Andrew Garnham

Exercise increases serum Hsp72 in humans

Abstract Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87 ± 0.24 and 1.02 ± 0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

Skeletal muscle adaptation and performance responses to once a day versus twice every second day endurance training regimens

We determined the effects of a cycle training program in which selected sessions were performed with low muscle glycogen content on training capacity and subsequent endurance performance, whole body substrate oxidation during submaximal exercise, and several mitochondrial enzymes and signaling proteins with putative roles in promoting training adaptation. Seven endurance-trained cyclists/triathletes trained daily (High) alternating between 100-min steady-state aerobic rides (AT) one day, followed by a high-intensity interval training session (HIT; 8 x 5 min at maximum self-selected effort) the next day. Another seven subjects trained twice every second day (Low), first undertaking AT, then 1-2 h later, the HIT. These training schedules were maintained for 3 wk. Forty-eight hours before and after the first and last training sessions, all subjects completed a 60-min steady-state ride (60SS) followed by a 60-min performance trial. Muscle biopsies were taken before and after 60SS, and rates of substrate oxidation were determined throughout this ride. Resting muscle glycogen concentration (412 +/- 51 vs. 577 +/- 34 micromol/g dry wt), rates of whole body fat oxidation during 60SS (1,261 +/- 247 vs. 1,698 +/- 174 micromol.kg(-1).60 min(-1)), the maximal activities of citrate synthase (45 +/- 2 vs. 54 +/- 1 mmol.kg dry wt(-1).min(-1)), and beta-hydroxyacyl-CoA-dehydrogenase (18 +/- 2 vs. 23 +/- 2 mmol.kg dry wt(-1).min(-1)) along with the total protein content of cytochrome c oxidase subunit IV were increased only in Low (all P < 0.05). Mitochondrial DNA content and peroxisome proliferator-activated receptor-gamma coactivator-1alpha protein levels were unchanged in both groups after training. Cycling performance improved by approximately 10% in both Low and High. We conclude that compared with training daily, training twice every second day compromised high-intensity training capacity. While selected markers of training adaptation were enhanced with twice a day training, the performance of a 1-h time trial undertaken after a 60-min steady-state ride was similar after once daily or twice every second day training programs.

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.

β-adrenergic stimulation of skeletal muscle HSL can be overridden by AMPK signaling

Hormone‐sensitive lipase (HSL), an important regulatory enzyme for triacylglycerol hydrolysis within skeletal muscle, is controlled by β‐adrenergic signaling as well as intrinsic factors related to contraction and energy turnover. In the current study, we tested the capacity of 5′AMP‐ activated protein kinase (AMPK) to suppress β‐adrenergic stimulation of HSL activity. Eight male subjects completed 60 min of cycle exercise at 70% VO2 peak on two occasions: either with normal (CON) or low (LG) pre‐exercise muscle glycogen content, which is known to enhance exercise‐induced AMPK activity. Muscle samples were obtained before and immediately after exercise. Pre‐exercise glycogen averaged 375 ± 35 and 163 ± 27 mmol•kg–1 dm for CON and LG, respectively. AMPK α‐2 was not different between trials at rest and was increased (3.7‐fold, P<0.05) by exercise during LG only. HSL activity did not differ between trials at rest and increased (0 min: 1.67 ± 0.13; 60 min: 2.60 ± 0.26 mmol•min–1•kg–1 dm) in CON. The exercise‐induced increase in HSL activity was attenuated by AMPK α‐2 activation in LG. The attenuated HSL activity during LG occurred despite higher plasma epinephrine levels (60 min: CON, 1.96 ± 0.29 vs LG, 4.25 ± 0.60 nM, P<0.05) compared with CON. Despite the attenuated HSL activity in LG, IMTG was decreased by exercise (0 min: 27.1 ± 2.0; 60 min: 22.5 ± 2.0 mmol.kg–1 dm, P<0.05), whereas no net reduction occurred in CON. To confirm the apparent effect of AMPK on HSL activity, we performed experiments in muscle cell culture. The epineprine‐induced increase in HSL activity was totally attenuated (P<0.05) by AICAR administration in L6 myotubes. These data provide new evidence indicating that AMPK is a major regulator of skeletal muscle HSL activity that can override β‐adrenergic stimulation. However, the increased IMTG degradation in LG suggests factors other than HSL activity are important for IMTG degradation.

BDNF, metabolic risk factors, and resistance training in middle-aged individuals

INTRODUCTION AND PURPOSE Brain-derived neurotrophic factor (BDNF) and physical inactivity contribute to the development of the metabolic syndrome (MetS). There appears to be an association between BDNF and risk factors for MetS, and the effects of resistance training (RT) on BDNF and metabolic risk in middle-aged individuals with high and low numbers of metabolic risk factors (HiMF and LoMF, respectively) are unclear and are the focus of this research. METHODS Forty-nine men (N = 25) and women (N = 24) aged 50.9 +/- 6.2 yr were randomized to four groups, HiMF training (HiMFT), HiMF control (HiMFC), LoMF training (LoMFT), and LoMF control (LoMFC). Before and after 10 wk of RT, participants underwent tests for muscle strength and anthropometry, and a fasting blood sample was taken. Data were analyzed using Spearman correlations and repeated-measures ANOVA. RESULTS BDNF was positively correlated with plasma triglycerides, glucose, HbA1C, and insulin resistance. BDNF was elevated in HiMF compared with LoMF (904.9 +/- 270.6 vs 709.6 +/- 239.8 respectively, P = 0.01). Training increased muscle strength and lean body mass but had no effect on BDNF levels or any examined risk factors. CONCLUSION BDNF levels correlated with risk factors for MetS and were elevated in individuals with HiMF. RT had no effect on BDNF levels or other risk factors for MetS. As RT has an effect on muscle strength and lean body mass, it should be added to other nonpharmacological interventions for middle-aged individuals with HiMF such as aerobic and/or diet.

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

Abstract. 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.

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.

Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle.

Characterization of expression of, and consequently also the acute exercise effects on, Na+,K+‐ATPase isoforms in human skeletal muscle remains incomplete and was therefore investigated. Fifteen healthy subjects (eight males, seven females) performed fatiguing, knee extensor exercise at ∼40% of their maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue and 3 and 24 h postexercise, and analysed for Na+,K+‐ATPase α1, α2, α3, β1, β2 and β3 mRNA and crude homogenate protein expression, using Real‐Time RT‐PCR and immunoblotting, respectively. Each individual expressed gene transcripts and protein bands for each Na+,K+‐ATPase isoform. Each isoform was also expressed in a primary human skeletal muscle cell culture. Intense exercise (352 ± 69 s; mean ±s.e.m.) immediately increased α3 and β2 mRNA by 2.4‐ and 1.7‐fold, respectively (P < 0.05), whilst α1 and α2 mRNA were increased by 2.5‐ and 3.5‐fold at 24 h and 3 h postexercise, respectively (P < 0.05). No significant change occurred for β1 and β3 mRNA, reflecting variable time‐dependent responses. When the average postexercise value was contrasted to rest, mRNA increased for α1, α2, α3, β1, β2 and β3 isoforms, by 1.4‐, 2.2‐, 1.4‐, 1.1‐, 1.0‐ and 1.0‐fold, respectively (P < 0.05). However, exercise did not alter the protein abundance of the α1–α3 and β1–β3 isoforms. Thus, human skeletal muscle expresses each of the Na+,K+‐ATPase α1, α2, α3, β1, β2 and β3 isoforms, evidenced at both transcription and protein levels. Whilst brief exercise increased Na+,K+‐ATPase isoform mRNA expression, there was no effect on isoform protein expression, suggesting that the exercise challenge was insufficient for muscle Na+,K+‐ATPase up‐regulation.

Inflammation, hepatic enzymes and resistance training in individuals with metabolic risk factors.

Aims  Increases in inflammatory markers, hepatic enzymes and physical inactivity are associated with the development of the metabolic syndrome (MetS). We examined whether inflammatory markers and hepatic enzymes are correlated with traditional risk factors for MetS and studied the effects of resistance training (RT) on these emerging risk factors in individuals with a high number of metabolic risk factors (HiMF, 2.9 ± 0.8) and those with a low number of metabolic risk factors (LoMF, 0.5 ± 0.5).

Fat adaptation followed by carbohydrate restoration increases AMPK activity in skeletal muscle from trained humans.

We have previously reported that 5 days of a high-fat diet followed by 1 day of high-carbohydrate intake (Fat-adapt) increased rates of fat oxidation and decreased rates of muscle glycogenolysis during submaximal cycling compared with consumption of an isoenergetic high-carbohydrate diet (HCHO) for 6 days (Burke et al. J Appl Physiol 89: 2413-2421, 2000; Stellingwerff et al. Am J Physiol Endocrinol Metab 290: E380-E388, 2006). To determine potential mechanisms underlying shifts in substrate selection, eight trained subjects performed Fat-adapt and HCHO. On day 7, subjects performed 1-h cycling at 70% peak O2 uptake. Muscle biopsies were taken immediately before and after exercise. Resting muscle glycogen content was similar between treatments, but muscle triglyceride levels were higher after Fat-adapt (P < 0.05). Resting AMPK-alpha1 and -alpha2 activity was higher after Fat-adapt (P = 0.02 and P = 0.05, respectively), while the phosphorylation of AMPKs downstream target, acetyl-CoA carboxylase (pACC at Ser221), tended to be elevated after Fat-adapt (P = 0.09). Both the respiratory exchange ratio (P < 0.01) and muscle glycogen utilization (P < 0.05) were lower during exercise after Fat-adapt. Exercise increased AMPK-alpha1 activity after HCHO (P = 0.03) but not Fat-adapt. Exercise was associated with an increase in pACC at Ser221 for both dietary treatments (P < 0.05), with postexercise pACC Ser221 higher after Fat-adapt (P = 0.02). In conclusion, compared with HCHO, Fat-adapt increased resting muscle triglyceride stores and resting AMPK-alpha1 and -alpha2 activity. Fat-adapt also resulted in higher rates of whole body fat oxidation, reduced muscle glycogenolysis, and attenuated the exercise-induced rise in AMPK-alpha1 and AMPK-alpha2 activity compared with HCHO. Our results demonstrate that AMPK-alpha1 and AMPK-alpha2 activity and fuel selection in skeletal muscle in response to exercise can be manipulated by diet and/or the interactive effects of diet and exercise training.