Exercise and myocyte enhancer factor 2 regulation in human skeletal muscle

Overexpression of GLUT4 in skeletal muscle enhances whole-body insulin action. Exercise increases GLUT4 gene and protein expression, and a binding site for the myocyte enhancer factor 2 (MEF-2) is required on the GLUT4 promoter for this response. However, the molecular mechanisms involved remain elusive. In various cell systems, MEF-2 regulation is a balance between transcriptional repression by histone deacetylases (HDACs) and transcriptional activation by the nuclear factor of activated T-cells (NFAT), peroxisome proliferator-activated receptor- coactivator 1 (PGC-1), and the p38 mitogen-activated protein kinase. The purpose of this study was to determine if these same mechanisms regulate MEF-2 in contracting human skeletal muscle. Seven subjects performed 60 min of cycling at 70% of Vo2peak. After exercise, HDAC5 was dissociated from MEF-2 and exported from the nucleus, whereas nuclear PGC-1 was associated with MEF-2. Exercise increased total and nuclear p38 phosphorylation and association with MEF-2, without changes in total or nuclear p38 protein abundance. This result was associated with p38 sequence-specific phosphorylation of MEF-2 and an increase in GLUT4 mRNA. Finally, we found no role for NFAT in MEF-2 regulation. From these data, it appears that HDAC5, PGC-1, and p38 regulate MEF-2 and could be potential targets for modulating GLUT4 expression.

The effect of water deprivation on the tonicity responsive enhancer binding protein (TonEBP) and TonEBP-regulated genes in the kidney of the spinifex hopping mouse, Notomys alexis

In desert rodents, the production of concentrated urine is essential for survival in xeric environments in order to conserve water. Reabsorption of water in the kidney is dependent on large osmotic gradients in the renal medulla. This causes the renal cells to be bathed in a hypertonic extracellular fluid that can compromise cellular function. In response to hypertonicity, kidney cells accumulate compatible, non-ionic osmolytes that lower the ionic strength within the cells to isotonic levels by replacing intracellular ionic electrolytes. The tonicity-responsive enhancer binding protein (TonEBP) is a transcription factor that regulates the expression of genes that encode proteins that catalyse the accumulation of compatible osmolytes. We investigated the expression of TonEBP mRNA and protein and compatible osmolyte genes in the Spinifex hopping mouse, Notomys alexis, an Australian desert rodent that produces a highly concentrated urine. TonEBP mRNA expression was unchanged after 3 days of water deprivation but was significantly increased after 7 and 14 days of water deprivation. Immunohistochemistry showed that during water deprivation TonEBP had translocated from the cytoplasm into the nucleus of cells in the renal medulla and papilla. In addition, 3, 7 and 14 days of water deprivation caused a significant increase in aldose reductase (AR), myo-inositol (SMIT), betaine/GABA (BGT-1) and taurine (TauT) transporter mRNA expression, which is indicative of an increase in TonEBP activity. In desert rodents, TonEBP regulation of gene transcription is probably an important mechanism to protect renal cells in the face of the large corticomedullary gradient that is required to concentrate urine and conserve water.

Two novel missense mutations in hairy-and-enhancer-of-split-7 in a family with spondylocostal dysostosis

Spondylocostal dysostosis (SCD) is an inherited disorder with abnormal vertebral segmentation that results in extensive hemivertebrae, truncal shortening and abnormally aligned ribs. It arises during embryonic development by a disruption of formation of somites (the precursor tissue of the vertebrae, ribs and associated tendons and muscles). Four genes causing a subset of autosomal recessive forms of this disease have been identified: DLL3 (SCDO1: MIM 277300), MESP2 (SCDO2: MIM 608681), LFNG (SCDO3: MIM609813) and HES7 (SCDO4). These genes are all essential components of the Notch signalling pathway, which has multiple roles in development and disease. Previously, only a single SCD-causative missense mutation was described in HES7. In this study, we have identified two new missense mutations in the HES7 gene in a single family, with only individuals carrying both mutant alleles being affected by SCD. In vitro functional analysis revealed that one of the mutant HES7 proteins was unable to repress gene expression by DNA binding or protein heterodimerization.

Decreased expression of adipogenic genes in obese subjects with type 2 diabetes

Objective: Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.

Research Methods and Procedures: We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [ CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPdelta, GATA domain-binding protein 3 (GATA3), C/EBPbeta, peroxisome proliferator-activated receptor (PPAR) gamma2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor alpha, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARitalic gamma coactivator 1alpha, PPARitalic gamma coactivator 1beta, and PPARdelta] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.

Results: Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 plusminus 1.9 vs. 7.5 plusminus 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 plusminus 0.26 vs. 0.78 plusminus 0.17 mum; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARitalic gamma2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPbeta only tended to be lower (p = 0.07).

Discussion: This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.

Soluble manganese(IV) as a chemiluminescence reagent for the determination of opiate alkaloids, indoles and analytes of forensic Interest

We present the results of our investigations into the use of soluble manganese(IV) as a chemiluminescence reagent, which include a significantly faster method of preparation and a study on the effect of formaldehyde and orthophosphoric acid concentration on signal intensity. Chemiluminescence detection was applied to the determination of 16 analytes, including opiate alkaloids, indoles and analytes of forensic interest, using flow injection analysis methodology. The soluble manganese(IV) reagent was less selective than either acidic potassium permanganate or tris(2,2′-bipyridyl)ruthenium(III) and therefore provided a more universal chemiluminescence detection system for HPLC. A broad spectral distribution with a maximum at 730 ± 5 nm was observed for the reaction between the soluble manganese(IV) and a range of analytes, as well as the background emission from the reaction with the formaldehyde enhancer. This spectral distribution matches that reported for chemiluminescence reactions with acidic potassium permanganate, where a manganese(II) emitting species was elucidated. This provides further evidence that the emission evoked in reactions with soluble manganese(IV) also emanates from a manganese(II) species, and not bimolecular singlet oxygen as suggested by previous authors.

Stimulation of calcineurin Aα activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin A transgene (CnA) was overexpressed in skeletal muscles of mdx (mdx CnA*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnA* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnA* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnA* than mdx mice. In the diaphragm, despite a slower phenotype and a 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnA* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.

Calcineurin-Aα alpha activation enhances the structure and function of regenerating muscles after myotoxic injury

Calcineurin signaling is essential for successful muscle regeneration. Although calcineurin inhibition compromises muscle repair, it is not known whether calcineurin activation can enhance muscle repair after injury. Tibialis anterior (TA) muscles from adult wild-type (WT) and transgenic mice overexpressing the constitutively active calcineurin-Aα transgene under the control of the mitochondrial creatine kinase promoter (MCK-CnAα*) were injected with the myotoxic snake venom Notexin to destroy all muscle fibers. The TA muscle of the contralateral limb served as the uninjured control. Muscle structure was assessed at 5 and 9 days postinjury, and muscle function was tested in situ at 9 days postinjury. Calcineurin stimulation enhanced muscle regeneration and altered levels of myoregulatory factors (MRFs). Recovery of myofiber size and force-producing capacity was hastened in injured muscles of MCK-CnAα* mice compared with control. Myogenin levels were greater 5 days postinjury and myocyte enhancer factor 2a (MEF2a) expression was greater 9 days postinjury in muscles of MCK-CnAα* mice compared with WT mice. Higher MEF2a expression in regenerating muscles of MCK-CnAα* mice 9 days postinjury may be related to an increase of slow fiber genes. Calcineurin activation in uninjured and injured TA muscles slowed muscle contractile properties, reduced fatigability, and enhanced force recovery after 4 min of intermittent maximal stimulation. Therefore, calcineurin activation can confer structural and functional benefits to regenerating skeletal muscles, which may be mediated in part by differential expression of MRFs.

Heterotrimeric G proteins facilitate arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gbg dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Ga or Gb subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gb-deficient mutants while Ga-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gb-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gb-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Ga-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by Ga. We hypothesize that Gbg acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Exercise increases MEF2- and GEF DNA-binding activity in human skeletal muscle

Diabetes is quickly reaching epidemic proportions, with 216 million people worldwide predicted to be diagnosed with the disease by 2010. While it appears that the expression of the insulin responsive glucose transporter isoform 4 (GLUT4) is not reduced in diabetic populations, overexpression of GLUT4 exclusively in muscle enhances insulin action and improves glucose homeostasis. Consequently, understanding the regulation of GLUT4 expression is considered important in identifying potential therapeutic targets for the treatment and management of insulin resistance and related disorders such as type 2 diabetes. Using transgenic mice, we have identified two conserved regions on the GLUT4 gene promoter that are required for normal skeletal muscle GLUT4 expression. The first region contains a binding site for the myocyte enhancer factor 2 (MEF2) transcription factor, between –464 and –473 bp, and it appears that a MEF2A/D heterodimer binds this sequence. However, this site is not sufficient to support full GLUT4 expression, and another region between –712 and –742 bp, termed Domain 1, is also required. A novel transcription factor, named the GLUT4 enhancer factor (GEF), was found to bind to this region. It appears that MEF2 and GEF physically interact in order to induce GLUT4 expression. A single bout of exercise is sufficient to increase both GLUT4 transcription and mRNA abundance. However, the molecular mechanisms underpinning this response remain largely unexplored, particularly in human skeletal muscle. Therefore, the aim of this study was to determine whether a single, acute bout of exercise increases the DNA-binding activity of both MEF2 and GEF in human skeletal muscle.

Exercise and skeletal muscle glucose transporter 4 expression: molecular mechanisms

1.      Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT-4) expression in human skeletal muscle.

2.      Glucose transporter isoform 4 gene expression is increased immediately following a single bout of exercise, and the GLUT-4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. Glucose transporter isoform enhancer factor and MEF2 DNA binding activities are increased following exercise, and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined.

3.      These studies find possible roles for histone deacetylase 5 (HDAC5), adenosine monophosphate–activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and p38 mitogen-activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation.

4.      Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterized by mitochondrial dysfunction and insulin resistance in skeletal muscle.

Exercise and MEF2–HDAC interactions.

Exercise increases the metabolic capacity of skeletal muscle, which improves whole-body energy homeostasis and contributes to the positive health benefits of exercise. This is, in part, mediated by increases in the expression of a number of metabolic enzymes, regulated largely at the level of transcription. At a molecular level, many of these genes are regulated by the class II histone deacetylase (HDAC) family of transcriptional repressors, in particular HDAC5, through their interaction with myocyte enhancer factor 2 transcription factors. HDAC5 kinases, including 5′-AMP-activated protein kinase and protein kinase D, appear to regulate skeletal muscle metabolic gene transcription by inactivating HDAC5 and inducing HDAC5 nuclear export. These mechanisms appear to participate in exercise-induced gene expression and could be important for skeletal muscle adaptations to exercise.

Effect of sex differences on human MEF2 regulation during endurance exercise

Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% VO2peak in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-{alpha} ({alpha}AMPK) Thr172 (P < 0.001), histone deacetylase 5 (HDAC5) Ser498 (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors. mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC1{alpha}) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGC1{alpha} on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex.

Emitting species in chemiluminescence reactions with acidic potassium permanganate : a re-evaluation based on new spectroscopic evidence

The reaction of acidic potassium permanganate with a wide range of compounds is known to produce a broad red emission, and there is strong evidence for an excited manganese(II) emitting species. Nevertheless, numerous researchers have proposed other emitters for reactions with acidic potassium permanganate, particularly for systems where fluorescent compounds were present, either as enhancers or reaction products. We have examined many reactions of this type and found that, in most cases, the same red emission was produced. There were, however, some exceptions, including the oxidation of dihydralazine, certain thiols and sulphite (each in the presence of an enhancer).

Direct methods for distinction between endogenous and exogenous erythropoietin

Since the commercialization of the first recombinant human erythropoietin (rhEPO) product (epoetin-a) in 1989 as a treatment for acute anemia, rhEPO detection has represented a continuous challenge for the anti-doping fight. Indeed, it appeared rapidly that this ergogenic hormone would be abused by athletes looking for an artificial performance enhancer. Hemoglobin is one of the principal modulators of aerobic power [1, 2] and, consequently, of performance in endurance sports [3]. By stimulating the red blood cells production, EPO is known to raise hemoglobin concentration in a dose-dependant and predictable way. Therefore, this hormone soon became one of the athletes most popular doping agent. Since 1984, all forms of blood doping in sport have been officially banned. In 1990, the IOC medical commission, which was in charge of the anti-doping regulations, added rhEPO to the list of the prohibited drugs in sports, even if a direct test allowing to detect the molecule became available a decade after only.