Modeling Transmembrane 6 Superfamily Member 2 p.Glu167Lys-associated Steatotic Liver Disease Using Androgen-treated Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells.

Background and Aims: Transmembrane 6 superfamily member 2 (TM6SF2) p.Glu167Lys (E167K) is associated with hepatic steatosis and male-predominant type 2 diabetes (T2D) presumably due to defective secretion of very low density lipoprotein (VLDL) particles. The causal relationship between secreted VLDL density and insulin sensitivity in the context of the E167K variant remains unclear. Methods: E167K-carrying induced pluripotent stem cells were generated using a genome-editing method. Guided by targeted phospholipidomic analysis, we established an improved protocol to derive hepatocyte-like cells (HLCs) from induced pluripotent stem cells with significantly improved VLDL secretory profiles after linoleic acid, arachidonic acid and Liver X receptor agonist exposure. Furthermore, the E167K-defined steatosis and metabolic phenotype of HLCs treated with or without dihydrotestosterone were analyzed. In public database, we stratified male subjects by testosterone level and analyzed the impact of TM6SF2 on the incidence of hepatic steatosis and T2D. Results: E167K hepatocytes showed defective VLDL secretion and an exacerbated lipid accumulation phenotype, accompanied by a decrease in arachidonic acid-containing phosphatidylcholine. Furthermore, dihydrotestosterone supplementation worsened the steatotic phenotype of E167K HLCs without altering insulin sensitivity. In the clinical data, E167K-defined hepatic steatosis was testosterone-dosage dependent unlike the other genetic variant, while T2D was not. Conclusion: We report an improved human hepatocyte differentiation model to investigate metabolic dysfunction with functional VLDL secretory profiles influenced by TM6SF2 gene variant. Our combinatorial analysis of in vitro and human data indicates that male testosterone modifies the effect of E167K on hepatic steatosis but not insulin sensitivity. These data support the hypothesis that male-predominant T2D in E167K carriers is not causative but rather a consequence of persistent steatosis.