Figure 5
PAR 2 -induced AMPK activation in primary fat is enhanced in the absence of β-arrestin-2. A. β-arrestin expression levels in extracts from wt mice, β-arrestin-1-/- mice and β-arrestin-2-/- mice were examined using antibody that recognizes both β-arrestin-1 and 2 (preferentially reacts with β-arrestin-1). Epidydimal fat pads from male wild type (wt) and β-arrestin-2-/- (B) or wt and β-arrestin-1-/- (C) mice were harvested and treated with or without 2fAP for 0-120 minutes, and phosphor-AMPK determined as described in Figure 1. A total of 15 mice from each experimental group were analyzed. Representative westerns are shown in B and C. Levels of pAMPK normalized to tAMPK were determined as described in Figure 1 and mean μ SEM pAMPK/tAMPK across all samples is presented as a bar graph of for all mice (D). The same data is also presented as dot plots showing fold changes over baseline in pAMPK levels for individual mice (E, note 0 minutes = baseline and is not included in the plot) and showing normalized phospho-AMPK levels of individual mice (F) *Statistically significant differences in mean pAMPK compared with wt mice (p < .005, n = 15). Statistically significant differences between 2fAP-treated and baseline pAMPK were only observed in β-arrestin-2-/- mice (p = .001, n = 15). This experiment was repeated in liver samples, included as Supplemental Figure 1. G, H. AMPK activity was determined as described in Figure 1 for AMPK immunoprecipitated from wt and β-arrestin-2-/- fat and is depicted as fold change over baseline (G) and pmoles of ATP incorporated per mg of immunoprecipitated AMPK (H). I. Fat from β-arrestin-2-/- mice was prepared as described in A, but was pretreated with STO-609 prior to activation of PAR2 with 2fAP for 0, 5 and 120 minutes. Shown are representative westerns of phospho-AMPK and total AMPK (upper panel) and a bar graph of normalized pAMPK levels in the presence and absence of STO-609 at each time point(lower panel).