SUMMARY Activation of AMP-activated protein kinase (AMPK) is thought to convey many of the beneficial effects of exercise via its inhibitory effect on acetyl-CoA carboxylase 2 (ACC2) and promotion of fatty acid oxidation. Hence, AMPK and ACC have become major drug targets for weight loss and improved insulin action. However, it remains unclear if or how activation of the fatty acid oxidation pathway without a concomitant increase in energy expenditure could be beneficial. In this study we have used either pharmacological (administration of the AMPK agonist 5′ aminoimidazole-4-carboxamide-riboside (AICAR)) or genetic means (mutation of the ACC2 gene in mice) to manipulate fatty acid oxidation to determine if this is sufficient to promote leanness. Both of these strategies increased whole body fatty acid oxidation without altering energy expenditure or adiposity. We conclude that negative energy balance is a pre-requisite for weight reduction and increased fatty acid oxidation per se has little, if any, effect to reduce adiposity.

Glucose infusion in rats for 1-4 days results in insulin resistance and increased triglyceride, whole tissue long-chain fatty acyl-CoA (LCA-CoA), and malonyl-CoA content in red skeletal muscle. Despite this, the relation between these alterations and the onset of insulin resistance has not been defined. We aimed to 1) identify whether the changes in these lipids and of diacylglycerol (DAG) precede or accompany the onset of insulin resistance in glucose-infused rats, 2) determine whether the insulin resistance is associated with alterations in AMP-activated protein kinase (AMPK), and 3) assess whether similar changes occur in liver and in muscle. Hyperglycemia (17-18 mM) was maintained by intravenous glucose infusion in rats for 3 or 5 h; then euglycemia was restored and a 2-h hyperinsulinemic clamp was performed. Significant (P < 0.01) muscle and liver insulin resistance first appeared in red quadriceps and liver of the glucose-infused group at 5 h and was associated with a twofold increase in DAG and malonyl-CoA content and a 50% decrease in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and AMPK activity. White quadriceps showed qualitatively similar changes but without decreases in AMPK or ACC phosphorylation. Triglyceride mass was increased at 5 h only in liver, and whole tissue LCA-CoA content was not increased in liver or either muscle type. We conclude that the onset of insulin resistance induced by glucose oversupply correlates temporally with increases in malonyl-CoA and DAG content in all three tissues and with reduced AMPK phosphorylation and activity in red muscle and liver. In contrast, it was not associated with increased whole tissue LCA-CoA content in any tissue or triglyceride in muscle, although both are observed at later times.

(TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity Ͼ10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 Ϯ 3 vs. 67 Ϯ 2 nmol/mg protein in controls, P Ͻ 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 Ϯ 6 vs. 100 Ϯ 6 nmol/g protein in controls, P Ͻ 0.05) and increased [ 14 C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 Ϯ 4 vs. 20 Ϯ 2 mol/g wet wt in controls, P Ͻ 0.05), and overexpression in rat liver increased [1-14 C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.acyl-coenzyme A synthetase; hepatic triglyceride synthesis; adenovirus; fatty acid metabolism OBESITY AND ITS ASSOCIATED COMPLICATIONS of insulin resistance, cardiovascular disease, hypertension, hepatic steatosis, and type 2 diabetes have become a major health problem worldwide (50). All of these metabolic disorders exhibit altered regulation of lipid metabolism, which results in the intracellular accumulation of lipid in nonadipose tissues, but the intracellular mechanisms that control partitioning of fatty acids into different metabolic pathways are still being elucidated.Fatty acids can be metabolized in the pathways of ␤-oxidation, triglyceride synthesis, phospholipid synthesis, cholesterol ester synthesis, fatty acid elongation, and protein acylation and can also serve as signaling molecules (7,10,40). It is believed that the partitioning of intracellular fatty acids between storage pathways and ␤-oxidation is controlled by regulation of the mitochondrial acyl-CoA transporter carnitine palmitoyltransferase-1 (32), by regulation of metabolic gene expression (39), and by hormones including insulin. A number of recent studies suggest that partitioning might also involve the different isoforms of acyl-CoA synthetase (ACSL) (7,20,49).ACSL catalyzes the ATP-dependent acylation of fatty acids into long-chain acyl CoAs (LCA-CoAs) and is the first step in lipid metabolism after fatty acid entry into the cell. The LCA-CoAs can then enter the ␤-oxidation pathway for energy production or undergo further esterification for production of phospholipids, cholesterol esters, and triglycerides. Th...

Aims/hypothesis We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. Methods Prkce −/− and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes.Results While fat-fed WT mice became glucose intolerant after 1 week, Prkce −/− mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce −/− animals. Liver triacylglycerols were increased in fat-fed Prkce −/− mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce −/− mice. Prkce −/− hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce −/− mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. Conclusions/interpretation Prkce deletion ameliorates dietinduced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails.

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. This study investigated the impact of restricting feeding to the daylight hours and feeding a high fat diet on circadian clock (bmal1, dbp, tef and e4bp4) and metabolic (pepck, fas, ucp3, pdk4) gene expression and markers of energy metabolism in muscle and liver of rats. The results show that in chow-fed rats switched to daylight feeding, the peak diurnal expression of genes in liver was shifted by 6-12h while expression of these genes in muscle remained in a similar phase to rats feeding ad libitum. High fat feeding during the daylight hours had limited effect on clock gene expression in liver or muscle but shifted the peak expression of metabolic genes (pepck, fas) in liver by 6-12h. The differential effects of daylight feeding on gene and protein expression in muscle and liver were accompanied by an 8% reduction in whole body energy expenditure, a 20-30% increased glycogen content during the light phase in muscle of day-fed rats and increased adipose tissue deposition per gram food consumed. These data demonstrate that a mismatch of feeding and light/dark cycle disrupts tissue metabolism in muscle with significant consequences for whole body energy homeostasis.