Increased fructose consumption has been implicated in many diseases, including type 2 diabetes, nonalcoholic fatty liver disease and cardiovascular disease. Pharmaceutical companies are designing inhibitors against ketohexokinase (KHK), the primary enzyme for fructose metabolism. Most studies have used higher doses of fructose than humans consume. We do not fully understand the mechanisms by which moderate fructose consumption impact metabolism and how KHK inhibition would improve metabolism in these conditions. Preliminary data suggest that modest dietary fructose metabolism may primarily impair adipose, not hepatic, function. Using an antisense oligonucleotide (ASO) against KHK, we studied rats fed: a low-fat “regular” chow providing 15% calories from fructose and a high-fat diet providing 8% calories from fructose. KHK ASO improved adipose insulin action in both conditions. Our overarching hypothesis is modest amounts of dietary fructose increases hepatic VLDL production which can impair adipose insulin sensitivity leading to adipose dysfunction. Fructose induced adipose dysfunction can then indirectly impact hepatic glucose and lipid metabolism. We will leverage our expertise in in vivo metabolism and explorations of lipid mediated insulin resistance –that the accumulation of sn 1,2 diacyglycerol (DAG) in the plasma membrane recruits protein kinase C ε (PKCε) which then phosphorylates and impairs insulin receptor kinase (IRK) activation—to probe this novel aspect of fructose metabolism. Aim 1) To determine the mechanism of fructose mediated adipose insulin resistance. We will teAbsst the hypothesis that fructose mediated hepatic lipogenesis into VLDL triglyceride exceeds the capacity for WAT esterification, leading to the accumulation of adipose sn 1,2 diacylglycerol and WAT insulin resistance. Rats will be fed isocaloric low-fat diets with increasing sucrose content (accounting for 0, 5, 10 and 15% calories from fructose) to determine the threshold at which adipose sn 1,2 DAG accumulates and activates PKCε in relation to insulin signaling and insulin action in vivo. Using KHK ASO, we will establish that blocking fructose metabolism prevents adipose insulin resistance. We will further demonstrate that blocking hepatic KHK is sufficient to prevent adipose insulin resistance using a liver-specific GalNAc modified ASO. Aim 2) To determine the role of high-fat diets in potentiating fructose mediated adipose dysfunction. We will test the hypothesis that the addition of dietary fat reduces the “safe threshold” for dietary fructose. Dietary fats may constrain the ability of adipose tissue to handle fructose derived VLDL triglyceride. In addition, preliminary data suggests the combination of fat and fructose increase expression of 11β hydroxysteroid dehydrogenase 1, potentially another mechanism accounting for fructose induced adipose dysfunction and asses changes after a long term (24 week) exposure to high-fat, fructose diets. Aim 3) To assess the ...