Polyunsaturated fatty acids (PUFA) and a number of drugs (metformin, thiazolidinediones) and hormones (leptin, adiponectin) that activate AMP-activated protein kinase (AMPK) have been reported to improve insulin sensitivity. To determine whether PUFA activate AMPK, Sprague-Dawley rats were adapted to a 3 h meal-feeding regimen using a fat-free diet (FFD) supplemented with fish oil (n - 3) or triolein (n -9) for 7 days. No differences in hepatic AMPK activity were observed between the groups after 21 h of fasting. On the other hand, hepatic AMPK phosphorylation was decreased in rats refed the FFD, the FFD + triolein, and the FFD + PUFA by 80%, 75%, and 50%, respectively, when assessed 2 h after completion of a meal. In keeping with these changes, decreases in acetyl-CoA carboxylase phosphorylation and carnitine palmitoyl transferase-1 mRNA and increases in fatty acid synthase gene expression were greatest in rats fed the FFD and least in the PUFA-fed rats. The results indicate that dietary PUFA enhance hepatic AMPK activity in vivo, and implicate AMPK as a component of the nutrient-sensing mechanism through which dietary fatty acids and especially PUFA influence the regulation of hepatic lipid metabolism dand gene expression. (C) 2004 Elsevier Inc. All rights reserved.