1A). To test whether increased TACE activity is a downstream effector of metabolic toxicity to impaired insulin action, we overexpressed TACE by way of adenoviral vectors. This resulted in increased TACE activity (Fig. 1B).

Inhibition of JNK activity by SP600125 partially reversed the effect of palmitic acid and TACE overexpression on TACE activity (Fig. 1C,D). In a preliminary set of results, we observed that TACE overexpression impairs ligand-dependent phosphorylation of the insulin receptor β subunit at different insulin concentrations (10−9M and 10−7M) and time points (Supporting Fig. 1). Next, we analyzed CAL-101 price downstream elements of insulin signaling involved in the control of glucose and lipid metabolism. We found that phosphorylation of AKT on serine 473, FoxO1 on serine 256, and GSK3α/β on serine 9/21 were all consistently reduced by increased TACE activity (Fig. 1E). To identify tissues in which

TACE activity may affect glucose and lipid metabolism, we analyzed its activation in white adipose tissue (WAT), muscle, and liver of C57/BL6 mice fed either a high-fat diet (HFD) or chow for 5, 10, and 20 weeks after weaning. We found that TACE activity was significantly increased by HFD first in liver at 10 weeks and continued to be increased after 20 weeks of HFD compared with chow (Fig. 2A). Both WAT and muscle also displayed increased TACE activity by this time point. Next, we analyzed the expression levels of TACE and its inhibitor Timp3 in all three tissues and found that whereas increased TACE activation associated with a mild increase of TACE expression in mTOR inhibitor WAT and liver, a more significant decrease of Timp3 expression occurs at both messenger RNA (mRNA) and protein levels in all three tissues (Fig. 2B,C). Overall,

these results suggest that prolonged metabolic stress is associated with increased TACE activity and decreased Timp3 expression. Timp3−/− mice manifest increased TACE activity, especially in the liver.16 However, we have previously shown that metabolic homeostasis in Timp3−/− mice is similar to that of WT littermates at 24 weeks of age, when both are fed chow, offering the ideal scenario to study the interaction between increased TACE activity and the prolonged metabolic stress caused by a diet rich in lipids. Timp3−/− mice fed a HFD for 20 weeks exhibited a weight this website similar to that of WT mice (Fig. 3A); however, Timp3−/− animals showed significantly increased fasting and fed glucose and insulin levels (Fig. 3B,C), increased aminotransferases (Fig. 3D), and worsened glucose tolerance (Fig. 3E) and insulin sensitivity (Fig. 3F) compared with WT littermates. Analysis of liver function and histology revealed that after 20 weeks of HFD, Timp3−/− mice manifested increased TACE activity (Fig. 4A) and macrovesicular steatosis with features of ballooning degeneration as seen in grade 2 human steatohepatitis (Fig.