Background: Roux-en-Y gastric bypass (RYGB) reduces liver weight, hepatic triglycerides and oxidative stress in obese rats. AMP-activated kinase (AMPK) has been targeted in the treatment of the metabolic syndrome because of its role in reducing fatty acid synthesis and oxidative stress as well as increasing beta oxidation. LKB1 (a serine/threonine kinase also called STK11) can activate AMPK by phosphorylation. PKC-ζ is known to phosphorylate LKB1and plays a role in cellular stress and cytokine production. We, therefore, hypothesize that the improvement in hepatic steatosis after RYGB in obese rats is associated with an upregulation of the LKB1-AMPK signaling pathway.Methods: Obese Sprague-Dawley male rats underwent RYGB or sham. Liver tissue was obtained at 9 weeks postoperatively. Protein levels of LKB1, p-LKB1, AMPKα, p-AMPKα and p-PKC-ζ were measured. PKC-ζ mRNA levels were also measured. Protein associations of LKB1 with each of AMPKα and PKC-ζ were determined by both co-immunoprecipitation and co-immunofluorescent staining. Data are mean±SD; for t-test, p<0.05 was significant.Results: RYGB increased protein levels of both hepatic AMPKα and phosphorylated-AMPKα (p-AMPKα ) as compared to sham (5,431±150 vs 2,323±117; 3,665±120 vs 1,534±60; all p<0.001). While protein levels of hepatic LKB1 did not increase, phosphorylated-LKB1 increased significantly after RYGB (6,574±125 vs 3,265±89; p<0.001 vs. sham).PKC-ζ mRNA and phosphorylated-PKC-ζ did not change after RYGB (data not shown). However, interactions between LKB1 and AMPK were increased after RYGB (6,325±142 vs 2,132±87; p<0.001 vs. sham) as well as interactions of LKB1 and PKC-ζ (4,356±102 vs 2,354±78; p<0.001 vs. sham). Both LKB1-AMPK and LKB1-PKC-ζ co-localized most strongly in the cytoplasm of liver cells by co-immunofluoresence; LKB1-AMPK also co-localized in the nucleus to a lesser extent.Conclusion: RYGB increased hepatic levels of AMPK and p-AMPK. Increased phosphorylation of LKB1 after RYGB is associated with increased LKB1-AMPK interaction and co-localization within liver cells. While PKC-ζ levels were not increased after RYGB, PKC-ζ interaction and co-localization with LKB1 was increased. Further elucidation upstream signaling of the LKB1-AMPK pathway may provide greater clarity into the benefits of RYGB on Non-Alcoholic Fatty Liver Disease.