Fetal growth restriction shortens cardiac telomere length, but this is attenuated by exercise in early life.

BACKGROUND AND AIMS: Fetal and postnatal growth restriction cause a predisposition to cardiovascular disease (CVD) in adulthood. Telomeres are repetitive DNA-protein structures that protect chromosome ends, and the loss of these repeats (a reduction in telomere length) is associated with CVD. As exercise preserves telomere length and cardiovascular health, the aim of this study was to determine the effects of growth restriction and exercise training on cardiac telomere length and telomeric genes. METHODS AND RESULTS: Pregnant Wistar Kyoto rats underwent bilateral uterine vessel ligation to induce uteroplacental insufficiency and fetal growth restriction ("Restricted"). Sham-operated rats had either intact litters ("Control") or their litters reduced to five pups with slowed postnatal growth ("Reduced"). Control, Restricted, and Reduced male rats were assigned to Sedentary, Early exercise (5-9 wk of age), or Late exercise (20-24 wk of age) groups. Hearts were excised at 24 wk of age for telomere length and gene expression measurements by quantitative PCR. Growth restriction shortened cardiac telomere length ( P < 0.001), but this was rescued by early exercise ( P < 0.001). Early and Late exercise increased cardiac weight index ( P < 0.001), but neither this nor telomere length was associated with expression of the telomeric genes Tert, Terc, Trf2, Pnuts, or Sirt1. DISCUSSION AND CONCLUSIONS: Growth restriction shortens cardiac telomere length, reflecting the cardiac pathologies associated with low birth weight. Exercise in early life may offer long-term protective effects on cardiac telomere length, which could help prevent CVD in later life.

Sustained cardiac programming by short-term juvenile exercise training in male rats.

KEY POINTS: Cardiac hypertrophy following endurance-training is thought to be due to hypertrophy of existing cardiomyocytes. The benefits of endurance exercise on cardiac hypertrophy are generally thought to be short-lived and regress to sedentary levels within a few weeks of stopping endurance training. We have now established that cardiomyocyte hyperplasia also plays a considerable role in cardiac growth in response to just 4 weeks of endurance exercise in juvenile (5-9 weeks of age) rats. The effect of endurance exercise on cardiomyocyte hyperplasia diminishes with age and is lost by adulthood. We have also established that the effect of juvenile exercise on heart mass is sustained into adulthood. ABSTRACT: The aim of this study was to investigate if endurance training during juvenile life 'reprogrammes' the heart and leads to sustained improvements in the structure, function, and morphology of the adult heart. Male Wistar Kyoto rats were exercise trained 5 days week-1 for 4 weeks in either juvenile (5-9 weeks of age), adolescent (11-15 weeks of age) or adult life (20-24 weeks of age). Juvenile exercise training, when compared to 24-week-old sedentary rats, led to sustained increases in left ventricle (LV) mass (+18%; P < 0.05), wall thickness (+11%; P < 0.05), the longitudinal area of binucleated cardiomyocytes (P < 0.05), cardiomyocyte number (+36%; P < 0.05), and doubled the proportion of mononucleated cardiomyocytes (P < 0.05), with a less pronounced effect of exercise during adolescent life. Adult exercise training also increased LV mass (+11%; P < 0.05), wall thickness (+6%; P < 0.05) and the longitudinal area of binucleated cardiomyocytes (P < 0.05), despite no change in cardiomyocyte number or the proportion of mono- and binucleated cardiomyocytes. Resting cardiac function, LV chamber dimensions and fibrosis levels were not altered by juvenile or adult exercise training. At 9 weeks of age, juvenile exercise significantly reduced the expression of microRNA-208b, which is a known regulator of cardiac growth, but this was not sustained to 24 weeks of age. In conclusion, juvenile exercise leads to physiological cardiac hypertrophy that is sustained into adulthood long after exercise training has ceased. Furthermore, this cardiac reprogramming is largely due to a 36% increase in cardiomyocyte number, which results in an additional 20 million cardiomyocytes in adulthood.

Growing healthy muscles to optimise metabolic health into adult life.

The importance of skeletal muscle for metabolic health and obesity prevention is gradually gaining recognition. As a result, interventions are being developed to increase or maintain muscle mass and metabolic function in adult and elderly populations. These interventions include exercise, hormonal and nutritional therapies. Nonetheless, growing evidence suggests that maternal malnutrition and obesity during pregnancy and lactation impede skeletal muscle development and growth in the offspring, with long-term functional consequences lasting into adult life. Here we review the role of skeletal muscle in health and obesity, providing an insight into how this tissue develops and discuss evidence that maternal obesity affects its development, growth and function into adult life. Such evidence warrants the need to develop early life interventions to optimise skeletal muscle development and growth in the offspring and thereby maximise metabolic health into adult life.

Growth restriction in the rat alters expression of cardiac JAK/STAT genes in a sex-specific manner.

Uteroplacental insufficiency resulting in intrauterine growth restriction has been associated with the development of cardiovascular disease, coronary heart disease and increased blood pressure, particularly in males. The molecular mechanisms that result in the programming of these phenotypes are not clear. This study investigated the expression of cardiac JAK/STAT signalling genes in growth restricted offspring born small due to uteroplacental insufficiency. Bilateral uterine vessel ligation was performed on day 18 of pregnancy to induce growth restriction (Restricted) or sham surgery (Control). Cardiac tissue at embryonic day (E) 20, postnatal day (PN) 1, PN7 and PN35 in male and female Wistar (WKY) rats (n=7-10 per group per age) was isolated and mRNA extracted. In the heart, there was an effect of age for males for all genes examined there was a decrease in expression after PN1. With females, JAK2 expression was significantly reduced after E20, while PI3K in females was increased at E30 and PN35. Further, mRNA expression was significantly altered in JAK/STAT signalling targets in Restricteds in a sex-specific manner. Compared with Controls, in males, JAK2 and STAT3 were significantly reduced in the Restricted, while in females SOCS3 was significantly increased and PI3K significantly decreased in the Restricted offspring. Finally, there were specific differences in the levels of gene expression within the JAK/STAT pathway when comparing males to females. Thus, growth restriction alters specific targets in the JAK/STAT signalling pathway, with altered JAK2 and STAT3 potentially contributing to the increased risk of cardiovascular disease in the growth restricted males.

Xanthine oxidase inhibition attenuates skeletal muscle signaling following acute exercise but does not impair mitochondrial adaptations to endurance training.

The aim of this research was to examine the impact of the xanthine oxidase (XO) inhibitor allopurinol on the skeletal muscle activation of cell signaling kinases' and adaptations to mitochondrial proteins and antioxidant enzymes following acute endurance exercise and endurance training. Male Sprague-Dawley rats performed either acute exercise (60 min of treadmill running, 27 m/min, 5% incline) or 6 wk of endurance training (5 days/wk) while receiving allopurinol or vehicle. Allopurinol treatment reduced XO activity to 5% of the basal levels (P < 0.05), with skeletal muscle uric acid levels being almost undetectable. Following acute exercise, skeletal muscle oxidized glutathione (GSSG) significantly increased in allopurinol- and vehicle-treated groups despite XO activity and uric acid levels being unaltered by acute exercise (P < 0.05). This suggests that the source of ROS was not from XO. Surprisingly, muscle GSSG levels were significantly increased following allopurinol treatment. Following acute exercise, allopurinol treatment prevented the increase in p38 MAPK and ERK phosphorylation and attenuated the increase in mitochondrial transcription factor A (mtTFA) mRNA (P < 0.05) but had no effect on the increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor-2, GLUT4, or superoxide dismutase mRNA. Allopurinol also had no impact on the endurance training-induced increases in PGC-1α, mtTFA, and mitochondrial proteins including cytochrome c, citrate synthase, and ß-hydroxyacyl-CoA dehydrogenase. In conclusion, although allopurinol inhibits cell signaling pathways in response to acute exercise, the inhibitory effects of allopurinol appear unrelated to exercise-induced ROS production by XO. Allopurinol also has little effect on increases in mitochondrial proteins following endurance training.

On the HORYZON: moderated online social therapy for long-term recovery in first episode psychosis.

BACKGROUND: Early intervention services have demonstrated improved outcomes in first episode psychosis (FEP); however, recent evidence shows that treatment benefits may not be sustainable over time. These findings have resulted in repeated recommendations for the implementation of longer term treatment programs. An Internet-based intervention specifically designed for young people with psychosis may provide a cost-effective alternative to prevent loss of treatment benefits from early intervention. METHODS: Our multi-disciplinary team has developed a highly novel online intervention (HORYZONS) in regular consultation with stakeholders within a specialist early psychosis program. HORYZONS integrates: i) peer-to-peer social networking, ii) individually tailored interactive psychosocial interventions, and iii) expert interdisciplinary and peer-moderation in a coherent platform designed to improve long-term outcomes in FEP. The acceptability, safety and initial clinical benefits of HORYZONS were examined through a 1-month pilot study with 20 participants with FEP. RESULTS: There were no dropouts during the pilot study. Seventy per cent of participants utilised the system for at least 3weeks, 95% used the social networking features, and 60% completed at least 3 therapy modules. System usage was high during the study. There were no incidents and the majority of participants reported feeling safe, empowered and more socially connected using HORYZONS. Analysis revealed a significant reduction in depressive symptoms at follow-up. CONCLUSIONS: Our results indicate that HORYZONS is feasible, engaging and safe and may augment social connectedness and empowerment in FEP. These findings have significant implications for the enhancement of specialist FEP services. The potential of HORYZONS to improve long-term recovery is worthy of further investigation.

Internet-based interventions for psychosis: a sneak-peek into the future.

The Internet and mobile technologies are becoming ubiquitous. However, the potential of these technologies to support people with psychosis has been unexplored and the development of innovative e-based interventions is overdue. Research suggests the acceptability and effectiveness of such interventions in psychosis. Internet-based technologies have the potential to transform psychosis treatment by enhancing the accessibility of evidence-based interventions, fostering engagement with mental health services, and maintaining treatment benefits over the long term. This article reviews the current evidence on Internet-based interventions for psychosis, including potential benefits, risks, and future challenges. Recommendations are proposed for developing future online interventions for psychosis.

Stage of perinatal development regulates skeletal muscle mitochondrial biogenesis and myogenic regulatory factor genes with little impact of growth restriction or cross-fostering.

Foetal growth restriction impairs skeletal muscle development and adult muscle mitochondrial biogenesis. We hypothesized that key genes involved in muscle development and mitochondrial biogenesis would be altered following uteroplacental insufficiency in rat pups, and improving postnatal nutrition by cross-fostering would ameliorate these deficits. Bilateral uterine vessel ligation (Restricted) or sham (Control) surgery was performed on day 18 of gestation. Males and females were investigated at day 20 of gestation (E20), 1 (PN1), 7 (PN7) and 35 (PN35) days postnatally. A separate cohort of Control and Restricted pups were cross-fostered onto a different Control or Restricted mother and examined at PN7. In both sexes, peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), cytochrome c oxidase subunits 3 and 4 (COX III and IV) and myogenic regulatory factor 4 expression increased from late gestation to postnatal life, whereas mitochondrial transcription factor A, myogenic differentiation 1 (MyoD), myogenin and insulin-like growth factor I (IGF-I) decreased. Foetal growth restriction increased MyoD mRNA in females at PN7, whereas in males IGF-I mRNA was higher at E20 and PN1. Cross-fostering Restricted pups onto a Control mother significantly increased COX III mRNA in males and COX IV mRNA in both sexes above controls with little effect on other genes. Developmental age appears to be a major factor regulating skeletal muscle mitochondrial and developmental genes, with growth restriction and cross-fostering having only subtle effects. It therefore appears that reductions in adult mitochondrial biogenesis markers likely develop after weaning.

High-dose antioxidant vitamin C supplementation does not prevent acute exercise-induced increases in markers of skeletal muscle mitochondrial biogenesis in rats.

High doses of the antioxidant vitamin C prevent the increases in skeletal muscle mitochondrial biogenesis after exercise training. Since exercise training effects rely on the acute stimulus of each exercise bout, we examined whether vitamin C supplementation also attenuates the increases in skeletal muscle metabolic signaling and mitochondrial biogenesis in response to an acute exercise bout. Male Sprague-Dawley rats performed 60 min of treadmill running (27 m/min, 5% grade) or remained sedentary. For 7 days before this, one-half of the rats received water containing 500 mg/kg body wt vitamin C. Acute exercise significantly (P<0.05) increased the phosphorylation of p38 MAPK, AMP-activated kinase-alpha, and activating transcription factor (ATF)-2 and the ratio of oxidized to total glutathione (GSSG/TGSH) in the gastrocnemius. However, vitamin C had no effect on these increases. Similarly, vitamin C did not prevent the exercise-induced increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, nuclear respiratory factor (NRF)-1, NRF-2, mitochondrial transcription factor A, glutathione peroxidase-1, MnSOD, extracellular SOD, or glucose transporter 4 (P<0.05) mRNA after exercise. Surprisingly, vitamin C supplementation significantly increased the basal levels of GSSG/TGSH, NRF-1, and NRF-2 mRNA and basal ATF-2 phosphorylation. In summary, despite other studies in rats showing that vitamin C supplementation prevents increases in skeletal muscle mitochondrial biogenesis and antioxidant enzymes with exercise training, vitamin C had no affect on the acute exercise-induced increases of these markers.

Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes.

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.

Growth restriction before and after birth increases kinase signaling pathways in the adult rat heart.

To investigate the mechanisms for the previously reported development of adult cardiac hypertrophy in male rats following growth restriction, the levels of oxidative stress and activation of signaling kinases were measured in the left ventricle (LV) of adult rat offspring. In experiment one, bilateral uterine vessel ligation to induce uteroplacental insufficiency and growth restriction in the offspring (Restricted) or sham surgery was performed during pregnancy. Litters from sham mothers had litter size either reduced (Reduced Litter), which also restricted postnatal growth, or were left unaltered (Control). In males, Reduced Litter offspring had increased LV phosphorylation of AMPKα, p38 MAPK and Akt compared with Restricted and Controls (P < 0.05). In females, both Restricted and Reduced Litter adult offspring had increased LV phosphorylation of p38 MAPK and Akt, however, only Restricted offspring had increased phosphorylation of AMPKα (P < 0.05). In addition, only Restricted male offspring displayed LV oxidative stress (P < 0.05). Experiment two investigated in mothers exposed to uteroplacental insufficiency or sham surgery the effects of cross-fostering offspring at birth, and therefore the effects of the postnatal lactational environment. Surprisingly, the cross-fostering itself resulted in increased LV phosphorylation of AMPKα and Akt in females and increased phosphorylation of Akt in males compared with Control non-cross-fostered offspring (P < 0.05). In conclusion, kinase signaling in the adult LV can be programmed by uteroplacental insufficiency induced growth restriction in a gender-specific manner. In addition, the heart of adult rats is also sensitive to programming following the postnatal intervention of cross-fostering alone as well as by postnatal growth restriction.

Differential attenuation of AMPK activation during acute exercise following exercise training or AICAR treatment.

Short-term exercise training in humans attenuates AMP-activated protein kinase (AMPK) activation during subsequent exercise conducted at the same absolute workload. Short-term 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) administration in rats mimics exercise training on skeletal muscle in terms of increasing insulin sensitivity, mitochondrial enzymes, and GLUT4 content, but it is not known whether these adaptations are accompanied by reduced AMPK activation during subsequent exercise. We compared the effect of 10 days of treadmill training (60 min/day) with 10 days of AICAR administration (0.5 mg/g body weight ip) on subsequent AMPK activation during 45 min of treadmill exercise in male Sprague-Dawley rats. Compared with nonexercised control rats, acute exercise significantly (P < 0.05) increased AMPKalpha Thr172 phosphorylation (p-AMPKalpha; 1.6-fold) and ACCbeta Ser218 phosphorylation (p-ACCbeta; 4.9-fold) in the soleus and p-ACCbeta 2.2-fold in the extensor digitorum longus. Ten days of exercise training abolished the increase in soleus p-AMPKalpha and attenuated the increase in p-ACCbeta (nonsignificant 2-fold increase). Ten days of AICAR administration also attenuated the exercise-induced increases in AMPK signaling in the soleus although not as effectively as 10 days of exercise training (nonsignificant 1.3-fold increase in p-AMPKalpha; significant 3-fold increase in p-ACCbeta). The increase in skeletal muscle 2-deoxyglucose uptake during exercise was greater after either 10 days of exercise training or AICAR administration. In conclusion, 10 days of AICAR administration substantially mimics the effect of 10 days training on attenuating skeletal muscle AMPK activation in response to subsequent exercise.

NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle.

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS-/-) or neuronal NOS (nNOS-/-) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS-/-, nNOS-/- and C57Bl/6 (CON) mice (16.3 +/- 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min(-1), 5% grade) for 60 min (n = 8 per group) and were killed 6 h after exercise. Other eNOS-/-, nNOS-/- and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS-/- mice had significantly higher nNOS protein and nNOS-/- mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2alpha and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS-/- mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1alpha mRNA higher in the EDL. Also, eNOS-/- mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1alpha mRNA in soleus and EDL and NRF2alpha mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle.

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no l-NAME ingestion and acute exercise, rest plus l-NAME, and rest without l-NAME. The exercised rats ran on a treadmill for 53 +/- 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-gamma coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.

L-Arginine infusion increases glucose clearance during prolonged exercise in humans.

Nitric oxide synthase (NOS) inhibition has been shown in humans to attenuate exercise-induced increases in muscle glucose uptake. We examined the effect of infusing the NO precursor L-arginine (L-Arg) on glucose kinetics during exercise in humans. Nine endurance-trained males cycled for 120 min at 72+/-1% Vo(2 peak) followed immediately by a 15-min "all-out" cycling performance bout. A [6,6-(2)H]glucose tracer was infused throughout exercise, and either saline alone (Control, CON) or saline containing L-Arg HCL (L-Arg, 30 g at 0.5 g/min) was confused in a double-blind, randomized order during the last 60 min of exercise. L-Arg augmented the increases in glucose rate of appearance, glucose rate of disappearance, and glucose clearance rate (L-Arg: 16.1+/-1.8 ml.min(-1).kg(-1); CON: 11.9+/- 0.7 ml.min(-1).kg(-1) at 120 min, P<0.05) during exercise, with a net effect of reducing plasma glucose concentration during exercise. L-Arg infusion had no significant effect on plasma insulin concentration but attenuated the increase in nonesterified fatty acid and glycerol concentrations during exercise. L-Arg infusion had no effect on cycling exercise performance. In conclusion, L-Arg infusion during exercise significantly increases skeletal muscle glucose clearance in humans. Because plasma insulin concentration was unaffected by L-Arg infusion, greater NO production may have been responsible for this effect.

Effect of exercise intensity and hypoxia on skeletal muscle AMPK signaling and substrate metabolism in humans.

We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O(2), 111 +/- 12 W, 72 +/- 3% hypoxia Vo(2 peak); 72% Hypoxia) or under normoxic conditions (20.9% O(2)) matched to the same absolute (111 +/- 12 W, 51 +/- 1% normoxia Vo(2 peak); 51% Normoxia) or relative (to Vo(2 peak)) intensity (171 +/- 18 W, 73 +/- 1% normoxia Vo(2 peak); 73% Normoxia). Increases (P < 0.05) in AMPK activity, AMPKalpha Thr(172) phosphorylation, ACCbeta Ser(221) phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats.

AIMS/HYPOTHESIS: Recruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3'-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle. METHODS: Lean and obese Zucker rats were randomly assigned to one of three treatment groups: (i). control, (ii). insulin-stimulated or (iii). acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured. RESULTS: Insulin receptor phosphorylation was increased 12-fold after insulin stimulation ( p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment ( p<0.001). CONCLUSIONS/INTERPRETATION: We also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats ( p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.

UCP3 protein expression is lower in type I, IIa and IIx muscle fiber types of endurance-trained compared to untrained subjects.

Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower ( P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups ( P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects ( P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.

Slow component of [V]O(2) kinetics: the effect of training status, fibre type, UCP3 mRNA and citrate synthase activity.

OBJECTIVE: In healthy individuals performing constant-load exercise at intensities above the lactate threshold a secondary rise in pulmonary oxygen uptake ([V]O(2)) occurs. [V]O(2) reaches a maximum and exhaustion rapidly prevails for a range of work rates lower than the maximal work rate achieved during a conventional rapid-incremental test. This phenomenon is called the slow component (SC) of [V]O(2) kinetics and represents an increase in [V]O(2) without an increase in work rate. Although still under debate, the magnitude of the SC is believed to be associated with the percentage of type II muscle fibres and their recruitment. In this study we investigated the relationship between the magnitude of the relative SC, citrate synthase activity, UCP2 and UCP3 mRNA levels and muscle fibre composition in both endurance-trained and recreationally active subjects. METHOD: The magnitude of the relative SC was measured in 12 endurance-trained (Tr) and 15 recreationally active (RA) male subjects. The magnitude of the relative SC was determined as the difference between the end-exercise [V]O(2) and 3 min [V]O(2) divided by the difference between end-exercise [V]O(2) and baseline [V]O(2). UCP2 and UCP3 mRNA expression in the vastus lateralis was measured by RT-PCR with beta-actin mRNA used as an internal control. These values were also normalized against cytochrome-b mRNA to control for training induced changes in mitochondria when comparing the Tr and RA groups. Type I, IIa and IIx skeletal muscle fibre composition was determined using a routine myosin ATPase histochemical staining technique. Citrate synthase (CS) activity was measured using spectrophotometric detection. RESULTS: The magnitude of the relative SC of the Tr group had the highest correlation with citrate synthase activity (r=-0.90, P<0.001) and that of the RA group with [V]O(2) peak (r=-0.68, P<0.01). For the Tr group other correlations with the magnitude of the relative SC included UCP3 mRNA levels (r=0.69, P<0.05) and the percentage of type I fibres (r=-0.58, P<0.05), while for the RA group they included UCP3 mRNA (r=0.58, P<0.05) and the percentage of type IIa muscle fibres (r=0.59, P<0.05). The Tr subjects had a lower relative SC (P=0.04) and a lower expression of UCP2 (P=0.04), and UCP3 mRNA (P=0.01) than the RA subjects. When the groups were combined the magnitude of the relative SC correlated with UCP3 mRNA (r=0.70, P<0.01), percentage of type IIa muscle fibres (r=0.60, P<0.01) and [V]O(2) peak (r=-0.73, P<0.01). Additionally UCP3 mRNA correlated with the percentage of type IIa muscle fibres (r=0.63, P<0.001). CONCLUSION: Citrate synthase activity and [V]O(2) peak are indicators of aerobic fitness. The high negative correlations between the magnitude of the relative SC and citrate synthase activity and [V]O(2) peak, of the Tr and RA subjects, respectively, suggests that the magnitude of the relative SC is inversely correlated with aerobic fitness. Additionally the correlations between UCP3 mRNA and the magnitude of the relative SC for both groups individually and combined suggest that the uncoupling activity of the UCP3 protein may also influence the magnitude of the relative SC.