During the basal equilibrium period (0 -150 min), fasting dogs (n ϭ 8) were infused with [3-3 H]glucose followed by either 2-h saline or AICAR (1.5-2.0 mg ⅐ kg Ϫ1 ⅐ min Ϫ1 ) infusions. SkM was biopsied at completion of each study. On a separate day, the same protocol was undertaken after 48-h in vivo FA OX blockade. The AICAR and AICAR ϩ MP studies were repeated in three chronic alloxan-diabetic dogs. AICAR produced a transient fall in plasma glucose and increase in insulin and a small decline in free fatty acid (FFA). Parallel increases in hepatic glucose production (HGP), glucose disappearance (R d tissue), and glycolytic flux (GF) occurred, whereas metabolic clearance rate of glucose (MCR g) did not change significantly. Intracellular SkM glucose, glucose 6-phosphate, and glycogen were unchanged. Acetyl-CoA carboxylase (ACCϳpSer 221 ) increased by 50%. In the AICAR ϩ MP studies, the metabolic responses were modified: the glucose was lower over 120 min, only minor changes occurred with insulin and FFA, and HGP and R d tissue responses were markedly attenuated, but MCR g and GF increased significantly. SkM substrates were unchanged, but ACCϳpSer 221 rose by 80%. Thus low-dose AICAR leads to increases in HGP and SkM glucose uptake, which are modified by prior FA ox blockade.5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside; 5-aminoimidazole-4-carboxamide-1-␤-D-ribosyl monophosphate; acetyl-coenzyme A carboxylase; phosphorylation; skeletal muscle; muscle biopsy; dogs AMPK ACTIVATION PLAYS A CENTRAL ROLE in the regulation of intracellular metabolism as the master switch regulating cellular ATP energy supplies by shutting down high energy-consuming pathways, such as fatty acid synthesis and cholesterol synthesis, and by activating ATP-generating pathways such as fatty acid oxidation (42, 58). Thus, through these actions, AMPK influences fuel usage and selection in and to various tissues (42). Endogenous AMPK can be activated pharmacologically by 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR), which is phosphorylated in vivo to the AMP analog AICAR monophosphate (ZMP) (18). However, many of those studies employed high doses of AICAR, resulting in high tissue levels of ZMP (5,6,25,26), which might have allosterically activated glycogen phosphorylase (7), certainly in cardiac muscle (32) and in the liver (45). Nevertheless, when skeletal muscle (SkM) AMPK is activated by upstream kinases and/or by AMP allosteric effects (60), the downstream substrate acetyl-CoA carboxylase (ACC) is phosphorylated in SkM (37, 58), at Ser 221 (ACCϳpSer 221 ) (11, 52) and thereby inactivated (37, 58). Therefore, the formation of ACCϳpSer 221 represents a key sensitive marker of biological activation of AMPK in vivo (3,58). Note that the corresponding SkM site (Ser 221 ) recognized by the ACC phosphospecific rat antibody used in the assay is raised against ACC␣ϳSer 79 (11, 36, 52). The inactivation of ACC leads to reduced malonyl-CoA levels and the activation of mitochondrial carnitine palmitoyltransferase I (CPT I), which ...