for 24 h, followed by stimulation with 100 nM insulin for 10 min. (E) The IRS1, p-IR, p-AMPK, p-ERK, and LC-3AB expression levels were analyzed using a western blot. (G) Pretreatment with Fen for 24 h followed by stimulation with 100 nM insulin for different times. The p-IR and p-AKT expression levels were analyzed using a western blot. Average values of the ratio of phosphorylated to beta-actin (B,F,H) were quantified in each group. (B,F,H) were performed using a two-way ANOVA. *P < 0.05 and **P < 0.01 vs. the control group. #P < 0.05 reagent pretreatment group vs. the 20 μM Fen combined group. Other statistical analysis was performed using one-way ANOVA (Dunnett’est). *P < 0.05, **P <0.01, and ***P < 0.001 vs. the control group.

FIGURE 4

Fen-driven ROS generation induced mitochondrial dysfunction and sustained the Warburg-like effect. (A) Intracellular O₂ generation was measured using flow cytometry with DHE staining. (B) MMP was determined using flow cytometry with TMRM staining. (C) HepG2 cells were either untreated or pretreated with ROS scavengers for 1 h before incubation with Fen for the indicated times. (D) After 24 h Fen treatment, extracellular glucose uptake was measured using a glucose (GO) assay kit. (E) Cellular ATP levels were measured using a cell ATP assay reagent. (F) Glycolysis was determined using a glycolysis assay kit. (G) After deltamethrin or cyclosporin A or okadaic acid pretreatment for 24 h, followed by stimulation with 100 nM insulin for 10 min, the p-IR, p-AKT, and p-ERK expression levels were analyzed using Western blot; β-actin was the loading control. (I) HepG2 cell glucose uptake was assessed by analyzing 2-NBDG uptake using flow cytometry. Average values of the ratio of phosphorylated to beta-actin (H) was quantified in each group. (C, E, G) were performed using a two-way ANOVA. *P < 0.05 and **P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 reagent pretreatment group vs. the 20 μM Fen combined group. Other analysis was performed using a one-way ANOVA (Dunnett’s test). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group; #P < 0.05 and ##P < 0.01 for the pretreatment group vs. the 20 μM Fen combined group; n.s = non-significant .

FIGURE 5

Chronic Fen exposure protected HepG2 cells against metformin-induced cell death. (A) Chronic Fen-HepG2 cell cultures were treated with 20 mM metformin for 24 h. Cell culture medium were sampled, and cytotoxicity was determined using an LDH assay. (B) Expression levels of p-mTOR, p-AMPK, p-AKT^Thr308, p-AKT^Ser473, p-GSK-3-β, p-ERK, and LC-3AB were analyzed using Western blotting; β-actin was used as the loading control. (D) Co-expressed Mito-Tracker and Lyso-Tracker were stained with Mito-Tracker staining (red), Lyso-Tracker staining (green), and DAPI (blue). Scale bar = 10 µm. (E) In chronic Fen-treated HepG2 cell cultures, Lyso-Tracker levels were measured using flow cytometry with Lyso-Tracker staining. (F) Intracellular O₂ generation was measured using flow cytometry with DHE staining. (G) Chronic Fen-treated HepG2 cell cultures were starved at different times. Cell culture medium were sampled, and cytotoxicity was determined using the LDH assay. (H) Cellular ATP levels were measured using a cell ATP assay reagent. (I) Schematic diagram depicting the Fen-sustained Warburg-like effect and enhanced glucose metabolism (Created in BioRender. Sun, L. (2025) https://BioRender.com/x86k997). Average values of the ratio of phosphorylated to beta-actin (C) was quantified in each group. (C, G) was performed using a two-way ANOVA. *P < 0.05 vs. the control or starvation group; #P < 0.05 for the pretreatment group vs. the metformin group. The other analysis was performed using a one-way ANOVA (Dunnett’s test). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the control group; n.s = non-significant .