Józef Langfort

Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle.

The enzymic regulation of triacylglycerol breakdown in skeletal muscle is poorly understood. Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying demonstrated the presence of immunoreactive hormone-sensitive lipase (HSL), with the concentrations in soleus and diaphragm being more than four times the concentrations in extensor digitorum longus and epitrochlearis muscles. Neutral lipase activity determined under conditions optimal for HSL varied directly with immunoreactivity. Expressed relative to triacylglycerol content, neutral lipase activity in soleus muscle was about 10 times that in epididymal adipose tissue. In incubated soleus muscle, both neutral lipase activity against triacylglycerol (but not against a diacylglycerol analogue) and glycogen phosphorylase activity increased in response to adrenaline (epinephrine). The lipase activation was completely inhibited by anti-HSL antibody and by propranolol. The effect of adrenaline could be mimicked by incubation of crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase, while no effect of the kinase subunit was seen with supernatant from adrenaline-treated muscle. The results indicate that HSL is present in skeletal muscle and is stimulated by adrenaline via beta-adrenergic activation of cAMP-dependent protein kinase. The concentration of HSL is higher in oxidative than in glycolytic muscle, and the enzyme is activated in parallel with glycogen phosphorylase.

Stimulation of hormone-sensitive lipase activity by contractions in rat skeletal muscle

Because the enzymic regulation of muscle triglyceride breakdown is poorly understood we studied whether neutral lipase in skeletal muscle is activated by contractions. Incubated soleus muscles from 70 g rats were electrically stimulated for 60 min. Neutral lipase activity against triacylglycerol increased after 1 and 5 min of contractions [0.36 +/- 0.02 (basal) versus 0.49 +/- 0.05 (1 min) and 0.54 +/- 0.05 (5 min) m-unit.mg of protein(-1), means +/- S.E.M., P < 0.05]. After 10 min the neutral lipase activity (0.40 +/- 0.05 m-unit.mg of protein(-1)) had decreased to basal values (P > 0.05). The contraction-mediated increase in lipase activity was increased by approximately 110% when muscle was stimulated in the presence of okadaic acid. Conversely, treatment of muscle homogenate with alkaline phosphatase completely reversed the contraction-mediated lipase activation. Lipase activity did not change during contractions when analysed in the presence of anti-hormone-sensitive-lipase (HSL) antibody [0.17 +/- 0.02 (basal) versus 0.21 +/- 0.02 (5 min) m-unit.mg of protein(-1), P > 0.05]. Furthermore, immunoprecipitation with affinity-purified anti-HSL antibody reduced muscle-HSL protein concentration by 81+/-4% and caused similar reductions in lipase activity against triacylglycerol and in the contraction-induced increase in this activity. Neither prior sympathectomy [0.33+/- 0.02 (basal) versus 0.53 +/- 0.06 (5 min) m-unit.mg of protein(-1), P < 0.05] nor propranolol impaired the lipase response to contractions. Glycogen phosphorylase activity in the absence of AMP increased after 1 min [27.3 +/- 3.1 versus 8.9 +/- 1.8% (activity without AMP/total activity with AMP), P < 0.05] and returned to basal levels after 5 min. In conclusion, skeletal-muscle-immunoreactive HSL is transiently stimulated by contractions and the mechanism probably involves phosphorylation. The time course of HSL activation is similar to that of glycogen phosphorylase. Apparently, the two enzymes are regulated in parallel by contraction-induced as well as hormonal mechanisms, allowing simultaneous recruitment of all major extra- and intra-muscular energy stores.

Expression profiling reveals differences in metabolic gene expression between exercise‐induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise‐induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise‐induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in β‐oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise‐induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise‐induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.

Anaerobic threshold in rats

1. The aim of this study was to find out whether the anaerobic threshold (AT) can be estimated in rats running at increasing speed and if so what is the reproducibility of the measurements. 2. Lactate (LA) concentrations in blood taken from 11 rats were determined during a discontinued, multistage treadmill exercise test repeated four times in each animal. 3. It was found that blood LA changes vs speed have an exponential pattern with a distinct, rapid rise at the speed above 25 m/min which corresponds to blood LA of approx. 4 mmol/l. 4. The variation coefficient of the speed at which AT occurred in individual animals ranged between 10 and 20%. 5. These results offer a potential application of AT determination in the animal studies concerning mechanisms controlling exercise metabolism.

Adrenaline and glycogenolysis in skeletal muscle during exercise: a study in adrenalectomised humans

1 The role of adrenaline in regulating muscle glycogenolysis and hormone‐sensitive lipase (HSL) activity during exercise was examined in six adrenaline‐deficient bilaterally adrenalectomised, adrenocortico‐hormonal‐substituted humans (Adr) and in six healthy control individuals (Con). 2 Subjects cycled for 45 min at ∼70 % maximal pulmonary O2 uptake (V̇O2,max) followed by 15 min at ∼86 %V̇O2,max either without (−Adr and Con) or with (+Adr) adrenaline infusion that elevated plasma adrenaline levels (45 min, 4.49 ± 0.69 nmol l−1; 60 min, 12.41 ± 1.80 nmol l−1). Muscle samples were obtained at 0, 45 and 60 min of exercise. 3 In −Adr and Con, muscle glycogen was similar at rest (−Adr, 409 ± 19 mmol (kg dry wt)−1; Con, 453 ± 24 mmol (kg dry wt)−1) and following exercise (−Adr, 237 ± 52 mmol (kg dry wt)−1; Con, 227 ± 50 mmol (kg dry wt)−1). Muscle lactate, glucose‐6‐phosphate and glucose were similar in −Adr and Con, whereas glycogen phosphorylase (a/a+b× 100%) and HSL (% phosphorylated) activities increased during exercise in Con only. Adrenaline infusion increased activities of phosphorylase and HSL as well as blood lactate concentrations compared with those in −Adr, but did not enhance glycogen breakdown (+Adr, glycogen following exercise: 274 ± 55 mmol (kg dry wt)−1) in contracting muscle. 4 The present findings demonstrate that during exercise muscle glycogenolysis can occur in the absence of adrenaline, and that adrenaline does not enhance muscle glycogenolysis in exercising adrenalectomised subjects. Although adrenaline increases the glycogen phosphorylase activity it is not essential for glycogen breakdown in contracting muscle. Finally, a novel finding is that the activity of HSL in human muscle is increased in exercising man and this is due, at least partly, to stimulation by adrenaline.

HORMONE-SENSITIVE LIPASE (HSL) EXPRESSION AND REGULATION IN SKELETAL MUSCLE

Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration of lipase in adipose tissue. In agreement with the in vitro findings, in adrenalectomized patients an increase in muscle neutral lipase activity was found at the end of prolonged exercise only if epinephrine was infused. In accordance with feedforward regulation of substrate mobilization in exercise, our studies have shown that HSL is present in skeletal muscle cells and is stimulated in parallel with glycogen phosphorylase by both epinephrine and contractions. HSL adapts differently to training in muscle compared with adipose tissue.

The effect of exercise training on hormone-sensitive lipase in rat intra-abdominal adipose tissue and muscle

1 Adrenaline‐stimulated lipolysis in adipose tissue may increase with training. The rate‐limiting step in adipose tissue lipolysis is catalysed by the enzyme hormone‐sensitive lipase (HSL). We studied the effect of exercise training on the activity of the total and the activated form of HSL, referred to as HSL (DG) and HSL (TG), respectively, and on the concentration of HSL protein in retroperitoneal (RE) and mesenteric (ME) adipose tissue, and in the extensor digitorum longus (EDL) and soleus muscles in rats. 2 Rats (weighing 96 ± 1 g, mean ±s.e.m.) were either swim trained (T, 18 weeks, n= 12) or sedentary (S, n= 12). Then RE and ME adipose tissue and the EDL and soleus muscles were incubated for 20 min with 4.4 μm adrenaline. 3 HSL enzyme activities in adipose tissue were higher in T compared with S rats. Furthermore, in RE adipose tissue, training also doubled HSL protein concentration (P < 0.05). In ME adipose tissue, the HSL protein levels did not differ significantly between T and S rats. In muscle, HSL (TG) activity as well as HSL (TG)/HSL (DG) were lower in T rats, whereas HSL (DG) activity did not differ between groups. Furthermore, HSL protein concentration in muscle did not differ between T and S rats (P > 0.05). 4 In conclusion, training increased the amount of HSL and the sensitivity of HSL to stimulation by adrenaline in intra‐abdominal adipose tissue, the extent of the change differing between anatomical locations. In contrast, in skeletal muscle the amount of HSL was unchanged and its sensitivity to stimulation by adrenaline reduced after training.

Skiing across the Greenland icecap: divergent effects on limb muscle adaptations and substrate oxidation

SUMMARY This study investigates the adaptive response of the lower limb muscles and substrate oxidation during submaximal arm or leg exercise after a crossing of the Greenland icecap on cross-country skies. Before and after the 42-day expedition, four male subjects performed cycle ergometer and arm-cranking exercise on two separate days. On each occasion, the subjects exercised at two submaximal loads (arm exercise, 45 W and 100 W; leg exercise, 100 W and 200 W). In addition, peak oxygen uptake (V̇O2max) was determined for both leg and arm exercise. Before and after the crossing, a muscle biopsy was obtained from the vastus lateralis and the triceps brachii muscles prior to exercise (N=3). After the crossing, body mass decreased by 5.7±0.5 kg (in four of four subjects), whereas V̇O2max was unchanged in the arm (3.1±0.2 l min-1) and leg (4.0±0.1 l min-1). Before the crossing, respiratory exchange ratio (RER) values were 0.84±0.02 and 0.96±0.02 during submaximal arm exercise and 0.82±0.02 and 0.91±0.01 during submaximal leg exercise at the low and high workloads, respectively. After the crossing, RER was lower (in three of four subjects) during arm exercise (0.74±0.02 and 0.81±0.01) but was higher (in three of four subjects) during leg exercise (0.92±0.02 and 0.96±0.01) at the low and high workloads, respectively. Citrate synthase andβ -hydroxy-acyl-CoA-dehydrogenase activity was decreased by approximately 29% in vastus lateralis muscle and was unchanged in triceps brachii muscle. Fat oxidation during submaximal arm exercise was enhanced without a concomitant increase in the oxidative capacity of the triceps brachii muscle after the crossing. This contrasted with decreased fat oxidation during leg exercise, which occurred parallel to a decreased oxidative capacity in vastus lateralis muscle. Although the number of subjects is limited, these results imply that the adaptation pattern after long-term, prolonged, low-intensity, whole body exercise may vary dramatically among muscles.

Time course of GLUT4 and AMPK protein expression in human skeletal muscle during one month of physical training

Endurance training elicits profound adaptations of skeletal muscle, including increased expression of several proteins. The 5′‐AMP activated protein kinase (AMPK) may be one of these, considering the fact that acute exercise increases AMPK activity. Eight young (26 ± 1 year) lean, healthy males endurance trained one leg (while the other leg remained resting) on an ergometer bicycle for 30 min/day for four weeks (workload corresponding to ∼70% of maximal oxygen uptake). Muscle biopsies were obtained ∼18 h after the previous training session. On day eight GLUT4 protein expression was 36% higher in trained (T) compared with untrained (UT) (P < 0.05), but no further increase was seen at day 14 and 30 despite continuously increasing absolute workloads. Expression of AMPKα2 and actin did not change with training. In contrast, expression of AMPKα1 was 27% higher in T vs. UT muscle (P < 0.05) (measured only on day 30). Conclusions: GLUT4 protein expression increases substantially after seven days of endurance training with no further increase with prolonged training at progressively increasing workloads. AMPKα1 and α2 behave differently in their expression in response to endurance training. AMPKα1 protein content is increased after one month of training, while no change in AMPKα2 and actin expression was detected over the time course of the training period.

Arginine and ornithine supplementation increases growth hormone and insulin-like growth factor-1 serum levels after heavy-resistance exercise in strength-trained athletes.

Zając, A, Poprzęcki, S, Żebrowska, A, Chalimoniuk, M, and Langfort, J. Arginine and ornithine supplementation increases GH and IGF-1 serum levels after heavy-resistance exercise in strength-trained athletes. J Strength Cond Res 24(4): 1082-1090, 2010-This placebo-controlled double-blind study was designed to investigate the effect of arginine and ornithine (arg and orn) supplementation during 3-week heavy-resistance training on serum growth hormone/insulin-like growth factor-1/insulin-like growth factor-binding protein 3 (GH/IGF-1/IGFBP-3), testosterone, cortisol, and insulin levels in experienced strength-trained athletes. The subjects were randomly divided between a placebo group (n = 8) and the l-Arg/l-Orn-supplemented group (n = 9), and performed pre and posttraining standard exercise tests with the same absolute load, which consisted of the same exercise schedule as that applied in the training process. Fasting blood samples were obtained at rest, 2 minutes after the cessation of the strength exercise protocol, and after 1 hour of recovery. The resting concentrations of the investigated hormones and IGFBP-3 did not differ significantly between the study groups. In response to exercise test, all the hormones were elevated (p < 0.05) at both time points. Significant increases (p < 0.05) were observed in both GH and IGF-1 serum levels after arg and orn supplementation at both time points, whereas a significant decrease was seen in IGFBP-3 protein during the recovery period. Because there was no between-group difference in the remaining hormone levels, it appears that the GH/IGF-1/IGFBP-3 complex may be the major player in muscle tissue response to short-term resistance training after arg and orn supplementation.