IL-6 Gene Expression in Human Adipose Tissue in Response to Exercise – Effect of Carbohydrate Ingestion

Interleukin-6 (IL-6) is a cytokine involved in a number of immunological processes, but it is also linked to exercise and possibly energy status. During exercise, muscle IL-6 mRNA levels and plasma IL-6 levels are increased and further augmented when intramuscular glycogen levels are low. In contrast, the increase in plasma IL-6 is blunted if carbohydrate is administered, indicating a substrate-regulated induction of IL-6 in human skeletal muscle. Recent studies have demonstrated that IL-6 is also released from adipose tissue in response to an exercise bout. Furthermore, IL-6 has been demonstrated to have a lipolytic effect, thus possibly playing a role in mobilisation of energy as free fatty acids (FFA) in response to exercise. The purpose of the present study was to investigate the gene expression pattern of IL-6 in adipose tissue in response to exercise, and to determine whether gene expression was affected by the ingestion of carbohydrate. Eight male subjects performed 3 h of bicycling with ingestion of a carbohydrate drink or placebo. Fat biopsy samples and blood samples were obtained before, during and in the recovery phase of exercise. Both plasma IL-6 and adipose IL-6 mRNA levels increased in response to exercise. IL-6 gene expression was lower (   P < 0.05  ) in the CHO trial (1.98-fold increase, confidence interval (CI) 1.16–3.83) compared with the control (6.49-fold increase, CI 3.57–13.91) at end of exercise. Furthermore, CHO ingestion blunted the increase in plasma IL-6 levels (   P < 0.05  ) at end of exercise (26.0 ± 3.7 pg ml⁻¹ in the control vs. 15.6 ± 2.4 pg ml⁻¹ in the CHO trial). In conclusion, exercise results in an increase in IL-6 gene expression in adipose tissue in response to exercise, an effect that is significantly blunted by ingestion of carbohydrate.     

Interleukin-6 production in human subcutaneous abdominal adipose tissue: the effect of exercise

The interleukin-6 (IL-6) output from subcutaneous, abdominal adipose tissue was studied in nine healthy subjects before, during and for 3 h after 1 h two-legged bicycle exercise at 60 % maximal oxygen consumption. Seven subjects were studied in control experiments without exercise. The adipose tissue IL-6 output was measured by direct Fick technique. An artery and a subcutaneous vein on the anterior abdominal wall were catheterized. Adipose tissue blood flow was measured using the ¹³³Xe-washout method. In both studies there was a significant IL-6 output in the basal state and no significant change was observed during exercise. Post-exercise the IL-6 output began to increase after 30 min. Three hours post-exercise it was 58.6 ± 22.2 pg (100 g)⁻¹ min⁻¹. In the control experiments the IL-6 output also increased, but it only reached a level of 3.5 ± 0.8 pg (100 g)⁻¹ min⁻¹. The temporal profile of the post-exercise change in the IL-6 output closely resembles the changes in the outputs of glycerol and fatty acids, which we have described previously in the same adipose tissue depot. The difference is that it begins to increase ≈30 min before the glycerol and fatty acid outputs begin to increase. Thus, we suggest that the enhanced IL-6 production post-exercise in abdominal, subcutaneous adipose tissue may act locally via autocrine/paracrine mechanisms influencing lipolysis and fatty acid mobilization rate from this lipid depot.     

Exhaustive exercise causes an anti-inflammatory effect in skeletal muscle and a pro-inflammatory effect in adipose tissue in rats

It is well known that exhaustive exercise increases serum and skeletal muscle IL-6 concentrations. However, the effect of exhaustive exercise on the concentrations of other cytokines in the muscle and in the adipose tissue is controversial. The purpose of this study was to evaluate the effect of exhaustive exercise on mRNA and protein expression of IL-10, TNF-α and IL-6 in different types of skeletal muscle (EDL, soleus) and in two different depots of white adipose tissue (mesenteric–MEAT and retroperitoneal–RPAT). Rats were killed by decapitation immediately (E0 group, n = 6), 2 (E2 group, n = 6) and 6 (E6 group, n = 6) hours after the exhaustion protocol, which consisted of running on a treadmill (approximately 70% VO_2max for 50 min and then subsequently at an elevated rate that increased at 1 m/min every minute, until exhaustion). The control group (C group, n = 6) was not subjected to exercise. Cytokine protein expression increased in EDL, soleus, MEAT and RPAT from all exercised groups, as detected by ELISA. EDL IL-10 and TNF-α expression was higher than that of the soleus. The IL-10/TNF-α ratio was increased in the skeletal muscle, especially in EDL, but it was found to be decreased in the adipose tissue. These results show that exhaustive exercise presents a different effect depending on the tissue which is analysed: in the muscle, it induces an anti-inflammatory effect, especially in type 2 fibres, while the pro-inflammatory effect prevails in adipose tissue, possibly contributing to increased lipolysis to provide energy for the exercising muscle.     

Muscle as an endocrine organ: focus on muscle-derived interleukin-6

Skeletal muscle has recently been identified as an endocrine organ. It has, therefore, been suggested that cytokines and other peptides that are produced, expressed, and released by muscle fibers and exert paracrine, autocrine, or endocrine effects should be classified as "myokines." Recent research demonstrates that skeletal muscles can produce and express cytokines belonging to distinctly different families. However, the first identified and most studied myokine is the gp130 receptor cytokine interleukin-6 (IL-6). IL-6 was discovered as a myokine because of the observation that it increases up to 100-fold in the circulation during physical exercise. Identification of IL-6 production by skeletal muscle during physical activity generated renewed interest in the metabolic role of IL-6 because it created a paradox. On one hand, IL-6 is markedly produced and released in the postexercise period when insulin action is enhanced but, on the other hand, IL-6 has been associated with obesity and reduced insulin action. This review focuses on the myokine IL-6, its regulation by exercise, its signaling pathways in skeletal muscle, and its role in metabolism in both health and disease.     

Physical activity and modulation of systemic low-level inflammation

It has been recognized for some time that cardiovascular disease and type 2 diabetes are, to a major extent, inflammatory disorders associated with an environment characterized by a sedentary lifestyle together with abundant intakes of calories. Systemic low-level inflammation is suggested to be a cause as well as consequence of pathological processes with local tumor necrosis factor alpha production as an important biological driver. It is hypothesized that physical inactivity contributes to an enhanced proinflammatory burden independently of obesity, as regular muscle contractions mediate signals with myokines/cytokines as important messengers, which suppress proinflammatory activity at distant sites as well as within skeletal muscle. Muscle-derived interleukin (IL)-6 is considered to possess a central role in anti-inflammatory activities and health beneficial effects in relation to physical exercise. It is discussed how this fits the consistent observation that enhanced plasma levels of IL-6 represent a strong risk marker in chronic disorders associated with systemic low-level inflammation and all-cause mortality.     

Interstitial glycerol concentrations in human skeletal muscle and adipose tissue during graded exercise

AIM: It is not clear how lipolysis changes in skeletal muscle and adipose tissue during exercise of different intensities. We aimed at estimating this by microdialysis and muscle biopsy techniques. METHODS: Nine healthy, young men were kicking with both legs at 25% of maximal power (Wmax) for 45 min and then simultaneously with one leg at 65% and the other leg at 85% Wmax for 35 min. RESULTS: Glycerol concentrations in skeletal muscle and adipose tissue interstitial fluid and in arterial plasma increased (P<0.001) during low intensity exercise and increased (P<0.05) even more during moderate intensity exercise. The difference between interstitial muscle and arterial plasma water glycerol concentration, which indicates the direction of the glycerol flux, was positive (P<0.05) at rest (21 +/- 9 microM) and during exercise at 25% Wmax (18 +/- 6 microM). The difference decreased (P<0.05) with increasing exercise intensity and was not significantly different from zero during exercise at 65% (-11 +/- 17 microM) and 85% (-12 +/- 13 microM) Wmax. In adipose tissue, the difference between interstitial and arterial plasma water glycerol increased (P<0.001) with increasing intensity. The net triacylglycerol breakdown, measured chemically from the biopsy, did not differ significantly from zero at any exercise intensity although directional changes were similar to microdialysis changes. CONCLUSIONS: Skeletal muscle releases glycerol at rest and at low exercise intensity but not at higher intensities. This can be interpreted as skeletal muscle lipolysis peaking at low exercise intensities but could also indicate that glycerol is taken up in skeletal muscle at a rate which is increasing with exercise intensity.     

Letter to the Editor

The early pioneers in the field of metabolism during exercise such as Lindhard and Krogh understood the importance of fat as a fuel for muscle contraction. But they could not have understood the details of the pathways involved, as neither the metabolic role of adipose tissue nor the transport role of non-esterified fatty acids (NEFA) in the plasma was clearly understood at the time. We now recognize that the onset of muscular contraction coincides with an increase in the delivery of NEFA from adipose tissue, probably coordinated by the sympatho-adrenal system. During light exercise, adipose tissue-derived NEFA make up the majority of the oxidative fuel used by muscle. As exercise is prolonged, the importance of NEFA increases. The onset of exercise is marked by an increased proportion of NEFAs entering β-oxidation rather than re-esterification and recycling. At moderate intensities of exercise, other sources of fat, potentially plasma- and intramyocellular-triacylglycerol, supplement the supply of plasma NEFA. The delivery of NEFA is augmented by increased adipose tissue blood flow and by other stimuli such as atrial natriuretic peptide. Only during high-intensity exercise is there a failure of adipose tissue to deliver sufficient fatty acids for muscle (which is coupled with an inability of muscle to use them, even when fatty acids are supplied artificially). This limitation of adipose tissue NEFA delivery may reflect some feedback inhibition of lipolysis, perhaps via lactate, or possibly α-adrenergic inhibition of lipolysis at very high catecholamine concentrations.     

Calprotectin is released from human skeletal muscle tissue during exercise

Skeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle. IL-6 was infused for 3 h into healthy young males ( n = 7) and muscle biopsies obtained at time points 0, 3 and 6 h in these individuals and in resting controls. Affymetrix microarray analysis of gene expression changes in skeletal muscle biopsies identified a small set of genes changed by IL-6 infusion. RT-PCR validation confirmed that S100A8 and S100A9 mRNA were up-regulated 3-fold in skeletal muscle following IL-6 infusion compared to controls. Furthermore, S100A8 and S100A9 mRNA levels were up-regulated 5-fold in human skeletal muscle following cycle ergometer exercise for 3 h at ∼60% of     in young healthy males ( n = 8). S100A8 and S100A9 form calprotectin, which is known as an acute phase reactant. Plasma calprotectin increased 5-fold following acute cycle ergometer exercise in humans, but not following IL-6 infusion. To identify the source of calprotectin, healthy males ( n = 7) performed two-legged dynamic knee extensor exercise for 3 h with a work load of ∼50% of peak power output and arterial–femoral venous differences were obtained. Arterial plasma concentrations for calprotectin increased 2-fold compared to rest and there was a net release of calprotectin from the working muscle. In conclusion, IL-6 infusion and muscle contractions induce expression of S100A8 and S100A9 in skeletal muscle. However, IL-6 alone is not a sufficient stimulus to facilitate release of calprotectin from skeletal muscle.     

Absence of humoral mediated 5'AMP-activated protein kinase activation in human skeletal muscle and adipose tissue during exercise

5'AMP-activated protein kinase (AMPK) exists as a heterotrimer comprising a catalytic α subunit and regulatory β and γ subunits. The AMPK system is activated under conditions of cellular stress, indicated by an increase in the AMP/ATP ratio, as observed, e.g. in muscles during contractile activity. AMPK was originally thought to be activated only by local intracellular mechanisms. However, recently it has become apparent that AMPK in mammals is also regulated by humoral substances, e.g. catecholamines. We studied whether humoral factors released during exercise regulate AMPK activity in contracting and resting muscles as well as in abdominal subcutaneous adipose tissue in humans. In resting leg muscle and adipose tissue the AMPK activity was not up-regulated by humoral factors during one-legged knee extensor exercise even when arm cranking exercise, inducing a ∼20-fold increase in plasma catecholamine level, was added simultaneously. In exercising leg muscle the AMPK activity was increased by one-legged knee extensor exercise eliciting a whole body respiratory load of only 30%     but was not further increased by adding arm cranking exercise. In conclusion, during exercise with combined leg kicking and arm cranking, the AMPK activity in human skeletal muscle is restricted to contracting muscle without influence of marked increased catecholamine levels. Also, with this type of exercise the catecholamines or other humoral factors do not seem to be physiological regulators of AMPK in the subcutaneous adipose tissue.     

Metabolic effects of interleukin-6 in human splanchnic and adipose tissue

Interleukin-6 (IL-6) was infused intravenously for 2.5 h in seven healthy human volunteers at a dose giving rise to a circulating IL-6 concentration of ≈35 ng l⁻¹. The metabolic effects of this infusion were studied in subcutaneous adipose tissue on the anterior abdominal wall and in the splanchnic tissues by the Fick principle after catheterizations of an artery, a subcutaneous vein draining adipose tissue, and a hepatic vein, and measurements of regional adipose tissue and splanchnic blood flows. In control studies without IL-6 infusion subcutaneous adipose tissue metabolism was studied by the same technique in eight healthy subjects. The net release of glycerol and fatty acids from the subcutaneous abdominal adipose tissue remained constant in the control experiment. IL-6 infusion gave rise to increase in net glycerol release in subcutaneous adipose tissue while the net release of fatty acids did not change significantly. In the splanchnic region IL-6 elicited a pronounced vasodilatation, and the uptake of fatty acids and the gluconeogenic precursors glycerol and lactate increased significantly. The splanchnic net output of glucose and triacylglycerol did not change during the IL-6 infusion. It is concluded that IL-6 elicits lipolytic effects in human adipose tissue in vivo , and that IL-6 also has effects on the splanchnic lipid and carbohydrate metabolism.     

Muscle and bone,two interconnected tissues

As bones are levers for skeletal muscle to exert forces, both are complementary and essential for locomotion and individual autonomy. In the past decades, the idea of a bone–muscle unit has emerged. Numerous studies have confirmed this hypothesis from in utero to aging works. Space flight, bed rest as well as osteoporosis and sarcopenia experimentations have allowed to accumulate considerable evidence. Mechanical loading is a key mechanism linking both tissues with a central promoting role of physical activity. Moreover, the skeletal muscle secretome accounts various molecules that affect bone including insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-2), interleukin-6 (IL-6), IL-15, myostatin, osteoglycin (OGN), FAM5C, Tmem119 and osteoactivin. Even though studies on the potential effects of bone on muscle metabolism are sparse, few osteokines have been identified. Prostaglandin E2 (PGE2) and Wnt3a, which are secreted by osteocytes, osteocalcin (OCN) and IGF-1, which are produced by osteoblasts and sclerostin which is secreted by both cell types, might impact skeletal muscle cells. Cartilage and adipose tissue are also likely to participate to this control loop and should not be set aside. Indeed, chondrocytes are known to secrete Dickkopf-1 (DKK-1) and Indian hedgehog (Ihh) and adipocytes produce leptin, adiponectin and IL-6, which potentially modulate bone and muscle metabolisms. The understanding of this system will enable to define new levers to prevent/treat sarcopenia and osteoporosis at the same time. These strategies might include nutritional interventions and physical exercise.     

Physical activity and exercise in the regulation of human adipose tissue physiology

Physical activity and exercise are key components of energy expenditure and therefore of energy balance. Changes in energy balance alter fat mass. It is therefore reasonable to ask: What are the links between physical activity and adipose tissue function? There are many complexities. Physical activity is a multifaceted behavior of which exercise is just one component. Physical activity influences adipose tissue both acutely and in the longer term. A single bout of exercise stimulates adipose tissue blood flow and fat mobilization, resulting in delivery of fatty acids to skeletal muscles at a rate well-matched to metabolic requirements, except perhaps in vigorous intensity exercise. The stimuli include adrenergic and other circulating factors. There is a period following an exercise bout when fatty acids are directed away from adipose tissue to other tissues such as skeletal muscle, reducing dietary fat storage in adipose. With chronic exercise (training), there are changes in adipose tissue physiology, particularly an enhanced fat mobilization during acute exercise. It is difficult, however, to distinguish chronic "structural" changes from those associated with the last exercise bout. In addition, it is difficult to distinguish between the effects of training per se and negative energy balance. Epidemiological observations support the idea that physically active people have relatively low fat mass, and intervention studies tend to show that exercise training reduces fat mass. A much-discussed effect of exercise versus calorie restriction in preferentially reducing visceral fat is not borne out by meta-analyses. We conclude that, in addition to the regulation of fat mass, physical activity may contribute to metabolic health through beneficial dynamic changes within adipose tissue in response to each activity bout.     

Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid oxidation: an overview

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.     

Surgical manipulation,but not moderate exercise,is associated with increased cytokine mRNA expression in the rat soleus muscle

Interleukin (IL)-6 production in contracting skeletal muscle and IL-6 concentration in plasma are increased after prolonged and strenuous exercise. However, as tissue stress or damage are unspecific triggers of increased cytokine levels, we examined whether moderate muscle activity is an independent stimulus for cytokine expression, and to which extent invasive procedures might affect the results. Soleus muscles were isolated from sedentary rats or from rats that had been running on a treadmill at moderate intensity (70% of maximal oxygen uptake) for 1 h. In another group the soleus muscle was prepared in situ and stimulated intermittently at 5 Hz for 1 h, so that maximal developed force declined by 30%. In situ prepared soleus muscles not subjected to electrical stimulation were used as controls. Messenger RNA (mRNA) expression of 11 cytokines was analysed in the soleus muscles using multiprobe RNAse protection assay, and IL-6 plasma concentration was measured by enzyme-linked immunosorbent assay. Treadmill exercise did not affect the mRNA expression of any of the measured cytokines in the soleus muscle. Irrespective of electrical stimulation, mRNA expression of IL-6 and IL-1beta were significantly increased in the surgically manipulated soleus muscles. Interleukin-6 plasma concentration was not affected by treadmill running or electrical stimulation. Conclusion, gentle surgical manipulation is a strong stimulus for IL-6 and IL-1beta mRNA synthesis in skeletal muscle, whereas exercise or electrical muscle stimulation at moderate intensity does not independently affect cytokine mRNA levels in the contracting soleus.     

Lipoprotein-lipase activity of human skeletal-muscle and adipose tissue after intensive physical exercise.

Seven young, healthy subjects performed bicycle exercise with a working load leading to exhaustion after one hour of work. The tests were done in the afternoon in the fed state. The serum insulin concentrations decreased from 22 to 4 mU/l and plasma glucagon increased from 241 to 340 pg/l already after 30 min of work. The level of adipose tissue lipoprotein lipase activity (LPLA) did not fall as had been expected, but increased. The skeletal muscle LPLA was unchanged. The results indicate that during the first hour of heavy exercise the heparin-releasable LPLA in tissues is not influenced by the work induced changes in serum hormone levels.     

Lipoprotein-lipase activity of human skeletal-muscle and adipose tissue after intensive physical exercise

Seven young, healthy subjects performed bicycle exercise with a working load leading to exhaustion after one hour of work. The tests were done in the afternoon in the fed state. The serum insulin concentrations decreased from 22 to 4 mU/1 and plasma glucagon increased from 241 to 340 pg/1 already after 30 min of work. The level of adipose tissue lipoprotein lipase activity (LPLA) did not fall as had been expected, but increased. The skeletal muscle LPLA was unchanged. The results indicate that during the first hour of heavy exercise the heparin-releasable LPLA in tissues is not influenced by the work induced changes in serum hormone levels.     

Blood flow in different adipose tissue depots during prolonged exercise in dogs

Adipose tissue blood flow was measured by the microsphere technique in all major adipose tissue depots in dogs during exercise. The measurements were done during rest, after 1 and 2 h of exercise and after a postexercise rest period. It was found that the blood flow to the inguinal, subcutaneous adipose tissue increased from about 6 ml/(100g·min) during rest to about 10 ml/(100 g·min) during exercise. This increase in flow was significantly smaller than the increase found in the perirenal, the mesenteric and the pericardial depots. In these depots the resting blood flow was about 10 ml/(100 g·min) increasing to about 30 ml/(100 g·min) during exercise. It is concluded that the increase in adipose tissue blood flow during exercise is a general phenomenon for all major adipose tissue depots. The increase in flow in the inguinal, subcutaneous fat pad was comparable to the previously described increase in flow in abdominal, subcutaneous tissue in man. Blood flow to abdominal skin was constant during exercise, while the flow in tissues from the gastrointestinal canal and in the kidneys decreased. The flow in the tongue and in the Achilles tendon significantly increased during exercise.     

Foreword

Aim: It is not clear how lipolysis changes in skeletal muscle and adipose tissue during exercise of different intensities. We aimed at estimating this by microdialysis and muscle biopsy techniques. Methods: Nine healthy, young men were kicking with both legs at 25% of maximal power (W_max) for 45 min and then simultaneously with one leg at 65% and the other leg at 85%W_max for 35 min. Results: Glycerol concentrations in skeletal muscle and adipose tissue interstitial fluid and in arterial plasma increased (P < 0.001) during low intensity exercise and increased (P < 0.05) even more during moderate intensity exercise. The difference between interstitial muscle and arterial plasma water glycerol concentration, which indicates the direction of the glycerol flux, was positive (P < 0.05) at rest (21 ± 9 μm ) and during exercise at 25%W_max (18 ± 6 μm ). The difference decreased (P < 0.05) with increasing exercise intensity and was not significantly different from zero during exercise at 65% (−11 ± 17 μM) and 85% (−12 ± 13 μm ) W_max. In adipose tissue, the difference between interstitial and arterial plasma water glycerol increased (P < 0.001) with increasing intensity. The net triacylglycerol breakdown, measured chemically from the biopsy, did not differ significantly from zero at any exercise intensity although directional changes were similar to microdialysis changes. Conclusions: Skeletal muscle releases glycerol at rest and at low exercise intensity but not at higher intensities. This can be interpreted as skeletal muscle lipolysis peaking at low exercise intensities but could also indicate that glycerol is taken up in skeletal muscle at a rate which is increasing with exercise intensity.