Fig 2: SHBG modulates miRNA expression in liver sections ex vivo. To evaluate the expression of miRNA related to apoptosis and ER stress, liver fragments acquired from tissue samples post mortem were cultured in the presence of 50 and 100 nM SHBG for 6 and 24 h. Next, samples were subjected to RT-qPCR analysis and the expression of miR-103a (A), miR-107 (B), miR-30c (C), and miR-34c (D) was established. U6 snRNA was used as a housekeeping gene. The results are presented as mean ± SEM; n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: SHBG protects against PA-induced endoplasmic reticulum (ER) stress. HepG2 cells were incubated with SHBG in two concentrations (50 or 100 nM) before exposition to PA for 24 h. ER localization in cells was visualized with ER-TrackerTM Green (A). mRNA levels of IRE1A (B), ATF6 (C), CHOP (D), and HSPA5 (E) were determined using the RT-qPCR method. The phosphorylation of eIF2a at Ser51 and IRE1a at Ser724, as well as the cleavage activation of ATF6 were estimated using the Western blot technique (F). Relative intensities of ATF6 full length (G), ATF6 cleaved (H), phosphorylated IRE1a (I), and eIF2a (J) were calculated with Image Lab software after normalization to ß-actin as a control housekeeping protein. Intracellular localization of XBP1 investigated with confocal microscopy (K). Expression levels (L) of spliced and unsliced XBP1 established by RT-qPCR followed by gel electrophoresis. The results are presented as mean ± SEM; n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: MS individuals are characterized by decreased liver function and suffer from lipotoxicicty, ER stress, and decreased SHBG levels. Biochemical analysis revealed increased levels of GGTP (A) and AST (B) in the circulating blood of MS subjects. Furthermore, using ELISA, the amount of IL-6 (C) and PPARG (D) in serum was evaluated. RT-qPCR revealed an increased expression of CHOP (E) and ultrastructural alternations including lipid overload (F) in metabolic syndrome-derived liver samples. These specimens were also characterized by decreased SHBG levels (G). The results are presented as mean ± SEM; n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: Correlation between lipid overload and sex hormone-binding globulin (SHBG) levels in palmitate (PA) treated HepG2 cells. Oil Red O staining (A) and its quantification (B) revealed time-depended lipid overload. PA treatment significantly upregulated the SHBG expression (C); however, the protein amount remained unchanged (D). The results are presented as mean ± SEM; n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001.