Environmental factors affecting transcription of the human L1 retrotransposon. II. Stressors

Retrotransposons have clearly molded the structure of the human genome. The reverse transcriptase coded for by long interspersed nuclear elements (LINEs) accounts for 35% of the human genome, with 8–9 x 105 copies of the most common human LINE element, L1Hs. Retrotransposons cycle through an RNA intermediate with transcription as the rate limiting step. Because various retrotransposons have been demonstrated to be induced by environmental stimuli, we investigated the response of the L1Hs promoter to various agents. L1Hs promoter activity was analyzed by transfecting an L1Hs-expressing cell line with plasmids containing one of two L1Hs promoters fused to the LacZ reporter gene. L1Hs promoter activity was then monitored with a ß-galactosidase assay. Treatment with UV light and heat shock resulted in a small increase in ß-galactosidase activity from one promoter, while treatment with tetradecanoylphorbol 13-acetate resulted in small increases in ß-galactosidase activity from both promoters. No increase in ß-galactosidase activity was observed after exposure to X-rays or hydrogen peroxide.

Effect of 2 weeks of endurance training on uncoupling protein3 content in untrained human subjects

Aim: The mitochondrial uncoupling protein-3 (UCP3) is able to lower the proton gradient across the inner mitochondrial membrane, thereby uncoupling substrate oxidation from ATP production and dissipating energy as heat. What the effect of endurance training on UCP3 is, is still  controversial. Endurance-trained athletes are characterized by lower levels of UCP3, but longitudinal studies in rodents reported no effect of endurance training on muscular UCP3 levels. Here, we examined the effect of a 2-week training programme on skeletal muscle UCP3 protein content in untrained human subjects, and hypothesized that UCP3 will be reduced after the training programme. Methods: Nine untrained men [age: 23.3±3.2 years; BMI: 22.6±2.6 kg m^-2; maximal power output (^Wmax): 3.8±0.6 W kg^-1 body weight] trained for 2 weeks. Before and at least 72 h after the training period, muscle biopsies were taken for determination of UCP3 protein content. Results: UCP3 protein content tended to be lower after the training programme [95±10 vs. 109±12 arbitrary units (AU), P= 0.08]. Cytochrome c content tended to increase with 33% in response to endurance training (52± 6 vs. 39± 6 AU, P = 0.08). The ratio UCP3 relative to cytochrome c tended to decrease significantly upon endurance training (2.0±0.4 vs. 3.2±0.6 AU, P = 0.01). Conclusion: A short-term (2-week) endurance training programme decreased UCP3 protein levels and significantly reduced the ratio of UCP3 to cytochrome c.

Mitofusions1/2 and ERRα expression are increased in human skeletal muscle after physical exercise.

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of insulin resistance. Mitofusin (Mfn) proteins regulate the biogenesis and maintenance of the mitochondrial network, and when inactivated, cause a failure in the mitochondrial architecture and decreases in oxidative capacity and glucose oxidation. Exercise increases muscle mitochondrial content, size, oxidative capacity and aerobic glucose oxidation. To address if Mfn proteins are implicated in these exercise-induced responses, we measured Mfn1 and Mfn2 mRNA levels, pre-, post-, 2 and 24 h post-exercise. Additionally, we measured the expression levels of transcriptional regulators that control mitochondrial biogenesis and functions, including PGC-1α, NRF-1, NRF-2 and the recently implicated ERRα. We show that Mfn1, Mfn2, NRF-2 and COX IV mRNA were increased 24 h post-exercise, while PGC-1α and ERRα mRNA increased 2 h post-exercise. Finally, using in vitro cellular assays, we demonstrate that Mfn2 gene expression is driven by a PGC-1α programme dependent on ERRα. The PGC-1α/ERRα-mediated induction of Mfn2 suggests a role of these two factors in mitochondrial fusion. Our results provide evidence that PGC-1α not only mediates the increased expression of oxidative phosphorylation genes but also mediates alterations in mitochondrial architecture in response to aerobic exercise in humans.

Human Sulfotransferases and Their Role in Chemical Metabolism

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some
recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual’s response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract

CPE is an aqueous extract of the edible micro alga Chlorella pyrenoidosa, which has been shown to have immunostimulatory effects in vivo. In the present study, CPE was evaluated for an ability to stimulate cytokine production by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were treated ex vivo for 24 hours with 1, 10 and 100 μg/mL CPE. This resulted in a marked increase in the level of IL-10, a regulatory cytokine, and strong stimulation of the T-helper-1 (Th1) cell cytokines, IFN-γ and TNF-α. In contrast, stimulation of representative T-helper-2 (Th2) cell cytokines, IL-4 and IL-13, was minor. CPE (1, 10 or 100 μg/mL) did not cause a proliferation of human PBMC suggesting that enhanced secretion of cytokines was not secondary to an increase in cell number. We conclude that CPE stimulation of human PBMC induces a Th1-patterned cytokine response and a strong anti-inflammatory regulatory cytokine response, observations that await confirmation in vivo.

Regulation of STARS and its downstream target suggest a novel pathway involved in human skeletal hypertrophy and atrophy

Skeletal muscle atrophy is a severe consequence of ageing, neurological disorders and chronic disease. Identifying the intracellular signalling pathways controlling changes in skeletal muscle size and function is vital for the future development of potential therapeutic interventions. Striated activator of Rho signalling (STARS), an actin-binding protein, has been implicated in rodent cardiac hypertrophy; however its role in human skeletal muscle has not been determined. This study aimed to establish if STARS, as well as its downstream signalling targets, RhoA, myocardin-related transcription factors A and B (MRTF-A/B) and serum response factor (SRF), were increased and decreased respectively, in human quadriceps muscle biopsies taken after 8 weeks of both hypertrophy-stimulating resistance training and atrophy-stimulating de-training. The mRNA levels of the SRF target genes involved in muscle structure, function and growth, such as α-actin, myosin heavy chain IIa (MHCIIa) and insulin-like growth factor-1 (IGF-1), were also measured. Following resistance training, STARS, MRTF-A, MRTF-B, SRF, α-actin, MHCIIa and IGF-1 mRNA, as well as RhoA and nuclear SRF protein levels were all significantly increased by between 1.25- and 3.6-fold. Following the de-training period all measured targets, except for RhoA, which remained elevated, returned to base-line. Our results show that the STARS signalling pathway is responsive to changes in skeletal muscle loading and appears to play a role in both human skeletal muscle hypertrophy and atrophy.

RTKN2 induces NF-KappaB dependent resistance to intrinsic apoptosis in HEK cells and REgulates BCL-2 genes in human CD4+ lymphocytes

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4+ T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNFα. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4+ T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4+ T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.

The effect of nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells

Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 ± 2 years, BMI 31.7 ± 1.2 kg/m², fasting plasma glucose 9.52 ± 0.80 mmol/L) and eight healthy individuals (aged 46 ± 2 years, BMI 27.1 ± 1.5 kg/m², fasting plasma glucose 4.69 ± 0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 μM) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) was also examined at a concentration of 50 μM. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P = 0.03) but not the therapeutic SNP doses (P = 0.60) or SNAP (P = 0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.

Reconstruction of in vivo time-evolving neuroendocrine dose - response properties unveils admixed deterministic and stochastic elements

Homeostasis in the intact organism is achieved implicitly by repeated incremental feedback (inhibitory) and feedforward (stimulatory) adjustments enforced via intermittent signal exchange. In separated systems, neurohormone signals act deterministically on target cells via quantifiable effector-response functions. On the other hand, in vivo interglandular signaling dynamics have not been estimable to date. Indeed, experimentally isolating components of an interactive network definitionally disrupts time-sensitive linkages. We implement and validate analytical reconstruction of endogenous effector-response properties via a composite model comprising (i) a deterministic basic feedback and feedforward ensemble structure; (ii) judicious statistical allowance for possible stochastic variability in individual biologically interpretable dose–response properties; and (iii) the sole data requirement of serially observed concentrations of a paired signal (input) and response (output). Application of this analytical strategy to a prototypical neuroendocrine axis in the conscious uninjected horse, sheep, and human (i) illustrates probabilistic estimation of endogenous effector dose–response properties; and (ii) unmasks statistically vivid (2- to 5-fold) random fluctuations in inferred target-gland responsivity within any given pulse train. In conclusion, balanced mathematical formalism allows one to (i) reconstruct deterministic properties of interglandular signaling in the intact mammal and (ii) quantify apparent signal-response variability over short time scales in vivo. The present proof-of-principle experiments introduce a previously undescribed means to estimate time-evolving signal-response relationships without isotope infusion or pathway disruption.

Patient safety ethics and human error management in ED contexts Part II : Accountability and the challenge to change

In recent years there has been increasing recognition internationally that health care is not as safe as it ought to be and that patient safety outcomes need to be improved. To this end, patient safety has become the focus of a world-wide endeavour – endorsed by the World Health Organisation – to reduce the incidence and impact of preventable human errors and related adverse events in health care domains. The emergency department has been identified as a significant site of preventable human errors and adverse events in the health care system, raising important questions about the nature of human error management and patient safety ethics in rapidly changing environments, of which the Emergency Department is a prime example. In Part I of this article series, an overview of the incidence and impact of preventable adverse events in Emergency Department contexts and the development of the global patient safety movement was presented. In this second article brief attention is given to examining some of the ethical tensions that have arisen in response to the patient safety movement and their possible implications for Emergency Department contexts and staff.

A novel zebrafish jak2a V581F model shared features of human JAK2 V617F polycythemia vera

Objective : The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2^V617F) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2^V617F (referred herewith jak2a^V581F) by site-directed mutagenesis and examined its relevance as a model of human PV.

Materials and Methods : Zebrafish embryos at one-cell stage were injected with jak2a^V581F mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.

Results : Injection with jak2a^V581F mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp+ population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% ± 0.08%; jak2a^V581F injected: 5.71% ± 0.07%, coinjecting jak2a^V581F mRNA and stat5.1 morpholino: 4.66% ± 0.13%; p < 0.01). jak2a^V581F mRNA also upregulated gata1 (1.83 ± 0.08 fold; p = 0.005), embryonic α-hemoglobin (1.61 ± 0.12 fold; p = 0.049), and β-hemoglobin gene expression (1.65 ± 0.13–fold; p = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a^V581F mRNA significantly reduced erythropoietin gene (0.24 ± 0.03 fold; p = 0.006) and protein expression (control: 0.633 ± 0.11; jak2a^V581F mRNA: 0.222 ± 0.07 mIU/mL; p = 0.019).

Conclusion : The zebrafish jak2a^V581F model shared many features with human PV and might provide us with mechanistic insights of this disease.

Zinc and DHA have opposing effects on the expression levels of histones H3 and H4 in human neuronal cells

Zn and DHA have putative neuroprotective effects and these two essential nutrients are known to interact biochemically. We aimed to identify novel protein candidates that are differentially expressed in human neuronal cell line M17 in response to Zn and DHA that would explain the molecular basis of this interaction. Two-dimensional gel electrophoresis and MS were applied to identify major protein expression changes in the protein lysates of human Ml7 neuronal cells that had been grown in the presence and absence of Zn and DHA. Proteomic findings were further investigated using Western immunoblot and real-time PCR analyses. Four protein spots, which had significant differential expression, were identified and selected for in-gel trypsin digestion followed by matrix-assisted laser desorption ionisation MS analysis. The resultant peptide mass fingerprint for each spot allowed their respective identities to be deduced. Two human histone variants H3 and H4 were identified. Both H3 and H4 were downregulated by Zn in the absence of DHA (Zn effect) and upregulated by DHA (DHA effect) in the presence of Zn (physiological condition). These proteomic findings were further supported by Western immunoblot and real-time PCR analyses using H3- and H4-specific monoclonal antibodies and oligonucleotide primers, respectively. We propose that dietary Zn and DHA cause a global effect on gene expression, which is mediated by histones. Such novel information provides possible clues to the molecular basis of neuroprotection by Zn and DHA that may contribute to the future treatment, prevention and management of neurodegenerative diseases such as Alzheimer's disease.

Studies of the human lymphocyte P2Z receptor and its activation of phospholipase D

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

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.

Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK [alpha]2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK [alpha]2 activity reduced the association of AMPK with glycogen and increased AMPK [alpha]2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% [VO.sub.2] peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK [alpha]2 activity and acetyl-CoA carboxylase [beta] phosphorylation, increased translocation of AMPK [alpha]2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK [alpha]2 under conditions of low muscle glycogen enhances AMPK [alpha]2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle.