Project description:Background: Beta-adrenergic receptor agonists (BA) induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. We chose to evaluate global changes in gene expression by utilizing the Affymetrix platform to identify gene expression changes in mouse skeletal muscle. Changes in gene expression were evaluated 24 h (1D) and 10 days (10D) after administration of the BA clenbuterol. Results: Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P < 0.01) and increased body weight gain (P < 0.05) 24 h or 10 d, respectively, after initiation of clenbuterol treatment. A total of 22,787 probesets were evaluated with 113 probesets defined as differentially expressed based on a false discovery rate of 10%. Differential mRNA abundance of four of these genes was validated in an independent experiment by quantitative PCR. Patterns of gene expression change observed from the microarray data suggested more genes are differentially regulated after 24 h than 10 d of BA administration. Functional characterization of differentially expressed genes revealed several categories that participate in biological processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signaling pathways, and genes involved in polyamine metabolism. Included within these lists were several genes previously associated with myogenic differentiation, along with multiple genes known to promote a general up-regulation of translation. Conclusions: Global evaluation of gene expression after administration of clenbuterol identified changes gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in the mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on the proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Additionally, increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators, rather than a sustained up-regulation of gene expression at the transcriptional level. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle. Experiment Overall Design: A total of 27 mice were used to represent three treatments. Hybridizations were done using RNA pools from 3 mice, with 3 pools (replicates) per treatment. Treatments included control, 1 day of clenbuterol administration, and 10 days of clenbuterol administration. Data represent skeletal muscle gene expression.

Project description:Background: Clenbuterol, 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. Steady state clenbuterol concentrations were achieved at approximately 50 hours 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. Conclusion: This 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.

Project description:Background: Beta-adrenergic receptor agonists (BA) induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. We chose to evaluate global changes in gene expression by utilizing the Affymetrix platform to identify gene expression changes in mouse skeletal muscle. Changes in gene expression were evaluated 24 h (1D) and 10 days (10D) after administration of the BA clenbuterol. Results: Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P < 0.01) and increased body weight gain (P < 0.05) 24 h or 10 d, respectively, after initiation of clenbuterol treatment. A total of 22,787 probesets were evaluated with 113 probesets defined as differentially expressed based on a false discovery rate of 10%. Differential mRNA abundance of four of these genes was validated in an independent experiment by quantitative PCR. Patterns of gene expression change observed from the microarray data suggested more genes are differentially regulated after 24 h than 10 d of BA administration. Functional characterization of differentially expressed genes revealed several categories that participate in biological processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signaling pathways, and genes involved in polyamine metabolism. Included within these lists were several genes previously associated with myogenic differentiation, along with multiple genes known to promote a general up-regulation of translation. Conclusions: Global evaluation of gene expression after administration of clenbuterol identified changes gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in the mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on the proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Additionally, increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators, rather than a sustained up-regulation of gene expression at the transcriptional level. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle. Keywords: Compound administration, time course

Project description:Background: Clenbuterol, 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 Î²2-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. Steady state clenbuterol concentrations were achieved at approximately 50 hours post administration of the first dose for the low dose regimen and at approximately 18-19 days (10 days post administration of 3.2 Î¼g/kg) for the escalating dosing regimen. Following chronic administration of the low dose (0.8 Âµg/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 Î²2-adrenergic receptor were noted. For most genes, expression levels returned towards baseline levels following cessation of drug administration. Conclusion: This 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 Î²2-adrenergic receptor. Five healthy horses were studied. Twenty-two of the horses received 0.8 Âµg/kg clenbuterol PO BID for 30 days and an additional 4 horses received 0.8 Âµg/kg, BID x 3 days; 1.6 Âµg/kg, BID x 3 days; 2.4 Âµg/kg, BID x 3 days; 3.2 Âµg/kg, BID for 21 days. Muscle biopsy samples were collected one day prior to administration of the first dose of clenbuterol and at a number of times post drug administration. A final sample was collected one-week post administration of the final dose (35 days post administration of the first dose). The tissue was transferred to a cryovial containing RNAlater (Qiagen Inc, Valencia, CA) and stored at -20Â° C until processed. Total RNA was purified using a miRNeasy Mini kit (RNeasy Mini, Qiagen Inc, Valencia, CA) and following the manufacturerâs instructions. Total RNA integrity was assessed using the Experion Automated Electrophoresis System (Bio-rad, Hercules, CA). Only RNA samples with RIN â¥ 8 and 260/280 ratios between 1.7 and 2.1 were used. Equine specific microarrays (EquGene-1.0-st; Affymetrix, Santa Clara, CA), containing expression profiling of 30,559 well-characterized genes using 504,603 probes were utilized. To reduce biological noise as a result of genetic variability, each horse was analyzed separately and served as their own control for comparison of baseline samples to day 14 (low dose administration) or day 28 (escalating dose regimen). Five biological replicates per time point were tested. Purified total RNA (5 Âµg) was used for cDNA synthesis in accordance with the AmbionÂ® WT Expression assay kit (Affymetrix, Santa Clara, CA) manufacturerâs protocol. In vitro transcription was used to incorporate biotin labels using the GeneChipÂ® WT Terminal Labeling system (Affymetrix, Santa Clara, CA) and samples hybridized to the Equine microarray. Arrays were washed and stained on a Fluidics Station 450 (Affymetrix, Santa Clara, CA) and scanned on a GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA) in accordance with manufacturerâs protocols. The microarrays were evaluated for differential gene expression using Transcriptome Analysis Console (TAC) and for hybridization quality control using Expression Console Software (Affymetrix, Santa Clara, CA). In brief, a total of five Cell Intensity Files were generated per time point, uploaded and normalized under the following conditions: PM (perfect match)-only as a PM intensity adjustment and the Robust Multichip Analysis (RMA) quantification method. For evaluation of the assays performance the number of differentially expressed genes detected between baseline and day 14 (low dose administration) or day 28 (escalating dose regimen) were assessed. Based on the TAC software userâs manual, genes with mean transformed ratios significantly less than -2 and larger than +2 were considered significantly regulated. A number of the significant genes were selected by filtering the genes using an ANOVA (p value < 0.05). A Pearson's correlation coefficient was used to calculate linear dependence between time point and baseline samples to evaluate the correlation coefficient, where 1 was a positive correlation and 0 was no correlation. For each probeset, expression at day 14 (low dose administration) or day 28 (escalating dose regimen) was compared to expression at baseline in the same horse using a paired t-test. Fold changes and their confidence intervals were calculated by exponentiating (base 2) the mean within-horse difference in expression for each gene and the associated t confidence intervals. P-values were adjusted for multiple testing using the False Discovery Rate (FDR) method. Analyses were conducted using the statistical software environment R, version 3.0.2 (R Core Team, 2013).

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Mus musculus

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