This renewal application is for a project that had as its overall goal to gain understanding of the molecular bases for retinol-binding protein 4 (RBP4)-induced metabolic disease. We found from investigations of a newly generated transgenic mouse model (adi-hRBP4 mice) that modest increases in RBP4 expression in adipocytes (in both white and brown adipocytes) results in significantly increased inflammation within adipose tissue. This gives rise to a redistribution of unesterified fatty acids from adipocytes to the liver where they accumulated as triglyceride. We also identified effects of RBP4 in brown adipocytes that we propose contribute to the excessive body weight gain observed in adi-RBP4 mice in response to high fat diet feeding. Our findings from the previous period led us to conclude that adipocyte RBP4 is an important autocrine/paracrine factor whose adverse actions in metabolic disease causation are largely confined to the adipose tissue compartment. We are now proposing new research that grows directly out of published and preliminary findings obtained during the previous period. This research will involve animal models, mature brown adipocytes and brown adipocyte precursor cells in culture, and in vitro studies. Our preliminary data indicate that RBP4 acts to modulate BAT thermogenic capacity and energy expenditure and that this involves effects on all-trans-retinoic acid (ATRA)- signaling in BAT. In Specific Aims 1 and 2, we propose to explore the molecular bases for these observations. Specific Aim 1 will investigate the role that RBP4 expression in brown adipocytes has on obesity development. In complementary studies in Specific Aim 2, we will identify how ATRA signaling in brown adipocytes influences obesity development. We have shown that adipocyte-synthesis of RBP4 leads to excessive fat accumulation in the liver. We now wish to study further the progression of RBP4-induced hepatic disease. Specifically, we wish to establish that adipose inflammation induced by RBP4, in the setting of further insults to the liver (second “hits”), sensitizes the liver to non-alcoholic steatohepatitis with accompanying fibrosis. This question will be the focus of Specific Aim 3. It has been suggested in the recent literature that RBP4 may carry ligands other than retinol and that this may account for the observed relationships between adipocyte RBP4 expression and the RBP4-induced metabolic phenotypes. We are proposing in Specific Aim 4 to undertake a systematic investigation to identify novel bioactive ligands that bind RBP4 with high affinity (equivalent to or better than retinol). Here, we will employ a new high throughput method that we recently developed and used to identify novel high affinity ligands for two other retinol-binding proteins, RBP1 and RBP2. We consider Specific Aim 4 to be one with high risks but potentially very great gains for understanding RBP4 actions in metabolic disease.