Effects of high-intensity intermittent swimming on PGC-1alpha protein expression in rat skeletal muscle

AIM: The aim of the present investigation was to elucidate the effects of exercise intensity on exercise-induced expression of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) protein in rat skeletal muscle. METHODS: We measured PGC-1alpha content in the skeletal muscles of male Sprague-Dawley rats (age: 5-6 weeks old; body weight: 150-170 g) after a single session of high-intensity intermittent exercise (HIE) or low-intensity prolonged swimming exercise (LIE). During HIE, the rats swam for fourteen 20-s periods carrying a weight (14% of body weight), and the periods of swimming were separated by a 10-s pause. LIE rats swam with no load for 6 h in two 3-h sessions, separated by 45 min of rest. RESULTS: After HIE, the PGC-1alpha protein content in rat epitrochlearis muscle had increased by 126, 140 and 126% at 2, 6 and 18 h, respectively, compared with that of the age-matched sedentary control rats' muscle. Immediately, 6 and 18-h after LIE, the PGC-1alpha protein content in the muscle was significantly elevated by 84, 95 and 67% respectively. The PGC-1alpha protein content observed 6 h after HIE tended to be higher than that observed after LIE. However, there was no statistically significant difference between the two values (P = 0.12). CONCLUSION: The present investigation suggests that irrespective of the intensity of the exercise, PGC-1alpha protein content in rat skeletal muscle increases to a comparable level when stimuli induced by different protocols are saturated. Further, HIE is a potent stimulus for enhancing the expression of PGC-1alpha protein, which may induce mitochondrial biogenesis in exercise-activated skeletal muscle.     

Increased FXYD1 and PGC‐1α mRNA after blood flow‐restricted running is related to fibre type‐specific AMPK signalling and oxidative stress in human muscle

Aim

This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC‐1α (total, 1α1, and 1α4) and Na⁺,K⁺‐ATPase isoforms (NKA; α_1‐3, β_1‐3, and FXYD1) to an interval running session and determined whether these effects were related to increased oxidative stress, hypoxia, and fibre type‐specific AMPK and CaMKII signalling, in human skeletal muscle.

Methods

In a randomized, crossover fashion, 8 healthy men (26 ± 5 year and 57.4 ± 6.3 mL kg^?1 min^?1) completed 3 exercise sessions: without (CON) or with blood flow restriction (BFR), or in systemic hypoxia (HYP, ~3250 m). A muscle sample was collected before (Pre) and after exercise (+0 hour, +3 hours) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α‐AMPK Thr¹⁷²/α‐AMPK, ACC Ser²²¹/ACC, CaMKII Thr²⁸⁷/CaMKII, and PLBSer¹⁶/PLB ratios in type I and II fibres.

Results

Muscle hypoxia (assessed by near‐infrared spectroscopy) was matched between BFR and HYP, which was higher than CON (~90% vs ~70%; P < .05). The mRNA levels of FXYD1 and PGC‐1α isoforms (1α1 and 1α4) increased in BFR only (P < .05) and were associated with increases in indicators of oxidative stress and type I fibre ACC Ser²²¹/ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling.

Conclusion

Blood flow restriction augmented exercise‐induced increases in muscle FXYD1 and PGC‐1α mRNA in men. This effect was related to increased oxidative stress and fibre type‐dependent AMPK signalling, but unrelated to the severity of muscle hypoxia, lactate accumulation, and modulation of fibre type‐specific CaMKII signalling.

     

Interaction of exercise and diet on GLUT‐4 protein and gene expression in Type I and Type II rat skeletal muscle

We determined the interaction of exercise and diet on glucose transporter (GLUT‐4) protein and mRNA expression in type I (soleus) and type II [extensor digitorum longus (EDL)] skeletal muscle. Forty‐eight Sprague Dawley rats were randomly assigned to one of two dietary conditions: high‐fat (FAT, n=24) or high‐carbohydrate (CHO, n=24). Animals in each dietary condition were allocated to one of two groups: control (NT, n=8) or a group that performed 8 weeks of treadmill running (4 sessions week^–1 of 1000 m @ 28 m min^–1, RUN, n=16). Eight trained rats were killed after their final exercise bout for determination of GLUT‐4 protein and mRNA expression: the remainder were killed 48 h after their last session for measurement of muscle glycogen and triacylglycerol concentration. GLUT‐4 protein expression in NT rats was similar in both muscles after 8 weeks of either diet. However, there was a main effect of training such that GLUT‐4 protein was increased in the soleus of rats fed with either diet (P < 0.05) and in the EDL in animals fed with CHO (P < 0.05). There was a significant diet–training interaction on GLUT‐4 mRNA, such that expression was increased in both the soleus (100% ↑P < 0.05) and EDL (142% ↑P < 0.01) in CHO‐fed animals. Trained rats fed with FAT decreased mRNA expression in the EDL (↓ 45%, P < 0.05) but not the soleus (↓ 14%, NS). We conclude that exercise training in CHO‐fed rats increased both GLUT‐4 protein and mRNA expression in type I and type II skeletal muscle. Despite lower GLUT‐4 mRNA in muscles from fat‐fed animals, exercise‐induced increases in GLUT‐4 protein were largely preserved, suggesting that control of GLUT‐4 protein and gene expression are modified independently by exercise and diet.     

Reduced AMPKα2 protein expression restores glucose‐induced insulin secretion in islets from calorie‐restricted rats

In a state of caloric restriction (CR), improved insulin action was associated with the activation of AMP‐activated kinase (AMPK). Here, we verified whether AMPK was involved in impaired β‐cell function in islets from rats subjected to CR for 21 days. Eight‐week‐old male rats were distributed into a control (CTL) group that was fed an isocaloric diet ad libitum or a CR group that received 60% of the food consumed by the CTL group. From days 18–21, CTL and CR rats were treated with sense (CTLS and CRS) or antisense (CTLAS and CRAS) AMPKα2 oligonucleotides. Caloric restriction was associated with decreased body weight, perigonadal fat pads and insulinaemia, while higher glucose tolerance was observed in CRS rats. Antisense treatment normalized insulinaemia and glucose tolerance in CRAS rats and increased cholesterolaemia in CRAS and CTLAS groups. These effects were associated with reduced pAMPK/AMPK protein expression in the liver of rats treated with antisense oligonucleotides. Additionally, CRS islets showed higher pAMPK/AMPK content and lower glucose‐induced insulin release. As expected, antisense oligonucleotides against AMPKα2 efficiently reduced pAMPK/AMPK protein in CRAS and CTLAS islets. The lower AMPK content in CRAS islets normalized the insulin secretion in islets exposed to 16.7 mM glucose. In addition, CTLAS islets presented higher insulin secretion at 2.8 and 16.7 mM glucose. These findings support the hypothesis that higher AMPK protein expression is involved in impaired β‐cell function in islets from rats subjected to CR for 21 days.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

DHEA improves impaired activation of Akt and PKC ζ/λ‐GLUT4 pathway in skeletal muscle and improves hyperglycaemia in streptozotocin‐induced diabetes rats

Aim: Addition of dehydroepiandrosterone (DHEA) to a cultured skeletal muscle locally synthesizes 5α‐dihydrotestosterone (DHT). It induced activation of glucose metabolism‐related signalling pathway via protein kinase B (Akt) and protein kinase C zeta/lambda (PKC ζ/λ)‐glucose transporter‐4 (GLUT4) proteins. However, such an effect of DHEA in vivo remains unclear. Methods: Using streptozotocin (STZ)‐induced rats with type 1 diabetes mellitus, we tested the hypothesis that a single bout of DHEA injection in the rats improves hyperglycaemia and muscle GLUT4‐regulated signalling pathway. After 1 week of STZ injection (55 mg kg^?1) with male Wistar rats, fasting glucose concentrations were determined in a blood sample taken from the tail vein. Blood glucose levels were then monitored for 180 min after DHEA or sesame oil (control) was injected (n = 10 for each group). Results: Blood glucose levels decreased significantly for 30–150 min after 2 mg DHEA injection in the STZ rats. In the skeletal muscle, expression and translocation of GLUT4 protein, phosphorylation of Akt and PKC ζ/λ, and phosphofructokinase and hexokinase enzyme activities increased significantly by DHEA injection. However, DHEA‐induced improvements in Akt and PKC ζ/λ‐GLUT4 pathways were blocked by a DHT inhibitor. Conclusion: These results suggest that a single bout of DHEA injection can improve hyperglycaemia and activate the glucose metabolism‐related signalling pathway via Akt and PKC ζ/λ‐GLUT4 proteins of skeletal muscles in rats. Moreover, these results show that a DHEA‐induced increase in muscle glucose uptake and utilization might contribute to improvement in hyperglycaemia in type 1 diabetes mellitus.     

IgE detection to α/β/γ‐gliadin and its clinical relevance in wheat‐dependent exercise‐induced anaphylaxis

Wheat‐dependent exercise‐induced anaphylaxis (WDEIA) is characterized by anaphylactic reactions after wheat ingestion and physical exercise. IgE antibodies to recombinant ω₅‐gliadin are detectable in a majority of WDEIA patients, but other wheat allergens may also play a role in elicitation of WDEIA. Here, we performed a comprehensive analysis of IgE reactivity to different wheat proteins in 17 patients with confirmed WDEIA by ImmunoCAP research prototypes and a semi‐quantitative microarray immunoassay with α/β/γ‐gliadin, high‐molecular‐weight (HMW) glutenin, alpha‐amylase inhibitor (AAI) dimer, and wheat lipid transfer protein (LTP). By ImmunoCAP, IgE to recombinant ω₅‐gliadin was detectable in 14/17 patients (82%), to α/β/γ‐gliadin in 82% including the three patients lacking IgE to ω₅‐gliadin, and to HMW glutenin in 59%. The microarray revealed specifically γ‐gliadin as the second most important allergen. These results demonstrate the additional diagnostic value of α/β‐ and γ‐gliadin in particular in ω₅‐gliadin‐negative patients in the diagnosis of WDEIA.     

Effects of nonexhaustive bouts of high-intensity intermittent swimming training on GLUT-4 expression in rat skeletal muscle

We previously reported that 14 bouts of exhaustive high-intensity intermittent training [20 s periods of swimming while carrying a weight (14% of body weight), separated by pauses of 10 s] is the highest stimuli in terms of exercise training-induced glucose transporter 4 (GLUT-4) expression in rat epitrochlearis (EPI) muscles. In the present study, we found that the GLUT-4 protein content in the skeletal muscle of male Sprague-Dawley rats (age 5 weeks old; body weight 90–110 g) that underwent intermittent exercise training of 3 and 14 bouts of 20 s swimming for 5 days was increased over age-matched sedentary control rats by 75 and 71%, respectively, 18 h after the last bout of exercise. These results suggest that GLUT-4 content in rat EPI muscle increases dramatically after very short (60 s) and nonexhaustive high-intensity intermittent exercise training.     

Mutations and polymorphisms of the skeletal muscle α‐actin gene (ACTA1)

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations ( http://waimr.uwa.edu.au ). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of oestrogen receptor α and β is higher in skeletal muscle of highly endurance‐trained than of moderately active men

Aim: Two known oestrogen receptors (ERs), ERα and the recently cloned ERβ, are expressed in the human skeletal muscle of both males and females. The effects of oestrogen and the role of ERs in skeletal muscle tissue are not well known. Oestrogen receptors and some of their target genes are involved in angiogenic processes. It was hypothesized that ERs are expressed at a higher level in a group with higher oxidative capacity, and that such an enhanced expression would parallel expression of the angiogenic factor – vascular endothelial growth factor (VEGF). Method: Muscle biopsies were taken from vastus lateralis in 10 highly endurance‐trained males and 10 moderately active males and analysed for the expression of ERs and VEGF. Results: The major findings in the present study were the higher mRNA levels of ERα, ERβ and VEGF in the highly endurance‐trained than in the moderately active group. Conclusion: These data suggest that the greater mRNA expression of ERα and ERβ and the oestrogen‐associated angiogenic factor VEGF support the hypothesis of an involvement of ERs in the adaptation of skeletal muscle to endurance training.     

Recyclable Vinyl‐Functionalized Polyesters via Chemoselective Organopolymerization of Bifunctional α‐Methylene‐δ‐Valerolactone

α‐Methylene‐δ‐valerolactone (MVL) is a bifunctional monomer comprising of a highly stable six‐membered δ‐valerolactone ring and highly reactive C?C bond. Previously, the vinyl‐addition polymerization (VAP) product, namely P(MVL)_VAP, has been formed exclusively. In this study, this conventional chemoselectivity is reversed, wherein organic catalysts are used to enable the first ring‐opening polymerization (ROP) of MVL, exclusively affording a metal‐free, unsaturated polyester, namely P(MVL)_ROP. This challenging goal is achieved by investigating different catalysts, initiators, and reaction conditions. In addition, the formation of two polymers, namely P(MVL)_ROP and P(MVL)_VAP, is easily regulated by varying the polymerization solvent. The resulting P(MVL)_ROP can be easily post‐functionalized to form crosslinked or sulfurized materials; notably, it can be almost completely converted into its monomer thermochemically.     

Networks Based on Poly(α‐methylene‐δ‐valerolactone‐co‐δ‐valerolactone): Crosslinking Through Free‐Radical Vinyl Copolymerization

Unsaturated polyesters are synthesized via ring‐opening copolymerization of α‐methylene‐δ‐valerolactone and δ‐valerolactone. These polyesters 4a–c are mixed with ethyl methacrylate (EMA), 2‐hydroxyethyl methacrylate (HEMA), and α‐methylene‐δ‐valerolactone (α‐MVL), respectively. Then, crosslinking is carried out by free radical polymerization initiated by an azo‐initiator. A second glass transition is found with incorporation of HEMA and α‐MVL. These findings indicate the formation of phase‐separated polyester blocks crosslinked with the poly(meth)‐acrylic‐segments, respectively poly(α‐methylene‐δ‐valerolactone) segments.

     

DL4‐mediated Notch signaling is required for the development of fetal αβ and γδ T cells

T‐cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta‐like 4 (DL4) on TECs by Notch1 expressed by blood‐borne BM‐derived precursors is essential for T‐cell commitment in the adult thymus. In contrast to the adult, the earliest T‐cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T‐cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T‐cell lymphopoiesis, we show here that DL4‐mediated Notch signaling is essential for the development of both αβ and γδ T‐cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T‐cell development and a dramatic reduction of all γδ T‐cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T‐cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4‐mediated Notch signaling in fetal and adult thymopoiesis.     

Organ‐specific lymphangiectasia,arrested lymphatic sprouting,and maturation defects resulting from gene‐targeting of the PI3K regulatory isoforms p85α, p55α, and p50α

The phosphoinositide 3‐kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85α, p55α, and p50α impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage‐dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell‐origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up‐regulation of the transforming growth factor‐β co‐receptor endoglin, and reduced levels of mature vascular endothelial growth factor‐C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis. Developmental Dynamics 238:2670–2679, 2009. © 2009 Wiley‐Liss, Inc.     

Chronic aerobic exercise enhances components of the classical and novel insulin signalling cascades in Sprague–Dawley rat skeletal muscle

Aim: The aim of this study was to provide a more extensive evaluation of the effects of chronic aerobic exercise on various components of the insulin signalling cascade in normal rodent skeletal muscle because of the limited body of literature that exists in this area of investigation. Methods: Male Sprague–Dawley rats were assigned to either control (n = 7) or chronic aerobic exercise (n = 7) groups. Aerobic exercise animals were run 3 day week^?1 for 45 min on a motor‐driven treadmill (32 m min^?1, 15% grade) for a 12 week period. Following the training period, all animals were subjected to hind limb perfusion in the presence of 500 μU mL^?1 insulin to determine what effect chronic aerobic training had on various components of the insulin signalling cascade, c‐Cbl protein concentration and c‐Cbl phosphorylation. Results: Twelve weeks of aerobic training did not alter skeletal muscle Akt 1/2 protein concentration, Akt Ser 473 phosphorylation, Akt Thr 308 phosphorylation, Akt 1 activity, aPKC‐ζ protein concentration, aPKC‐λ protein concentration or c‐Cbl protein concentration. In contrast, chronic aerobic exercise increased insulin‐stimulated phosphatidylinositol 3‐kinase, Akt 2 kinase and aPKC‐ζ/λ kinase activities, as well as c‐Cbl tyrosine phosphorylation, in a fibre type specific response to aerobic training. In addition, chronic aerobic exercise enhanced insulin‐stimulated plasma membrane glucose transporter 4 (GLUT4) protein concentration. Conclusion: Collectively, these findings suggest that chronic aerobic exercise enhances components of both the classical and novel insulin signalling cascades in normal rodent skeletal muscle, which may contribute to an increased insulin‐stimulated plasma membrane GLUT4 protein concentration.