Ameliorative effects of mangiferin derivative TPX on insulin resistance via PI3K/AKT and AMPK signaling pathways in human HepG2 and HL-7702 hepatocytes

Background: As a multifaceted metabolic disorder, insulin resistance is accompanied by the preceding onset of type 2 diabetes mellitus, hyperinsulinemia, metabolic dysfunction-associated fatty liver disease (MAFLD) and other metabolic syndromes. Currently, the number of existing drugs and mechanism-based strategies is limited to alleviate insulin resistance in clinics. As a natural polyphenol product derivative, 1,3,6,7-tetrapropylene acyloxy-ketone (TPX) showed a significant hypoglycemic effect in our previous studies. However, whether TPX could improve hepatic insulin sensitivity was unknown. Purpose: To explore whether insulin sensitivity can be improved by the treatment with TPX and further inves-tigate its mechanism(s) of activity. Methods: To mimic hyperglycemia and insulin resistance in vitro, human HepG2 and HL-7702 hepatocytes were exposed to high glucose. Cellular glucose uptake, glucose consumption, glycogen synthesis, and glucose pro-duction were quantified after TPX treatment. The effects of TPX on AMP-activated protein kinase (AMPK) phosphorylation, glucose metabolism, and insulin signal transduction were evaluated by western blotting and network pharmacology analysis. The eGFP-membrane of glucose transporter type 4 (GLUT4) lentivirus trans-fected cells were constructed to investigate the effects of TPX on GLUT4 mobilization. Reactive oxygen species activity in high glucose-induced insulin-resistant cells was measured by DCFH-DA to show oxidative stress. Results: Treatment with TPX improved glycogen synthesis and inhibited gluconeogenesis by regulating GSK3 beta, G6Pase, and PEPCK. Furthermore, high glucose-induced inhibition of glucose consumption, glucose uptake, and GLUT4-mediated membrane translocation were reverted by TPX. Accordingly, mechanistic investigations revealed that TPX interacted with AMPK protein and activated the phosphorylation of AKT, thereby improving energy homeostasis and further ameliorating hepatic insulin resistance. Network pharmacology analysis and molecular docking further confirmed AMPK as an active target of TPX. Concordantly, the pharmacological ac-tivity of TPX was reversed by the AMPK inhibitor compound C when hepatocytes were exposed to high glucose stimulation. Conclusion: In summary, our study confirmed TPX contributions to insulin resistance improvements by targeting AMPK and PI3K/AKT to restore the insulin signaling pathway, which may be an important potential treatment strategy for insulin-resistance-related diseases, including MAFLD and diabetes.