Lactate elimination and glycogen resynthesis after intense bicycling.

OBJECTIVE: Muscles break down glycogen to lactate during intense exercise, and in the recovery period, glycogen reappears while lactate disappears. The purpose of this study was to examine to what extent lactate is resynthesized to glycogen within the formerly active muscles themselves in man. MATERIAL AND METHODS: Fifteen healthy young men cycled for 2 min to exhaustion. Muscle biopsies were taken from the knee extensor muscle before the exercise, just after the ride, and again after 45 min of recovery. In addition, blood samples were taken from the femoral artery and vein, and the leg blood flow was measured using the ultrasound Doppler technique. The muscle biopsies were analysed for glycogen, lactate and other metabolites, and the blood samples were analysed for lactate and glucose. The exchanges of lactate and glucose of the leg were assessed by multiplying the measured arterio-venous (a-v) differences by the blood flow. RESULTS: During the exercise the muscles broke down 20+/-4 mmol glycogen kg(-1) wet muscle mass and produced 26+/-1 mmol lactate kg(-1). In the recovery period after 24+/-1 mmol lactate kg(-1) had disappeared, of which 48 % was released to the blood, 52 % disappeared within the muscle. An R-value of 0.62 across the leg suggests that none of the lactate was oxidized. Altogether, 10+/-3 mmol glycogen kg(-1) reappeared during recovery. Glucose uptake accounted for 2 mmol kg(-1) and glycolytic intermediates (G-6-P and free glucose) accounted for 4 mmol kg(-1); 4 mmol glycogen kg(-1) (42 %) reappeared from unknown sources. CONCLUSIONS: The present data are compatible with the idea that around half of the lactate produced during intense bicycling is resynthesized to glycogen within the working muscles themselves in the recovery period after the bicycling.

Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation.

AIM: Effects of in vivo adrenaline infusion on subsequent insulin-stimulated glucose uptake and glycogen synthase activation was investigated in slow-twitch (soleus) and fast-twitch (epitrochlearis) muscles. Furthermore, role of glycogen content and Protein kinase B (PKB) phosphorylation for modulation insulin sensitivity was investigated. METHODS: Male Wistar rats received adrenaline from osmotic mini pumps ( approximately 150 microg kg(-1) h(-1)) for 1 or 12 days before muscles were removed for in vitro studies. RESULTS: Glucose uptake at physiological insulin concentration was elevated in both muscles after 1 and 12 days of adrenaline infusion. Insulin-stimulated glycogen synthase activation was also improved in both muscles. This elevated insulin sensitivity occurred despite the muscles were exposed to hyperglycaemia in vivo. After 1 day of adrenaline infusion, glycogen content was reduced in both muscles; insulin-stimulated PKB ser(473) phosphorylation was increased in both muscles only at the highest insulin concentration. After 12 days of adrenaline infusion, glycogen remained low in epitrochlearis, but returned to normal level in soleus; insulin-stimulated PKB phosphorylation was normal in both muscles. CONCLUSION: Insulin-stimulated glucose uptake and glycogen synthase activation were increased after adrenaline infusion. Increased insulin-stimulated glucose uptake and glycogen synthase activation after adrenaline infusion cannot be explained by a reduction in glycogen content or an increase in PKB phosphorylation. The mechanisms for the improved insulin sensitivity after adrenaline treatment deserve particular attention as they occur in conjunction with hyperglycaemia.

Leg gas exchange, release of glycerol, and uptake of fats after two minutes bicycling to exhaustion.

It is not known to what extent the muscles use fats and carbohydrates as substrate for oxidation after intense, anaerobic types of bicycling. Six healthy young men therefore bicycled at constant power for 2 min to exhaustion. Blood was drawn from indwelling catheters in the femoral artery and vein at intervals during the 1-h postexercise recovery. The blood samples were analysed for concentrations of O2 and CO2, and for free fatty acids (FFA), triacylglycerols (TG), and glycerol in plasma. The blood flow was also measured, and the rate of leg uptake of FFA, TG, and O2 and the release of CO2 and glycerol as well as its gas exchange ratio were calculated and integrated over the recovery period. The leg gas exchange ratio integrated over the exercise plus 1-h recovery period was 0.67 +/- 0.06 (mean +/- SEM ), suggesting pure fat oxidation. There was no statistically significant arterial-femoral-venous difference of FFA across the leg. The concentration of TG in plasma fell by 0.18 +/- 0.09 mmol L(-1) (32%) during the first 10 min of the recovery period, and the leg took up 18 +/- 8 micromol TG kg(-1) body mass (bm) during the whole 1-h recovery period. Free glycerol was released from the leg throughout the recovery period in excess of that released from hydrolysis of TG from plasma, suggesting that 30 +/- 10 micromol TG kg (-1) bm was hydrolysed, probably from intra-muscular stores. If fully oxidized, the triacylglycerols hydrolysed can account for 101% of the measured O2 uptake. Thus, muscle seems to use only triacylglycerols as substrate for its oxidative energy release after intense exercise.

Apoptotic effects of dichloro stearic and dichloro myristic acid in human hepatoma cells (HepG2).

Chlorinated fatty acids represent the major fraction of extractable organically bound chlorine in fish. After dietary intake such fatty acids may accumulate in adipose tissue, and even be transferred from mother to child via breast milk. We have previously reported that 9,10-dichloro stearic acid and 5,6-dichloro myristic acid inhibited cell growth. The aim of the present work was to investigate whether the growth inhibitory effect of these modified fatty acids might involve apoptosis. Human hepatoma cells (HepG2) were cultured for 4 days before addition of chlorinated fatty acids, and then cultured for another day before harvested. Morphological analysis was mainly done by light microscopy. In addition, fluorescence microscopy and electrophoretic analysis of DNA were carried out. The effect of 0.3 and 0.6 mmol/l was studied. Both chlorinated fatty acids seemed to cause a concentration-dependent increase in the relative abundance of pycnotic and broken nuclei, as well as nuclear fragments, with the strongest effect of dichloro stearic acid. Apoptosis by the chlorinated fatty acids was however less than that of docosahexaenoic acid, a known apoptosis effector. In conclusion, chlorinated fatty acids seem to possess the ability to cause apoptosis.

Effect of strenuous strength training on the Na-K pump concentration in skeletal muscle of well-trained men.

This study examined how strenuous strength training affected the Na-K pump concentration in the knee extensor muscle of well-trained men and whether leg muscle strength and endurance was related to the pump concentration. First, the pump concentration, taken as 3H-ouabain binding, was measured in top alpine skiers since strength training is important to them. Second, well-trained subjects carried out strenuous eccentric resistance training either 1, 2, or 3 times.week-1 for 3 months. The Na-K pump concentration, the maximal muscle strength in a full squat lift (one repetition maximum, 1 RM), and the muscle endurance, taken as the number of full squat lifts of a mass of 70% of the 1 RM load, were measured before and after the training period. The mean pump concentration of the alpine skiers was 425 (SEM 11) nmol.kg-1 wet muscle mass. The subjects in part two increased their maximal strength in a dose-dependent manner. The muscle endurance increased for all subjects but independently of the training programme. From a mean starting value of 356 (SEM 6) nmol.kg-1 the mean Na-K pump concentration increased by 54 (SEM 15) nmol.kg-1 (+15%, P < 0.001) when the results for all subjects were pooled. The effect was larger for those who had trained twice a week than for those who had trained only once a week (P = 0.025), suggesting that the effect of strength training depended on the amount of training carried out. The muscle strength and endurance were not related to the pump concentration, suggesting that the pumping power of this enzyme did not limit the performance during heavy lifting. However, the individual improvements in the endurance test during the training period correlated with the individual changes in the pump concentration (rSpearman = 0.5; P = 0.01) which could mean that a common factor both increases the pump concentration and makes the muscles more adapted to repeated heavy lifting.

Arterio-venous differences of blood acid-base status and plasma sodium caused by intense bicycling.

After intense exercise muscle may give off hydrogen ions independently of lactate, perhaps by a mechanism involving sodium ions. To examine this possibility further five healthy young men cycled for 2 min to exhaustion. Blood was drawn from catheters in the femoral artery and vein during exercise and at 1-h intervals after exercise. The blood samples were analysed for pH, blood gases, lactate, haemoglobin, and plasma proteins and electrolytes. Base deficit was calculated directly without using common approximations. The leg blood flow was also measured, thus allowing calculations of the leg's exchange of metabolites. The arterial blood lactate concentration rose to 14.2 +/- 1.0 mmol L-1, the plasma pH fell to 7. 18 +/- 0.02, and the base deficit rose 22% more than the blood lactate concentration did. The femoral-venous minus arterial differences peaked at 1.8 +/- 0.2 mmol L-1 (lactate), -0.24 +/- 0.01 (pH), and 4.5 +/- 0.4 mmol L-1 (base deficit), and -2.5 +/- 0.7 mmol L-1 (plasma sodium concentration corrected for volume changes). Thus, near the end of the exercise and for the first 10 min of the recovery period the leg gave off more hydrogen ions than lactate ions to the blood, and sodium left plasma in proportion to the extra hydrogen ions appearing. The leg's integrated excess release of hydrogen ions of 0.88 +/- 0.45 mmol kg-1 body mass was 67% of the integrated lactate release. Base deficit calculated by the traditional approximate equations underestimated the true value, but the error was less than 10%. We conclude that intense exercise and lactic acidosis may lead to a muscle release of hydrogen ions independent of lactate release, possibly by a Na+,H+ exchange. Hydrogen ions were largely buffered in the red blood cells.

Effect of training on the activity of five muscle enzymes studied on elite cross-country skiers.

This study examines the effect of training intensity on the activity of enzymes in m. vastus lateralis. Elite junior cross-country skiers of both sexes trained 12-15 h weeks-1 for 5 months at either moderate (60-70% of VO2max, MIG) or high training intensity (80-90% of the VO2max, close to the lactate threshold; HIG). Muscle biopsies for enzyme analyses and fibre typing were taken before and after the training period. Histochemical analyses on single fibres were done for three enzymes (succinate dehydrogenase [SDH], hydroxybutyrate dehydrogenase [HBDH], glycerol-3-phosphate dehydrogenase [GPDH]), while the activity of citrate synthase [CS] and phosphofructokinase [PFK] was measured on whole biopsies. The activity of GPDH was low in ST fibres and high in FT fibres. The activity of SDH and HBDH was high in both ST and FTa fibres but low in the FTb fibres. The HIG increased their performance more than the MIG did during the training period as judged from scores on a 20-min run test. The SDH activity rose by 6% for the HIG (P < 0.02). No effects of training were found in the activities of CS, HBDH or GPDH, neither in the two training groups nor for the two genders (P > or = 0.16). The PFK activity fell by 10% for the HIG (P=0.02), while no change was found for the MIG. For GPDH, CS and SDH the women's activity was approximately 20% less than the value for the men (P < 0.03). For PFK and HBDH there was no sex difference (P > or = 0.27). There were positive correlations between the activity of three of the enzymes (CS, SDH and GPDH) and the performance parameters (VO2max, cross-country skiing and running performance; r > or = 0.6, P < 0.01). No correlations were found between the PFK or HBDH activities and the performance parameters (r < or = 0.16, P > 0.05). This study suggests that intensities near the lactate threshold affect biochemical and physiological parameters examined in this study as well as the performance of elite skiers, and that the rate-limiting enzymes may be more sensitive to training than non-rate-limiting enzymes.

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content.

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.

Growth-modulating effects of dichloro myristic and dichloro stearic acid in cell cultures.

Chloro-containing fatty acids are a major fraction of extractable, organically bound chlorine in fish. It has been suggested that dichloro stearic acid (9,10-dichlorooctadecanoic acid) (C18) is metabolized to dichloro myristic acid (5,6-dichlorotetradecanoic acid) (C14) which accumulates in tissues. Hence, the biological effects of the C18 dichloro fatty acid could be due to formation of the C14 dichloro fatty acid. In this study we have compared the effects of dichloro stearic and dichloro myristic acid on growth of three widely differing cell lines. Both fatty acids inhibited cell growth; however, dichloro myristic acid had a weaker growth inhibitory effect than dichloro stearic acid. Dichloro myristic acid had a biphasic effect (i.e. growth was stimulated at low concentrations, followed by inhibition at higher concentrations) on the growth of human hepatoma cells and immortalized human kidney epithelial cells, but no such effect on human microvascular endothelial cells. The order of potency for growth inhibition by dichloro myristic acid was consistently human hepatoma cells>immortalized human kidney epithelial cells >human microvascular endothelial cells, whereas the relative potency of dichloro stearic acid was variable. Albumin alone stimulated cell growth and had a stronger protective effect against growth inhibition by dichloro myristic acid than against that of dichloro stearic acid. It seems unlikely that a major part of the effect of dichloro stearic acid on cell growth is caused by conversion to dichloro myristic acid.

Growth modulating effects of chlorinated oleic acid in cell cultures.

Chlorinated fatty acids represent a major fraction of extractable, organically bound chlorine in fish. After dietary intake such fatty acids may be transferred from the mother to the foetus through the placenta, and via breast milk to the child. In the present work we have studied the effect of chlorinated oleic acid on the growth of three widely differing types of cells in culture. Chlorinated oleic acid inhibited growth of Human Microvascular Endothelial Cells (HMVEC), Immortilized Human Kidney Epithelial (IHKE) cells, and human Hepatoma cells (HepG2). The order of potency was: HMVEC > IHKE > HepG2. Vitamin E counteracted the inhibitory effect of chlorinated oleic acid on HepG2 cells, but did not significantly affect the fatty acid effect on HMVEC or IHKE. Defatted serum albumin stimulated the growth of HMVEC and IHKE. With HMVEC there was no major interaction between the effect of albumin and chlorinated oleic acid on cell growth. In contrast, with IHKE albumin at low concentration abolished the growth inhibiting effect of chlorinated oleic acid and appreciably counteracted growth inhibition by the fatty acid of HepG2. We conclude that the growth modulation by chlorinated oleic acid and its interaction with vitamin E and albumin are cell specific.

Hard training for 5 mo increases Na(+)-K+ pump concentration in skeletal muscle of cross-country skiers.

To study how training affects the Na(+)-K+ pump concentration, 11 male and 9 female elite junior cross-country skiers trained 12-15 h/wk at 60-70% (moderate-intensity group) or 80-90% (high-intensity group) of their maximal O2 uptake for 5 mo. Muscle biopsies taken from the vastus lateralis muscle before and after the training period were analyzed for Na(+)-K+ pump concentration by the [3H]ouabain-binding technique. Before training, the concentration was 343 +/- 11 nmol/kg wet muscle mass (mean +/- SE) for the men and 281 +/- 14 nmol/kg for the women (18% less than for the men, P = 0.003). The Na(+)-K+ pump concentration rose by 49 +/- 11 nmol/kg (16%, P < 0.001) for all subjects pooled during the training period, and there was no difference between the two training groups (P = 0.3) or the sexes (P = 0.5) in this increase. The Na(+)-K+ pump concentration correlated with the maximal O2 uptake (r = 0.6, P = 0.003), with the performance during a 20-min treadmill run (r = 0.6, P = 0.003), and to the rank of the subjects' performance as cross-country skiers (Spearman's rank correlation coefficient = 0.76, P < 0.001). These data could mean that for elite cross-country skiers the performance is related to the Na(+)-K+ pump concentration. However, other studies have shown an equally high pump concentration for far less fit subjects, suggesting that the pump concentration may not be a limiting factor.

Blood reticulocyte count and plasma lactate dehydrogenase activity are positively related to the free fatty acid/albumin ratio in geriatric patients.

The objective of this study was to investigate the in vivo relationship between plasma free fatty acid (FFA)/albumin molar ratio and indicators of cellular damage. A case series study was carried out in 20 geriatric patients in a stable clinical condition. Their plasma albumin concentration was in the range 26-42 g l-1. There was a significant positive correlation between the FFA/albumin ratio and (a) reticulocyte count (r = 0.61, p = 0.006), (b) lactate dehydrogenase activity (r = 0.69, p = 0.002), and (c) haptoglobin concentration (r = 0.46, p = 0.05). The haemoglobin concentration was inversely related to relative reticulocyte count (r = -0.55, p = 0.01). Absolute and relative reticulocyte counts were positively associated (r = 0.92, p < 0.0001). The results are in accordance with the contention that a high FFA/albumin ratio in vivo may elicit cellular damage. Further studies are required to elucidate to what extent a high FFA/albumin ratio might be causally related to diseases.

Changes in Na+, K(+)-adenosinetriphosphatase, citrate synthase and K+ in sheep skeletal muscle during immobilization and remobilization.

The K+ balance and muscle activity seem to interact in a complex way with regard to regulating the muscle density of Na(+)-K+ pumps. The effect of immobilization was examined in ten sheep that had low muscle K+ content. Three additional sheep served as untreated controls. After being brought from pasture to sheep stalls one hindlimb was immobilized in a plaster splint for 9 weeks, and in five of the animals remobilization was carried out for a further 9 weeks. The weight bearing of the leg in plaster was recorded by a force plate. Open muscle biopsies from the vastus lateralis muscle were obtained before the study, after 9 weeks of immobilization, and after another 9 weeks of remobilization. The Na(+)-K+ pump density was measured as [3H]-ouabain binding to intact tissue, and citrate synthase activity was measured in tissue homogenate. The tissue content of K+ was measured in fat-free dried tissue. Muscle K+ content increased linearly by almost 70% through the 18-week period independent of intervention. Immobilization reduced thigh circumference by 8% (P < 0.05). A slight decrease in the area of type I fibres at 9 weeks and a slight increase at 18-weeks was found. The [3H]-ouabain binding was reduced by 39% and 22% in the immobilized and control legs, respectively, whereas citrate synthase activity was reduced by about 30% in both legs after 9 weeks of immobilization. During remobilization both the [3H]-ouabain binding and the citrate synthase activity increased to the same level as in the control animals. The plaster cast significantly reduced mass bearing of the immobilized leg, and a corresponding reduction in muscle activity must be assumed to have occurred in both legs as judged from citrate synthase activity. We concluded from this study that the reduction in the [3H]-ouabain binding during immobilization independent of an increase in muscle K+ content points to muscle activity as a strong stimulus for control of Na(+)-K+ pump density.

Enzymatic microdetermination of plasma and serum free fatty acids.

A simple and sensitive enzymatic method for determination of plasma and serum fatty acids (FAs) is described. The method is based on acylation of long chain FAs by a bacterial acyl-CoA synthetase (ACS) producing equivalent amounts of acyl-CoA and AMP. AMP production was measured using the coupled reaction of myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) allowing fluorinate detection of NADH. Two moles of NAD were produced per mole of FA acylated. Concentrations of substrates and enzymes were kept as low as possible maintaining the ACS reaction as rate limiting. Addition of fat-free human serum albumin (HSA) to standards reduced initial reaction rates but did not affect end-point fluorescence levels. Triton X-100 partly counteracted the inhibition by HSA. To keep albumin concentration low, plasma or serum samples were diluted by 1:400. Duplicate measurements of plasma or serum FA concentrations between 0 and 2 mmol l-1 can then be performed on 5 microliters samples with intra- and inter-assay variation coefficients of 1.7 and 4% respectively.

Phase-shifts of apparent circadian rhythms due to west and east transmeridian flights or to corresponding night-shift sleep displacements.

Phase movements of apparent circadian rhythms during 2 wk of forward or backward displacement of the sleep-wake cycle were investigated in four experimental series in a subject. The 7-hr delay or advance of sleep due to a westward or an eastward transmeridian flight was duplicated by corresponding sleep displacements during experimental night shifts. Sudden phase advances (or delays) by several hours were observed in the rhythms of continuously recorded rectal temperature and urinary excretion rates (4-hr collection intervals) of adrenaline, noradrenaline and aldosterone the first day after sleep-wake displacement. The desired 7-hr phase-shifts were reached more quickly and completely when the phase was delayed than when it was advanced. In addition, the best-fitting period of these rhythms became shorter than 24 hr when the phase was delayed, and longer than 24 hr when it was advanced. The apparent rhythms of urine flow and electrolyte excretions (potassium, sodium, zinc) were much weaker and their phase movements more irregular than those of hormonal excretion. It is concluded that the sudden phase-shifts resulted from the immediate adaptation of the exogenous components of the rhythms to the demands of the displaced sleep-wake patterns (masking effects) and that the true phase-shifts of the endogenous components followed more slowly and gradually.

Apparent phase-shifts of circadian rhythms (masking effects) during rapid shift rotation.

Six subjects worked an experimental 8 h rapidly rotating shift system in which 6 shifts were compressed into 5 d (work two mornings - 8 h interval - work two nights - 8 h interval - work two afternoons). Rectal temperature was continuously recorded and urinary excretion rates of adrenaline, noradrenaline, K+, Na+ and Hg+ were assessed in samples collected at 4 h intervals. Higher rectal temperatures and higher excretion rates of the two each catecholamines than those expected from the normal circadian rhythms were observed during night work, while lower than expected values were recorded forenoon sleep. In these three rhythms, immediate, significant 3-4 h delay phase-shifts occurred in the two days following night shifts. The acrophases reverted suddenly to their initial positions when the subjects returned to their normal sleep/wake schedule. The circadian rhythms of the excretion of electrolytes were not perceptibly disturbed. It is concluded that the apparent phase-shifts were due to a direct influence (masking effect) of the changes in body functions during sleep and wakefulness on the measured parameters, and that no true phase-shifts (entrainment) occurred in the endogenous circadian regulation. The evidence of such an immediate, partial adaptation to unconventional sleep/wake patterns supports the adoption of rapidly rotating shift systems which would not greatly interfere with the endogenous oscillators.

Circadian rhythmicity of the urinary excretion of mercury, potassium and catecholamines in unconventional shift-work systems.

The round the clock urinary excretion rates of mercury were assessed for two series of unconventional patterns of activity and sleep in subjects who were not exposed to occupational, medical, or other obvious sources of mercury. In the first series the urine was collected in 3-h periods from six subjects during the first and last 2 d of a four-week, continuous 6-h shift (car ferry, watches either 0800--1400 and 2000--0200 or 1400--2000 and 0200--0800). In the second series the urine was collected in 4-h periods from five subjects working an 8-h experimental rotation shift compressed into 5 d (work two mornings--8-h interval--work two nights--8-h interval--work two afternoons). The mean daily excretion rate of the 11 subjects (48 investigation days, 334 urine samples) was 14.5 pmol of mercury/min (range 5.5--24.4 pmol of mercury/min). The mercury excretion oscillated regularly during 24 h by +/- 20--25% of the individual's daily mean excretion rates. The peak excretion rates were found at 0652 in the first and 0642 in the second series (cosinor treatment). Due to the circadian rhythm the mean 24-h excretion rates were best represented (correlation coefficient 0.92) by analyses of urine produced around noon (spot samples, collection periods 1100--1400 and 1000-1400, respectively). The circadian oscillations of mercury excretion were not influenced by the widely different and varying activity-sleep patterns of the two series. The rhythmicity of potassium excretion (peaks at around 1400) was more irregular. The stable oscillations of mercury excretion contrasted most with the excretion of adrenaline and noradrenaline, which, without losing the basic 24-h rhythmicity, closely followed the unconventional patterns of activity and sleep.