The effect of the hyperbaric environment on heat shock protein 72 expression in vivo.

Heat shock protein 72 (HSP72) is expressed in response to stress and has been demonstrated to follow a diurnal expression pattern within monocytes and is sensitive to changes in core temperature. Numerous studies have shown changes in HSP72 expression within cell lines exposed to hyperbaric conditions. No studies have investigated changes in HSP72 expression in vivo. Six males participated in the study and were exposed to hyperbaric air and hyperbaric oxygen a week apart. Monocyte HSP72 was analyzed by flow cytometry at 09:00, 13:00, 17:00, 21:00 with hyperbaric oxygen or hyperbaric air breathing commencing at 15:00 for 78 min at a pressure of 2.8 ATA. HSP72 under normoxia followed the established trend; however, following the hyperbaric air or oxygen exposure a reduction in detectable HSP72 was observed at 17:00 and 21:00. No changes in core temperature were observed between 13:00 and 21:00 for any condition. The data show that HSP72 expression is impaired following hyperbaric air (HA) exposure, when compared with control or hyperbaric oxygen (HO) exposure.

Variation in basal heat shock protein 70 is correlated to core temperature in human subjects.

Heat shock proteins are highly conserved proteins and play an important chaperone role in aiding the folding of nascent proteins within cells. The heat shock protein response to various stressors, both in vitro and in vivo, is well characterised. However, basal levels of heat shock protein 70 (Hsp70) have not previously been investigated. Monocyte-expressed Hsp70 was determined every 4 h, over a 24 h time period, in 17 healthy male subjects (177 +/- 6.4 cm, 75.7 +/- 10.9 kg, 19.8 +/- 4.3 years) within a temperature and activity controlled environment. Core temperature was measured at 5-min intervals during the 24 h period. Hsp70 showed significant diurnal variation (F = 7.4; p < 0.001), demonstrating peaks at 0900 and 2100 hours, and a nadir at 05.00. Core temperature followed a similar temporal trend (range = 35.96-38.10 degrees C) and was significantly correlated with Hsp70 expression (r(s) = 0.44; p < 0.001). These findings suggest a high responsiveness of Hsp70 expression in monocytes to slight variations in core temperature.

The effect of 15 consecutive days of heat-exercise acclimation on heat shock protein 70.

The purpose of this study was to investigate the alterations in serum heat shock protein (Hsp) 70 levels during a 15-consecutive-day intermittent heat-exercise protocol in a 29-year-old male ultra marathon runner. Heat acclimation, for the purpose of physical activities in elevated ambient temperatures, has numerous physiological benefits including mechanisms such as improved cardiac output, increased plasma volume and a decreased core temperature (T (c)). In addition to the central adaptations, the role of Hsp during heat acclimation has received an increasing amount of attention. The acclimation protocol applied was designed to correspond with the athlete's tapering period for the 2007 Marathon Des Sables. The subject (VO(2)max = 50.7 ml.kg(-1).min(-1), peak power output [PPO] = 376 W) cycled daily for 90 min at a workload corresponding to 50% of VO(2)max in a temperature-controlled room (average WBGT = 31.9 +/- 0.9 degrees C). Venous blood was sampled before and after each session for measurement of serum osmolality and serum Hsp70. In addition, T (c), heart rate (HR) and power output (PO) was measured throughout the 90 min to ensure that heat acclimation was achieved during the 15-day period. The results show that the subject was successfully heat acclimated as seen by the lowered HR at rest and during exercise, decreased resting and exercising T (c) and an increased PO. The heat exercise resulted in an initial increase in Hsp70 concentrations, known as thermotolerance, and the increase in Hsp70 after exercise was inversely correlated to the resting values of Hsp70 (Spearman's rank correlation = -0.81, p < 0.01). Furthermore, the 15-day heat-exercise protocol also increased the basal levels of Hsp70, a response different from that of thermotolerance. This is, as far as we are aware, the first report showing Hsp70 levels during consecutive days of intermittent heat exposure giving rise to heat acclimation. In conclusion, a relatively longer heat acclimation protocol is suggested to obtain maximum benefit of heat acclimation inclusive of both cellular and systemic adaptations.

Microparticle-associated vascular adhesion molecule-1 and tissue factor follow a circadian rhythm in healthy human subjects.

An increased risk of death or severe injury due to late-morning thrombotic events is well established. Tissue factor (TF) is the initiator of the coagulation cascade, and endothelial stresses, coupled with production of pro-coagulant microparticles (MP) are also important factors in loss of haemostasis. TF and vascular cell adhesion molecule-1 (VCAM-1) -positive cell microparticles were assessed periodically over a 24-hour (h) period in healthy human subjects to ascertain if they followed a circadian rhythm. Eleven healthy male subjects were assessed in a temperature-controlled environment with dietary intake consistent between subjects. Blood samples were taken every 4 h by venipuncture, and TF and VCAM-1 positive microparticles were quantified by flow cytometry. A significant circadian rhythm was observed in VCAM-1 MP (p=or<0.0001), and a trend was shown, although not statistically significant (p=0.065) in TF microparticles. A peak was observed at 9 a.m. for VCAM-1 positive MP, followed by a decrease and subsequent peak at 9 p.m. and a minimum at 5 a.m. TF-positive MP followed a strikingly similar trend in both variation and absolute numbers with a delay. A circadian rhythm was observed in VCAM-1 and less so TF-positive MP. This has significant implications in terms of the well known increased risk of cardiovascular thrombotic events matching this data. To our knowledge this is the first such report of quantified measurements of these MP over a 24-h period and the only measurement of a 24-h variation of in-vivo blood-borne TF.

Inducible heat shock protein 70 and its role in preconditioning and exercise.

Heat shock proteins (Hsp) are well known to be expressed in response to a range of cellular stresses. They are known to convey protection against protein denaturation and a subsequent immediate stress. Inducible heat shock protein 70 (Hsp70) is among the most studied of these stress proteins and its role and function are discussed here in terms of thermal and in particular exercise preconditioning. Preconditioning has been shown to confer cellular protection via expression Hsp, which may be of benefit in preventing protein damage following subsequent periods of exercise. Many studies have used animal models to gather data on Hsp70 and these and the most recent human studies are discussed.

A1 receptor deficiency causes increased insulin and glucagon secretion in mice.

Adenosine influences metabolism and the adenosine receptor antagonist caffeine decreases the risk of type 2 diabetes. In this study the metabolic role of one adenosine receptor subtype, the adenosine A(1)R, was evaluated in mice lacking this receptor [A(1)R (-/-)]. The HbA1c levels and body weight were not significantly different between wild type [A(1)R (+/+)] and A(1)R (-/-) mice (3-4 months) fed normal lab chow. At rest, plasma levels of glucose, insulin and glucagon were similar in both genotypes. Following glucose injection, glucose tolerance was not appreciably altered in A(1)R (-/-) mice. Glucose injection induced sustained increases in plasma insulin and glucagon levels in A(1)R (-/-) mice, whereas A(1)R (+/+) control mice reacted with the expected transient increase in insulin and decrease in glucagon levels. Pancreas perfusion experiments showed that A(1)R (-/-) mice had a slightly higher basal insulin secretion than A(1)R (+/+) mice. The first phase insulin secretion (initiated with 16.7 mM glucose) was of the same magnitude in both genotypes, but the second phase was significantly enhanced in the A(1)R (-/-) pancreata compared with A(1)R (+/+). Insulin- and contraction-mediated glucose uptake in skeletal muscle were not significantly different between in A(1)R (-/-) and A(1)R (+/+) mice. All adenosine receptors were expressed at mRNA level in skeletal muscle in A(1)R (+/+) mice and the mRNA A(2A)R, A(2B)R and A(3)R levels were similar in A(1)R (-/-) and A(1)R (+/+) mice. In conclusion, the A(1)R minimally affects muscle glucose uptake, but is important in regulating pancreatic islet function.

Activation of aconitase in mouse fast-twitch skeletal muscle during contraction-mediated oxidative stress.

Aconitase is a mitochondrial enzyme that converts citrate to isocitrate in the tricarboxylic acid cycle and is inactivated by reactive oxygen species (ROS). We investigated the effect of exercise/contraction, which is associated with elevated ROS production, on aconitase activity in skeletal muscle. Humans cycled at 75% of maximal workload, followed by six 60-s bouts at 125% of maximum workload. Biopsies were taken from the thigh muscle at rest and after the submaximal and supramaximal workloads. Isolated mouse extensor digitorum longus (EDL; fast twitch) and soleus (slow twitch) muscles were stimulated to perform repeated contractions for 10 min. Muscles were analyzed for enzyme activities and glutathione status. Exercise did not affect aconitase activity in human muscle despite increased oxidative stress, as judged by elevated levels of oxidized glutathione. Similarly, repeated contractions did not alter aconitase activity in soleus muscle. In contrast, repeated contractions significantly increased aconitase activity in EDL muscle by approximately 50%, despite increased ROS production. This increase was not associated with a change in the amount of immunoreactive aconitase (Western blot) but was markedly inhibited by cyclosporin A, an inhibitor of the protein phosphatase calcineurin. Immunoprecipitation experiments demonstrated that aconitase was phosphorylated on serine residues. Aconitase in cell-free extracts was inactivated by the addition of the ROS hydrogen peroxide. In conclusion, the results suggest that aconitase activity can be regulated by at least two mechanisms: oxidation/reduction and phosphorylation/dephosphorylation. During contraction, a ROS-mediated inactivation of aconitase can be overcome, possibly by dephosphorylation of the enzyme. The dual-control system may be important in maintaining aerobic ATP production during muscle contraction.

Mechanical load plays little role in contraction-mediated glucose transport in mouse skeletal muscle.

The factors responsible for control of glucose transport during exercise are not fully understood. We investigated the role of mechanical load in contraction-mediated glucose transport in an isolated muscle preparation. Mouse extensor digitorum longus muscles were stimulated with repeated contractions for 10 min with or without N-benzyl-p-toluene sulphonamide (BTS, an inhibitor of myosin II ATPase) to block crossbridge activity. BTS inhibited force production during repeated contraction to approximately 5% of control. In contrast, BTS had little effect on glucose transport in the basal state (control = 0.55 +/- 0.04; BTS = 0.47 +/- 0.09 micromol (20 min)(-1) ml(-1)) or after contraction (control = 2.27 +/- 0.15; BTS = 2.10 +/- 0.16 micromol (20 min)(-1) ml(-1)). BTS did not significantly alter the contraction-mediated changes in high-energy phosphates, glutathione status (a measure of oxidant status) or AMP-activated protein kinase activity. In conclusion, these data show that mechanical load plays little role in contraction-mediated glucose transport. Instead, it is likely that the increased glucose transport during contraction is a consequence of the increase in myoplasmic Ca(2+) and the subsequent alterations in metabolism, e.g. increased energy turnover and production of reactive oxygen species.

Respiratory chain dysfunction in skeletal muscle does not cause insulin resistance.

Insulin resistance in skeletal muscle is a characteristic feature of diabetes mellitus type 2 (DM2). Several lines of circumstantial evidence suggest that reduced mitochondrial oxidative phosphorylation capacity in skeletal muscle is a primary defect causing insulin resistance and subsequent development of DM2. We have now experimentally tested this hypothesis by characterizing glucose homeostasis in tissue-specific knockout mice with progressive respiratory chain dysfunction selectively in skeletal muscle. Surprisingly, these knockout mice are not diabetic and have an increased peripheral glucose disposal when subjected to a glucose tolerance test. Studies of isolated skeletal muscle from knockout animals show an increased basal glucose uptake and a normal increase of glucose uptake in response to insulin. In summary, our findings indicate that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in DM2.

The role of Ca2+ influx for insulin-mediated glucose uptake in skeletal muscle.

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.

Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle.

Exercise increases glucose transport into skeletal muscle via a pathway that is poorly understood. We investigated the role of endogenously produced reactive oxygen species (ROS) in contraction-mediated glucose transport. Repeated contractions increased 2-deoxyglucose (2-DG) uptake roughly threefold in isolated, mouse extensor digitorum longus (fast-twitch) muscle. N-Acetylcysteine (NAC), a non-specific antioxidant, inhibited contraction-mediated 2-DG uptake by approximately 50% (P < 0.05 versus control values), but did not significantly affect basal 2-DG uptake or the uptake induced by insulin, hypoxia or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR, which mimics AMP-mediated activation of AMP-activated protein kinase, AMPK). Ebselen, a glutathione peroxidase mimetic, also inhibited contraction-mediated 2-DG uptake (by almost 60%, P < 0.001 versus control values). Muscles from mice overexpressing Mn2+-dependent superoxide dismutase, which catalyses H2O2 production from superoxide anions, exhibited a approximately 25% higher rate of contraction-mediated 2-DG uptake versus muscles from wild-type control mice (P < 0.05). Exogenous H2O2 induced oxidative stress, as judged by an increase in the [GSSG]/[GSH + GSSG] (reduced glutathione + oxidized glutathione) ratio to 2.5 times control values, and this increase was substantially blocked by NAC. Similarly, NAC significantly attenuated contraction-mediated oxidative stress as judged by measurements of glutathione status and the intracellular ROS level with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (P < 0.05). Finally, contraction increased AMPK activity and phosphorylation approximately 10-fold, and NAC blocked approximately 50% of these changes. These data indicate that endogenously produced ROS, possibly H2O2 or its derivatives, play an important role in contraction-mediated activation of glucose transport in fast-twitch muscle.

Cross bridges account for only 20% of total ATP consumption during submaximal isometric contraction in mouse fast-twitch skeletal muscle.

It is generally believed that cross bridges account for >50% of the total ATP consumed by skeletal muscle during contraction. We investigated the effect of N-benzyl-p-toluene sulfonamide (BTS), an inhibitor of myosin ATPase, on muscle force production and energy metabolism under near-physiological conditions (50-Hz stimulation frequency at 30 degrees C results in 35% of maximal force). Extensor digitorum longus muscles from mice were isolated and stimulated to perform continuous isometric tetanic contractions. Metabolites of energy metabolism were analyzed with fluorometric techniques. ATP turnover was estimated from the changes in phosphocreatine (PCr), ATP, and lactate (-2DeltaATP - DeltaPCr + [1.5Deltalactate]). During contractions (2-10 s), BTS decreased force production to approximately 5% of control. Under these conditions, BTS inhibited ATP turnover by only 18-25%. ATP turnover decreased markedly and similarly with and without BTS as the duration of contraction progressed. In conclusion, cross bridges (i.e., actomyosin ATPase) account for only a small fraction (approximately 20%) of the ATP consumption during contraction in mouse fast-twitch skeletal muscle under near-physiological conditions, suggesting that ion pumping is the major energy-consuming process.

Insulin-independent glycogen supercompensation in isolated mouse skeletal muscle: role of phosphorylase inactivation.

Glycogen supercompensation (increase in muscle glycogen content above basal) is an established phenomenon induced by unknown mechanisms. It consists of both insulin-dependent and -independent components. Here, we investigate insulin-independent glycogen supercompensation in isolated, intact extensor digitorum longus muscles from mice. Muscles were stimulated electrically, incubated in vitro with 5.5 mM glucose for up to 16 h and then analysed for glycogen, glucose uptake and enzyme activities. Basal glycogen was 84+/-6 micro mol glucosyl units/g dry muscle and was depleted by 80% after 10 min contraction. Glycogen increased after contraction, reaching a peak value of 113+/-9 micro mol glucosyl units/g dry muscle ( P<0.05 vs. basal) by 6 h, and returned to basal values by 16 h (84+/-8). Maximal activities of glycogen synthase, phosphorylase and alpha-glucosidase were not significantly altered by contraction or during the 6-h recovery period. Glycogen synthase fractional activity (0.17/7.2 mM glucose-6-P; inversely related to phosphorylation state of the enzyme) was increased about twofold early after contraction but then decreased and was slightly lower than baseline during the period of supercompensation (4-6 h). Phosphorylase fractional activity (+/-adenosine monophosphate; directly related to phosphorylation state of the enzyme) decreased to 60% of basal after contraction and decreased further during the initial 4 h of recovery to 40% of basal ( P<0.01 vs. basal). After 4 h recovery, glucose uptake was slightly (50%) higher in the stimulated than in the non-stimulated muscle ( P<0.01). Thus, insulin-independent glycogen supercompensation involves inactivation of phosphorylase and hence an inhibition of glycogen breakdown.

Contraction-mediated glycogenolysis in mouse skeletal muscle lacking creatine kinase: the role of phosphorylase b activation.

Skeletal muscle that is deficient in creatine kinase (CK-/-) exhibits accelerated glycogenolysis during contraction. Understanding this phenomenon could provide insight into the control of glycogenolysis during contraction. Therefore, glycogen breakdown was investigated in isolated extensor digitorum longus CK-/- muscle. Muscles were stimulated to produce repeated tetani for 20 s in the presence of sodium cyanide to block mitochondrial respiration. Accumulation of lactate after stimulation was similar in wild-type (WT) and CK-/- muscles, whereas accumulation of glucose-6-phosphate was twofold higher in CK-/- muscles, indicating greater glycogenolysis in CK-/- muscles. Total phosphorylase activity was decreased by almost 30 % in CK-/- muscle (P < 0.001). Phosphorylase fractional activity (-/+ 3.3 mM AMP) was similar in both groups in the basal state (about 10 %), but increased to a smaller extent in CK-/- muscles after stimulation (39 +/- 4 % vs. 52 +/- 4 % in WT, P < 0.05). Inorganic phosphate, the substrate for phosphorylase, increased marginally in CK-/- muscles after stimulation (basal = 25.3 +/- 2.2 micromol (g dry muscle)-1; stimulated = 33.9 +/- 2.3 micromol (g dry muscle)-1), but substantially in WT muscles (basal = 11.4 +/- 0.7 micromol (g dry muscle)-1; stimulated = 54.2 +/- 4.5 micromol (g dry muscle)-1). Kinetic studies of phosphorylase b (dephosphorylated enzyme) from muscle extracts in vitro demonstrated higher relative activities in CK-/- muscles (60-135 %) in response to low AMP concentrations (up to 50 microM) in both the basal state and after stimulation (P < 0.05), whereas no differences in activity between CK-/- and WT muscles were observed at high AMP concentrations (> 100 microM). These data indicate that allosteric activation of phosphorylase b accounts for the accelerated glycogenolysis in CK-/- muscle during contraction.