Figure 4.

Dihydrotestosterone treatment augments the steatosis phenotype in TM6SF2 E167K-edited HLC dissociated from insulin sensitivity. (A) Schematic overview shows the protocol for HLCs differentiation with AALAT and 200nM DHT. (B, D, C, E) B and D show representative images of EE or KK HLCs ± DHT stained for lipid droplets and nuclei. Quantification for male (YD5-1, YD3-1) and female (YD8-1) donor HLCs is shown in C and E, respectively (∗P < .05, ∗∗∗P < .001, Tukey’s post hoc tests). (F) The enrichment plot shows the correlation with the male-biased gene set in the DHT-treated HLCs. (G) The arachidonic acid (20:4) content of PC in EE or KK HLCs ± DHT treatment was measured (∗∗P < .01, ∗P < .05, Student’s t-test). (H) The phosphorylation of Akt induced by insulin in DHT-treated EE or KK HLCs was measured by western blotting. Akt phosphorylation was measured after cells were starved overnight in insulin-free medium followed by 100 nM insulin for 20 minutes. Cells were also treated with OA and PA (200 μM each) for steatotic induction for 48 hours. The band intensity quantification data is shown in (I). (J) Genes related to sugar and lipid metabolism were analyzed to assess the effect of DHT on insulin sensitivity in KK HLCs. After overnight insulin starvation, the cells were treated with 100 nM insulin, and RNA was collected after 4 hours followed by bulk-RNA-seq. The TPM of the control condition was taken as 1.0, and the fold change was analyzed (Tukey’s post hoc tests). All data represent mean ± SD. OA, oleic acid.