OR01-01 Novel Anti-obesity Phytohormone Promotes Cellular Insulin Sensitivity in Hypothalamic Neurons

Abstract C.X. Zhang: None. C. Lieu: None. W. He: None. D.D. Belsham: None. Obesity is a global epidemic and is associated with dangerous comorbidities, such as type 2 diabetes mellitus (T2DM). Insulin is sensed by insulin receptor (INSR)-expressing neurons in the hypothalamus, exerts anorexigenic effects to suppress feeding and promote weight loss. In obesity and T2DM, elevated insulin levels induce cellular insulin resistance in hypothalamic neurons, further dysregulating energy homeostasis. Our lab recently discovered that a plant-based phytohormone, compound X (CX), demonstrates in vivo efficacy for weight loss and maintenance. Notably, oral feeding of CX remarkably decreased body weight and food intake in obese CD-1 mice while maintained on a 60% high-fat diet. Moreover, CX-fed mice had lower basal serum insulin levels and improved glucose tolerance, indicating a protective role of CX against obesity-induced insulin resistance. Given the critical interplay between central insulin resistance and obesity, we hypothesized that CX enhances cellular insulin signalling in hypothalamic neurons. A clonal mouse hypothalamic cell line, mHypoE-46, was treated with 100 µM CX or DMSO for 24 hours. The mRNA/protein expression of INSR was measured, and insulin sensitivity was determined upon insulin re-exposure by phosphorylation levels of protein kinase B (AKT) at Ser473. RNA-sequencing was also performed on mHypoE-46 cells after 4 or 16 hours of 100 µM CX treatment to identify pathways altered by CX. We determined that CX robustly increased mRNA and protein expression of INSR and enhanced insulin-indued AKT phosphorylation. Furthermore, CX upregulated the mRNA expression of phosphatidylinositol 3-kinase regulatory subunit 1 (Pik3r1), a downstream mediator of INSR, in CX-treated cells, further demonstrating an enhanced INSR/PI3K/AKT response. Concurrently, we conducted pathway analysis of the RNA-seq data using GSEA and ErimeR. The mammalian target of rapamycin complex 1 (mTORC1) pathway was identified as a top candidate pathway inhibited by CX, which was then validated by assessing the expression and phosphorylation changes of its downstream effectors. The inhibition of mTORC1 is likely responsible for INSR induction by CX, as chronic mTORC1 activation induces cellular insulin resistance. Accordingly, we hypothesize that CX relieves central insulin resistance via modulation of INSR expression and downstream signalling components, such as PI3K and mTORC1. Selective activators and inhibitors of PI3K and mTORC1 will be used to confirm their involvement in CX signalling. Overall, our study delineates the molecular mechanisms involved in the CX-mediated enhancement of insulin signalling and promotion of cellular insulin sensitivity in hypothalamic neuronal models, advancing our knowledge of central insulin resistance and paving the way for future clinical trials of a new T2DM and obesity plant-based therapeutic for humans. Sunday, June 2, 2024