Fig. 2.

Glycogen synthase kinase (GSK)-3 mediates toxic lipid-induced proinflammatory responses in liver sinusoidal endothelial cells (LSECs).

(A) Primary human LSECs were treated with vehicle (Veh) or 500 μM palmitate (PA) ± 20 nM LY2090314 (LY) for 16 h, and gene expression profiling was performed using the NanoString nCounter system (1). LSECs were isolated from chow- and fat, fructose, and cholesterol (FFC)-fed mice and subjected to RNA-sequencing (RNA-seq) (2). Venn diagram (bottom) of genes classified as ‘lipotoxic stress-dependent genes’ and ‘GSK3-dependent genes’ in NanoString analysis and ‘Metabolic dysfunction-associated steatohepatitis (MASH)-dependent genes’ in mouse LSEC RNA-seq. Primary human LSECs were treated with Veh or 500 μM of PA (B) or 10 ng/ml tumor necrosis factor (TNF)-α (C) ± 20 nM LY for 16 h, and mRNA expression of C-X-C motif chemokine ligand 2 (CXCL2) and intracellular adhesion molecule 1 (ICAM1) was examined. (D) Primary human LSECs were transfected with siGSK3β or non-target small interfering (si)RNA for 72 h, then treated with Veh or 500 μM PA for 24 h, and the mRNA expression of GSK3B, CXCL2, and ICAM1 was examined (n = 3 per group). (E) Phospho-GSK3α/β (Y279/216) and GSK3β protein levels were assessed in normal controls and patients with steatosis, and MASH by Western blotting with quantification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control (n = 4–8 per group). Bar graphs represent mean ± SEM; ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001, ns, non-significant (one-way ANOVA with Bonferroni’s multiple comparison).