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Centre of Inflammation and Metabolism, Department of Infectious Diseases and The Copenhagen Muscle Research Center, Faculty of Health Sciences, University Hospital of Copenhagen, Copenhagen, Denmark
Submitted 10 June 2004 ; accepted in final form 5 January 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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IL-6 infusion; catecholamines; cytokines; physical activity; appetite
The level of circulating interleukin-6 (IL-6) increases dramatically in response to exercise (27), with IL-6 being produced by working muscle (28, 32) and adipose tissue (16, 17, 22), whereas CHO ingestion attenuates the exercise-induced increase in plasma IL-6 (10). IL-6 seems to have several important roles in metabolism, including induction of lipolysis (22, 33) and enhancement of insulin sensitivity when injected into IL-6-deficient mice (34). Thus it is possible that the effects of acute exercise on leptin may be dependent on IL-6.
Here, we investigate the regulation of the adipose-derived cytokine leptin by studying various metabolic conditions in lean, healthy men: acute exercise, CHO supplementation during acute exercise, IL-6 infusion, and epinephrine infusion. We expected acute exercise of high-energy expenditure (>1,000 kcal) to acutely decrease leptin in adipose tissue and plasma and that this effect would be opposed by increased substrate availability in terms of intake of carbohydrate during the exercise bout. Because exercise markedly increases IL-6 and epinephrine, we investigated their regulatory role in mediating the leptin response to exercise.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Thirty-nine subjects of normal weight who were physically active at least three times a week participated in the study. The study was approved by the Ethical Committee of the Copenhagen and Frederiksberg Communities, Denmark, and performed according to the Declaration of Helsinki. Subjects were informed about possible risks and discomfort before giving their informed, written consent to participate. The volunteers did not use any medication and did not have any febrile illness in the fortnight preceding the study and abstained from heavy exercise 2 days in advance of the experiments.
Subjects participated in one of the following protocols.
Exercise With or Without CHO Supplementation
Eight healthy men [means ± SE: age 24 ± 1 yr, height 186 ± 2 cm, weight 82 ± 2 kg, body mass index (BMI) 23.7 ± 0.5 kg/m2] participated in the study. Subjects underwent 3 h of bicycling on two different occasions separated by at least 1 wk. Both trials were identical in design and differed only during the exercise bout, in which either a carbohydrate drink (Gatorade, 6% carbohydrate) or an artificially sweetened placebo was ingested. The trials were performed in a randomized order.
One week before the first trial, subjects performed a maximal oxygen uptake test on a cadence independent cycle ergometer (Monark 839E, Monark, Varberg, Sweden). After an 8-min warm-up at 100 W, workload was increased by 50 W every 3 min until a workload of 200 W was reached. Subsequently, the workload was increased 25 W every minute until a cadence of 60 rpm could no longer be maintained. The highest workload the person could maintain for 1 min was set as the maximal workload.
On the experimental day, subjects reported to the laboratory at 0800 after an overnight fast. A catheter was placed in the antecubital vein for blood sampling. Adipose tissue biopsies were sampled from the abdominal subcutaneous adipose tissue by the percutaneous needle biopsy technique with suction, preceded by a subcutaneous injection of lidocaine. A preexercise biopsy and blood sample were obtained after 10 min of supine rest. Subjects commenced 3 h of bicycling at 60% of their maximal workload (means ± SE: 214 ± 7 W) equivalent to an energy expenditure of 
2,200 ± 80 kcal (calculated from workload in watts, time, and assuming an efficiency of 25%). During this period they ingested 250 ml of either the carbohydrate drink or placebo every 15 min equivalent to a 750-kcal intake in the carbohydrate trial. Adipose tissue biopsies and blood samples were obtained at the following time points: preexercise, at 3 h of exercise, and in the recovery period at 1.5 h and 5 h postexercise. The biopsies were cleaned of connective tissue and blood and quickly frozen in liquid nitrogen. Subjects consumed an isocaloric diet 2 days preceding experimental days. Free access to water was permitted throughout the experiment.
IL-6 Infusion Study With Adipose Tissue Biopsies
Twenty-one healthy men (means ± SE: age 26 ± 1 yr, height 185 ± 2 cm, weight 80 ± 4 kg, BMI 23.5 ± 1 kg/m2) participated in the study. To accommodate for interindividual differences, subjects who participated were selected for normal weight and being physically active at least three times a week. Eleven persons received recombinant human (rh) IL-6 and 10 received saline (controls). The infusion trials were performed in a randomized order. On the experimental day, subjects arrived at the laboratory at 0800 after an overnight fast. An antecubital vein of one limb was cannulated and used for the infusion. Subjects (n = 11) were infused with rhIL-6 administered in 20% albumin for 3 h at a rate of 5 µg/h, in a volume of 25 ml/h. Control persons (n = 10) were infused with 20% albumin for 3 h. Blood samples were collected from a catheter placed in the antecubital vein before infusion, at the cessation of infusion, and 1.5 h and 5 h postinfusion (n = 11 + 10). Biopsies (n = 5 + 4) were obtained from the abdominal subcutaneous adipose tissue before infusion, at the cessation of infusion, as well as 1.5 h and 5 h postinfusion. Subjects were permitted to consume only water during the experiment. Persons infused with saline were used as resting controls and compared with the exercise trial.
Epinephrine-Infusion Study With Adipose Tissue Biopsies
Ten healthy men participated in the study (means ± SE: age 27 ± 1 yr, height 184 ± 2 cm, weight 83 ± 3 kg, BMI 24.4 ± 0.7 kg/m2), receiving either epinephrine or saline (controls); six persons served as their own controls. The infusion trials were performed in a randomized order. On the experimental day, subjects arrived at the laboratory at 0800 after an overnight fast. An antecubital vein of one limb was cannulated and used for the infusion. To mimic a level of epinephrine seen during intense exercise, subjects (n = 8) were infused with epinephrine at a rate of 24 ng·kg–1·min–1 for the first 1.5 h and then at 42 ng·kg–1·min–1 for the final 1.5 h. Epinephrine was diluted in saline such that each subject received a total volume of 80 ml, whereas controls received saline alone. Biopsies were obtained from abdominal subcutaneous adipose tissue before infusion, at the cessation of infusion, and 1.5 h and 5 h postinfusion. Blood samples were collected before infusion, at the cessation of infusion, and 1.5 h and 5 h postinfusion. Subjects were permitted to consume only water during the experiment.
RNA extraction. RNA was extracted by using Trizol (Life Technologies) according to the manufacturer's protocol. In short, 1 ml of Trizol was added to 30–50 mg of adipose tissue and homogenized using a Polytron (PT-MR2100, Kinematica) on setting 25–30 for 20–30 s and placed on ice. After careful removal of the upper triglyceride layer, 100 µl of chloroform were added to all samples, shaken vigorously, and incubated for 5 min on ice. Samples were spun at 12,000 g for 15 min at 4°C, and the upper aqueous phase was placed in a fresh Eppendorf tube. The same volume of isopropanol was added, and samples were placed at –20°C for 1 h followed by centrifugation at 12,000 g for 15 min at 4°C. The resulting RNA pellet was washed with 75% ethanol in diethylpyrocarbonate-treated water and spun at 6,000 g for 10 min at 4°C. The pellets were dissolved in diethylpyrocarbonate-treated water.
Reverse transcription. One microgram of total RNA was reverse transcribed in a 50-µl reaction according to the manufacturer's protocol (Applied Biosystems, Taqman reverse transcription reagents) with the use of random hexamer primers. The reactions were run in a Perkin-Elmer GeneAmp PCR system 9700 with conditions at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min.
Analysis of Gene Expression Levels in Adipose Tissue
Samples were analyzed for leptin mRNA levels by real-time PCR by using an ABI PRISM 7700 or 7900 sequence detector (PE Biosystems). Samples were run in triplicates under standard real-time PCR conditions; 50°C for 2 min, 95°C for 10 min followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. The gene expression levels were normalized to the housekeeping gene 18S (obtained from Applied Biosystems). The leptin and 18S reactions were run under singleplex conditions. Human leptin primers and Taqman probe were obtained from Applied Biosystems (AOD, no. Hs00174877m1). Samples were analyzed using the comparative cycle threshold (Ct) method: the Ct values of the samples are compared with the Ct value of the corresponding resting sample after normalization to the housekeeping gene 18S.
Measurement of Plasma Leptin, IL-6, Insulin, and Blood Metabolites
Blood samples for measurement of cytokines, insulin, and metabolites were drawn into glass tubes containing EDTA. The tubes were spun immediately at 3,500 g for 15 min at 4°C. The plasma was stored at –80°C until analyses were performed. Leptin plasma levels were measured using the human Leptin Quantikine kit obtained from R&D systems (catalog no. DLP00). The sensitivity is 7.8 pg/ml, and the intra- and interassay coefficients are 3.3 and 5.4%, respectively. Before measurements, samples were diluted 1:50 or 1:20 in Calibrator diluent provided by the manufacturer. Plasma IL-6 levels were measured by high-sensitivity ELISA kits from R&D Systems, Minneapolis, MN. The sensitivity is 0.094 pg/ml, and the intra- and interassay coefficients are 6.9 and 9.6%, respectively. This kit does not distinguish between soluble and receptor-bound IL-6 and therefore gives a measure of the total IL-6 content in the sample. Plasma insulin was measured using an ELISA kit from Dako (catalog no. K6219) with a sensitivity of 3 pmol/l and intra- and interassay coefficients of 7.5 and 9.3%, respectively. Free fatty acids (FFAs) and glucose in plasma were determined by COBAS analysis (Fara Roche). For measuring circulating epinephrine levels, blood was drawn into ice-cold glass tubes containing glutathione (1.3 mg/ml blood) and EGTA (1.5 mg/ml blood), pH 6–7, and analyzed by high-performance liquid chromatography (Hewlett-Packard, Waldbronm, Germany) with electrochemical detection.
Body temperature and heart rate did not change in the IL-6 experiments compared with the control studies. Subjects did not suffer from any known adverse side effects of IL-6 such as headache or nausea.
Statistics
Leptin mRNA and plasma data in the CHO and exercise trial were normally distributed after log transformation. However, when leptin mRNA in the exercising trial was compared with the resting trial, data could not be normally distributed and nonparametric statistics was used. Plasma levels were normally distributed after log transformation. In the rhIL-6 infusion study leptin data were normally distributed. In the epinephrine study, plasma leptin data were normally distributed, whereas leptin mRNA data were normally distributed after log transformation.
Plasma IL-6 data were normally distributed in the exercise and CHO study and were normally distributed after log transformation in the rhIL-6 and epinephrine infusion studies.
Epinephrine plasma levels were normally distributed in the epinephrine infusion study after log transformation. In the exercise and CHO study, epinephrine data could not be normally distributed for a two-way ANOVA analysis; therefore, changes over time were analyzed by using a one-way ANOVA and changes between groups were analyzed with a t-test with Bonferroni correction for comparison of multiple time points.
Glucose and FFA were normally distributed in the exercise trial with CHO ingestion, whereas insulin levels were normally distributed after square root transformation. In the rhIL-6 infusion trial, glucose and insulin plasma data were normally distributed, whereas FFA plasma levels were log transformed to obtain a normal distribution.
In the epinephrine infusion study, FFA levels were normally distributed. Insulin and glucose levels could not be normally distributed for a two-way ANOVA analysis; therefore, changes over time were analyzed with a one-way ANOVA and changes between groups were analyzed with a t-test with Bonferroni correction for comparison of multiple time points.
Data in the figures are presented as means ± SE or as geometric means ± SE with respect to log-transformed data. According to leptin mRNA data when exercise was compared with the resting trial, a normal distribution could not be obtained, data are thus presented as medians and 75% and 25% quartiles.
For analysis of data in the exercise and infusion trials, a two-way repeated-measures ANOVA was used to detect changes over time or between groups, followed by Student-Newman-Keuls t-test or the Bonferroni t-test for post hoc analysis to detect changes over time from resting values or differences between groups. A one-way ANOVA was run for each group, to detect changes over time. According to the nonparametric statistics, the Friedman repeated-measures ANOVA on ranks was run for each group, and data at different time points were compared by using a Wilcoxon signed rank sum test.
P values < 0.05 were considered significant. Statistical calculations were performed using Sigma Stat 3.0 (SPSS, Chicago, IL).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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When exercising persons were compared with resting persons, there was no difference in leptin mRNA levels in adipose tissue, although leptin mRNA decreased over time (P < 0.05; Fig. 1A) in the exercise trial only. Plasma leptin levels decreased in both exercising and resting persons (P < 0.001), but the two groups differed significantly (P < 0.05), as exercising persons displayed an 50–60% decrease at all time points, whereas this decrease was only 30% in the resting persons (Fig. 1B; at rest, mean plasma leptin levels were 2.9 ± 0.3 ng/ml in the resting trial and 2.2 ± 0.6 ng/ml in the exercise trial). Leptin mRNA levels in adipose tissue were not influenced by CHO supplementation during exercise (see Table 1). In the CHO trial, leptin plasma levels decreased (P < 0.001); however, CHO supplementation attenuated the exercise effect at all time points compared with exercise with placebo ingestion (P < 0.001) (Fig. 2; at rest, mean plasma leptin levels were 1.9 ± 0.4 ng/ml in the CHO trial).
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Leptin mRNA levels decreased at the 5-h postinfusion time point in the epinephrine-infused group (one-way ANOVA, P < 0.01); however, compared with the saline-infused group, there was no difference between the two groups (Fig. 3A). Circulating leptin levels diminished after infusion of epinephrine (P < 0.001) compared with placebo infusion, reaching 60% of preinfusion levels at the cessation of the epinephrine infusion. This effect leveled off 1.5 h after the end of the epinephrine infusion (Fig. 3B; 2-way ANOVA: P < 0.001) (at rest, mean plasma leptin levels were 4.3 ± 1.3 ng/ml in the control group and 3.2 ± 0.8 ng/ml in the epinephrine infusion group).
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As shown in Table 2, glucose and insulin levels were higher in the CHO trial and in the epinephrine-infused group. Although insulin levels decreased over time in the rhIL-6 infusion group, whereas glucose levels decreased over time in the control trial, there were no significant differences between the two groups. Levels of FFA were elevated in the groups infused with rhIL-6 (P < 0.001) or epinephrine and in response to exercise but were lower in the CHO trial. As shown in Table 3, plasma IL-6 levels were increased by exercise (repeated-measures ANOVA on ranks, P < 0.001) but markedly decreased in the CHO trial immediately after exercise (paired t-test, P < 0.01). rhIL-6 infusion increased plasma IL-6 levels by 100-fold (2-way ANOVA, P < 0.001), whereas there was no change in the placebo-infused group. Epinephrine levels increased in the epinephrine infusion trial only (2-way ANOVA, P < 0.001). Epinephrine also increased in the exercising group, with CHO counteracting this increase at the postexercise and 1.5-h postinfusion time point (paired t-test, P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Circulating leptin levels declined 50% at the end of exhaustive acute exercise, a significantly greater decline than in the fasting-induced control trial. Despite subjects displaying high variability of plasma leptin concentrations, there was a very consistent 60% decrease with exercise at the 5-h time point for each person. Surprisingly, we found no effect of exhaustive acute exercise on leptin mRNA levels in adipose tissue. The finding that exercise reduced circulating levels of leptin but not mRNA levels compared with resting persons suggests that exercise mainly influences leptin at the posttranscriptional level. Our results are consistent with previous findings that plasma leptin decreases after long-duration intense exercise (8, 9, 19, 20, 26). The exercise-induced decrease in plasma leptin was counteracted by CHO ingestion, which indicates sensitivity to energy availability or to hormones affected by the presence of CHO, as previously shown in rodents (35) and in humans (1, 15). From our epinephrine infusion study, we obtained indirect evidence to suggest that the exercise effect on leptin was mediated by epinephrine or epinephrine-induced changes in FFA, insulin, or glucose concentrations. Epinephrine decreased plasma leptin in a manner similar to exercise and also decreased with CHO ingestion; thus epinephrine or other metabolic parameters, such as plasma insulin, glucose, or FFA, affected by epinephrine may be involved in the regulation of circulating leptin levels. IL-6 is regulated by CHO in a parallel manner to leptin (10): in working muscle, CHO has no effect on the IL-6 transcription level but blunts the release of IL-6, and IL-6 could possibly mediate the exercise-induced effects on leptin. However, leptin mRNA and plasma levels were unaffected by infusion of rhIL-6; thus it is highly likely that the decrease in plasma leptin seen with exercise occurs via an IL-6-independent mechanism, although there is a remote possibility that muscle-derived IL-6 has different effects than rhIL-6 owing to posttranslational modifications (24). Our finding that epinephrine or epinephrine-induced hormonal changes decreased leptin mRNA and plasma in healthy men is in accordance with findings in women (7) and obese persons (4). Furthermore, the decrease in plasma leptin we saw with a 3-h exercise bout was mimicked by the 3-h epinephrine infusion, suggesting a link between the two, either direct or indirect possibly via insulin, glucose, or FFA. However, glucose and insulin levels were higher in the epinephrine trial compared with the exercise trial, but leptin decreased to comparable levels during these two experimental conditions. Because insulin and glucose levels differed in the two experimental settings, we cannot discriminate between single effects of either of the two. Thus glucose and insulin are probably not alone responsible for the regulation of leptin, as leptin decreased in both situations; thus possibly epinephrine plays a more direct role.
Plasma levels of FFA increased after both exercise and epinephrine infusion but were reduced when subjects ingested CHO, thus allowing for the possibility that plasma FFA affects leptin by acting as a mediator of the signal for substrate availability. This is consistent with the inhibition of both leptin mRNA and protein levels in rat adipocyte cell cultures by FFAs (31). Infusion of intralipid/heparin, which led to an increase in FFA comparable to the levels we saw in our studies, suppresses plasma leptin by 30% (12), which is much less compared with our exercise or epinephrine data. Thus it seems that FFA does not regulate leptin alone; other studies suggest that glucose (2, 18) and insulin (2) are more important in linking energy intake and leptin production. During fasting when plasma insulin is low, leptin levels are reduced in humans (2) and increase in response to feeding when insulin and glucose levels also increase and FFA levels are reduced (18). In humans, insulin regulates plasma leptin at physiological doses (30) and leptin mRNA levels at nonphysiological levels (29). Furthermore, in mice, in vivo and in vitro studies show that treatment with insulin increases both leptin mRNA and protein levels (21). The importance of both glucose and insulin levels on leptin is further demonstrated by the finding that the rise in leptin plasma levels is reduced when a meal low in carbohydrate is consumed (14). In cultured rat adipocytes, the amount of glucose taken up by the adipocytes, rather than insulin levels, regulates leptin production, thus pointing to an important role of glucose transport in the release of leptin (25). Because insulin levels were increased in the epinephrine-infusion study and the CHO trial in this study, but with opposite effects on plasma leptin levels, it is more likely that insulin is not the sole regulator of circulating leptin levels but that glucose and FFA are also important factors and that epinephrine may play a more direct role. However, because opposing findings exist on this subject, further studies are needed to clarify this question. Thus we show that exercise decreases leptin levels, an effect that is highly likely initiated by increased epinephrine levels, possibly mediated via changes in FFA, glucose, and insulin.
Here, we report that exercise decreases plasma leptin without affecting the gene expression level in adipose tissue in humans. Substrate availability during exercise affects leptin with CHO ingestion attenuating the exercise-induced decline in plasma leptin. rhIL-6 infusion had no effect on leptin, whereas epinephrine decreased leptin mRNA and plasma in a manner similar to exercise.
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GRANTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Data on IL-6 plasma levels from the subjects participating in the exercise study with or without CHO supplementation have previously been published by C. Keller et al. in J Physiol 550: 927–931, 2003.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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  L. Frydelund-Larsen, T. Akerstrom, S. Nielsen, P. Keller, C. Keller, and B. K. Pedersen Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E24 - E31. [Abstract] [Full Text] [PDF]
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, but there is not a significant difference between the 2 groups. Data are expressed as fold change from Pre value and presented as median and quartiles. B: leptin plasma levels in response to exercise or rest (n = 8 and 6). There is a decrease in leptin plasma levels at all time points in both trials (P < 0.001) denoted by 
