Acyl CoA:monoacylglycerol acyltransferase (MGAT) enzymes catalyze the formation of diacylglycerols (DG) from monoacylglycerol (MG) and fatty acyl CoA precursors and are key enzymes in triacylglycerol (TG) biosynthesis. MGAT-catalyzed reactions are thought to be important in many essential biological processes in metazoans, including the absorption of dietary fat, the preservation of unsaturated (and essential) fatty acids in tissues, and the assembly and secretion of TG-rich lipoproteins. Although MGAT activity in tissues was reported decades ago, genetic tools for investigating MGAT became available only recently. Two murine MGAT genes have been identified. Mgatl is expressed mainly in the stomach and kidney of mice, and preliminary studies have localized this expression to epithelial cells of these tissues. We hypothesize that MGAT1 plays a role in the uptake and absorption of specific lipids in these organs. Mgat2 is expressed highly in the small intestine and at lower levels in the liver and white adipose tissue (WAT). We hypothesize that MGAT2, together with acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which catalyzes the synthesis of TG from DG and fatty acyl CoA, is crucial for the absorption of dietary TG and for chylomicron assembly and secretion in enterocytes. We also hypothesize that MGAT2 contributes to reassembly of TG for hepatic storage or secretion and to the preservation of polyunsaturated fatty acids in the liver and WAT. The goal of this proposal is to elucidate the in vivo functions of MGAT enzymes. In Aim 1, we will determine the physiological functions of MGAT1 by studying its expression pattern and regulation and by studying the phenotype of mice lacking MGAT1. In Aim 2, we will assess the physiological functions of MGAT2 by studying the phenotype of mice that lack MGAT2, in particular defining its role in intestinal lipid absorption, and by studying mice that lack MGAT2 and DGAT1. In Aim 3, we will test the hypothesis that MGAT enzymes serve to conserve polyunsaturated fatty acids by analyzing mice lacking MGATi orMGAT2, and we will determine ifoverexpressing MGAT2 in human hepatoma cells alters TG storage or VLDL assembly and secretion. Completion of these aims should greatly enhance our understanding of the physiological roles of MGAT enzymes in triacylglycerol metabolism. The proposed studies are also important for determining whether MGAT enzymes, particularly MGAT2, are suitable targets for the treatment of obesity or type 2 diabetes. Drugs that block intestinal MGAT2 offer the possibility of blocking fat absorption within the enterocytes in a manner that does not result in significant side effects. Additionally, if MGAT2 deficiency protects against diet-induced obesity, it may also protect against diet-induced insulin resistance and type 2 diabetes.