IGF-1 phosphorylates AMPK-alpha subunit in ATM-dependent and LKB1-independent manner

Serine/threonine protein kinase AMP-activated protein kinase (AMPK) is a key metabolic stress-responsive factor that promotes the adaptation of cells to their microenvironment. Elevated concentrations of intracellular AMP, caused by metabolic stress, are known to activate AMPK by phosphorylation of the catalytic subunit. Recently, the tumor suppressor serine/threonine protein kinase LKB1 was identified as an upstream kinases, AMPKKs. In the current study, we found that stimulation with growth factors also caused AMPK-alpha subunit phosphorylation. Interestingly, even an LKB1-nonexpressing cancer cell line, HeLa, exhibited growth factor-stimulated AMPK-alpha subunit phosphorylation, suggesting the presence of an LKB1-independent pathway for AMPK-alpha subunit phosphorylation. In the human pancreatic cancer cell line PANC-1, AMPK-alpha subunit phosphorylation promoted by IGF-I was suppressed by antisense ataxia telangiectasia mutated (ATM) expression. We found that IGF-1 also induced AMPK-alpha subunit phosphorylation in the human normal fibroblast TIG103 cell line, but failed to do so in a human fibroblast AT2-KY cell line lacking ATM. Immunoprecipitates of ATM collected from IGF-1-stimulated cells also caused the phosphorylation of the AMPK-alpha subunit in vitro. IGF-1-stimulated ATM phosphorylation at both threonine and tyrosine residues, and our results demonstrated that the phosphorylation of tyrosine in the ATM molecule is important for AMPK-alpha subunit phosphorylation during IGF-1 signaling. These results suggest that IGF-1 induces AMPK-alpha subunit phosphorylation via an ATM-dependent and LKB1-independent pathway. (C) 2004 Elsevier Inc. All rights reserved.

An adaptogenic role for omega-3 fatty acids in stress; a randomised placebo controlled double blind intervention study (pilot) [ISRCTN22569553]

Background There is evidence for an adaptive role of the omega -3 fatty acid, docosahexaenoic acid (DHA) during stress. Mechanisms of action may involve regulation of stress mediators, such as the catecholamines and proinflammatory cytokines. Prevention of stress-induced aggression and hostility were demonstrated in a series of clinical trials. This study investigates whether perceived stress is ameliorated by DHA in stressed university staff. Methods Subjects that scored ≥ 17 on the Perceived Stress Scale were randomised into a 6-week pilot intervention study. The diet reactive group was supplemented with 6 g of fish oil containing 1.5 g per day DHA, while the placebo group was supplemented with 6 g a day of olive oil. The groups were compared with each other and a wider cross sectional study population that did not receive either active or placebo intervention. Results There was a significant reduction in perceived stress in both the fish oil and the placebo group from baseline. There was also a significant between-group difference between the fish oil group and the no-treatment controls in the rate of stress reduction (p < 0.05). However, there was not a significant between-group difference between the fish oil and the placebo group, nor the placebo group and the control group. These results are discussed in the context of several methodological limitations. The significant stress reductions in both the fish oil and the placebo group are considered in view of statistical regression, an effect likely to have been exaggerated by the time course of the study, a large placebo effect and the possibility of an active effect from the placebo. Conclusion There were significant differences (p < 0.05) in the fish oil group compared with no-treatment controls. This effect was not demonstrated in the placebo group. As a pilot study, it was not sufficiently powered to find the difference between the fish oil group and the placebo group significant. Further work needs to be undertaken to conclusively demonstrate these data trends. However, the findings from this research support the literature in finding a protective or 'adaptogenic' role for omega-3 fatty acids in stress.

Cyclopropyl containing fatty acids as mechanistic probes for cytochromes P450

The mechanism of aliphatic hydroxylation by cytochromes P450 has been the subject of intense debate with several proposed mechanistic alternatives. Various cyclopropyl containing compounds (radical clocks), which can produce both unrearranged and ring opened products upon oxidation, have been key tools in these investigations. In this study, we introduce several cyclopropyl containing fatty acids 1a-4a with which to probe the mechanism of P450s capable of fatty acid hydroxylation. The probes are shown to be capable of distinguishing radical from cationic intermediates due to the rapid equilibration of isomeric cyclopropyl cations. Ring opening of a radical intermediate in an oxidative transformation is expected to yield a single rearranged alcohol, whereas a cation isomerizes prior to ring opening, leading to two isomeric homoallylic alcohols. Oxidation of these probes by P450(BM3) and P450(Biol) gives results consistent with a radical but not a cationic intermediate in fatty acid hydroxylation by these enzymes. Quantitation of the unrearranged and ring opened products gives remarkably homogeneous rates for oxygen rebound of (2-3) x 10(10) s(-1). The effects of introduction of a cyclopropane ring into a fatty acid upon the regiochemistry of hydroxylation are discussed.

Nur77 regulates lipolysis in skeletal muscle cells - Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for similar to 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)- mediated adaptive thermogenesis. beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30 - 60 min of beta-AR agonist ( isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (> 100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma 3, UCP3, CD36,adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.