Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective ß2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a ß2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

ß-adrenoceptors of the infra-limbic cortex mediate corticosterone-induced enhancement of acquisition and consolidation of fear memory extinction in rats.

The extinction of auditory fear conditioning (AFC) refers to reducing the fear responses induced following repeated presentation of a conditioned stimulus (tone) in the absence of an unconditioned stimulus (electric foot shock). Glucocorticoid receptors (GRs) play an important role in extinction, but the underlying neurobiological mechanisms are unclear. This study aimed to investigate the interaction between glucocorticoids and ß-adrenoceptors of the infra-limbic cortex (IL) in regulating the acquisition and consolidation of fear memory extinction in rats. Male rats were trained to AFC and received three trial tones (30 s, 4 kHz, 80 dB) co-terminated with a footshock (0.8 mA, 1 s; unconditioned stimulus). Extinction trials were conducted over 3 days after training (Ext 1-3). In experiment 1, rats received clenbuterol (0.25 mg/kg/2 ml, IP) as a ß2-adrenoceptor agonist or propranolol (2.5 mg/kg/2 ml, IP) as a ß-adrenoceptors antagonist before Ext 1 and immediately after Ext 1 and Ext 2 followed by systemic injection of corticosterone (3 mg/kg/2 ml, IP). In Experiment 2, separate groups of rats received a bilateral intra-IL injection of clenbuterol (50 ng/0.5 µl/side) or propranolol (500 ng/0.5 µl/side) followed by a systemic injection of corticosterone (3 mg/kg/2 ml) before Ext 1 and immediately after Ext 1 and Ext 2. Results indicated that systemic and intra-IL injections of clenbuterol and propranolol inhibited and increased the facilitative effects of corticosterone on fear memory extinction, respectively. These findings show that activating ß-adrenergic receptors in the IL mediates glucocorticoid effects on the acquisition and consolidation of auditory-conditioned fear memory extinction.

Adult and regenerating planarians respond differentially to chronic drug exposure.

There is a lack of data on the effects of chronic exposure to common drugs and stimulants on the developing nervous system. Freshwater planarians have emerged as a useful invertebrate model amenable to high-throughput behavioral phenotyping to assay chemical safety in adult and developing brains. Here, we leverage the unique strength of the system to test in parallel for effects on the adult and developing nervous system, by screening ten common drugs and stimulants (forskolin, clenbuterol, LRE-1, MDL-12,330A, adenosine, caffeine, histamine, mianserin, fluoxetine and sertraline) using the asexual freshwater planarian Dugesia japonica. The compounds were tested up to 100 µM nominal concentration for their effects on planarian morphology and behavior. Quantitative phenotypic assessments were performed on days 7 and 12 of exposure using an automated screening platform. The antidepressants sertraline and fluoxetine were the most potent to induce lethality, with significant lethality observed at 10 µM. All ten compounds caused sublethal morphological and/or behavioral effects, with the most effects, in terms of potency and breadth of endpoints affected, seen with mianserin and fluoxetine. Four of the compounds (forskolin, clenbuterol, mianserin, and fluoxetine) were developmentally selective, causing effects at lower concentrations in regenerating planarians. Of these, fluoxetine showed the greatest differences between the two developmental stages, inducing many behavioral endpoints in regenerating planarians but only a few in adult planarians. While some of these behavioral effects may be due to neuroefficacy, these results substantiate the need for better evaluation of the safety of these common drugs on the developing nervous system.

Clenbuterol attenuates immune reaction to lipopolysaccharide and its relationship to anhedonia in adolescents.

While inflammation has been implicated in psychopathology, relationships between immune-suppressing processes and psychiatric constructs remain elusive. This study sought to assess whether ß2-agonist clenbuterol (CBL) would attenuate immune activation in adolescents with mood and anxiety symptoms following ex vivo exposure of whole blood to lipopolysaccharide (LPS). Our focus on adolescents aimed to target a critical developmental period when psychiatric conditions often emerge and prior to chronicity effects. To capture a diverse range of immunologic and symptomatologic phenotypes, we included 97 psychotropic-medication free adolescents with mood and anxiety symptoms and 33 healthy controls. All participants had comprehensive evaluations and dimensional assessments of psychiatric symptoms. Fasting whole-blood samples were collected and stimulated with LPS in the presence and absence of CBL for 6 hours, then analyzed for 41 cytokines, chemokines, and hematopoietic growth factors. Comparison analyses used Bonferroni-corrected nonparametric tests. Levels of nine immune biomarkers-including IL-1RA, IL-1ß, IL-6, IP-10, MCP-1, MIP-1α, MIP-1ß, TGF-α, and TNF-α-were significantly reduced by CBL treatment compared to LPS alone. Exploratory factor analysis reduced 41 analytes into 5 immune factors in each experimental condition, and their relationships with psychiatric symptoms were examined as a secondary aim. CBL + LPS Factor 4-comprising EGF, PDGF-AA, PDGF-AB/BB, sCD40L, and GRO-significantly correlated with anticipatory and consummatory anhedonia, even after controlling for depression severity. This study supports the possible inhibitory effect of CBL on immune activation. Using a data-driven method, distinctive relationships between CBL-affected immune biomarkers and dimensional anhedonia were reported, further elucidating the role of ß2-agonism in adolescent affective symptomatology.

Effects of long-term treatment with dietary theobromine on rat skeletal muscles.

BACKGROUND: Plastic changes of skeletal muscles, such as hypertrophy and atrophy, are dependent on physiological activities and regulated by a variety of signaling pathways, including cyclic adenosine monophosphate (cAMP) pathway. The cAMP inducing agents, such as the ß2-adrenergic agonist clenbuterol, are known to induce muscle hypertrophy, and has been reported to induce slow-to-fast transitions in rat soleus muscle. Theobromine, one of the active components of cacao, functions as an inhibitor of phosphodiesterase and increases cAMP. This study hypothesized that theobromine, like clenbuterol, can induce muscle hypertrophy and influence contractile properties. METHODS AND RESULTS: Male Wistar rats were fed a normal diet or a diet containing 0.05% theobromine for 20 weeks. Using biochemical, anatomical, and physiological techniques, effects of dietary theobromine on skeletal muscles (soleus, extensor digitorum longus, plantaris, and gastrocnemius) were examined. There were no significant differences in body weight, serum levels of proteins and lipids, muscle weights, dry/wet ratio of muscle weights, mitochondrial oxidation enzyme activity of muscles, isometric contractile properties of muscles, and muscle fatigue between control and theobromine-fed rats. Quantitative analysis of mRNA, however, revealed upregulation of myosin heavy chain 2x and myogenic differentiation 1, as previously reported in clenbuterol-treated muscles. CONCLUSION: The long-term theobromine (0.05%) diet in rats had no effect in inducing muscle hypertrophy and in changing contractile properties, although it had some similar effects of clenbuterol on muscle gene expression.

Systemic administration of a ß2-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 (ß2 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 ß2-AR mimetics reported in several pain states. In the current study, we examined the therapeutic efficacy of the ß2-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 ß2-AR stimulation in this model are largely peripherally mediated. Lastly, we examined ß2-AR distribution in the spinal cord and skin using in-situ hybridization and IHC. These data add to our understanding of the role of ß2-ARs in the nervous system on hypersensitivity after surgical incision and extend previously observed anti-inflammatory actions of ß2-AR agonists to models of surgical injury.

Role of PDK1 in skeletal muscle hypertrophy induced by mechanical load.

Phosphatidylinositol 3-kinase (PI3K) plays an important role in protein metabolism and cell growth. We here show that mice (M-PDK1KO mice) with skeletal muscle-specific deficiency of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key component of PI3K signaling pathway, manifest a reduced skeletal muscle mass under the static condition as well as impairment of mechanical load-induced muscle hypertrophy. Whereas mechanical load-induced changes in gene expression were not affected, the phosphorylation of ribosomal protein S6 kinase (S6K) and S6 induced by mechanical load was attenuated in skeletal muscle of M-PDK1KO mice, suggesting that PDK1 regulates muscle hypertrophy not through changes in gene expression but through stimulation of kinase cascades such as the S6K-S6 axis, which plays a key role in protein synthesis. Administration of the ß2-adrenergic receptor (AR) agonist clenbuterol activated the S6K-S6 axis in skeletal muscle and induced muscle hypertrophy in mice. These effects of clenbuterol were attenuated in M-PDK1KO mice, and mechanical load-induced activation of the S6K-S6 axis and muscle hypertrophy were inhibited in mice with skeletal muscle-specific deficiency of ß2-AR. Our results suggest that PDK1 regulates skeletal muscle mass under the static condition and that it contributes to mechanical load-induced muscle hypertrophy, at least in part by mediating signaling from ß2-AR.

ß2-Adrenergic Receptor Stimulation Upregulates Cx43 Expression on Glioblastoma Multiforme and Olfactory Ensheathing Cells.

Glioblastoma multiforme (GBM) is described as an invasive astrocytic tumor in adults. Despite current standard treatment approaches, the outcome of GBM remains unfavorable. The downregulation of connexin 43 (Cx43) expression is one of the molecular transformations in GBM cells. The Cx43 levels and subsequently gap junctional intercellular communication (GJIC) have an important role in the efficient transfer of cytotoxic drugs to whole tumor cells. As shown in our previous study, the stimulation of the ß2-adrenergic receptor (ß2-AR) leads to the modulation of Cx43 expression level in the GBM cell line. Here we further examine the effect of clenbuterol hydrochloride as a selective ß2-AR agonist on the Cx43 expression in human GBM-derived astrocyte cells and human olfactory ensheathing cells (OECs) as a potent vector for future gene therapy. In this experiment, first we established a primary culture of astrocytes from GBM samples and verified the purity using immunocytofluorescent staining. Western blot analysis was performed to evaluate the Cx43 protein level. Our western blot findings reveal that clenbuterol hydrochloride upregulates the Cx43 protein level in both primary human astrocyte cells and human OECs. Conversely, ICI 118551 as a ß2-AR antagonist inhibits these effects. Moreover, clenbuterol hydrochloride increases the Cx43 expression in primary human astrocyte cells and OECs co-culture systems, and ICI 118551 reverses these effects. To confirm the western blot results, immunocytofluorescent staining was performed to evaluate the ß2-AR agonist effect on Cx43 expression. Our immunocytofluorescent results supported western blot analysis in primary human astrocyte cells and the OECs co-culture system. The results of this study suggest that the activation of ß2-AR with regard to Cx43 protein levels enhancement in GBM cells and OECs might be a promising approach for GBM treatment in the future.

Beta2 -adrenergic agonist clenbuterol increases energy expenditure and fat oxidation, and induces mTOR phosphorylation in skeletal muscle of young healthy men.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 µg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Exacerbation of ozone-induced pulmonary and systemic effects by ß2-adrenergic and/or glucocorticoid receptor agonist/s.

Agonists of ß2 adrenergic receptors (ß2AR) and glucocorticoid receptors (GR) are prescribed to treat pulmonary diseases. Since ozone effects are mediated through the activation of AR and GR, we hypothesized that the treatment of rats with relevant therapeutic doses of long acting ß2AR agonist (LABA; clenbuterol; CLEN) and/or GR agonist (dexamethasone; DEX) would exacerbate ozone-induced pulmonary and systemic changes. In the first study, male 12-week-old Wistar-Kyoto rats were injected intraperitoneally with vehicle (saline), CLEN (0.004 or 0.02 mg/kg), or DEX (0.02 or 0.1 mg/kg). Since dual therapy is commonly used, in the second study, rats received either saline or combined CLEN + DEX (each at 0.005 or 0.02 mg/kg) one day prior to and on both days of exposure (air or 0.8ppm ozone, 4 hr/day x 2-days). In air-exposed rats CLEN, DEX or CLEN + DEX did not induce lung injury or inflammation, however DEX and CLEN + DEX decreased circulating lymphocytes, spleen and thymus weights, increased free fatty acids (FFA) and produced hyperglycemia and glucose intolerance. Ozone exposure of vehicle-treated rats increased bronchoalveolar lavage fluid protein, albumin, neutrophils, IL-6 and TNF-α. Ozone decreased circulating lymphocytes, increased FFA, and induced hypeerglycemia  and glucose intolerance. Drug treatment did not reverse ozone-induced ventillatory changes, however, lung effects (protein and albumin leakage, inflammation, and IL-6 increase) were exacerbated by CLEN and CLEN + DEX pre-treatment in a dose-dependent manner (CLEN > CLEN + DEX). Systemic effects induced by DEX and CLEN + DEX but not CLEN in air-exposed rats were analogous to and more pronounced than those induced by ozone. These data suggest that adverse air pollution effects might be exacerbated in people receiving LABA or LABA plus glucocorticoids.

Thyrotoxicosis Involves ß2-Adrenoceptor Signaling to Negatively Affect Microarchitecture and Biomechanical Properties of the Femur.

Background: Thyrotoxicosis increases bone turnover, resulting in net bone loss. Sympathetic nervous system (SNS) activation, via ß2-adrenoceptor (ß2-AR) signaling, also has osteopenic effects. Because thyroid hormones (TH) interact with the SNS to regulate several physiological processes, we hypothesized that this interaction also occurs to regulate bone mass. Previous studies support this hypothesis, as α2-AR knockout (KO) mice are less susceptible to thyrotoxicosis-induced osteopenia. Here, we evaluated whether TH-SNS interactions in bone involve ß2-AR signaling. Methods: Thyrotoxicosis was induced in 120-day-old female and male mice with ß2-AR gene inactivation (ß2-AR-/-) by daily treatment with supraphysiological doses of triiodothyronine (T3) for 12 weeks. The impact of thyrotoxicosis on femoral bone microarchitecture, remodeling, fracture risk, and gene expression of the receptor activator of nuclear factor-kappa-B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) pathway was evaluated. In addition, the effect of the ß2-AR-specific agonist clenbuterol (CL) on cAMP accumulation was determined in osteoblastic (MC3T3-E1) cells treated with T3 and/or 17ß-estradiol (E2). Results: Thyrotoxicosis negatively affected trabecular bone microarchitecture in wild-type (WT) females, but this effect was milder or nonexistent in ß2-AR-/- animals, whereas the opposite was seen in males. T3 treatment increased the femoral RANKL/OPG mRNA ratio and the endosteal perimeter and medullary area of the diaphysis in WT females and males, but not in ß2-AR-/- mice, suggesting that T3 promotes endosteal resorption in cortical bone, in a mechanism that involves ß2-AR signaling. T3 treatment increased endocortical mineral apposition rate only in WT females but not in ß2-AR-/- mice, suggesting that TH also induce bone formation in a ß2-AR signaling-dependent mechanism. T3 treatment decreased femoral resistance to fracture only in WT females, but not in KO mice. E2 and CL similarly increased cAMP accumulation in MC3T3-E1 cells; whereas T3 alone had no effect, but it completely blocked E2-stimulated cAMP accumulation, suggesting that some T3 effects on bone may involve E2/cAMP signaling in osteoblasts. Conclusions: These findings sustain the hypothesis that T3 interacts with the SNS to regulate bone morphophysiology in a ß2-AR signaling-dependent mechanism. The data also reveal sex as an important modifier of skeletal manifestations of thyrotoxicosis, as well as a modifier of the TH-SNS interactions to control bone microarchitecture, remodeling, and resistance to fracture.

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.

Inhibitory effect of mabuterol on proliferation of rat ASMCs induced by PDGF-BB via regulating [Ca2+]i and mitochondrial fission/fusion.

This study is aimed to investigate whether Mabuterol (Mab) inhibits proliferation of airway smooth muscle cells (ASMCs) induced by platelet-derived growth factor BB (PDGF-BB) and how far it is related to mitochondrial fission/fusion and intracellular calcium if it comes into play. To explore the mechanism of Mab's antagonizing the proliferation, Mdivi-1, DRP1 inhibitor, which has an inhibitory effect on mitochondrial fission, is used to compare with Mab. Cell viability was measured by either MTT or CCK-8. The inhibitory effect of Mab on S phase of ASM cell cycle induced by PDGF-BB was analyzed by flow cytometry (FCM). Fluo-3/AM, Ca2+ fluorescent probe, was used to detect Ca2+ fluorescence intensity by inverted microscope and flow cytometry. The gene expression of Drp-1 and Mfn-2 was observed with Real time PCR and the proteins of Drp-1, Mfn-2, PCNA and cyclin D1 were assessed by Western Blot. Mab and Mdivi-1 both suppressed the proliferation induced by PDGF-BB. The results from inverted microscope and flow cytometry showed that Mab inhibited [Ca2+]i in rat ASMCs induced by PDGF-BB. Cell cycle concept map illustrated that Mab significantly controlled the S phase of ASM cell cycle induced by PDGF-BB. As a consequence, Real time PCR and Western blot revealed the fact that Mab decreased the expression of Drp-1 mRNA and protein, and promoted the expression of Mfn-2 mRNA and protein. These findings suggested that Mab placed restrictions on the proliferation of rat ASMCs induced by PDGF-BB and the mechanism might be associated with the intracellular calcium inhibited and the mitochondrial fission/fusion regulated by Mab.

Structural and pharmacological basis for the induction of mitochondrial biogenesis by formoterol but not clenbuterol.

Mitochondrial dysfunction is associated with numerous acute and chronic degenerative diseases. The beta-2 adrenergic receptor (ß2AR) agonist formoterol induces mitochondrial biogenesis (MB), but other ß2AR agonists, such as clenbuterol, do not. We sought to identify the MB signaling pathway of formoterol and the differences in signaling between these two ligands that result in the differential induction of MB. While formoterol and clenbuterol increased cAMP, only formoterol increased the phosphorylation of Akt and its downstream target eNOS. The increase in Akt phosphorylation was Gßγ- and PI3K-dependent, and the increase in eNOS phosphorylation was Gßγ- and Akt-dependent. Only formoterol increased cGMP. Formoterol induced MB as measured by increases in uncoupled cellular respiration and PGC-1α and NDUFS1 mRNA expression and was blocked by inhibitors of Gßγ, Akt, NOS, and soluble guanylate cyclase. To identify distinct receptor-ligand interactions leading to these differences in signaling, we docked formoterol and clenbuterol to six structures of the ß2AR. Compared to clenbuterol, the methoxyphenyl group of formoterol interacted more frequently with V114 and F193, while its formamide group interacted more frequently with C191. These data indicate that the unique structural features of formoterol allow it to interact with the ß2AR to activate the Gßγ-Akt-eNOS-sGC pathway to induce MB.

Clenbuterol Attenuates hERG Channel by Promoting the Mature Channel Degradation.

Clenbuterol, a ß2-selective adrenergic receptor agonist, is illicitly used in weight loss and performance enhancement and animal production. Increasing evidence demonstrates that clenbuterol induces various kinds of arrhythmias and QTc interval prolongation. However, little is known about the underlying mechanism. Most drugs are associated with QTc prolongation through interfering with human ether-a-go-go-related gene (hERG) K+ channels. The present study aims to investigate the effects and underlying mechanisms of clenbuterol on the hERG channel. HEK 293 cells were transfected with wild type and Y652A or F656A mutants of the hERG channel and treated with clenbuterol. The hERG current was recorded using whole-cell patch-clamp technique, and protein level was evaluated by Western blot. We found that clenbuterol decreases the mature form of the hERG protein at the cell membrane in a concentration- and time-dependent manner, without affecting the immature form. Correspondingly, clenbuterol chronic treatment reduced hERG current to a greater extent compared to acute treatment. In the presence of Brefeldin A (BFA), which was used to block hERG channel trafficking to cell membrane, clenbuterol reduced hERG on plasma membrane to a greater extent than BFA alone. In addition, the hERG channel's drug binding sites mutant Y652A and F656A abolished clenbuterol-mediated hERG reduction and current blockade. In conclusion, clenbuterol reduces hERG channel expression and current by promoting the channel degradation. The effect of clenbuterol on the hERG channel is related to the drug-binding sites, Tyr-652 and Phe-656, located on the S6 domain. This biophysical mechanism may underlie clenbuterol-induced QTc prolongation or arrhythmia.

Beta-agonist stimulation ameliorates the phenotype of spinal and bulbar muscular atrophy mice and patient-derived myotubes.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of ß-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the ß-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that ß-agonist stimulation is a novel therapeutic strategy for SBMA.

Metabolomic investigation of porcine muscle and fatty tissue after Clenbuterol treatment using gas chromatography/mass spectrometry.

Clenbuterol is a ß-adrenergic agonist used as additive to increase the muscle mass of meat-producing animals. Previous studies were limited to evaluations of animal growth performance and determination of the residues. Several studies have focused on urine samples. Little information about the underlying molecular mechanisms that can explain Clenbuterol metabolism and promote energy repartition in animal muscle and fatty tissue is available. Therefore, this research aims to detect the metabolite variations in muscle and fatty tissue acquired from Chinese pigs fed with Clenbuterol using gas chromatography/mass spectrometry (GC/MS). Ten two-month old Enshi black pigs were fed under the same condition; five of which were fed with basic ration containing Clenbuterol for one month, whereas the other five pigs were fed only with basic ration. Muscle and fatty tissue were subjected to metabolomics analysis using GC/MS. Differences in metabolomic profiles between the two groups were characterized by multivariate statistical analysis. The muscle samples showed that 15 metabolites were significantly different in the Clenbuterol-treated group compared with the control group; 13 potential biomarkers were found in the fatty tissue. Most of the metabolites were associated with fatty acid metabolism and amino acid metabolism. Glycerol, phenylalanine, and leucine were the common metabolites between the muscle and fatty tissue. These metabolites may provide a new clue that contributes to the understanding of the energy reassignment induced by Clenbuterol.

Enhanced noradrenergic activity in the amygdala contributes to hyperarousal in an animal model of PTSD.

Increased activity of the noradrenergic system in the amygdala has been suggested to contribute to the hyperarousal symptoms associated with post-traumatic stress disorder (PTSD). However, only two studies have examined the content of noradrenaline or its metabolites in the amygdala of rats previously exposed to traumatic stress showing inconsistent results. The aim of this study was to investigate the effects of an inescapable foot shock (IFS) procedure (1) on reactivity to novelty in an open-field (as an index of hyperarousal), and (2) on noradrenaline release in the amygdala during an acute stress. To test the role of noradrenaline in amygdala, we also investigated the effects of microinjections of propranolol, a ß-adrenoreceptor antagonist, and clenbuterol, a ß-adrenoreceptor agonist, into the amygdala of IFS and control animals. Finally, we evaluated the expression of mRNA levels of ß-adrenoreceptors (ß1 and ß2) in the amygdala, the hippocampus and the prefrontal cortex. Male Wistar rats (3 months) were stereotaxically implanted with bilateral guide cannulae. After recovering from surgery, animals were exposed to IFS (10 shocks, 0.86mA, and 6s per shock) and seven days later either microdialysis or microinjections were performed in amygdala. Animals exposed to IFS showed a reduced locomotion compared to non-shocked animals during the first 5min in the open-field. In the amygdala, IFS animals showed an enhanced increase of noradrenaline induced by stress compared to control animals. Bilateral microinjections of propranolol (0.5µg) into the amygdala one hour before testing in the open-field normalized the decreased locomotion observed in IFS animals. On the other hand, bilateral microinjections of clenbuterol (30ng) into the amygdala of control animals did not change the exploratory activity induced by novelty in the open field. IFS modified the mRNA expression of ß1 and ß2 adrenoreceptors in the prefrontal cortex and the hippocampus. These results suggest that an increased noradrenergic activity in the amygdala contributes to the expression of hyperarousal in an animal model of PTSD.

Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks.

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases.

ß2-Agonist clenbuterol hinders human monocyte differentiation into dendritic cells.

Clenbuterol (CLB) is a beta2-adrenergic agonist commonly used in asthma therapy, but is also a non-steroidal anabolic drug often abused in sport doping practices. Here we evaluated the in vitro impact of CLB on the physiology and function of human monocytes and dendritic cells (DCs), instrumental in the development of immune responses. We demonstrate that CLB inhibits the differentiation of monocytes into DCs and this effect is specific and dependent on ß2-adrenergic receptor (AR) activation. We found that CLB treatment reduced the percentage of CD1a(+) immature DCs, while increasing the frequency of monocytes retaining CD14 surface expression. Moreover, CLB inhibited tumor necrosis factor-alpha (TNF-alpha) enhanced IL-(interleukin)-10 and IL-6 production. In contrast, CLB did not modulate the phenotypic and functional properties of monocytes and DCs, such as the surface expression of HLA-DR, CD83, CD80 and CD86 molecules, cytokine production, immunostimulatory activity and phagocytic activity. Moreover, we found that CLB did not modulate the activation of NF-kB in DCs. Moreover, we found that the differentiation of monocytes into DCs was associated with a significant decrease of ß2-ARs mRNA expression. These results provide new insights on the effect of CLB on monocyte differentiation into DCs. Considering the frequent illegal use of CLB in doping, our work suggests that this drug is potentially harmful to immune responses decreasing the supply of DCs, thus subverting immune surveillance.