PROJECT SUMMARY Mounting evidence supports a central role for chronic inflammation in obesity and metabolic dysfunction, yet our understanding of actual causal links remains limited. We have identified and characterized numerous points of crosstalk between inflammation and the metabolic effects of overnutrition. Activation of the innate immune system triggers the robust production of interferon stimulated genes (ISGs), many of which have potent roles in metabolism. One such gene encodes for the ubiquitin-like modifier ISG15 that is conjugated to target proteins in a reversible process referred to as ISGylation. Our preliminary data show diet-induced obesity increases liver expression of Isg15 and protein ISGylation. Deficiency in liver Isg15 (ISG15LKO) renders mice resistant to diet- induced weight gain, and improves insulin sensitivity and hepatic steatosis. The liver, however, is not a thermogenic organ. Therefore, the impact of liver ISGylation on the regulation of body weight is likely mediated through endocrine signaling to other organs capable of influencing food intake, locomotor activity and/or energy expenditure. To identify ISGylated proteins, we engineered endogenously tagged Isg15 mice, and mass spectrometry proteomics data revealed robust enrichment of proteins involved in peroxisomal and mitochondrial oxidation. The overall goal of this proposal is to interrogate the role of liver ISGylation in regulating organismal energy balance. The central hypothesis is that in the liver, obesity triggers ISGylation of critical oxidative proteins that influence the hepatocyte secretome and promote weight gain, either by increasing food intake or decreasing energy expenditure. This hypothesis will be tested through the following two specific aims. Aim 1 will determine the effects of hepatic ISGylation on energy balance, and Aim 2 will identify liver ISGylation targets responsible for systemic metabolic regulation.