Differential expression of skeletal muscle genes following administration of clenbuterol to exercised horses

Abstract

BackgroundClenbuterol, a beta2-adrenergic receptor agonist, is used therapeutically to treat respiratory conditions in the horse. However, by virtue of its mechanism of action it has been suggested that clenbuterol may also have repartitioning affects in horses and as such the potential to affect performance. Clenbuterol decreases the percent fat and increases fat-free mass following high dose administration in combination with intense exercise in horses. In the current study, microarray analysis and real-time PCR were used to study the temporal effects of low and high dose chronic clenbuterol administration on differential gene expression of several skeletal muscle myosin heavy chains, genes involved in lipid metabolism and the β2-adrenergic receptor. The effect of clenbuterol administration on differential gene expression has not been previously reported in the horse, therefore the primary objective of the current study was to describe clenbuterol-induced temporal changes in gene expression following chronic oral administration of clenbuterol at both high and low doses.ResultsSteady state clenbuterol concentrations were achieved at approximately 50 h post administration of the first dose for the low dose regimen and at approximately 18–19 days (10 days post administration of 3.2 μg/kg) for the escalating dosing regimen. Following chronic administration of the low dose (0.8 μg/kg BID) of clenbuterol, a total of 114 genes were differentially expressed, however, none of these changes were found to be significant following FDR adjustment of the p-values. A total of 7,093 genes were differentially expressed with 3,623 genes up regulated and 3,470 genes down regulated following chronic high dose administration. Of the genes selected for further study by real-time PCR, down-regulation of genes encoding myosin heavy chains 2 and 7, steroyl CoA desaturase and the β2-adrenergic receptor were noted. For most genes, expression levels returned towards baseline levels following cessation of drug administration.ConclusionThis study showed no evidence of modified gene expression following chronic low dose administration of clenbuterol to horses. However, following chronic administration of high doses of clenbuterol alterations were noted in transcripts encoding various myosin heavy chains, lipid metabolizing enzymes and the β2-adrenergic receptor.

Clenbuterol plasma concentrations after therapeutic administration in fit Standardbred horses: threshold recommendations

“…If the terminal portion of the elimination curve is limited by either inability to collect sufficient samples, or lack of sensitivity of the testing methodology, the true slope of the terminal elimination curve cannot be determined. When the data presented are evaluated as mono-exponential, our pharmacokinetic parameters closely follow those of previous investigators, with our observed clenbuterol half-life of 12.9 h mirroring the findings of Soma et al (2004) at 12.9 h, Knych et al (2016) at 10.4 h, andLehner et al (2001) at 7 h. However, the LOQ of the methods used in previous studies was 13 pg/ml (Lehner et al, 2001;Soma et al, 2004) and 10 pg/ml (Knych et al, 2016), whereas the LOQ in this present study was 0.55 pg/ml permitting more accurate and definitive determination of the terminal elimination of clenbuterol. When just the 48 to 96 h data are evaluated, the terminal half-life of 16.9 h is significantly longer than previously reported.…”

“…Pharmacokinetics differ not only between Standardbreds and Thoroughbreds, but also between animals that are fit or sedentary (Khazaeinia et al, 2000). The relevant pharmacokinetic parameters from our study are similar to previous studies which evaluated sedentary horses (Lehner et al, 2001;Soma et al, 2004) and race fit Thoroughbreds (Knych et al, 2016), which suggests that fitness and breed characteristics do not influence clenbuterol metabolism in horses. However, because the previous studies lacked the more sensitive analytical methodology used in this study, no conclusions can be drawn about possible differences in the terminal pharmacokinetic parameters between breeds or levels of fitness.…”

Evaluation of clenbuterol-induced changes in blood biochemical parameters in white mice

“…Hye-Kyeong Kim et al reported that clenbuterol increased the rate of lipolysis and concomitantly decreased the rate of lipogenesis in adipose tissues 28 . In addition, it was found that clenbuterol down-regulated lipoprotein lipase, which hydrolyzes triglyceride in lipoproteins into glycerol and fatty acid 29 . In the present study, we observed that when orally administering clenbuterol to mice for 16 weeks at dosage 2 µg/kg, mean LDL-cholesterol concentrations were slightly increased and mean HDL-cholesterol concentrations were significantly decreased when comparing with control group, without changes in triglyceride and total cholesterol concentrations.…”

Molecular characterization of the apoptosis-related SH3RF1 and SH3RF2 genes and their association with exercise performance in Arabian horses

“…The expression levels of both genes were detected on a Quant Studio 7 flex real-time PCR system (Applied Biosystems) using AmpliQ 5× HOT EvaGreen® qPCR Mix Plus (ROX) (Novazym, Poland) in 3 replicates for each sample. The primers for the targeted genes and endogenous controls ( GAPDH and B2M ) [ 15 ] were designed using Primer3 version 4.0.0 based on the Ensemble reference sequence (Additional file 1 : Table S1). The primers for SH3RF2 gene were not affected by the identified c.796 T > C missense variant.…”