How You Can Grow To Be Fantastic With Ku-0059436

This acute treatment induced robust phosphorylation of AMPK��, ERK1, and ERK2 (Figure?2), well-known FGF21 downstream signaling events [19,22]. Therefore, we used 50?ng/mL rhFGF21 in all subsequent experiments. Figure?2 FGF21 signaling in differentiating hasc preadipocytes Cells from day 8 of the differentiation protocol were left untreated (?) or were treated (+) for 10?min with rhFGF21 (50?ng/mL). Immunoblotting was performed with primary antibodies ... 3.3. Effect of chronic FGF21 RG-7204 treatment on lipid accumulation As shown by qualitative Oil Red O staining (Figure?3A) and photometric quantification of the intracellular Oil Red O content over time (Figure?3B), triglyceride accumulation was significantly increased by rhFGF21 during the early stages of differentiation. This effect, however, was modest (+15%, days 4 and 8) and diminished to a statistical trend at later time-points (p?=?0.09, days 12 and 18; Figure?3B). Figure?3 FGF21 effect on lipid accumulation in differentiating hasc preadipocytes (A) Triglyceride accumulation was visualized by Oil Red O staining of proliferating preadipocytes (topmost panel) and of differentiating adipocytes (from days 4, 8, and 18) left ... 3.4. Effects of chronic FGF21 treatment on white and brown adipocyte marker gene expression Chronic rhFGF21 treatment significantly impaired the expression of the key adipogenic transcription factors peroxisome proliferator-activated receptor-�� (PPAR-��; -15%, day 18; Figure?4A) and CCAAT/enhancer-binding protein-�� (C/EBP-��; up to??40%, days 8�C18; Figure?4B). In addition, rhFGF21 reduced the expression of the adipokine adiponectin (up to??20%; days 4 and 18; Figure?4C), whereas leptin expression remained unaffected (p?>?0.08). Unexpectedly, the brown adipocyte marker genes encoding uncoupling protein-1 (UCP-1), PPAR-�� coactivator-1�� (PGC-1��), and cell death-inducing DNA fragmentation factor-like effector a (CIDEA) were differently regulated with UCP-1 being 2-fold induced by rhFGF21 (days 12 and 18; Figure?4D) and PGC-1�� and CIDEA being repressed by up to??40% and??50%, respectively (all time-points; Figure?4E,F). Similar regulations of the brown adipocyte marker genes were seen when rhFGF21 was sub-chronically added for three days to adipocytes (from day 18) that were differentiated in the absence of rhFGF21 (data not shown). It should be noted that the UCP-1 expression levels were very low (mean Cp-value on day 0: 35.9; mean Cp-value on day 18: 31.4). Moreover, we did not obtain higher expression levels using isoproterenol, a well-known and potent inducer of the gene. Therefore, we concluded that hasc preadipocytes represent a cell type with very limited capacity to brown. In accordance with this, we did not detect any convincing signal for UCP-1 protein by immunocytochemistry.