AMPK and transcriptional regulation

The AMP-activated protein kinase (AMPK) is an energy sensing enzyme that once activated, promotes energy production and limits energy utilisation to ensure cellular survival. In addition to targeting numerous metabolic enzymes for this purpose, it is becoming apparent that AMPK can also regulate a number of transcriptional processes. These processes ensure cell survival through the inhibition of cell cycle and growth mechanisms, and also prepare the cell for future perturbations in energy balance by increasing the capacity of the cell to produce ATP. While these adaptations might be inextricably linked through regulation of the proliferation-differentiation process, recent studies have identified a number of transcriptional regulators as AMPK substrates that give insights into the regulation of transcription by AMPK in a number of metabolically active tissues.

Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in Enterotoxigenic Escherichia coli

The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.

Regulation of insulin signalling by exercise in skeletal muscle

Regular physical activity improves insulin action and is an effective therapy for the treatment and prevention of type 2 diabetes. However, little is known of the mechanisms by which exercise improves insulin action in muscle. These studies investigate the actions of a single bout of exercise and short-term endurance training on insulin signalling. Twenty-four hours following the completion of a single bout of endurance exercise insulin action improved, although greater enhancement of insulin action was demonstrated following the completion of endurance training, implying that cumulative bouts of exercise substantially increase insulin action above that seen from the residual effects of an acute bout of prior exercise. No alteration in the abundance and phosphorylation of proximal members of the insulin-signalling cascade in skeletal muscle, including the insulin receptor and IRS-1 were found. A major finding however, was the significant increase in the serine phosphorylation of a known downstream signalling protein, Akt (1.5 fold, p ≤0.05) following an acute bout of exercise and exercise training. This was matched by the observed increase in protein abundance of SHPTP2 (1.6 fold, p ≤0.05) a protein tyrosine phosphatase, in the cytosolic fraction of skeletal muscle following endurance exercise. These data suggest a small positive role for SHPTP2 on insulin stimulated glucose transport consistent with transgenic mice models. Further studies were aimed at examining the gene expression following a single bout of either resistance or endurance exercise. There were significant transient increases in IRS-2 mRNA concentration in the few hours following a single bout of both endurance and resistance exercise. IRS-2 protein abundance was also observed to significantly increase 24-hours following a single bout of endurance exercise indicating transcriptional regulation of IRS-2 following muscular contraction. One final component of this PhD project was to examine a second novel insulin-signalling pathway via c-Cbl tyrosine phosphorylation that has recently been shown to be essential for insulin stimulated glucose uptake in adipocytes. No evidence was found for the tyrosine phosphorylation of c-Cbl in the skeletal muscle of Zucker rats despite demonstrating significant phosphorylation of the insulin receptor and Akt by insulin treatment and successfully immunoprecipitating c-Cbl protein. Surprisingly, there was a small but significant increase in c-Cbl protein expression following insulin-stimulation, however c-Cbl tyrosine phosphorylation does not appear to be associated with insulin or exercise-mediated glucose transport in skeletal muscle.

Neisseria meningitidis and Neisseria gonorrhoeae are differently adapted in the regulation of denitrification: single nucleotide polymorphisms that enable species-specific tuning of the aerobic–anaerobic switch

The closely related pathogenic Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. aniA (copper-containing nitrite reductase) is tightly regulated by four transcriptional regulators: FNR (fumarate and nitrate reductase), NarP, FUR (Ferric uptake regulator) and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show in the present study that FUR and NarP are both required for the induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-co-operative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase-binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic–anaerobic switch.

The effects of short-term sprint training on MCT expression in moderately endurance-trained runners

The purpose of this study was to assess the effects of short-term sprint training on transient changes in monocarboxylate lactate transporter 1 (MCT1) and MCT4 protein and mRNA content. Seven moderately endurance-trained runners (mean ± SE; age 27.7±2.9 years, body mass 81.1±5.9 kg, VO₂ max 58.1±2.0 ml kg⁻¹ min⁻¹) completed a VO₂ max and a supramaximal running test to exhaustion (RTE) before and after a 6-week period of sprint training. The sprint training was progressive and consisted of 18 sessions of near maximal short duration (5–15 s) sprints to compliment the athlete’s endurance training. Prior to the training period there was a significant (P<0.05) increase in MCT1, but not MCT4 protein, 2 h after the RTE. This occurred without any change in corresponding mRNA levels. After the training period, there was a significant increase in MCT1 protein but no significant change in the MCT4 isoform. Both MCT1 and MCT4 mRNA was significantly lower at rest and 2 h post-RTE after the completion of the training period. After the training period, there was a significant increase in the time to exhaustion and distance covered during the RTE. This study demonstrates that sprint training of this length and type results in an upregulation of MCT1 protein, but not MCT4 content. Additionally, this study shows conflicting adaptations in MCT1 and MCT4 protein and mRNA levels following training, which may indicate post-transcriptional regulation of MCT expression in human muscle.

Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1

The molecular mechanisms influencing muscle atrophy in humans are poorly understood. Atrogin-1 and MuRF1, two ubiquitin E3-ligases, mediate rodent and cell muscle atrophy and are suggested to be regulated by an Akt/Forkhead (FKHR) signaling pathway. Here we investigated the expression of atrogin-1, MuRF1, and the activity of Akt and its catabolic (FKHR and FKHRL1) and anabolic (p70s6k and GSK-3β) targets in human skeletal muscle atrophy. The muscle atrophy model used was amyotrophic lateral sclerosis (ALS). All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls as well as in G93A ALS mice. ALS patients had a significant increase in atrogin-1 mRNA and protein content, which was associated with a decrease in Akt activity. There was no difference in the mRNA and protein content of FKHR, FKHRL1, p70s6k, and GSK-3β. Similar observations were made in the G93A ALS mice. Human skeletal muscle atrophy, as seen in the ALS model, is associated with an increase in atrogin-1 and a decrease in Akt. The transcriptional regulation of human atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via another signaling pathway.

From transcriptome to biological function: environmental stress in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis

Background
Our understanding of the importance of transcriptional regulation for biological function is continuously improving. We still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. In this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. We exposed individuals of the coral reef fish Pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately 16,000 genes in liver. We also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures.
Results
We identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. These common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. In response to heat exposure over five days, a total of 324 gene loci were differentially expressed. Many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. We were also able to identify groups of co-regulated genes, the genes within which shared similar functions.
Conclusion
This is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. We have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. The functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. This study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future.

Muscle atrophy and hypertrophy signaling in patients with chronic obstructive pulmonary disease

Rationale: The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways.
Objectives: To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70^S6K glycogen synthase kinase-3ß (GSK-3ß), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass.
Methods: Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively.
Measurements and Main Results: The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70^S6K, GSK-3ß, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70^S6K, GSK-3ß, and 4E-BP1 compared with patients with COPD with preserved muscle mass.
Conclusions: An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle

AMP-activated protein kinase (AMPK) has recently emerged as a key signaling protein in skeletal muscle, coordinating the activation of both glucose and fatty acid metabolism in response to increased cellular energy demand. To determine whether AMPK signaling may also regulate gene transcription in muscle, rats were given a single subcutaneous injection (1 mg/g) of the AMP analog 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR). AICAR injection activated (P < 0.05) AMPK-α₂ (~2.5-fold) and transcription of the uncoupling protein-3 (UCP3, ~4-fold) and hexokinase II (HKII, ~10-fold) genes in both red and white skeletal muscle. However, AICAR injection also elicited (P < 0.05) an acute drop (60%) in blood glucose and a sustained (2-h) increase in blood lactate, prompting concern regarding the specificity of AICAR on transcription. To maximize AMPK activation in muscle while minimizing potential systemic counterregulatory responses, a single-leg arterial infusion technique was employed in fully conscious rats. Relative to saline-infused controls, single-leg arterial infusion of AICAR (0.125, 0.5, and 2.5 µg · g^-1 · min^-1 for 60 min) induced a dose-dependent increase (2- to 4-fold, P < 0.05) in UCP3 and HKII transcription in both red and white skeletal muscle. Importantly, AICAR infusion activated transcription only in muscle from the infused leg and had no effect on blood glucose or lactate levels. These data provide evidence that AMPK signaling is linked to the transcriptional regulation of select metabolic genes in skeletal muscle.

Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway

The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P < 0.001), while pSRF protein levels increased similarly by 48% with both CONC and ECC exercise (P < 0.001). Prolonged ECC and CONC training equally stimulated muscle hypertrophy and produced increases in MRTF-A protein of 125% and 99%, respectively (P < 0.001). No changes occurred for total SRF protein. There was no effect of whey protein supplementation. These results show that resistance exercise provides an acute stimulation of the STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations.

Affective dysfunction in a mouse model of Rett syndrome: therapeutic effects of environmental stimulation and physical activity

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2tm1Tam mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2+ / - mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2+ / - mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2+ / - mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise

Endurance exercise improves insulin sensitivity and increases fat oxidation, which are partly facilitated by the induction of metabolic transcription factors. Next to exercise, increased levels of FFA's also increase the gene expression of transcription factors, hence making it difficult to discern the effects from contractile signals produced during exercise, from those produced by increased circulatory FFA's. We aimed to investigate, in human skeletal muscle, whether acute exercise affects gene expression of metabolic transcriptional co-activators and transcription factors, including PGC-1α, PRC, PPARα, β/δ, and γ and RXR, SREBP-1c and FKHR, and to discern the effect of exercise per se from those of elevated levels of FFA. Two hours of endurance exercise was performed either in the fasted state, or following carbohydrate ingestion prior to and during exercise, thereby blunting the fasting-induced increase in FA availability and oxidation. Of the genes measured, PGC-1α and PRC mRNA increased immediately after, while PPARβ/δ and FKHR mRNA increased 1–4 h after exercise, irrespective of the increases in FFA's. Our results suggest that the induction in vivo of metabolic transcription factors implicated in mitochondrial biogenesis are under the control of inherent signals, (PGC-1α, PRC), while those implicated in substrate selection are under the control of associated signals (PPARβ/δ, FKHR) stimulated from the contracting skeletal muscle that are independent of circulating FFA levels.

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.

MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive gene expression in arabidopsis

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.

The regulation and function of the striated muscle activator of rho signaling (STARS) protein

Healthy living throughout the lifespan requires continual growth and repair of cardiac, smooth, and skeletal muscle. To effectively maintain these processes muscle cells detect extracellular stress signals and efficiently transmit them to activate appropriate intracellular transcriptional programs. The striated muscle activator of Rho signaling (STARS) protein, also known as Myocyte Stress-1 (MS1) protein and Actin-binding Rho-activating protein (ABRA) is highly enriched in cardiac, skeletal, and smooth muscle. STARS binds actin, co-localizes to the sarcomere and is able to stabilize the actin cytoskeleton. By regulating actin polymerization, STARS also controls an intracellular signaling cascade that stimulates the serum response factor (SRF) transcriptional pathway; a pathway controlling genes involved in muscle cell proliferation, differentiation, and growth. Understanding the activation, transcriptional control and biological roles of STARS in cardiac, smooth, and skeletal muscle, will improve our understanding of physiological and pathophysiological muscle development and function.