Supplementary Material for: Electroacupuncture Alleviates Obesity and Insulin Resistance via the GLP-1-VTA DA Reward Circuit

Objective: We investigated the effects of electroacupuncture (EA) on improving obesity and insulin resistance (IR) in high-fat-diet-induced (HFDI) obese rats by modulating the nucleus tractus solitarius (NTS) glucagon-like peptide-1 (GLP-1)-ventral tegmental area (VTA) dopamine (DA) neural reward circuit, thereby uncovering a possible central mechanism underlying EA’s actions in improving obesity and IR. Methods: We randomly allocated 45 Wistar male rats to five groups (normal, model, EA, chemogenetic activation, chemogenetic suppression + EA), with nine rats in each group. All interventions were conducted within 8 weeks after the model was established. We tested rats for obesity phenotypes included body mass, Lee's index, 24-h food intake, and glucose-metabolism parameters. We observed protein and gene expression for GLP-1 in the NTS and tyrosine hydroxylase (TH) in the VTA by western blotting (WB) and real-time polymerase chain reaction (RT-qPCR), as well as their localization by immunofluorescence. We also determined the DA content in the VTA using high-performance liquid chromatography (HPLC). Results: Obese rats exhibited marked hyperphagia, accompanied by increased excitability of DA neurons in the VTA region and reduced insulin sensitivity. After EA treatment, obese rats showed augmented excitability of NTS GLP-1 and suppression of VTADA neurons with a diminution in food intake, showing results similar to those in the chemogenetic-activation group. After EA treatment and while inhibiting GLP-1 neurons by chemogenetics, the effect of EA on activating GLP-1 neurons and inhibiting VTADA was partially abrogated. The effects of improving obesity and insulin sensitivity were likewise also suppressed. Conclusion: EA effectively activated GLP-1 neurons in the NTS, thereby inhibited the expression of DA in the VTA and improved obesity and insulin sensitivity in HFDI-obese rats