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.

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-ß1 (PLC-ß1) at 4 weeks post-transplantation. Administration of clenbuterol improved cardiac function, prevented myocardial atrophy, and restored expression of TRPC3 and PLC-ß1 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-ß1/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-ß1/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.

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.

Effect of ß2-agonist treatment on insulin-stimulated peripheral glucose disposal in healthy men in a randomised placebo-controlled trial.

ß2-agonist treatment improves skeletal muscle glucose uptake and whole-body glucose homeostasis in rodents, likely via mTORC2-mediated signalling. However, human data on this topic is virtually absent. We here investigate the effects of two-weeks treatment with the ß2-agonist clenbuterol (40 µg/day) on glucose control as well as energy- and substrate metabolism in healthy young men (age: 18-30 years, BMI: 20-25 kg/m2) in a randomised, placebo-controlled, double-blinded, cross-over study (ClinicalTrials.gov-identifier: NCT03800290). Randomisation occurred by controlled randomisation and the final allocation sequence was seven (period 1: clenbuterol, period 2: placebo) to four (period 1: placebo, period 2: clenbuterol). The primary and secondary outcome were peripheral insulin-stimulated glucose disposal and skeletal muscle GLUT4 translocation, respectively. Primary analyses were performed on eleven participants. No serious adverse events were reported. The study was performed at Maastricht University, Maastricht, The Netherlands, between August 2019 and April 2021. Clenbuterol treatment improved peripheral insulin-stimulated glucose disposal by 13% (46.6 ± 3.5 versus 41.2 ± 2.7 µmol/kg/min, p = 0.032), whereas skeletal muscle GLUT4 translocation assessed in overnight fasted muscle biopsies remained unaffected. These results highlight the potential of ß2-agonist treatment in improving skeletal muscle glucose uptake and underscore the therapeutic value of this pathway for the treatment of type 2 diabetes. However, given the well-known (cardiovascular) side-effects of systemic ß2-agonist treatment, further exploration on the underlying mechanisms is needed to identify viable therapeutic targets.

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.

Effect of Clenbuterol on Muscle Activity During Exercise in Standardbred Horses.

Clenbuterol (ß2 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 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.

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 ß2-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 ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-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 ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

In vivo metabolic effects after acute activation of skeletal muscle Gs signaling.

OBJECTIVE: The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. METHODS: To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (ß2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). RESULTS: Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of ß2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. CONCLUSIONS: Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release.

Post-training intra-basolateral complex of the amygdala infusions of clenbuterol enhance memory for conditioned place preference and increase ARC protein expression in dorsal hippocampal synaptic fractions.

The basolateral complex of the amygdala (BLA) is critically involved in modulation of memory by stress hormones. Noradrenergic activation of the BLA enhances memory consolidation and plays a necessary role in the enhancing or impairing effects of stress hormones on memory. The BLA is not only involved in the consolidation of aversive memories but can regulate appetitive memory formation as well. Extensive evidence suggests that the BLA is a modulatory structure that influences consolidation of arousing memories through modulation of plasticity and expression of plasticity-related genes, such as the activity regulated cytoskeletal-associated (Arc/Arg 3.1) protein, in efferent brain regions. ARC is an immediate early gene whose mRNA is localized to the dendrites and is necessary for hippocampus-dependent long-term potentiation and long-term memory formation. Post-training intra-BLA infusions of the ß-adrenoceptor agonist, clenbuterol, enhances memory for an aversive task and increases dorsal hippocampus ARC protein expression following training on that task. To examine whether this function of BLA noradrenergic signaling extends to the consolidation of appetitive memories, the present studies test the effect of post-training intra-BLA infusions of clenbuterol on memory for the appetitive conditioned place preference (CPP) task and for effects on ARC protein expression in hippocampal synapses. Additionally, the necessity of increased hippocampal ARC protein expression was also examined for long-term memory formation of the CPP task. Immediate post-training intra-BLA infusions of clenbuterol (4 ng/0.2 µL) significantly enhanced memory for the CPP task. This same memory enhancing treatment significantly increased ARC protein expression in dorsal, but not ventral, hippocampal synaptic fractions. Furthermore, immediate post-training intra-dorsal hippocampal infusions of Arc antisense oligodeoxynucleotides (ODNs), which reduce ARC protein expression, prevented long-term memory formation for the CPP task. These results suggest that noradrenergic activity in the BLA influences long-term memory for aversive and appetitive events in a similar manner and the role of the BLA is conserved across classes of memory. It also suggests that the influence of the BLA on hippocampal ARC protein expression and the role of hippocampal ARC protein expression are conserved across classes of emotionally arousing memories.

Egg White Protein Feeding Facilitates Skeletal Muscle Gain in Young Rats with/without Clenbuterol Treatment.

Based on the Digestible Indispensable Amino Acid Score (DIAAS), egg white protein (EGG) has an excellent score, comparable to that of whey protein but with a lower amount of leucine. We examined the effect of EGG feeding on rat skeletal muscle gain in comparison to that of two common animal-derived protein sources: casein (CAS) and whey (WHE). To explore the full potential of EGG, this was examined in clenbuterol-treated young rats. Furthermore, we focused on leucine-associated anabolic signaling in response to EGG after single-dose ingestion and chronic ingestion, as well as clenbuterol treatment. Because EGG is an arginine-rich protein source, a portion of the experiment was repeated with diets containing equal amounts of arginine. We demonstrated that EGG feeding accelerates skeletal muscle gain under anabolism-dominant conditions more efficiently than CAS and WHE and this stronger effect with EGG is not dependent on the arginine-rich composition of the protein source. We also demonstrated that the plausible mechanism of the stronger muscle-gain effect with EGG is not detectable in the mechanistic target of rapamycin (mTOR) or insulin signaling under our experimental conditions. We conclude that EGG may have a superior efficiency in muscle gain compared to other common animal-based proteins.

Age-related neuroinflammation and pathology in the locus coeruleus and hippocampus: beta-adrenergic antagonists exacerbate impairment of learning and memory in aged mice.

The locus coeruleus (LC) provides the primary noradrenergic input to the forebrain and hippocampus, and may be vulnerable to degeneration and contribute to age-related cognitive decline and neuroinflammation. Additionally, inhibition of noradrenergic transmission by brain-permeable beta-blockers could exacerbate cognitive impairment. This study examined effects of age and acute beta-blocker administration on LC and hippocampus pathology, neuroinflammation and learning and memory behavior in mice. Male mice, 3 and 18 months old, were administered propranolol (beta-blocker) or mabuterol (beta-adrenergic agonist) acutely around behavioral assessment. Terminal inflammatory markers in plasma, hippocampus and LC were assessed alongside histopathology. An increase in hippocampal and LC microgliosis and inflammatory proteins in the hippocampus was detected in aged mice. We report pathological hyperphosphorylation of the postsynaptic NMDA receptor subunit 2B (NR2B) in the hippocampus, suggesting neuronal hyperexcitability. Furthermore, the aged proteome revealed an induction in proteins related to energy metabolism, and mitochondria dysfunction in the LC and hippocampus. In a series of hippocampal dependent behavioral assessment tasks acute beta-adrenergic agonist or beta blocker administration altered learning and memory behavior in both aged and young mice. In Y-maze, propranolol and mabuterol differentially altered time spent in novel versus familiar arms in young and aged mice. Propranolol impaired Novel Object Recognition in both young and aged mice. Mabuterol enhanced trace learning in fear conditioning. Aged mice froze more to context and less to cue. Propranolol impaired contextual recall in aged mice. Concluding, aged mice show LC and hippocampus pathology and heightened effects of beta-adrenergic pharmacology on learning and memory.

ß2-adrenergic receptor agonist counteracts skeletal muscle atrophy and oxidative stress in uremic mice.

In patients with chronic kidney disease, skeletal muscle dysfunction is associated with mortality. Uremic sarcopenia is caused by ageing, malnutrition, and chronic inflammation, but the molecular mechanism and potential therapeutics have not been fully elucidated yet. We hypothesize that accumulated uremic toxins might exert a direct deteriorative effect on skeletal muscle and explore the pharmacological treatment in experimental animal and culture cell models. The mice intraperitoneally injected with indoxyl sulfate (IS) after unilateral nephrectomy displayed an elevation of IS concentration in skeletal muscle and a reduction of instantaneous muscle strength, along with the predominant loss of fast-twitch myofibers and intramuscular reactive oxygen species (ROS) generation. The addition of IS in the culture media decreased the size of fully differentiated mouse C2C12 myotubes as well. ROS accumulation and mitochondrial dysfunction were also noted. Next, the effect of the ß2-adrenergic receptor (ß2-AR) agonist, clenbuterol, was evaluated as a potential treatment for uremic sarcopenia. In mice injected with IS, clenbuterol treatment increased the muscle mass and restored the tissue ROS level but failed to improve muscle weakness. In C2C12 myotubes stimulated with IS, although ß2-AR activation also attenuated myotube size reduction and ROS accumulation as did other anti-oxidant reagents, it failed to augment the mitochondrial membrane potential. In conclusion, IS provokes muscular strength loss (uremic dynapenia), ROS generation, and mitochondrial impairment. Although the ß2-AR agonist can increase the muscular mass with ROS reduction, development of therapeutic interventions for restoring skeletal muscle function is still awaited.

The Effect of the Clenbuterol-ß2-Adrenergic Receptor Agonist on the Peripheral Blood Mononuclear Cells Proliferation, Phenotype, Functions, and Reactive Oxygen Species Production in Race Horses In Vitro.

Clenbuterol, the ß2-adrenoceptor agonist, is gaining growing popularity because of its effects on weight loss (i.e., chemical liposuction). It is also popular in bodybuilding and professional sports, due to its effects that are similar to anabolic steroids. However, it is prohibited by anti-doping control. On the other hand, it is suggested that clenbuterol can inhibit the inflammatory process. The cells from 14 untrained and 14 well-trained race horses were collected after acute exercise and cultured with clenbuterol. The expressions of CD4, CD8, FoxP3, CD14, MHCII, and CD5 in PBMC, and reactive oxygen species (ROS) production, as well as cell proliferation, were evaluated by flow cytometry. In addition, IL-1ß, IL-4, IL-6, IL-10, IL-17, INF-γ and TNF-α concentrations were evaluated by ELISA. ß2-adrenoceptor stimulation leads to enhanced anti-inflammatory properties in well-trained horses, as do low doses in untrained animals. In contrast, higher clenbuterol doses create a pro-inflammatory environment in inexperienced horses. In conclusion, ß2-adrenoceptor stimulation leads to a biphasic response. In addition, the immune cells are more sensitive to drug abuse in inexperienced individuals under physical training.

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.

Prolonged ß2-adrenergic agonist treatment improves glucose homeostasis in diet-induced obese UCP1-/- mice.

Prolonged supplementation with the ß2-agonist clenbuterol improves glucose homeostasis in diabetic rodents, likely via ß2-adrenoceptor (ß2-AR)-mediated effects in the skeletal muscle and liver. However, since rodents have, in contrast to-especially diabetic-humans, substantial quantities of brown adipose tissue (BAT) and clenbuterol has affinity to ß1- and ß3-ARs, the contribution of BAT to these improvements is unclear. Therefore, we investigated clenbuterol-mediated improvements in glucose homeostasis in uncoupling protein 1-deficient (UCP1-/-) mice, lacking thermogenic BAT, versus wild-type (WT) mice. Anesthetized WT and UCP1-/- C57Bl/6 mice were injected with saline or clenbuterol and whole body oxygen consumption was measured. Furthermore, male WT and UCP1-/- C57Bl/6 mice were subjected to 17-wk of chow feeding, high-fat feeding, or high-fat feeding with clenbuterol treatment between weeks 13 and 17. Body composition was measured weekly with MRI. Oral glucose tolerance and insulin tolerance tests were performed in week 15 and 17, respectively. Clenbuterol increased oxygen consumption approximately twofold in WT mice. This increase was blunted in UCP1-/- mice, indicating clenbuterol-mediated activation of BAT thermogenesis. High-fat feeding induced diabetogenic phenotypes in both genotypes. However, low-dose clenbuterol treatment for 2 wk significantly reduced fasting blood glucose by 12.9% in WT and 14.8% in UCP1-/- mice. Clenbuterol treatment improved glucose and insulin tolerance in both genotypes compared with HFD controls and normalized to chow-fed control mice independent of body mass and composition alterations. Clenbuterol improved whole body glucose homeostasis independent of UCP1. Given the low human abundancy of BAT, ß2-AR agonist treatment provides a potential novel route for glucose disposal in diabetic humans.NEW & NOTEWORTHY Improvements in whole body glucose homeostasis of rodents upon prolonged ß2-adrenergic agonist supplementation could potentially be attributed to UCP1-mediated BAT thermogenesis. Indeed, we show that acute injection with the ß2-AR agonist clenbuterol induces BAT activation in mice. However, we also demonstrate that prolonged clenbuterol supplementation robustly improves whole body glucose and insulin tolerance in a similar way in both DIO WT and UCP1-/- mice, indicating that ß2-AR agonist supplementation improves whole body glucose homeostasis independent of UCP1-mediated BAT thermogenesis.

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.