High sugar consumption, and more specifically fructose intake, leads to lipid profiles associated with increased risk of type 2 diabetes (T2D), cardiovascular disease (CVD), and non-alcoholic fatty liver disease. The underlying metabolic mechanisms leading to these conditions remain largely unknown. The parent study for this proposal, R01DK116033, focuses on the role and metabolic fates of fructose in the liver, which historically was considered the main tissue handling fructose. This proposal takes into account our preliminary data supporting a major role of non-hepatic tissues as sites of fructose metabolism, and of apolipoprotein clearance factors influencing postprandial lipid profiles. We address three questions related to non-hepatic fructose metabolism using high- and low-fructose meals in pre-diabetic and control subjects enrolled in the parent trial. Our first question regards the role of the small intestine in fructose metabolism; specifically the conversion of fructose to fat by de novo lipogenesis (DNL) and the composition of apolipoproteins in chylomicrons (CM), namely apoCIII, and apoB, and their relative contribution to postprandial hyperlipidemia. We developed an immunoaffinity method to isolate CM that transport the fat packaged in the small intestine. This will allow us to quantify, for the first time, intestinal DNL and the apolipoprotein composition of CM without contamination from fat made in the liver and transported by very-low density lipoproteins (VLDL). Our second question addresses the amount of fructose that escapes intestinal and hepatic metabolism and appears in the circulation, thereby becoming available to other tissues. For this aim, we will use a recently published dual stable isotope method to measure plasma fructose levels. Additionally, this method will allow us to explore, for the first time, intestinal gluconeogenesis in humans. For our third question, we will measure how much fructose is used as fuel by determining the amount of fructose that is completely oxidized to produce labeled 13CO2. Recent studies have suggested that oxidation is another major route of fructose metabolism. The administration of oral and intravenous stable isotopes in 18 pre-diabetic and 18 control subjects enrolled in the parent trial and the application of a technique to fractionate TRL allows for the quantification of CM and VLDL apolipoproteins and intestinal- and hepatic- DNL, along with measurements of fructose in the peripheral circulation, estimation of intestinal gluconeogenesis, and fructose oxidation. By measuring DNL, clearance factors, circulating fructose, and fructose oxidation in conjunction with the measurements of the parent study (hepatic gluconeogenesis, TRL-DNL and glycogen storage), we will have a more complete characterization of fructose metabolism and answer longstanding questions regarding the fates of fructose in two distinct populations. Importantly, we will demonstrate how extra-hepatic fructose metabolism contributes to T2D and CVD risk.