Transcriptomics analysis of 5-dpf zebrafish larvae exposed to 5 μM PFHxS. (A) Venn diagram illustrating enriched genes within each selected term. (B) The ninety-four genes identified as core enrichment genes associated with NAFLD, predominantly showing upregulation upon exposure to 5 µM PFHxS. (C) A KEGG network diagram based on the 17 genes consistently detected in both NAFLD and IR.

Exposure to PFHxS triggers a cascade of events leading to NAFLD. Pro-inflammatory cytokines, activated in response to PFHxS exposure, initiate the ubiquitin-mediated proteasomal degradation of insulin receptor substrates 1 and 2 (IRS1/2), essential molecules for insulin transduction, via the phosphoinositide 3-kinase (PI3K)/Akt pathway. This degradation is facilitated through the activation of the suppressor of cytokine signaling (SOCS) by the signal transducer and activator of transcription 3 (STAT3). Alternatively, the proteasomal degradation of IRS1/2 can promote mTOR signaling. The depletion of IRS1/2 disrupts Akt signaling, impairing Akt-mediated phosphorylation and leading to the further ubiquitin-mediated proteasomal degradation of forkhead box protein O (FoxO). FoxO activation subsequently upregulates gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6P), resulting in increased hepatic glucose production. Conversely, the PFHxS activation of akt2 promotes the expression of sterol regulatory element-binding transcription factor 1 (srebf1, also known as sterol regulatory element-binding protein 1 (SREBP-1)), which in turn enhances lipid synthesis in the liver. Together, these mechanisms exacerbate the progression of PFHxS-induced NAFLD. Black, continuous lines: activation pathways. Red, dashed lines: inactivated pathways. Grey, dashed lines: possible stimulation/induction. Green triangle: upregulated genes. Red triangle: downregulated genes.

The mRNA expression levels of 10 selected genes (akt2, irs1, irs2a, socs31, pck1, srebf1, g6pca.1, il6, tnfa, and stat3) evaluated in PFHxS-exposed fish and compared to the control group at 5-dpf. Data are expressed as mean ± SEM. * indicates p < 0.05.