In an independent in vivo validation study, immunoassays were performed using commercially available antibodies against 5 proteins and mouse blood taken at the 24 h and 48 h time points. Consistent with the gene expression data, Ccl11 Kinase Inhibitor Library abmole bioscience protein levels were increased. The lack of increase in circulating Ccl11 at 24 hours post FGF21 treatment may reflect the specific differences in the kinetics of protein synthesis and/or secretion. Il1rn protein levels were significantly decreased at the 24 h time point following the high dose of PEG30-FGF21 Q108. This is in agreement with the microarray analysis where the Il1rn transcript was down-regulated by the 2.5 mg/kg dose of PEG30-FGF21 Q108 at the 2 day time point. The increase in Il1rn at 48 h post-dose may reflect feedback regulation, although this requires further experimentation. Immunoassays for the three other proteins did not show dose-dependent changes in the plasma after FGF21 treatment. There was, however, a significant decrease in the plasma levels of all three proteins at the highest dose of FGF21 tested at the 24 h time point in agreement with their transcript regulation. Further studies are required to fully validate these findings. In the context of secreted proteins, the insulin-sensitizing adipokine adiponectin is of particular relevance to FGF21 signaling. Two recent studies have shown that adiponectin is a downstream effector of FGF21. Furthermore, treatment with FGF21 enhanced both the expression and secretion of adiponectin in mouse adipocytes, and also increased the serum levels of adiponectin in mice. In the present study we did not see significant increases in adiponectin mRNA in adipose tissues in vivo. In parallel experiments, we treated 3T3L1 adipocytes with WT FGF21 at 0.3 and 1 ��g/ml and could not observe an increase in adiponectin protein in the media, despite increased Erk phosporylation, a robust indicator of FGF21 receptor activation. Moreover, rosiglitazone treatment did trigger a 65% increase in secreted adiponectin in the same experiment. Similarly, in mice treated with WT FGF21 a 2-fold increase in Erk phosphorylation, indicative of target engagement, was not associated with increased levels of plasma adiponectin after either 1 or 6 hours of treatment. Rosiglitazone was not used in this in vivo study since more chronic dosing has been 
reported to be required to observe an increase in circulating adiponectin levels. Further experiments are needed to determine the basis of these apparent inconsistencies. Changes in the phosphorylation of downstream signaling molecules following FGF21 treatment in adipose/adipocytes are expected to be the most proximal effects of receptor activation, and could potentially be used as TE biomarkers in adipose tissue biopsies. We used an unbiased phosphoproteomic SILAC approach in 3T3L1 mouse adipocytes treated with FGF21 to identify such changes. As a quality control, LC-MS profiling of forward and reverse labeled samples demonstrated that the heavy isotope incorporation as well as the equal Reversine purchase mixing of heavy and light samples were of good quality. We subsequently identified and quantified a total of 1186 phosphopeptides corresponding to 671 unique proteins from 3T3L1 adipocytes. Among them, 137 phosphopeptides showed a greater than 1.5 fold change between vehicle and FGF21 treatment. Of these phosphorylation events, 127 were up-regulated while 10 were down-regulated by FGF21 treatment compared to vehicle. Enriched canonical pathways from the Ingenuity Pathway Analysis tool included Insulin Receptor Signaling, IGF-1 Signaling, and Phospholipase C Signaling. The aim of the present study was to further interrogate the signaling events downstream of FGF21 receptor activation and, as an added bonus we identified a robust panel of target engagement biomarkers that may be useful for clinical development of FGF21-based therapeutics.