Results from unbiased genome-wide studies have increased our understanding of which pathways are activated by FGF21 in this primary target tissue. Moreover, the activation of the FGFR/Klb co-receptor complex triggered phosphorylation signaling cascades that could be tied in to the gene expression changes. Finally, by monitoring a subgroup of these events in whole blood, we were able to monitor FGF21 TE either acutely or sub-chronically in vivo. SILAC MS-based phosphoButenafine hydrochloride protein enrichment and profiling identified and quantified FGF21-dependent phosphoprotein changes in 3T3L1 adipocyte cell lysates obtained 10 minutes post-treatment. Erk1 and 2 were identified as two of the most robustly phosphorylated peptides in 3T3L1 adipocytes after FGF21 treatment compared to the vehicle, as expected. Other phosphorylation events occurred in pathways such as the Insulin Receptor Signaling pathway and the Phospholipase C Signaling pathway. Since these studies were carried out in adipocytes in vitro, it was important to validate these findings in adipose tissues in vivo. However, given that most of the phosphorylation sites we identified were novel, it was not feasible to use commercially available antibody reagents. Despite these limitations, we were able to replicate five of the FGF21-mediated phosphorylation events that were identified in vitro, in a visceral adipose depot in mice. Once additional reagents for the novel phosphorylation events become available, further validation studies will be possible. Robust and consistent downstream transcriptional responses in white adipose tissues in vivo were also identified. We first selected probe sets that were consistently regulated across the three WAT depots and across the three mouse models and then sub-selected those probe sets that were robustly regulated acutely in WT mice on chow diet, resulting in 1129 and 165 probe sets, respectively. Pathway and GO term enrichment analysis on the broader gene set identified metabolic pathways known to be affected by FGF21 as well as pathways not previously associated with this protein. Multiple metabolic and signaling pathways were enriched, such as Fgfr, Erk/Mapk, Pi3k/Akt, Igf-1, and mTor signaling, triglyceride synthesis and degradation, glucose uptake, amino acid transport and energy expenditure. Perhaps not surprisingly, the most robustly regulated genes were those involved in negative feedback regulation of Fgfr signaling, even at the lowest doses of FGF21 investigated. Depicted are phosphorylation, protein, or RNA changes after FGF21 treatment in vitro or in vivo. See Table S2, S3, S4, S9 for details.Most genes regulated by FGF21 identified by our studies have unknown biological significance in terms of beneficial consequences of FGF21 activation, but can nevertheless be used as robust TE biomarkers. Others have known Albaspidin-AA functions and their regulation by FGF21 either supports or seems to contradict a beneficial role of FGF21. Indeed, Sfrp5 expression was consistently downregulated by FGF21 across the three WAT depots and across the three mouse models used. In addition, Sfrp5 expression was higher in WAT depots from db/db mice when compared to WT mice suggesting a ��reversal�� of the disease phenotype by FGF21 treatment. This is in contrast to the reported decreased expression in WAT from ob/ob mice. Furthermore, changes in Sfrp5 expression following FGF21 treatment in WAT approached the level of 
expression of Sfrp5 in BAT under basal conditions, indicative of a white adipose tissue ��browning�� effect by FGF21. There are also conflicting reports on the role of Sfrp5 in human adipose biology, thus the biological impact of a down-regulation of this gene by FGF21 warrants further investigation. FGF21 expression has been shown to be up-regulated by PPAR�� agonist treatment in adipose tissue and adipocytes, and there is also evidence that FGF21 treatment.