The ß-amyloid peptide in Alzheimer's disease decreases adhesion of vascular muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Aβ) builds up. In this study, the possibility that Aβ disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Aβ in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Aβ was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Aβ that was fluorescently labelled at the N-terminus (fluo-Aβ) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Aβ. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Aβ1–40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Aβ treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Aβ on cell adhesion. The studies suggest that Aβ can decrease VSMC viability by disrupting VSMC–extracellular matrix (ECM) adhesion.

Brief intense interval exercise activates AMPK and p38 MAPK signaling and increases the expression of PGC-1α in human skeletal muscle

From a cell signaling perspective, short-duration intense muscular work is typically associated with resistance training and linked to pathways that stimulate growth. However, brief repeated sessions of sprint or high-intensity interval exercise induce rapid phenotypic changes that resemble traditional endurance training. We tested the hypothesis that an acute session of intense intermittent cycle exercise would activate signaling cascades linked to mitochondrialbiogenesis in human skeletal muscle. Biopsies (vastus lateralis) were obtained from six young men who performed four 30-s "all out" exercise bouts interspersed with 4 min of rest (<80 kJ total work). Phosphorylation of AMP-activated protein kinase (AMPK; subunits 1 and 2) and the p38 mitogen-activated protein kinase (MAPK) was higher (P  0.05) immediately after bout 4 vs. preexercise. Peroxisome proliferator-activated receptor- coactivator-1(PGC-1) mRNA was increased approximately twofold above rest after 3 h of recovery (P  0.05); however, PGC-1protein content was unchanged. In contrast, phosphorylation of protein kinase B/Akt (Thr³⁰⁸ and Ser⁴⁷³) tended to decrease, and downstream targets linked to hypertrophy (p70 ribosomal S6 kinase and 4E binding protein 1) were unchanged after exercise and recovery. We conclude that signaling through AMPK and p38 MAPK to PGC-1 may explain in part the metabolic remodeling induced by low-volume intense interval exercise, including mitochondrial biogenesis and an increased capacity for glucose and fatty acid oxidation.

ß2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the β2-adrenoceptor agonist (β2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.

Functional dairy protein supplements for elite athletes

Elite athletes require a greater dietary protein intake than recreationally active people to maintain optimal muscular function. The timing of protein ingestion relative to exercise is critical to maximizing its physiological impact on skeletal muscles. Sports protein supplements provide a convenient means of supplying athletes with an adequate and timely source of quality dietary protein. There is now strong evidence that not all dietary proteins are equipotent in their effects on various aspects of athletic performance and specific protein isolates can provide benefits to athletes beyond simple supply of nutritional amino acids. Thus, there is an opportunity to develop new functional protein supplements to maximize athletic performance. This paper outlines the clinical evidence for the benefits of dairy proteins in sports performance and describes the development of new dairy protein supplements to build muscle strength, and to expedite recovery of strength following muscle-damaging eccentric exercise.

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.

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.

AMP-activated protein kinase regulates GLUT4 transcription by phosphorylating histone deacetylase 5

Objective: Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms.

Research Design and Methods and Results: Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription.

Conclusions: These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.

Normal hypertrophy accompanied by phosphoryation and activation of AMP-activated protein kinase α1 following overload in LKB1 knockout mice

The activation of the AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1) is hypothesized to underlie the fact that muscle growth following resistance exercise is decreased by concurrent endurance exercise. To directly test this hypothesis, the capacity for muscle growth was determined in mice lacking the primary upstream kinase for AMPK in skeletal muscle, LKB1. Following either 1 or 4 weeks of overload, there was no difference in muscle growth between the wild type (wt) and LKB1−/− mice (1 week: wt, 38.8 ± 7.75%; LKB1−/−, 27.8 ± 12.98%; 4 week: wt, 75.8 ± 15.2%; LKB1−/−, 85.0 ± 22.6%). In spite of the fact that the LKB1 had been knocked out in skeletal muscle, the phosphorylation and activity of the α1 isoform of AMPK were markedly increased in both the wt and the LKB1−/− mice. To identify the upstream kinase(s) responsible, we studied potential upstream kinases other than LKB1. The activity of both Ca2+–calmodulin-dependent protein kinase kinase α(CaMKKα) (5.05 ± 0.86-fold) and CaMKKβ (10.1 ± 2.59-fold) increased in the overloaded muscles, and this correlated with their increased expression. Phosphorylation of TAK-1 also increased 10-fold following overload in both the wt and LKB1 mice. Even though the α1 isoform of AMPK was activated by overload, there were no increases in expression of mitochondrial proteins or GLUT4, indicating that the α1 isoform is not involved in these metabolic adaptations. The phosphorylation of TSC2, an upstream regulator of the TORC1 pathway, at the AMPK site (Ser1345) was increased in response to overload, and this was not affected by LKB1 deficiency. Taken together, these data suggest that the α1 isoform of AMPK is preferentially activated in skeletal muscle following overload in the absence of metabolic adaptations, suggesting that this isoform might be important in the regulation of growth but not metabolism.

Exercise-induced histone modifications in human skeletal muscle

Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP-activated protein kinase (AMPK) and the calcium–calmodulin-dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation-dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

Acute signalling responses to intense endurance training commenced with low or normal muscle glycogen

We have previously demonstrated that well-trained subjects who completed a 3 week training programme in which selected high-intensity interval training (HIT) sessions were commenced with low muscle glycogen content increased the maximal activities of several oxidative enzymes that promote endurance adaptations to a greater extent than subjects who began all training sessions with normal glycogen levels. The aim of the present study was to investigate acute skeletal muscle signalling responses to a single bout of HIT commenced with low or normal muscle glycogen stores in an attempt to elucidate potential mechanism(s) that might underlie our previous observations. Six endurance-trained cyclists/triathletes performed a 100 min ride at ∼70% peak O₂ uptake (AT) on day 1 and HIT (8 × 5 min work bouts at maximal self-selected effort with 1 min rest) 24 h later (HIGH). Another six subjects, matched for fitness and training history, performed AT on day 1 then 1–2 h later, HIT (LOW). Muscle biopsies were taken before and after HIT. Muscle glycogen concentration was higher in HIGH versus LOW before the HIT (390 ± 28 versus 256 ± 67 μmol (g dry wt)⁻¹). After HIT, glycogen levels were reduced in both groups (P < 0.05) but HIGH was elevated compared with LOW (229 ± 29 versus 124 ± 41 μmol (g dry wt)−1; P < 0.05). Phosphorylation of 5'AMP-activated protein kinase (AMPK) increased after HIT, but the magnitude of increase was greater in LOW (P < 0.05). Despite the augmented AMPK response in LOW after HIT, selected downstream AMPK substrates were similar between groups. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was unchanged for both groups before and after the HIT training sessions. We conclude that despite a greater activation AMPK phosphorylation when HIT was commenced with low compared with normal muscle glycogen availability, the localization and phosphorylation state of selected downstream targets of AMPK were similar in response to the two interventions.

AMPK-mediated transcription in skeletal muscle

Skeletal muscle phenotype plays a critical role in human performance and health, and skeletal muscle oxidative capacity is a key determinant of exercise tolerance. More recently, defective muscle oxidative metabolism has been implicated in a number of conditions associated with the metabolic syndrome, cardiovascular disease and muscle-wasting disorders. AMPK (AMP-activated protein kinase) is a critical regulator of cellular and organismal energy balance. AMPK has also emerged as a key regulator of skeletal muscle oxidative function, including metabolic enzyme expression, mitochondrial biogenesis and angiogenesis. AMPK mediates these processes primarily through alterations in gene expression. The present review examines the role of AMPK in skeletal muscle transcription and provides an overview of the known transcriptional substrates mediating the effects of AMPK on skeletal muscle phenotype.

Histone modifications and skeletal muscle metabolic gene expression

1.      Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases.

2.      Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression.

3.      Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response.

4.      The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified.

5.      Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases.

6.      Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

Creatine transporter expression in skeletal muscle

Creatine is an important molecule involved in providing energy to the body. Its major stores are in skeletal muscle. The creatine transporter protein (CreaT) mRNA is believed to be responsible for the uptake of the majority of creatine in skeletal muscle. This thesis examined factors that might have affected the expression of the creatine transporter in skeletal muscle.

PKC and CaMK in human skeletal muscle : effects of exercise

The effects of exercise on novel signalling enzymes in skeletal muscle of humans was investigated. It was shown that exercise increased the activity of a calcium and calmodulin activated kinase. High-intensity, but submaximal, exercise increased the activity of some but not all isoforms of protein kinase C, a lipid-activated kinase family. These findings suggest that these enzymes may be part of the signalling process leading to beneficial adaptation to repeated exercise as well as the control of function within skeletal muscle during exercise.

Hypertrophic signalling in human skeletal muscle

Ý-lactoglobulin enriched whey protein isolate, but not carbohydrate, increased growth signalling in human skeletal muscle when consumed in conjunction with resistance exercise. Ageing did not impair the anabolic signalling response; however this response was attenuated after training. These findings help identify strategies to prevent, or delay the onset of sarcopenia.