The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic ␣ subunit and noncatalytic ␤ and ␥ subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the ␣1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. Cterminal truncation of the 548-amino acid (aa) wild-type ␣1 protein to aa 312 or 392 abolishes the binding of the ␤/␥ subunits and dramatically increases protein expression. The full-length wild-type ␣1 subunit is only minimally active in the absence of co-expressed ␤/␥, and ␣1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. ␣-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated ␣ subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the ␣1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.

AMP-activated kinase (AMPK) is activated in response to metabolic stresses that deplete cellular ATP, and in both liver and skeletal muscle, activated AMPK stimulates fatty acid oxidation. To determine whether AMPK might reciprocally regulate glycerolipid synthesis, we studied liver and skeletal-muscle lipid metabolism in the presence of 5-amino-4-imidazolecarboxamide (AICA) riboside, a cell-permeable compound whose phosphorylated metabolite activates AMPK. Adding AICA riboside to cultured rat hepatocytes for 3 h decreased [14C]oleate and [3H]glycerol incorporation into triacylglycerol (TAG) by 50% and 38% respectively, and decreased oleate labelling of diacylglycerol by 60%. In isolated mouse soleus, a highly oxidative muscle, incubation with AICA riboside for 90 min decreased [14C]oleate incorporation into TAG by 37% and increased 14CO2 production by 48%. When insulin was present, [14C]oleate oxidation was 49% lower and [14C]oleate incorporation into TAG was 62% higher than under basal conditions. AICA riboside blocked insulin's antioxidative and lipogenic effects, increasing fatty acid oxidation by 78% and decreasing labelled TAG 43%. Similar results on fatty acid oxidation and acylglycerol synthesis were observed in C2C12 myoblasts, and in differentiated C2C12 myotubes, AICA riboside also inhibited the hydrolysis of intracellular TAG. These data suggest that AICA riboside might inhibit sn-glycerol-3-phosphate acyltransferase (GPAT), which catalyses the committed step in the pathway of glycerolipid biosynthesis. Incubating rat hepatocytes with AICA riboside for both 15 and 30 min decreased mitochondrial GPAT activity 22-34% without affecting microsomal GPAT, diacylglycerol acyltransferase or acyl-CoA synthetase activities. Finally, purified recombinant AMPKalpha1 and AMPKalpha2 inhibited hepatic mitochondrial GPAT in a time-and ATP-dependent manner. These data show that AMPK reciprocally regulates acyl-CoA channelling towards beta-oxidation and away from glycerolipid biosynthesis, and provide strong evidence that AMPK phosphorylates and inhibits mitochondrial GPAT.

AMP-activated kinase (AMPK) is activated in response to metabolic stresses that deplete cellular ATP, and in both liver and skeletal muscle, activated AMPK stimulates fatty acid oxidation. To determine whether AMPK might reciprocally regulate glycerolipid synthesis, we studied liver and skeletal-muscle lipid metabolism in the presence of 5-amino-4-imidazolecarboxamide (AICA) riboside, a cell-permeable compound whose phosphorylated metabolite activates AMPK. Adding AICA riboside to cultured rat hepatocytes for 3 h decreased [14C]oleate and [3H]glycerol incorporation into triacylglycerol (TAG) by 50% and 38% respectively, and decreased oleate labelling of diacylglycerol by 60%. In isolated mouse soleus, a highly oxidative muscle, incubation with AICA riboside for 90 min decreased [14C]oleate incorporation into TAG by 37% and increased 14CO2 production by 48%. When insulin was present, [14C]oleate oxidation was 49% lower and [14C]oleate incorporation into TAG was 62% higher than under basal conditions. AICA riboside blocked insulin's antioxidative and lipogenic effects, increasing fatty acid oxidation by 78% and decreasing labelled TAG 43%. Similar results on fatty acid oxidation and acylglycerol synthesis were observed in C2C12 myoblasts, and in differentiated C2C12 myotubes, AICA riboside also inhibited the hydrolysis of intracellular TAG. These data suggest that AICA riboside might inhibit sn-glycerol-3-phosphate acyltransferase (GPAT), which catalyses the committed step in the pathway of glycerolipid biosynthesis. Incubating rat hepatocytes with AICA riboside for both 15 and 30 min decreased mitochondrial GPAT activity 22-34% without affecting microsomal GPAT, diacylglycerol acyltransferase or acyl-CoA synthetase activities. Finally, purified recombinant AMPKalpha1 and AMPKalpha2 inhibited hepatic mitochondrial GPAT in a time-and ATP-dependent manner. These data show that AMPK reciprocally regulates acyl-CoA channelling towards beta-oxidation and away from glycerolipid biosynthesis, and provide strong evidence that AMPK phosphorylates and inhibits mitochondrial GPAT.

The complexity of initial resection and pancreatic fistula were independently associated with hospital readmissions after PD. Further efforts should be centered on preventing early readmissions, which constitute half of all readmissions.