AMP-activated protein kinase (AMPK) is a cellular energy sensor that conserves energy by inhibiting anabolic processes, such as de novo lipogenesis (DNL). Paradoxically, fructose metabolism activates AMPK by reducing the energy state (elevated AMP/ATP ratio), yet also promotes DNL. However, the comprehensive effects of fructose metabolism and AMPK activity on hepatic DNL has not been thoroughly investigated. Here, we examine how loss of AMPK function impacts DNL in response to fasting followed by refeeding a fructose or chow diet. After a 16 hour fast, 12 liver-specific AMPKα1α2 knock-out mice (AMPK-KO) and 12 AMPK α1α2 lox/lox mice (control) were fed either high fructose or chow diets (6 per a group) and administered D2O drinking water for 7 hours prior to blood collection and tissue harvest. After Folch extraction, fatty acids in liver triglyceride (TG-FA) were analyzed by proton and deuterium NMR spectroscopy to determine 2H incorporation and estimate DNL, elongation and re-esterification fluxes. Body water enrichment (~3.5%) was similar between groups. After correcting for food intake and body weight, preliminary data indicates that the AMP/ATP ratio was greater in AMPK-KO liver compared to controls, but neither chow nor fructose diets affected the energy state. Overall, neither hepatic steatosis nor DNL differed between groups. Furthermore, loss of AMPK function did not affect the amount of various FA species (i.e. saturated, unsaturated, DHA and ω3, linoleic, and non-essential). Instead, AMPK-KO liver had increased TG-FAs that were elongated compared to controls. This effect also tended to be greater in fructose fed mice (AMPK-KO and controls) compared to chow fed mice, even though fructose fed mice consumed fewer calories. In conclusion, loss of AMPK function resulted in increased elongation of hepatic TG-FAs, but not DNL, or the amount of FAs in the liver in response to fasting and refeeding with either fructose or chow diets. Disclosure J. A. Fletcher: None. S. Deja: None. S. C. Burgess: None. X. Fu: None. M. R. Inigo: None. J. Browning: None. B. Kucejova: None. Funding National Institutes of Health (K01AA030327)