Body composition is an important factor for both elite performance (15,16) and aerobic capacity (14) in horses. Fat mass, although detrimental for sports performance and health, is not merely a tissue of energy storage. It also plays a critical role in energy homeostasis (35) by secreting a variety of proteins that modulate many biological functions (8,9,22). Together, these proteins are known as "adipocytokines," and they include leptin, tumor necrosis factor-α, adipsin, resistin, and adiponectin. Recently, it has been shown in horses, as in humans, that plasma leptin concentrations are proportional to adiposity whereas plasma adiponectin concentrations are inversely proportional to adiposity (3). Limited data exist on these adipocytokines in horses and no data are found on the effects of changes in fat mass on plasma leptin or adiponectin levels.
Clenbuterol, a β2-adrenoceptor agonists, is most well known for its ability to elicit a muscle-directed protein anabolic response in young lambs (2,5), broiler chickens (6), steers (29), rats (21,28), and horses (17). Specifically, clenbuterol increases muscle mass and simultaneously decreases fat mass. To date, however, no published data are available on effects of clenbuterol-mediated repartitioning on any of the known adipocytokines in horses. It could be hypothesized that adiponectin levels would increase while leptin levels would decrease in direct proportion to changes in adiposity. The present study, therefore, tested the hypothesis that long-term administration of clenbuterol would alter plasma concentrations of adiponectin and leptin in horses as well as the relationship between body composition and circulating concentrations of those hormones.
MATERIALS AND METHODS
Animals and drug administration.
Twenty-three healthy, untrained, standard-bred mares (age: 10 ± 3 yr) were evaluated. The mares were unfit, but accustomed to the laboratory and running on the treadmill before the start of the experiment. During the trial, horses were housed as a group on pasture. Each mare was fed approximately 6kg·d−1 of alfalfa and grass hay (Semican, Pessiville, Quebec, Canada) and approximately 3 kg·d−1 of a commercially available grain ration (F. M. Brown's Sons, Birdsboro, PA); split into two feedings. Water was provided ad libitum. TheRutgers University institutional animal care review board approved all methods and procedures used in this experiment.
Horses were divided into four experimental groups: clenbuterol plus exercise (CLENEX; N = 6) and clenbuterol only (CLEN; N = 6) were given an oral dose of clenbuterol syrup (Boehringer Ingelheim, UK), 2.4 μg·kg−1 twice daily, (for an average volume of 20 mL) on a schedule of 5 d on and 2 d off for the duration of the 8-wk study. In addition to being given the drug, the CLENEX group also was trained aerobically for 3 d·wk−1. Five horses were assigned to the aerobic exercise-training group (EX) and aerobically trained for 3 d·wk−1. Another group of horses served as the sedentary non-drug-treated control group (CON; N = 6). Both EX and CON were administered volumes of molasses similar to the volume of clenbuterol twice daily on a 5-d on and 2-d off schedule.
The exercise program consisted of continuous treadmill running 3 d·wk−1 for 8 wk. Horses initially ran for 15 min·d−1 at a work rate of 50% V̇O2max (determined before the study). After 1 wk, run time duration was increased to 20 min·d−1 and was held at this duration for the entire study. During exercise training, the high-speed horse treadmill (Sato I, Equine Dynamics, Inc., Lexington, KY) was set at a fixed 6% grade. Jugular venous blood samples (30 mL) for the measurement of plasma concentrations of the adipocytokines were drawn into tubes containing EDTA and plasma samples were frozen and stored at −80°C until analysis. Samples were collected before and after the 8-wk treatment or training period.
Plasma leptin concentration was measured using a commercial RIA kit (Linco, St. Charles, MO) that had been previously validated for measuring leptin concentration in equine serum and plasma (7,13,24). In the absence of purified equine leptin, results were expressed as human equivalents of immunoreactive leptin (ir-leptin HE). The kit used 125I-labeled recombinant human leptin with a specific activity of 135 μCi·μg−1, a guinea pig multispecies leptin primary antibody, and a goat antiguinea pig IgG serum for the precipitating reagent. Purified recombinant human leptin was used for the kit standards and quality controls. Samples were run in duplicate and counted for 1 min in a gamma counter (Packard Instrument Company). In the absence of purified equine leptin, results were expressed as human equivalents of ir-leptin HE. Serial dilution of standards and equine plasma demonstrated both parallelism and linearity with a sensitivity of 0.5 ng·mL−1 HE. Samples were processed in one assay run where the within assay coefficient of variation (CV) for was ± 8.5%.
Plasma adiponectin concentration was measured using a RIA kit that was previously validated for measurement of adiponectin concentration in equine serum and plasma (13). In the absence of purified equine adiponectin, results were expressed as human equivalents of ir-adiponectin HE. The kit used 125I-labeled murine adiponectin with a specific activity of 67.7 μCi·μg−1, a multispecies adiponectin rabbit antiserum, and goat antirabbit IgG serum in the precipitating reagent. Purified recombinant human adiponectin was used for the kit standards and quality controls. Samples were run in duplicate and counted for 1min in a gamma counter (Packard Instrument Company). Data from the manufacturer indicated a specificity of 100% for human and mouse adiponectin with a fourfold affinity for mouse adiponectin compared with human and <0.01% for human C1q (type VIII collagen and hibernation-specific protein). Parallelism of the adiponectin assay kit was established using a serial dilution of horse plasma and the adiponectin standard from the assay kit and a serial dilution of the control spiked with horse plasma. Spiking and recovery studies demonstrated that the observed concentration, on average, was 97% of the expected concentration. Sensitivity of the assay was 0.39 ng·mL−1. Samples were processed in one assay run where the within assay CV for the adiponectin assay was ± 9.1%.
Body composition measurement.
Rump fat thickness was measured using B-mode ultrasonography and used to calculate percent body fat (%fat) as previously published (13,14,16,17,30) in horses. The site was determined by placing the probe over the rump at approximately 5 cm lateral from the midline at the center of the pelvic bone (34). The region was scanned and the position of maximal fat thickness was used as the measured site. The calculated average CV based on six animals for this rump fat thickness determination was 3.6 ± 0.7%. Percent fat was estimated from the equations of Kane and colleagues (12):
Fat mass was determined by multiplying %fat and total body mass. Fat-free mass (FFM) was derived by subtracting fat mass from total body mass. Rump fat was measured before the start and at the completion of the study.
Results are expressed as means ± SEM. For comparison by group and time a two-way ANOVA with repeated measures was used with the a priori level of statistical significance set a P < 0.05. Post hoc differences were determined using the Tukey test and correlation coefficients were derived using the Pearson product moment (Sigma Stat 2.0).
Body composition changes.
Data on the changes in body composition related to exercise training and clenbuterol administration for the horses used in the present study have been previously reported (18). Briefly, for the EX group, body fat was decreased (P < 0.05) at week 4 (−9.3%), %fat at week 6 (−6.9%), and FFM increased (P < 0.05) at week 8 (+3.2%) (18). In contrast, both the CLEN and CLENEX groups had an earlier and more pronounced repartitioning effect (P < 0.05). The CLEN group of horses had decreases (P < 0.05) in %fat (−15.4%) and fat mass (−14.7%), and an increase in FFM (+4.3%) at week 2 (18). The combination of clenbuterol and exercise (CLENEX) caused significant decreases in %fat (−17.6%) and fat mass (−19.5%) at week 2, which was similar to CLEN; however, they had a different FFM response that significantly increased (+4.4%) at week 6 (18). The control group showed no changes (P > 0.05) in any variable at any time (18).
Adiponectin and leptin: Clenbuterol or exercise treatment.
Following 8 wk of treatment, plasma ir-adiponectin HE concentration was increased (P < 0.05) in the CLENEX horses (+31.2%, P = 0.02) and in the CLEN horses (+24.4%, P < 0.05) (Fig. 1A). No significant changes were noted in plasma ir-adiponectin HE concentration in either the EX or the CON groups. Posttreatment plasma ir-leptin HE concentration was decreased compared with pretreatment in CLENEX (−30.5%, P < 0.05) and CLEN (−25.3%, P < 0.05) groups. No significant changes were seen in either the EX or CON groups (Fig. 1B).
The change in plasma ir-adiponectin HE concentration was negatively correlated (r = −0.520; P = 0.01) to the change in fat mass (Fig. 2A) and positively correlated (r = 0.446; P = 0.03) to the change in fat-free mass (Fig. 2B). The change in plasma ir-leptin HE concentration was positively correlated (r = 0.550; P = 0.02) to the change in fat mass (Fig. 2C) and negatively correlated (r = −0.473; P< 0.05) to the change in fat-free mass (Fig. 2D).
Before treatment, leptin was negatively correlated (r = −0.457; P = 0.03) to V̇O2max, whereas adiponectin was not correlated (r = 0.203; P > 0.05) to V̇O2max. After treatment, leptin was negatively correlated (r = −0.611; P= 0.02) to V̇O2max, whereas adiponectin was not correlated (r = 0.347; P = 0.11) to V̇O2max. Changes in leptin were not correlated (P > 0.05) to the changes in V̇O2max (r= 0.286) or fat mass (r = 0.126). Similarly, changes in adiponectin were not correlated to the changes in V̇O2max (r = −0.138; P >0.05) or fat mass (r = −0.404; P = 0.06).
It has been hypothesized that leptin and adiponectin can be regulated in opposite directions by common factors because both are involved in energy homeostasis (36). Supporting that speculation are data from descriptive studies showing that the adipocytokine leptin is proportional to fat mass, whereas adiponectin is inversely proportion to fat mass in horses (13) and humans (1,4,32). Those studies, however, did not attempt to manipulate body composition or fat mass. To that end, one would predict that either an exercise training-induced or pharmacological-induced change in fat mass and body composition would alter circulating concentrations of leptin and adiponectin. Data from previous studies of the horses used in the present experiment demonstrate that therapeutic levels of clenbuterol could cause repartitioning in as little as 2 wk (18). The present study findings extend those results by demonstrating that therapeutic levels of clenbuterol also affect the plasma concentrations of both adiponectin and leptin. Additionally, the present study demonstrates that exercise training alone does not alter the concentrations of either leptin or adiponectin. These data are consistent with previous findings in humans (10,11).
The finding that training did not affect mean values for circulating concentrations of leptin and adiponectin in the horses in the present study is consistent with findings from other species summarized in a recent review (19). It appears that exercise itself has little effect on either gene expression for the adipocytokines or the circulating levels of adipocytokines in the plasma (19). Mechanistically, the review suggests that changes in circulating leptin and adiponectin concentration are independent of exercise's effects on energy balance. Those papers and data from the horses in the present study suggest that chronic exercise training that does not induce the loss of fat mass does not affect the concentrations of adipocytokines. Exercise that results in a loss of fat mass, however, has been shown to increase adiponectin (11) or decrease leptin (19). Thus, a considerable expenditure of energy may alter leptin concentrations (10), but those data are less clear (for review, see (10)). A comparison of group means for the plasma concentration of leptin and adiponectin, however, does not give a full picture of the effect of training has on these adipocytokines. When looking further, regression analysis demonstrates that circulating concentrations of leptin and adiponectin were proportional to changes in fat mass. Thus, horses in the exercise group that lost the largest degree of fat mass also demonstrated changes in their adipocytokine concentrations.
Interestingly, leptin was correlated with V̇O2max, whereas adiponectin was not. This is surprising in lieu of the fact that fat mass is not correlated with V̇O2max (14) or 1-mile race performance in horses (16). The relationship between leptin and aerobic capacity, however, do appear to be similar, in part, to data seen in humans (26). In those studies, plasma leptin concentrations were negatively correlated to V̇O2peak in men but not in women (26). Adiponectin, on the other hand, was shown to be inversely proportional to V̇O2peak in boys (27). The reasons for this species difference are unclear, but they may be related to the fact that only mares were considered in the present study. A significant relationship between adipocytokines and V̇O2peak has consistently been reported in male subjects (26,27) but not in female subjects.
A new finding of the present study was that exercise and clenbuterol had an additive effect on both leptin and adiponectin. The clenbuterol plus exercise group had a larger change in both leptin (−30.5 vs −25.3%) and adiponectin (+31.2 vs +24.4%) than the clenbuterol group alone. Data from the present do not address the cellular mechanism for the effects of clenbuterol administration on the cytokines. Published papers, however, suggest that the effects of chronic β-agonists on adipocytokines can be a result of direct activation of fat tissue (3,31). The β3-adrenoceptors are found primarily in brown and white adipose tissue (3). Clenbuterol, on the other hand, is more specific for the β2-receptors found in skeletal muscle (3); however, the possibility of cross talk between pharmacological concentrations of β2-agonists and β3-receptor cannot be excluded (3,31). It has been shown also that β-adrenergic agonists have a role in the regulation of leptin (20) and adiponectin gene expression (25). Furthermore, β3-adrenergic activation decreases leptin gene expression and serum concentration and simultaneously increases adiponectin gene expression and serum concentrations (36). The results of the present study demonstrate that chronic clenbuterol administration alters the concentrations of the adipocytokines adiponectin and leptin in a similar manner and that the changes in these adipocytokines were proportional to the changes in fat mass. Such a change would be consistent with data from other adrenergic agonist studies that have demonstrated a direct or indirect action on adipose tissue receptors (3,25,31).
Chronic administration of clenbuterol has been shown to improve glucose tolerance in obese Zucker rats (3,31). The exact mechanism for the improvement is not known, but it is believed to be partly caused by the known repartitioning effects of clenbuterol (2,5,6,18,21,28,29) and the relative increase in muscle mass to total body weight. Hulver et al. (11) have suggested that exercise and weight loss improve insulin sensitivity through partially or completely different mechanisms (23,33). Although neither insulin nor glucose tolerance were measured in the present study, the changes in concentrations of the adipocytokines adiponectin and leptin were both in a direction that would suggest an improvement in glucose tolerance. Further investigation examining the interaction of clenbuterol and adipocytokines on insulin sensitivity is warranted.
SUMMARY AND CONCLUSION
Clenbuterol is widely prescribed for treatment of respiratory disease in the horse, despite data demonstrating that chronic administration of therapeutic doses causes a decline cardiac function, as well as decreases in V̇O2max and exercise capacity (17). Previous data from the horses of the present study also demonstrated that the drug also acts as a repartitioning agent with a substantial repartitioning effect and resultant decreases fat mass (18). Humans who misuse clenbuterol do so to alter body composition; however, studies to elucidate the mechanism behind those effects are not ethically feasible. Thus, the present study adds to information collected from prior studies of the same horses, giving insight into hormonal changes that may play an important role in mediating clenbuterol-induced changes in fat mass. Interestingly, exercise training alone was insufficient to change the plasma concentration of these adipocytokines; however, the combination of clenbuterol and exercise did have an additive effect on both leptin and adiponectin. Data from the present study suggest that adrenergic drugs that act as repartitioning agents may do so, in part, through concomitant alterations in the plasma concentrations of both leptin and adiponectin.
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