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and Ser 635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process. Endothelial nitric-oxide synthase (eNOS)1 is an important enzyme in the cardiovascular system producing nitric oxide (NO), a key regulator of blood pressure, platelet function, and vessel remodeling. Endothelial NOS is regulated by multiple mechanisms involving both protein-protein interactions with several different proteins, including caveolin-1 and Hsp90 (1), and post-translational modifications that include Nmyristoylation, cysteine palmitoylation, and multisite phosphorylation. The two most thoroughly studied phosphorylation sites have been the activation site, human Ser 1177

The AMP-activated protein kinase (AMPK) is an ␣␤␥ heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK ␤1 ؊/؊ mice on a C57Bl/6 background that are viable, fertile, survived greater than 2 years, and display no visible brain developmental defects. These mice have a 90% reduction in hepatic AMPK activity due to loss of the catalytic ␣ subunits, with modest reductions of activity detected in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, ␤1 ؊/؊ mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of ␤1 ؊/؊ mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of ␤1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet.The AMP-activated protein kinase (AMPK) 6 is an evolutionarily conserved serine/threonine protein kinase that functions as a metabolic regulatory enzyme at both the cellular and whole body level (1). AMPK is activated in response to physiological processes that raise intracellular levels of AMP, such as exercise and hypoxia. It restores cellular energy balance by switching off ATP-consuming anabolic pathways and switching on ATPgenerating catabolic pathways by direct phosphorylation of downstream targets. Modulation of AMPK activity by hormones adds an additional layer of control, allowing cellular energy supply and demand to be balanced with the energy requirements of the whole organism (2).AMPK functions as an ␣␤␥ heterotrimer. Different isoforms for each of the subunits exist (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3) as well as some splice variants, allowing more than 12 heterotrimeric combinations to be generated that may mediate unique tissue-specific functions (3, 4). The 63-kDa AMPK ␣ subunits, designated ␣1 and ␣2, contain a serine/threonine protein kinase catalytic domain that is activated by phosphorylation of Thr-172 in the activation loop (5, 6). We, and others have shown that the C terminus of the ␤ subunits are essential for AMPK heterotrimer assembly by anchoring the ␣ and ␥ subunits (7,8). The ␤1 and ␤2 subunits show 82% identity from residue 73 to 270, but only 43% identity for the N-terminal residues 1-72 (9). The ␤1 subunit is N-terminally myristoylated and is phosphorylated on multiple serines (10); however, the physiological importance of these phosphorylation sites is poorly understood. Northern blot analysis of human tissues revealed that AMPK ␤1 expression is highest in the liver and brain and low in kidney and skeletal muscle, whereas ␤2 is most highly expressed in skeletal muscle with lower expression in kidney, liver, and lung (11).Hepatic AMPK is thought to play important roles in regulating lipid metabolism, glucose homeostasis, and insulin sensitivity (1)....

In combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology to cells that are rapidly turned over, but brain cells might accumulate long-term damage leading ultimately to neurodegeneration and the loss of mitochondrial function observed post-mortem. Whether the ‘hyperactive’ state in lymphoblasts is a biomarker specifically of PD or more generally of neurodegenerative disease remains to be determined.

Both pharmacological intervention (i.e., thiazolidinediones [TZDs]) and lifestyle modification (i.e., exercise training) are clinically effective treatments for improving whole-body insulin sensitivity. However, the mechanism(s) by which these therapies reverse lipid-induced insulin resistance in skeletal muscle is unclear. We determined the effects of 4 weeks of rosiglitazone treatment and exercise training and their combined actions (rosiglitazone treatment and exercise training) on lipid and glucose metabolism in high-fat-fed rats. High-fat feeding resulted in decreased muscle insulin sensitivity, which was associated with increased rates of palmitate uptake and the accumulation of the fatty acid metabolites ceramide and diacylglycerol. Impairments in lipid metabolism were accompanied by defects in the Akt/AS160 signaling pathway. Exercise training, but not rosiglitazone treatment, reversed these impairments, resulting in improved insulinstimulated glucose transport and increased rates of fatty acid oxidation in skeletal muscle. The improvements to glucose and lipid metabolism observed with exercise training were associated with increased AMP-activated protein kinase ␣1 activity; increased expression of Akt1, peroxisome proliferator-activated receptor ␥ coactivator 1, and GLUT4; and a decrease in AS160 expression. In contrast, rosiglitazone treatment exacerbated lipid accumulation and decreased insulin-stimulated glucose transport in skeletal muscle. However, rosiglitazone, but not exercise training, increased adipose tissue GLUT4 and acetyl CoA carboxylase expression. Both exercise training and rosiglitazone decreased liver triacylglycerol content. Although both interventions can improve whole-body insulin sensitivity, our results show that they produce divergent effects on protein expression and triglyceride storage in different tissues. Accordingly, exercise training and rosiglitazone may act as complementary therapies for the treatment of insulin resistance.

The fragile X premutation (PM) allele contains a CGG expansion of 55–200 repeats in the FMR1 gene’s promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39–81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers – CGG repeat number and the levels of the FMR1 mRNA – were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85–90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings – suggestive of a role of this enzyme in the risk of FXTAS – need to be verified and expanded in future studies using larger samples and longitudinal assessment.

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