Clenbuterol Administration Research Articles

Effects on cerebral blood flow after single doses of the β2 agonist, clenbuterol, in healthy volunteers and patients with mild cognitive impairment or Parkinson's disease.

Cerebral hypometabolism occurs years prior to a diagnosis of neurodegenerative diseases and coincides with reduced cerebral perfusion and declining noradrenergic transmission from the locus coeruleus. In pre-clinical models, β-adrenoceptor (β-AR) agonists increase cerebrocortical glucose metabolism, and may have therapeutic potential for neurodegenerative diseases. This study investigated the safety and effects on regional cerebral blood flow (rCBF) of the oral, brain-penetrant β2-AR agonist, clenbuterol, in healthy volunteers (HV) and patients with mild cognitive impairment (MCI) or Parkinson's disease (PD). This study evaluated the safety and effects on cerebral activity of the oral, brain-penetrant, β2-AR agonist clenbuterol (20-160 μg) in healthy volunteers and patients with MCI or PD. Regional CBF, which is tightly coupled to glucose metabolism, was measured by arterial spin labelling MRI in 32 subjects (25 HV and 8 MCI or PD) across five cohorts. In some cohorts, low doses of nadolol (1-5mg), a β-AR antagonist with minimal brain penetration, were administered with clenbuterol to control peripheral β2-AR responses. Significant, dose-dependent increases in rCBF were seen in multiple brain regions, including hippocampus, amygdala and thalamus, following the administration of clenbuterol to HVs (mean changes from baseline in hippocampal rCBF of -1.7%, 7.3%, 22.9%, 28.4% 3h after 20, 40, 80 and 160 μg clenbuterol, respectively). In patients with MCI or PD, increases in rCBF following 80 μg clenbuterol were observed both without and with 5mg nadolol (in hippocampus, 18.6%/13.7% without/with nadolol). Clenbuterol was safe and well-tolerated in all subjects; known side effects of β2-agonists, including increased heart rate and tremor, were mild in intensity and were blocked by low-dose nadolol. The effects of clenbuterol on rCBF were evident both in the absence and presence of low-dose nadolol, suggesting central nervous system (CNS) involvement. Concomitant inhibition of the peripheral effects of clenbuterol by nadolol confirms that meaningful β2-AR antagonism in the periphery was achieved without interrupting the central effects of clenbuterol on rCBF.

Clenbuterol Prevents Mechanical Unloading-Induced Myocardial Atrophy via Upregulation of Transient Receptor Potential Channel-3.

Left ventricular assist device in combination with clenbuterol has been demonstrated to significantly improve heart function in patients with advanced heart failure. However, the roles of clenbuterol in mechanical unloading and its underlying mechanism are poorly understood. A rat abdominal heart transplantation model has been developed to mimic mechanical unloading of the heart. The recipient rats were randomly segregated into experimental groups for the daily administration of either saline (the "Trans" group; n = 13) or clenbuterol (2 mg/kg, the "Trans + CB" group; n = 12). Another group of 10 rats served as a treatment mimic control/sham animals (the "Sham" group). All interventions were performed via intraperitoneal injections once daily for 4 weeks. The Trans group animals exhibited myocardial atrophy and dysfunction with decreased expression levels of transient receptor potential channel 3 (TRPC3) and phospholipase C-β1 (PLC-β1) at 4 weeks post-transplantation. Administration of clenbuterol improved cardiac function, prevented myocardial atrophy, and restored expression of TRPC3 and PLC-β1 in the unloaded hearts of the "Trans + CB" animals at 4 weeks post-transplantation. Silencing of the TRPC3 gene by siRNA inhibited the pro-hypertrophic effect of clenbuterol in the rat primary cardiomyocytes in vitro. Furthermore, U73122, an inhibitor of the PLC-β1/diacylglycerol (DAG) pathway, significantly attenuated clenbuterol-induced upregulation of TRPC3 in cardiomyocytes. These findings suggest that the anti-atrophic effect of clenbuterol may be dependent on the upregulation of TRPC3 through the activation of the PLC-β1/DAG pathway during mechanical unloading. The results of our study reveal a potential target for the prevention and treatment of mechanical unloading-induced myocardial atrophy.

Production of a matrix certified reference material for measurement and risk monitoring of clenbuterol in mutton.

A novel matrix certified reference material (CRM) for clenbuterol in mutton (GBW 10216) was developed to assist measurement and risk monitoring of clenbuterol in mutton. The candidate CRM raw samples were obtained by oral administration of clenbuterol and investigating the pharmacokinetics of clenbuterol in sheep. A high-precision isotope dilution coupled with liquid chromatography tandem mass spectrometry (LC-ID-MS/MS) method was established and assigned the value of clenbuterol in mutton powder through combined detection of nine inter-laboratories. The certified value with expanded uncertainty was 21.1 ± 2.2μg/kg (k = 2, 95% confidence) for clenbuterol in mutton. The prepared matrix CRM was sufficiently homogeneous between and within bottles. The long-term stability of clenbuterol in mutton powder was evaluated for 12months at -20℃ and short-term stability for 7days at 4℃ and 50℃. The uncertainties originating from characterization, homogeneity, and stability were systematically analyzed and evaluated. The prepared matrix CRM can be applied for proficiency testing and nationwide risk monitoring programs to guarantee the accuracy and comparability of clenbuterol measurement results in mutton.

PSIII-A-8 Daily Clenbuterol Administration to Lambs Born with low Birthweight due to Intrauterine Growth Restriction Recovered Growth Rates and Improved Body Composition at 60 D of age

Abstract Low birthweight due to intrauterine growth restriction (IUGR) results from unfavorable in utero conditions (i.e., hypoxemia, hypoglycemia) that impairs growth capacity for muscle and other tissues. This programming effect reduces growth efficiency in IUGR offspring below their genetic potential. Specific programming mechanisms are not known, but recent evidence indicates reduced β2 adrenergic regulation of IUGR muscle. Thus, our objective was to determine if early-life growth deficits in IUGR juvenile lambs are improved by manipulating β2 adrenergic activity with daily IM administration of the β2 agonist, clenbuterol (Clen), from birth to 60 d of age. Maternal heat stress-induced IUGR (n=10) and IUGR+Clen (n=9) lambs were smaller (P< 0.05) than controls (n=11) at birth, weighed 18% and 11% less (P< 0.05), respectively, than controls at 30 d of age, and weighed 16% and 13% less (P< 0.05), respectively, at 60 d of age. Average daily gain at 30 and 60 d of age was reduced (P≤0.05) for IUGR but not IUGR+Clen lambs compared to controls. Bioelectrical impedance analysis was used to estimate body composition in live lambs at 58 d of age. Estimated fat-free lean mass and estimated muscle mass were less (P≤0.05) for IUGR but not IUGR+Clen lambs compared to controls. Estimated protein and fat content as well as protein:fat were likewise reduced (P< 0.05) for IUGR but not IUGR+Clen lambs. At necropsy, whole hindlimbs tended to be lighter (P≤0.09) and flexor digitorum superficialis muscle was lighter (P< 0.05) for IUGR but not IUGR+Clen lambs compared to controls. Loin-eye areas measured in chilled carcasses were 15% smaller (P< 0.05) for IUGR lambs and 19% larger (P< 0.05) for IUGR+Clen lambs compared to controls. From this study, we conclude that poor postnatal growth and body composition observed in IUGR juvenile lambs was improved when clenbuterol was used to target programmed deficits in β2 adrenergic regulation of muscle.

Effect of Clenbuterol on Muscle Activity During Exercise in Standardbred Horses

Clenbuterol (β2 agonist) is a commonly administered bronchodilator in race and performance horses. While long-term administration can alter exercise performance and muscle properties, little is known about its effects on these parameters following short-term administration. A single dose of clenbuterol (0.80 µg/kg) was expected to alter muscle activity of the extensor carpi radialis, semitendinosus, and longissimus dorsi during submaximal exercise. Eight mature Standardbred horses exercised for 2 minutes at 5 m/s on a high-speed treadmill following clenbuterol dosing (clenbuterol) or no dosing (control) in a crossover experimental design. Surface electromyography (sEMG) data were collected continuously from the muscles of interest and processed to determine average rectified value (ARV) and median frequency (MF) of the signal during peak muscle activation (100 ms period) during 15 strides. ARV data were log transformed. Data were analyzed with a mixed model ANOVA with fixed effects of period and treatment and a random effect of horse. No differences (P > .05) in amplitude (ARV) or frequency (MF) of the EMG signal were detected following clenbuterol administration. Thus, a one-time dose of clenbuterol had no statistically detectable effect on muscle activity during submaximal exercise. Further studies should be undertaken to confirm these results and examine the effects of long-term administration on muscle activity during exercise.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with β2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective β2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of β-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle β2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic β2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating β2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

Insight View on the Role of in Ovo Feeding of Clenbuterol on Hatched Chicks: Hatchability, Growth Efficiency, Serum Metabolic Profile, Muscle, and Lipid-Related Markers.

Simple SummaryThis study examined the effects of ovo injection of clenbuterol on fat deposition and growth performance in chickens, which is prejudicial to poultry consumers and muscle growth-related genes, egg hatchability, and fertility. The achieved result showed a definite effect of clenbuterol on body gain and hatchability. It decreased fat deposition and upregulation of muscle growth-related gene expressions accompanied by modulation of fatty and amino acid composition, reflecting a new insight into the intracellular pathways of clenbuterol supplementation on chicks.The present study aimed to assess the in ovo administration of clenbuterol on chick fertility, growth performance, muscle growth, myogenic gene expression, fatty acid, amino acid profile, intestinal morphology, and hepatic lipid-related gene expressions. In this study, 750 healthy fertile eggs from the local chicken breed Dokki-4 strain were analyzed. Fertile eggs were randomly divided into five experimental groups (150 eggs/3 replicates for each group). On day 14 of incubation, in addition to the control group, four other groups were established where 0.5 mL of worm saline (30 °C) was injected into the second group of eggs. In the third, fourth, and fifth groups, 0.5 mL of worm saline (30 °C), 0.9% of NaCl, and 10, 15, and 20 ppm of clenbuterol were injected into the eggs. Results suggested that clenbuterol increased growth efficiency up to 12 weeks of age, especially at 15 ppm, followed by 10 ppm, decreased abdominal body fat mass, and improved hatchability (p < 0.01). Clenbuterol also modulated saturated fatty acid levels in the breast muscles and improved essential amino acids when administered at 10 and 15 ppm. Additionally, clenbuterol at 15 ppm significantly decreased myostatin gene expression (p < 0.01) and considerably increased IGF1r and IGF-binding protein (IGFBP) expression. Clenbuterol administration led to a significant upregulation of hepatic PPARα, growth hormone receptor, and Lipoprotein lipase (LPL) mRNA expression with a marked decrease in fatty acid synthase (FAS) and sterol regulatory element-binding protein 1 (SREBP-1c) expression. In conclusion, the current study revealed that in ovo injection of clenbuterol showed positive effects on the growth of hatched chicks through reduced abdominal fat deposition, improved intestinal morphology, and modulation of hepatic gene expressions in myogenesis, lipogenesis, and lipolysis.

Clenbuterol-sensitive delayed outward potassium currents in a cell model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansions in the androgen receptor (AR) gene. SBMA is characterized by selective dysfunction and degeneration of motor neurons in the brainstem and spinal cord through still unclear mechanisms in which ion channel modulation might play a central role as for other neurodegenerative diseases. The beta2-adrenergic agonist clenbuterol was observed to ameliorate the SBMA phenotype in mice and patient-derived myotubes. However, the underlying molecular mechanism has yet to be clarified. Here, we unveil that ionic current alterations induced by the expression of polyQ-expanded AR in motor neuron-derived MN-1 cells are attenuated by the administration of clenbuterol. Our combined electrophysiological and pharmacological approach allowed us to reveal that clenbuterol modifies delayed outward potassium currents. Overall, we demonstrated that the protection provided by clenbuterol restores the normal function through the modulation of KV2-type outward potassium currents, possibly contributing to the protective effect on motor neuron toxicity in SBMA.

Profiling of transcriptional biomarkers in FFPE liver samples: PLS-DA applications for detection of illicit administration of sex steroids and clenbuterol in veal calves

Administering growth promoters to meat-producing animals is strictly regulated within the European Community to preserve consumers’ safety. Nevertheless, illicit misuse of steroids and other veterinary drugs to increase animal production has still been found by National Residues Control Plans (NRCPs), mainly based on expensive targeted multi-class/multi-residue analytical methods, used for confirming quantification analysis of these residues in tissues and biological fluids. The setup of complementary novel diagnostic methods, based on indirect biomarkers of exposure to anabolic substances is, therefore, needed to update current tests available in NRCPs at the screening level, where cheaper, sensitive and untargeted methods are required. Biological effects of illicit treatment transcriptomic analysis has reached a good grade of standardization, identified by microarray and RNAseq technologies of large panels of differentially expressed gene targets (DEG) related to abuse of different classes of anabolic molecules (e.g. sex steroids, thyreostats, β2-agonists, glucocorticoids, etc.). The aim of the work was to identify a minimum set of molecular markers able to detect illicit administration of two of the most recurrent classes of growth promoters (sex steroids and B2-agonists) on formalin-fixed paraffin-embedded (FFPE) tissues, being a potentially straightforward sampling strategy at the slaughterhouse level. A PCR array to allow quantitative profiling of 48 genetic targets was, therefore, developed on FFPE liver samples from veal calves. Conventional univariate and alternative approaches based on multivariate classification methods were considered for gene expression profiling of 92 samples collected from different animal trials, where effects of nandrolone, estradiol, their combination, and clenbuterol were studied.The developed Partial Least Squares – Discriminant Analysis (PLS-DA) models show the successful setup of a novel screening strategy based on multiple transcriptional biomarkers, able to expand tested sample sizes, and consequently strengthen current NRCPs.

Oxidized Hyaluronic Acid Hydrogels as a Carrier for Constant-Release Clenbuterol Against High-Fat Diet-Induced Obesity in Mice.

The global obesity population is increasing year-by-year, and the related cost is sharply increasing annually. There are several methods available to combat obesity; however, there is a lack of a single tool that is both safe and efficacious. The use of Clenbuterol in bodybuilding and by professional athletes is controversial owing to its side effects, including hepatotoxicity. This study administered Clenbuterol at a much lower dose than the established safety level, and rather than through oral administration, the treatments were delivered through controlled-release intra-adipose injection. The different dosing and mode of administration will lower the risk of side effects, increase the safety profile, and could facilitate use in the anti-obesity market. A thermo-sensitive hydrogel was used as the carrier uploaded with Clenbuterol to achieve controlled-release. In the in vitro study, the developed new formulae were not cytotoxic to 3T3-L1 cells and could inhibit lipogenesis effectively. In the animal study, the mice were fed a high-fat diet and treated with Clenbuterol by oral administration, or injected with Clenbuterol-modified hyaluronate hydrogel (HAC) regularly. Both groups showed reduction in whole-body, visceral, and gonadal fat contents and body weight. The abdominal fat was analyzed using MRI imaging in adipose mode and water mode. The abdominal fat ratio in the mice treated with normal diet and those given intra-adipose injections with HAC had the lowest value among the test groups. The mice treated with high-fat diet (HFD) showed the highest value of 53.78%. The chronic toxicity in-vivo test proved that controlled-release injections of 2–10 µg Clenbuterol daily were safe, as demonstrated in the blood elements and serological analyses. This study developed a new and promising method for anti-obesity treatment, using a monthly intra-adipose controlled-release injection of HAC. The developed new formulae of Clenbuterol not only effectively decreased body weight and body fat content but also inhibited lipogenesis on the harvested visceral tissue and reduced adipose tissue around the gonadal fat area. The side effects induced by traditional oral administration of Clenbuterol were not observed in this research; this has excellent potential to be a useful tool for future obesity treatment without safety concerns.

Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity.

Beta 2 adrenergic receptor (β2 AR) activation in the central and peripheral nervous system has been implicated in nociceptive processing in acute and chronic pain settings with anti-inflammatory and anti-allodynic effects of β2-AR mimetics reported in several pain states. In the current study, we examined the therapeutic efficacy of the β2-AR agonist clenbuterol in a rat model of persistent postsurgical hypersensitivity induced by disruption of descending noradrenergic signaling in rats with plantar incision. We used growth curve modeling of ipsilateral mechanical paw withdrawal thresholds following incision to examine effects of treatment on postoperative trajectories. Depletion of spinal noradrenergic neurons delayed recovery of hypersensitivity following incision evident as a flattened slope compared to non-depleted rats (-1.8 g/day with 95% CI -2.4 to -1.085, p < 0.0001). Chronic administration of clenbuterol reduced mechanical hypersensitivity evident as a greater initial intercept in noradrenergic depleted (6.2 g with 95% CI 1.6 to 10.8, p = 0.013) and non-depleted rats (5.4 g with 95% CI 1.2 to 9.6, p = 0.018) with plantar incision compared to vehicle treated rats. Despite a persistent reduction in mechanical hypersensitivity, clenbuterol did not alter the slope of recovery when modeled over several days (p = 0.053) or five weeks in depleted rats (p = 0.64). Systemic clenbuterol suppressed the enhanced microglial activation in depleted rats and reduced the density of macrophage at the site of incision. Direct spinal infusion of clenbuterol failed to reduce mechanical hypersensitivity in depleted rats with incision suggesting that beneficial effects of β2-AR stimulation in this model are largely peripherally mediated. Lastly, we examined β2-AR distribution in the spinal cord and skin using in-situ hybridization and IHC. These data add to our understanding of the role of β2-ARs in the nervous system on hypersensitivity after surgical incision and extend previously observed anti-inflammatory actions of β2-AR agonists to models of surgical injury.

Treatment with a \u03b2-2-adrenoceptor agonist stimulates glucose uptake in skeletal muscle and improves glucose homeostasis, insulin resistance and hepatic steatosis in mice with diet-induced obesity

Aims/hypothesisChronic stimulation of β2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of β2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of β2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect.MethodsC57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen.ResultsConsistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg−1 day−1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%, p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 ± 0.31%/min in comparison with 0.15 ± 0.36%/min in control mice, p < 0.05) and drastically reduced hepatic steatosis (by 40%, p < 0.01) and glycogen (by 23%, p < 0.05).Conclusions/interpretationClenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective β2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans.Graphical abstract

Clenbuterol accelerates recovery after immobilization-induced atrophy of rat hindlimb muscle

Using immunohistochemistry, we investigated whether daily administration of clenbuterol (CLE; 1 mg/kg body weight per day) accelerates recovery after casted immobilization(IMM)-induced atrophy of fast-twitch plantaris and slow-twitch soleus muscles. Adult male Sprague-Dawley rats were assigned to the control (CON), casted immobilization (IMM), casted immobilization following recovery control (RCON), and casted immobilization following recovery with CLE administration (RCLE) groups. Casted immobilization and recovery periods were 9 and 14days, respectively. Rats of the CON group were subjected to the experiment simultaneously with the IMM group.Nine days of immobilization induced muscle fiber atrophy, which was greater in the soleus muscle than in the plantaris muscle. After the 2-week recovery period, the cross-sectional areas of each fiber type in both muscles were higher in the RCON group than in the IMM group. The cross-sectional areas of each fiber type in both muscles in the RCLE group were larger than those in the RCON group. The myonuclear number of each fiber type of the plantaris muscle in the RCON and RCLE groups was higher than that in the CON group. In contrast, the myonuclear number per fiber of the soleus muscle was not affected by hindlimb immobilization, reloading, and clenbuterol administration regardless of muscle fiber type.These results suggest that CLE accelerates the recovery of atrophied plantaris and soleus muscles fibers and that their mechanisms of responses to CLE in both muscles may be different during recovery period after muscle atrophy.

Effects of clenbuterol administration on mitochondrial morphology and its regulatory proteins in rat skeletal muscle.

Clenbuterol induces a slow‐to‐fast fiber type transition in skeletal muscle. This muscle fiber transition decreased mitochondrial oxidative capacity and respiratory function. We hypothesized that the clenbuterol‐mediated reduction in oxidative capacity is associated with the alteration in mitochondrial morphology. To verify this hypothesis, we examined whether clenbuterol alters mitochondrial morphology and mitochondrial regulatory proteins in rat skeletal muscle. Clenbuterol was administered to rats via drinking water (30 mg/L) for 3 weeks. Myosin heavy chain (MHC) isoform composition, mitochondrial morphology, and fusion and fission regulatory protein levels in deep region and superficial region in tibialis anterior (TA) muscles were assessed. Clenbuterol induced the fiber type transition from slow to fast in both the regions of TA. The levels of optic atrophy protein 1, mitofusin 2, and mitochondrial fission 1, but not of dynamin‐related protein 1, significantly decreased in deep and superficial muscles after clenbuterol administration (P < 0.01). Also, observation using the transmission electron microscopy showed a decrease in mitochondrial volume (P < 0.05) and an increase in proportion of continuous or interacting mitochondria across Z‐lines (P < 0.05). We showed that clenbuterol administration induces a transition in the muscle fiber type composition toward fast phenotype and causes alterations in mitochondrial morphology with a concomitant decrease in mitochondrial fusion and fission regulatory protein levels. These mitochondrial morphological alterations may influence deleterious effects on skeletal muscle metabolism.

Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth.

We previously identified PEPCK-M (encoded by the Pck2 gene) to be highly up-regulated in skeletal muscle of pigs treated with Ractopamine, an anabolic beta-adrenergic receptor agonist. To determine whether PEPCK-M had a causative role in modulating the skeletal muscle growth response to Ractopamine, we used adeno-associated virus 1 (AAV1) to over-express Pck2 (AAV-Pck2) in murine skeletal muscle. A contralateral limb design was employed, such that each mouse served as its own control (injected with a GFP-only expressing AAV1, labelled AAV-GFP). Daily injections of Clenbuterol (1 mg/kg for 21 days) or vehicle control were also carried out to assess the effects of AAV-Pck2 overexpression on the anabolic response to a beta-adrenergic agonist. AAV-Pck2 overexpression in leg muscles of male C57BL6/J mice for 4 weeks (6–10 weeks of age) increased Pck2 mRNA (~100-fold), protein (not quantifiable) and enzyme activity (~3-fold). There was a trend (p = 0.0798) for AAV-Pck2 overexpression to reduce TA muscle weights, but there was no significant effect on muscle fibre diameters or myosin heavy chain isoform (MyHC) mRNA expression. When skeletal muscle growth was induced by daily administration of Clenbuterol (for 21 days), overexpression of AAV-Pck2 had no effect on the growth response, nor did it alter the expression of Phosphoserine Aminotransferase-1 (Psat1) or Asparagine Synthetase (Asns) mRNA or the Clenbuterol-induced decreases in MyHC IIa and IIx mRNA expression (p = 0.0065 and p = 0.0267 respectively). However AAV-Pck2 overexpression reduced TA muscle weights (p = 0.0434), particularly in the Control (vehicle treated) mice (p = 0.059 for AAV x Clenbuterol interaction) and increased the expression of Seryl-tRNA Synthetase (Sars) mRNA (p = 0.0477). Hence, contrary to the original hypothesis, AAV-Pck2 overexpression reduced TA muscle weights and did not mimic or alter the muscle hypertrophic effects of the beta-adrenergic agonist, Clenbuterol.

\u03b2-arrestin 1 regulates \u03b22-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility

Backgroundβ2-adrenergic receptors (β2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of β2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that β2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, β-arrestin, the precise role of β-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the β2AR by clenbuterol would engage β-arrestin as a key transducer of anabolic skeletal muscle growth.MethodsThe contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and β-arrestin 1 knockout mice (βarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and βarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak’s or Tukey’s multiple comparison test and the Student’s t test.ResultsChronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to βarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking β-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of β-arrestin 1.ConclusionsWe have identified a previously unappreciated role for β-arrestin 1 in mediating β2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

Chronic Beta2‐Adrenergic Receptor Stimulation Improves Whole‐Body Glucose Homeostasis through Skeletal Muscle Metabolic Reprogramming

Type 2 diabetes (T2D) has emerged as a major health problem worldwide. Since skeletal muscle (SKM) is the major site of insulin‐stimulated glucose disposal in the body, SKM insulin resistance plays a crucial role in the pathogenesis of this disorder. Drugs that could enhance or mimic insulin action in SKM hold great therapeutic potential for the treatment of obesity and T2D.Previous work carried out with cultured SKM cells indicates that activation of SKM β2‐adrenergic receptors (β2‐ARs) can promote glucose uptake. In rodent models of T2D, chronic administration of clenbuterol, a selective β2‐AR agonist, greatly improves glucose tolerance. However, the mechanism and in vivo tissue(s) responsible for the actions of clenbuterol are still unclear. This study was designed to test the hypothesis that the beneficial metabolic effects of clenbuterol observed in vivo are mediated by SKM β2‐ARs. For this purpose, we generated mice that lack β2‐ARs or the Gαs subunit selectively in SKM (SKM‐β2‐AR‐KO and SKM‐Gs‐KO mice, respectively). We also generated transgenic mice expressing a Gs‐DREADD selectively in SKM (SKM‐Gs‐DREADD mice).As expected, chronic administration of clenbuterol via drinking water improved glucose tolerance in wild‐type (WT) mice without affecting glucose‐stimulated insulin secretion or insulin tolerance. The SKM selectivity of this chronic clenbuterol effect was confirmed using SKM‐β2‐KO, SKM‐Gs‐KO, and SKM‐Gs‐DREADD mice. RNA‐seq and functional data indicated increases in SKM glucose accumulation, glycolysis, and glycogen synthesis following chronic treatment, suggesting a switch of SKM metabolism towards enhanced glucose utilization.Multiple components of known, but also less studied metabolic pathways seem to be simultaneously involved in mediating the beneficial metabolic effects of chronic β2‐AR stimulation.Our study represents a striking example of the complexity of β2‐AR signaling in vivo. Further analysis of β2‐AR signaling pathways in SKM could help identify potential drug targets for new T2D treatments.Support or Funding InformationThis research was funded by the Intramural Research Program of the NIDDK.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The β2-adrenoceptor agonist clenbuterol reduces the neuroinflammatory response, neutrophil infiltration and apoptosis following intra-striatal IL-1β administration to rats

Objectives: Clenbuterol is a brain penetrant β2-adrenoceptor agonist with anti-inflammatory and putative neuroprotective properties. In the present investigation, the effect of clenbuterol was assessed in a rat model of acute brain injury induced by intra-striatal administration of the pro-inflammatory cytokine IL-1β.Methods: Clenbuterol (0.5 mg/kg; i.p.) was administered one hour prior to stereotactically delivered IL-1β (100 ng) into the striatum. Four hours postinjection, rats were anesthetized, blood samples were collected for circulating cytokine and chemokine analysis, and the ipsilateral striatum and liver tissue were harvested for mRNA expression analysis of target genes.Results: Intrastriatal IL-1β provoked an inflammatory response with increased expression of IL-1β and the pro-inflammatory cytokine TNF-α. TNF-α expression was also increased in the liver and circulating concentrations of the chemokine cytokine-induced neutrophil chemoattractant 1 (CINC-1) were raised in response to intrastriatal IL-1β administration. The striatal response was accompanied by NFκB activation and 24 hours postinjection, increased immunoreactivity of the neutrophil marker MBS-2, indicative of cell infiltration and increased TUNEL staining, a cell marker of apoptosis. Treatment with clenbuterol attenuated all IL-1β-induced changes in the striatum including MBS-2 immunoreactivity and TUNEL + staining. Clenbuterol also attenuated IL-1β-induced expression of TNF-α in the liver and the increase in circulating CINC-1 concentrations.Conclusions: The results provide evidence that clenbuterol elicits anti-inflammatory effects, suppresses the peripheral acute phase response and reduces the infiltration of neutrophils and apoptotic response to acute IL-1β–induced brain injury. Suppression of both the central and peripheral response following clenbuterol administration may contribute to its protective properties following brain injury.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial – the potential of enantiomeric separation for doping control analysis

ABSTRACTThe differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes’ claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.

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

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.