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Urocortin II Treatment Reduces Skeletal Muscle Mass and Function Loss During Atrophy and Increases Nonatrophying Skeletal Muscle Mass and Function
Abstract
Two corticotropin-releasing factor 2 receptor (CRF2R)-selective peptides have been recently described, urocortin II (also known as stresscopin-related peptide) and urocortin III (stresscopin). We have used urocortin II to evaluate the effects of activation of the CRF2R on skeletal muscle-related physiological processes. Administration of urocortin II to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, urocortin II treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. Finally, we observed increased skeletal muscle mass and force in normal muscles when mice are treated with urocortin II. These results were confirmed using two additional CRF2R agonists, urocortin I and sauvagine. Thus, activation of the CRF2R modulates skeletal muscle mass in both normal and atrophying muscle. Therefore, CRF2R-selective agonists may find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia.
... Ucn2 is a myokine since it is produced, expressed, and released by muscle fibers and exerts either autocrine, paracrine or endocrine effects [1,2,13]. It has been demonstrated that acute treatment of Ucn2 increases muscle mass and strength [14], and prevents atrophy in rodent muscles submitted to models of sciatic motor denervation and treatment with dexamethasone [14,15]. A recent study demonstrated that modified Ucn2 peptide systemically administered enhances skeletal muscle mass and function in high-fat diet-induced obesity in mice [16]. ...
... Ucn2 is a myokine since it is produced, expressed, and released by muscle fibers and exerts either autocrine, paracrine or endocrine effects [1,2,13]. It has been demonstrated that acute treatment of Ucn2 increases muscle mass and strength [14], and prevents atrophy in rodent muscles submitted to models of sciatic motor denervation and treatment with dexamethasone [14,15]. A recent study demonstrated that modified Ucn2 peptide systemically administered enhances skeletal muscle mass and function in high-fat diet-induced obesity in mice [16]. ...
... CRF2R is a G as protein-coupled receptor (GPCR) that can activate adenylyl cyclase (AC), which enhances intracellular cAMP levels, and consequently activates cAMP-dependent protein kinase (PKA) [5,13]. Indeed, Ucn2 has been shown to stimulate the in vitro production of cAMP in C2C12 myotubes [17] and in isolated mouse muscles [14,15]. Cyclic AMP enhancers such as the nonspecific phosphodiesterase (PDE) inhibitor isobutylmethylxanthine and the selective PDE4 inhibitor rolipram have shown to suppress muscle protein degradation [18]. ...
Objective
Although it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive.
Methods
To elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro.
Results
Here, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt).
Conclusions
These data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.
... 19 Loss of skeletal muscle mass and function from nerve damage, corticosteroids, or disuse is prevented by sauvagine, a selective CRHR2 agonist, or human UCN2 in rodents. [20][21][22] Furthermore, in lean rodents, human UCN2 administration causes skeletal muscle hypertrophy. 20,21 The CRHR2 receptor is also involved in modulating skeletal muscle growth in chronic muscle wasting disorders. ...
... [20][21][22] Furthermore, in lean rodents, human UCN2 administration causes skeletal muscle hypertrophy. 20,21 The CRHR2 receptor is also involved in modulating skeletal muscle growth in chronic muscle wasting disorders. Treating mdx mice (a model for Duchenne muscular dystrophy) with a CRHR2 agonist prevents the progressive degeneration of diaphragm muscle, 23 while treatment with CRHR2 agonists in models of aging or chronic heart failure in rats and emphysema in hamsters improves skeletal muscle mass and power output. ...
... 4,61 Physiological loss of skeletal muscle mass and force production that occurs in models of chronic disease and disuse can be prevented by treating mice systemically with UCN2. 20,23,24 In the current study, Compound B treatment of obese mice increased the time to fatigue in the EDL muscle without altering its mass, while Compound A treatment increased skeletal muscle mass and preserved force production after casting in chow-fed mice. The greater muscle mass of the EDL did not translate to increased time to fatigue in Compound A-treated mice. ...
Background
Type 2 diabetes and obesity are often seen concurrently with skeletal muscle wasting, leading to further derangements in function and metabolism. Muscle wasting remains an unmet need for metabolic disease, and new approaches are warranted. The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin releasing factor receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance, glucose metabolism, and muscle mass. The aim of this study was to investigate the effects of modified UCN2 peptides as a pharmaceutical therapy to counteract the loss of skeletal muscle mass associated with obesity and casting immobilization.
Methods
High‐fat‐fed mice (C57Bl/6J; 26 weeks old) and ob/ob mice (11 weeks old) were injected daily with a PEGylated (Compound A) and non‐PEGylated (Compound B) modified human UCN2 at 0.3 mg/kg subcutaneously for 14 days. A separate group of chow‐fed C57Bl/6J mice (12 weeks old) was subjected to hindlimb cast immobilization and, after 1 week, received daily injections with Compound A. In vivo functional tests were performed to measure protein synthesis rates and skeletal muscle function. Ex vivo functional and molecular tests were performed to measure contractile force and signal transduction of catabolic and anabolic pathways in skeletal muscle.
Results
Skeletal muscles (extensor digitorum longus, soleus, and tibialis anterior) from high‐fat‐fed mice treated with Compound A were ~14% heavier than muscles from vehicle‐treated mice. Chronic treatment with modified UCN2 peptides altered the expression of structural genes and transcription factors in skeletal muscle in high‐fat diet‐induced obesity including down‐regulation of Trim63 and up‐regulation of Nr4a2 and Igf1 (P < 0.05 vs. vehicle). Signal transduction via both catabolic and anabolic pathways was increased in tibialis anterior muscle, with increased phosphorylation of ribosomal protein S6 at Ser235/236, FOXO1 at Ser²⁵⁶, and ULK1 at Ser³¹⁷, suggesting that UCN2 treatment modulates protein synthesis and degradation pathways (P < 0.05 vs. vehicle). Acutely, a single injection of Compound A in drug‐naïve mice had no effect on the rate of protein synthesis in skeletal muscle, as measured via the surface sensing of translation method, while the expression of Nr4a3 and Ppargc1a4 was increased (P < 0.05 vs. vehicle). Compound A treatment prevented the loss of force production from disuse due to casting. Compound B treatment increased time to fatigue during ex vivo contractions of fast‐twitch extensor digitorum longus muscle. Compound A and B treatment increased lean mass and rates of skeletal muscle protein synthesis in ob/ob mice.
Conclusions
Modified human UCN2 is a pharmacological candidate for the prevention of the loss of skeletal muscle mass associated with obesity and immobilization.
... Changes in muscle mass in the living animal have been assessed by a range of methods from gross measurements of whole muscle protein content [6][7][8][9] or inferences from muscle cross-sections [10][11][12], to low-throughput intensive analyses of confocalized z-stacks [13]. Such investigations are labor-and animal-intensive, largely restricting investigation to very specific mechanisms or modulatory agents. ...
... Ex vivo diminution of the myonuclear domain is not associated with loss or replacement of myonuclei Over two weeks of ex vivo maintenance we observe a decline in size of isolated myofibres and decreasing spacing of nuclei (Fig. 3A). Using f-actin signal per nucleus at various time-points following isolation as an indicator of atrophy (Fig. 3B), we observe a drop-off of~35% per week, similar to that observed for in vivo denervation, tenotomization, and hind-limb suspension studies of muscles predominant in type II myofibres [6][7][8]10,13]. Over this period, the number of nuclei per myofibre remains constant, averaging around 330, with considerable variation between individual myofibres (nuclear number per fibre ranged from less than 100 to over 700). ...
... We maintained myofibres of the murine EDL muscle in free-floating suspension in a 'low growth' culture medium, thereby denying a substrate for the long-term proliferation and fusion of associated myoblasts. The ex vivo rate of atrophy we observe is the same as that measured for in vivo denervation [6,13], tenotomization [8], and hindlimb suspension [7,10] of muscles predominant in type II myofibres. Type I myofibres can also be isolated from the soleus [3], and for those we would expect a faster rate of atrophy, as observed with in vivo tenotomization of the soleus [8]. ...
... Five groups of rats were generated, two of which received different concentrations of urocortin (referred to as "UCN low" and "UCN high"), one was treated with Estradiol (OVX-Estradiol), one was just ovariectomized (OVX) and one was not (SHAM). The treatment regimens were chosen according to preliminary tests after literature review (41,73). ...
... Hinkle et al. (41) reported that urocortin could prevent loss of skeletal muscle mass. In their rat model, they analyzed M. (41). ...
... Hinkle et al. (41) reported that urocortin could prevent loss of skeletal muscle mass. In their rat model, they analyzed M. (41). In our OVX model, we also observed enhanced muscle weight and muscle fiber size in M. soleus at the high dose and M. longissimus at the low dose. ...
Objective
In aging population, postmenopausal osteoporosis and decline of musculoskeletal function, referred to as “frailty syndrome” lead to loss of bone and muscle, causing falls and fall-related injuries. To limit the impact of this portentous duo, simultaneous treatment of both is needed. Urocortin (UCN) has been reported to improve osteoporotic bone properties while its effect on muscle has not been addressed yet.
Design & Methods
We aimed to investigate the effect of urocortin in vivo on skeletal muscle structure in osteopenic rats. Sixty Sprague-Dawley rats were divided into five groups: Four were ovariectomized (OVX) and one underwent sham operation (SHAM). One ovariectomized group was left untreated (OVX), while one was treated with urocortin s.c. in 3 µg/kg body weight (bw) (OVX+UCN low), one with 30 μg/kg (OVX+UCN high), while one group was treated with estradiol orally (OVX+E: 0.2 mg/kg bw), each for thirty-five days. Mm. gastrocnemius, longissimus and soleus were isolated and capillary density as well as diameters of type I and II fibers were measured. In addition, we examined the effect of UCN on tibia using biomechanical, micro-CT and ashing analysis and investigated the blood serum.
Results
We demonstrated a positive effect of UCN on M. soleus, in which fiber diameter was positively influenced. The biomechanical and structural parameters of bone were not changed in UCN treated rats. The higher cholesterol, glucose and triglyceride levels in the “UCN high” group raise concern about this treatment.
Conclusions
Our results portray urocortin as a substance that can be assessed for future therapeutic treatments of estrogen deficiency.
... This family of proteins plays different roles in metabolic functions, adaptive stress being one of them. Modulation of CRFR2 or its ligands may improve muscle mass and metabolism by activating the hypothalamic-pituitary-adrenal (HPA) axis [177]. Skeletal muscle has high levels of Ucn2 and CRFR2 [178]. ...
... Modulation of CRFR2 or its ligands may improve muscle mass and metabolism by activating the hypothalamic-pituitary-adrenal (HPA) axis [177]. Skeletal muscle has high levels of Ucn2 and CRFR2 [178]. ...
... Sinha-Hikim et al. [154] Traish et al. [160] Dalton et al. [161] Tiidus et al. [166] Myostatin inactivators ↑lean mass ↓fat mass ↑glucose homeostasis Sakuma et al. [1] Zhang et al. [171] Zhang et al. [172] Urocortins ↑muscle mass and metabolism ↑HPA axis ↑insulin signaling pathway Hinkle et al. [173] Roustit et al. [177] Vitamin D ↑muscle mass/force ↑insulin sensitivity ...
... This family of proteins plays different roles in metabolic functions, adaptive stress being one of them. Modulation of CRFR2 or its ligands may improve muscle mass and metabolism by activating the hypothalamic-pituitary-adrenal (HPA) axis [177]. Skeletal muscle has high levels of Ucn2 and CRFR2 [178]. ...
... Modulation of CRFR2 or its ligands may improve muscle mass and metabolism by activating the hypothalamic-pituitary-adrenal (HPA) axis [177]. Skeletal muscle has high levels of Ucn2 and CRFR2 [178]. ...
... Sinha-Hikim et al. [154] Traish et al. [160] Dalton et al. [161] Tiidus et al. [166] Myostatin inactivators ↑lean mass ↓fat mass ↑glucose homeostasis Sakuma et al. [1] Zhang et al. [171] Zhang et al. [172] Urocortins ↑muscle mass and metabolism ↑HPA axis ↑insulin signaling pathway Hinkle et al. [173] Roustit et al. [177] Vitamin D ↑muscle mass/force ↑insulin sensitivity ...
Although there are several reviews that report the interrelationship between sarcopenia and obesity and insulin resistance, the relation between sarcopenia and the other signs that compose the metabolic syndrome (MetS) has not been extensively revised. Here, we review the mechanisms underlying MetS-related sarcopenia and discuss the possible therapeutic measures proposed. A vicious cycle between the loss of muscle and the accumulation of intramuscular fat might be associated with MetS via a complex interplay of factors including nutritional intake, physical activity, body fat, oxidative stress, proinflammatory cytokines, insulin resistance, hormonal changes, and mitochondrial dysfunction. The enormous differences in lipid storage capacities between the two genders and elevated amounts of endogenous fat having lipotoxic effects that lead to the loss of muscle mass are discussed. The important repercussions of MetS-related sarcopenia on other illnesses that lead to increased disability, morbidity, and mortality are also addressed. Additional research is needed to better understand the pathophysiology of MetS-related sarcopenia and its consequences. Although there is currently no consensus on the treatment, lifestyle changes including diet and power exercise seem to be the best options.
... Many studies have shown, however, that cAMP-inducing agents or genetic modification of proteins involved in cAMP signaling can also have adaptive effects on skeletal muscle by increasing myofiber size and promoting fiber-type transitions to glycolytic fibers (9,18,42,43,57,63,86,91,101,121,128,154,155,163,190,193,194,215). The prohypertrophic actions of -adrenergic receptor (-AR) agonists and corticotropin-releasing factor receptor 2 (CRFR2) agonists have recently been harnessed to improve muscle function and ameliorate atrophy in several rodent models, including disuse (99,101,245), denervation (98, 100 -102, 127, 154, 253), aging (195,254), and muscular dystrophy (90,103,185,254,256). -AR agonists have also shown some promise in promoting muscle function in patients with muscular dystrophy (64,125,126,211,235). Despite many physiological studies, the molecular mechanisms underlying these effects in skeletal muscle are still being elucidated. ...
... The corticotropin-releasing factor receptor 2 (CRFR2) is also highly expressed in skeletal muscle (38), and activation of this receptor by a family of ligands called urocortins (Ucn1-3) stimulates cAMP production in differentiated myotubes (133) and isolated muscle tissue (99,101). Ucn2 is also strongly expressed in skeletal muscle (36), suggesting that it can serve as an autocrine signaling molecule. ...
... On the contrary, mice lacking G␣ s , which stimulates cAMP production, have smaller myofibers than littermate controls (42). In pathological settings, ligands for two GPCRs,  2 -AR and CRFR2, reduce atrophy or help sustain muscle function and strength in rodent models after denervation (100,101,123,127,154,253,255), unloading (99,101,123,127) or aging (35).  2 -AR agonists have also been shown to reduce muscle atrophy in animals with cancer cachexia (31,33,46). ...
Among organ systems, skeletal muscle is perhaps the most structurally specialized. The remarkable subcellular architecture of this tissue allows it to empower movement with instructions from motor neurons. Despite this high degree of specialization, skeletal muscle also has intrinsic signaling mechanisms that allow adaptation to long-term changes in demand and regeneration after acute damage. The second messenger adenosine 3',5'-monophosphate (cAMP) not only elicits acute changes within myofibers during exercise but also contributes to myofiber size and metabolic phenotype in the long term. Strikingly, sustained activation of cAMP signaling leads to pronounced hypertrophic responses in skeletal myofibers through largely elusive molecular mechanisms. These pathways can promote hypertrophy and combat atrophy in animal models of disorders including muscular dystrophy, age-related atrophy, denervation injury, disuse atrophy, cancer cachexia, and sepsis. cAMP also participates in muscle development and regeneration mediated by muscle precursor cells; thus, downstream signaling pathways may potentially be harnessed to promote muscle regeneration in patients with acute damage or muscular dystrophy. In this review, we summarize studies implicating cAMP signaling in skeletal muscle adaptation. We also highlight ligands that induce cAMP signaling and downstream effectors that are promising pharmacological targets.
... Changes in muscle mass in the living animal have been assessed by a range of methods from gross measurements of whole muscle protein content [6][7][8][9] or inferences from muscle crosssections [10][11][12], to low-throughput intensive analyses of confocalized z-stacks [13]. Such investigations are labor-and animal-intensive, largely restricting investigation to very specific mechanisms or modulatory agents. ...
... We maintained myofibres of the murine EDL muscle in free-floating suspension in a 'low growth' culture medium, thereby denying a substrate for the long-term proliferation and fusion of associated myoblasts. The ex vivo rate of atrophy we observe is the same as that measured for in vivo denervation [6,13], tenotomization [8], and hindlimb suspension [7,10] of muscles predominant in type II myofibres. Type I myofibres can also be isolated from the soleus [3], and for those we would expect a faster rate of atrophy, as observed with in vivo tenotomization of the soleus [8]. Figure 5 gives a schematic overview of PhAct. ...
... It has the advantage over existing approaches of providing information at the cellular (per myofibre) level. Existing approaches to the assay of change in muscle mass in vivo are limited to coarse measurement of whole muscle weight and protein content [6][7][8][9] or to indirect inferences from muscle cross-sections [10][11][12]. The major alternative, myotube tissue cultures, are valuable for analysis of molecular mechansisms but of questionable relevance to control of muscle size in vivo [14][15][16]. ...
Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can distinguish those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents.
... Recently, we have demonstrated that activation of the CRF2R reduces acute skeletal muscle atrophy resulting from disuse, nerve damage, corticosteroid treatment and causes hypertrophy of non-atrophying skeletal muscle in healthy animals [46][47][48]. CRF2R activation also reduces skeletal muscle wasting associated with two chronic disease conditions, cancer and muscular dystrophy [49][50][51]. We have not yet investigated if activation of the CRF2R will maintain skeletal muscle mass and force production in animals that have an underlying chronic condition that results in muscle wasting. ...
... Previously, we demonstrated that activation of the CRF2R can increase skeletal muscle mass and force production in young mice and rats [46][47][48]. In the present report we demonstrate that old rats, like young rats, respond to pharmacological activation of the CRF2R with greater absolute and/or relative skeletal muscle mass and force production when compared to age matched untreated rats. ...
... The results of this investigation provide suggestive evidence that CRF2R agonist may increase diaphragm muscle mass. This together with the observation that treatment with a CRF2R agonist is effective in preventing loss of non-actively contracting skeletal muscle [46,48] suggest that treatment with a CRF2R agonist may be an important therapeutic intervention for ameliorating the deleterious effects of MV on diaphragm mass and function. More work will be needed before we fully understand the potential of CRF2R agonist in treating MV weaning failure. ...
Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting.
We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals.
We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP.
In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals.
These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.
... UCN2 seems to play an important role in all three, skeletal, cardiac and smooth muscles. In the former, activation of the CRF-R2 by UCN2 increases contractile force and muscle mass, preventing the loss of skeletal muscle mass (Hinkle et al. 2003, Hinkle et al. 2004; it also allows skeletal muscle to contract and relax faster (Reutenauer-Patte et al. 2012). ...
... Moreover, in human myometrial cells, UCN2 is able to induce phosphorylation of myosin light chain (which is involved in the initiation of smooth muscle contraction), being the effect maximal with UCN2 at 10 -7 M after 5 min of treatment and returning to basal levels after 45 min (Karteris et al. 2004). Finally, in the skeletal muscle, activation of CRF-R2 by UCN2 increases contractile force and muscle mass (Hinkle et al. 2003, Hinkle et al. 2004. ...
Urocortin 2 (UCN2) is a peptide related to corticotropin-releasing factor, capable of activating CRF-R2. Among its multisystemic effects, it has actions in all 3 muscle subtypes. This study's aim was to determine its potential role in two of the intrinsic eye muscle kinetics. Strips of iris sphincter (rabbit) and ciliary (bovine) muscles were dissected and mounted in isometric force-transducer systems filled with aerated-solutions. Contraction was elicited using carbachol (10(-6) M for iris sphincter, 10(-5) M for ciliary muscle), prior adding to all testing substances. UCN2 induced relaxation in iris sphincter muscle, being the effect maximal at 10(-7) M concentrations (-12.2 % variation vs. control). This effect was abolished with incubation of indomethacin, antisauvagine-30, chelerytrine and SQ22536, but preserved with L-nitro-L-arginine. In carbachol pre-stimulated ciliary muscle, UCN2 (10(-5) M) enhanced contraction (maximal effect of 18.2 % increase vs. control). UCN2 is a new modulator of iris sphincter relaxation, dependent of CRF-R2 activation, synthesis of prostaglandins (COX pathway) and both adenylate cyclase and PKC signaling pathways, but independent of nitric oxide production. Regarding ciliary muscle, UCN2 enhances carbachol-induced contraction, in higher doses.
... UCN2 seems to play an important role in all three, skeletal, cardiac and smooth muscles. In the former, activation of the CRF-R2 by UCN2 increases contractile force and muscle mass, preventing the loss of skeletal muscle mass (Hinkle et al. 2003, Hinkle et al. 2004; it also allows skeletal muscle to contract and relax faster (Reutenauer-Patte et al. 2012). ...
... Moreover, in human myometrial cells, UCN2 is able to induce phosphorylation of myosin light chain (which is involved in the initiation of smooth muscle contraction), being the effect maximal with UCN2 at 10 -7 M after 5 min of treatment and returning to basal levels after 45 min (Karteris et al. 2004). Finally, in the skeletal muscle, activation of CRF-R2 by UCN2 increases contractile force and muscle mass (Hinkle et al. 2003, Hinkle et al. 2004. ...
Purpose
Urocortin 2 ( UCN 2) is a peptide related to corticotropin‐releasing factor, capable of activating CRF ‐R2. Among its multisystemic effects, it has actions in all 3 muscle subtypes, either increasing or decreasing contractility.
This study's aim was to determine its potential role in two of the intrinsic eye muscle kinetics and to study the likely subcellular pathways involved.
Methods
Strips of iris sphincter (rabbit) and ciliary (bovine) muscles were dissected and mounted in isometric force‐transducer systems filled with aerated‐solutions, subjected to 0.5 mN and 1.0 mN preloads, respectively. Contraction was elicited using carbachol (10–6M for iris sphincter, 10–5M for ciliary muscle), prior to all testing substances.
Results
UCN 2 induced relaxation in iris sphincter muscle, being the effect maximal at 10‐7M concentrations (‐12.2% variation, versus control, n = 6). This effect was abolished with incubation of indomethacin (n = 8), antisauvagine‐30 (n = 7), chelerytrine (n = 6) and SQ 22536 (n = 8), but preserved with L‐nitro‐L‐arginine (n = 9). In carbachol pre‐stimulated ciliary muscle, UCN 2 (10‐5M) enhanced contraction (maximal effect of 18.2% increase, versus control, n = 10 pairs); pre‐contraction studies were negative.
Conclusions
UCN 2 is a new neurohumoral modulator of iris sphincter relaxation, dependent on CRF ‐R2 activation, synthesis of prostaglandins ( COX pathway) and both adenylate cyclase and PKC signaling pathways, but independent of nitric oxide production. Regarding ciliary muscle, UCN 2 enhances carbachol‐induced contraction, in higher doses.
... Recently, we have shown that corticotrophin releasing factor receptor 2 (CRF2R) agonists can modulate skeletal muscle mass by increasing muscle mass (hypertrophy) and decreasing loss from atrophying/wasting of muscle mass [13][14][15]. These effects occur by decreasing proteolysis during atrophying conditions and activation of anabolic signalling pathways [13][14][15]. ...
... Recently, we have shown that corticotrophin releasing factor receptor 2 (CRF2R) agonists can modulate skeletal muscle mass by increasing muscle mass (hypertrophy) and decreasing loss from atrophying/wasting of muscle mass [13][14][15]. These effects occur by decreasing proteolysis during atrophying conditions and activation of anabolic signalling pathways [13][14][15]. Therefore, we have utilized potent CRF2R agonists in the mdx model in order to evaluate the therapeutic potential for these compounds in DMD. ...
Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.
Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.
We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.
Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.
... Previous data obtained with cultured myocytes suggest that acute treatment with UCN2 reduces insulin signaling in SKM [34]. Further, ex vivo experiments have shown that UCN2 dosedependently increases cAMP in mouse SKM [35]. To test the hypothesis that some of the observed metabolic effects of UCN2 involved SKM CRF2 receptors and stimulation of SKM Gs signaling, we studied mice that lack Gs (Gs) selectively in SKM (SKM-Gs-KO mice). ...
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 (β2-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. However, the acute metabolic effects following agonist activation of β2-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.
Conclusion
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.
... Personalized regenerative medicine lays the ground for developing effective therapies for these conditions. There are promising observations in using both stem cell and pharmacological approaches to treat musculoskeletal conditions like age-related muscle loss and sarcopenia in preclinical studies (14,15). There are numerous examples of successful stem cell clinical application in regenerative medicine (Table 1), the most known include: bone marrow transplantation to treat leukemia Drug discovery for long QT syndrome iPCS derived from skin cells with Timothy syndrome were used to model Long QT syndrome to assess the effects of different drugs (17). ...
Regenerative medicine is a multidisciplinary field that aims to determine different factors and develop various methods to regenerate impaired tissues, organs, and cells in the disease and impairment conditions. When treatment procedures are specified according to the individual's information, the leading role of personalized regenerative medicine will be revealed in developing more effective therapies. In this concept, endocrine disorders can be considered as potential candidates for regenerative medicine application. Diabetes mellitus as a worldwide prevalent endocrine disease causes different damages such as blood vessel damages, pancreatic damages, and impaired wound healing. Therefore, a global effort has been devoted to diabetes mellitus investigations. Hereupon, the preclinical study is a fundamental step. Up to now, several species of animals have been modeled to identify the mechanism of multiple diseases. However, more recent researches have been demonstrated that animal models with the ability of tissue regeneration are more suitable choices for regenerative medicine studies in endocrine disorders, typically diabetes mellitus. Accordingly, zebrafish has been introduced as a model that possesses the capacity to regenerate different organs and tissues. Especially, fine regeneration in zebrafish has been broadly investigated in the regenerative medicine field. In addition, zebrafish is a suitable model for studying a variety of different situations. For instance, it has been used for developmental studies because of the special characteristics of its larva. In this review, we discuss the features of zebrafish that make it a desirable animal model, the advantages of zebrafish and recent research that shows zebrafish is a promising animal model for personalized regenerative diseases. Ultimately, we conclude that as a newly introduced model, zebrafish can have a leading role in regeneration studies of endocrine diseases and provide a good perception of underlying mechanisms.
... Furthermore, an animal trial revealed that urocortin II, a corticotropin-releasing factor 2 receptor (CRF2R)-selective peptide, increased the SMM of mice with normal muscles. Hence, for patients with skeletal muscle wasting diseases, such as age-related muscle loss or spinal sarcopenia, CRF2R-selective agonists may be a treatment option [133]. Nonetheless, such pharmaceutical agents are still under investigation, and more evidence is needed to assess their benefits. ...
Spinal sarcopenia is a complex and multifactorial disorder associated with a loss of strength, increased frailty, and increased risks of fractures and falls. In addition, spinal sarcopenia has been associated with lumbar spine disorders and osteoporosis, which renders making decisions on treatment modalities difficult. Patients with spinal sarcopenia typically exhibit lower cumulative survival, a higher risk of in-hospital complications, prolonged hospital stays, higher postoperative costs, and higher rates of blood transfusion after thoracolumbar spine surgery. Several studies have focused on the relationships between spinal sarcopenia, appendicular muscle mass, and bonerelated problems—such as osteoporotic fractures and low bone mineral density—and malnutrition
and vitamin D deficiency. Although several techniques are available for measuring sarcopenia, each of them has its advantages and shortcomings. For treating spinal sarcopenia, nutrition, physical therapy, and medication have been proven to be effective; regenerative therapeutic options seem to be promising owing to their repair and regeneration potential. Therefore, in this narrative review, we summarize the characteristics, detection methodologies, and treatment options for spinal sarcopenia, as well as its role in spinal disorders.
... It has recently been suggested that even angiotensin-converting enzyme (ACE) inhibitors could have beneficial effects on muscle, increasing the number of mitochondria and serum IGF-1 (111), but further studies are needed to better understand the effects of these molecules on skeletal muscle. The urocortin 2 (Ucn2) is a peptide, commonly expressed in the central nervous system and other tissues, recently considered as a potential therapeutic option for sarcopenia, because of its relationship with muscle wasting, insulin resistance, obesity, and diabetes (112). Considering that glucose is an important energy source for skeletal muscle, the insulin resistance could have systemic effects for this tissue (113). ...
CITATION 1 READS 226 6 authors, including: Some of the authors of this publication are also working on these related projects: ICF in rehabilitation View project osteoarthritis View project Giovanni Iolascon Università degli Studi della Campania "Luigi Vanvitelli Summary Sarcopenia, defined as the loss of muscle mass and function, has important consequences in terms of increasing frailty, disability, and social and healthcare costs. The diagnosis of sarcopenia should be considered in all patients presenting a decline in physical function , muscle strength and general health conditions. Given that the progressive reduction of muscle mass and strength occurs also in aging, the switch towards a pathological condition has been established by combining diagnostic cutoffs and risk factors for reduced mobility , poor quality of life, and increased morbidity and mortality. On the other hand, the introduction of different criteria for the diagnosis of sarcopenia has hindered the development of guidelines for the management of this disorder. The objective of the treatment of muscle wasting is to maintain or improve muscle mass, and both mechanical and metabolic muscle functions. The management of sarcopenia should be multifactorial and interdisciplinary, including exercise, particularly muscle strengthening training, intake of proteins and vitamin D, and treatment of diseases causing muscle loss. To date there are no drugs that have been specifically approved for the treatment of sarcopenia (or other conditions causing reduced muscle function) although many substances are commonly used for this purpose. Several drugs have been studied to improve muscle mass and function, such as testosterone, estrogens, selective modulators of the androgen receptor (SARMs), Clinical Cases in Mineral and Bone Metabolism 2018; 15(3):407-415 407 ghrelin, anti-cytokines (IL-1, IL-6,TNF-α), and myostatin inhibitors. Identification of novel molecules targeting specific biological pathways whose stimulation or inhibition produces net anabolic effects on skeletal muscle might be a significant step forward for the treatment of muscle disorders.
... 9 Urocortin is a selective agonist of corticotropin-releasing factor 2 receptor (CRF2R), which exerts the effect on reducing agerelated sarcopenia via activating CRF2R. 10 Growth hormone replacement therapy is also applied to attenuating age-induced sarcopenia. It is beneficial to increase insulin-like growth factor 1 (IGF-1)-mediated protein synthesis and reduce oxidative damage. ...
Sarcopenia is commonly occurs in the elderly and patients with wasting diseases. The main reason is an imbalance in protein metabolism (protein degradation exceeding protein synthesis). It causes a serious decline in muscle strength and motion ability, even leading to long-term bed rest. Recent studies indicate that nutritional support is beneficial for ameliorating sarcopenia and restoring muscle function. This review will summarize the classical mechanisms of protein nutritional support for alleviating sarcopenia, such as modulating the ubiquitin-proteasome system, oxidative response, and cell autophagy, as well as the potential new mechanisms, including altering miRNA profiles and gut microbiota. In addition, the clinical application and outcome of protein nutritional support in the elderly and patients with wasting diseases are also introduced. Protein nutritional support is expected to provide new approaches for the prevention and adjuvant therapy of sarcopenia.
... β 2 -adrenergic agonists, such as clenbuterol and formoterol, have shown great promise in maintaining skeletal muscle mass in animal models, but have undesirable cardiovascular side effects [reviewed in (7,108,109)] that might be avoided by newer-generation agonists with functional selectivity for skeletal muscle (110). Similarly, stimulation of CRFR2 with chronic administration of urocortin 2 (94)(95)(96)(97) or transgenic expression of urocortin 3 (111) promotes muscle hypertrophy. However, CRFR2 signaling can either blunt (93) or augment (111) insulin action in skeletal muscle, depending on the ligand. ...
cAMP is one of the earliest described mediators of hormone action in response to physiologic stress that allows acute stress responses and adaptation in every tissue. The classic role of cAMP signaling in metabolic tissues is to regulate nutrient partitioning. In response to acute stress, such as epinephrine released during strenuous exercise or fasting, intramuscular cAMP liberates glucose from glycogen and fatty acids from triglycerides. In the long-term, activation of Gs-coupled GPCRs stimulates muscle growth (hypertrophy) and metabolic adaptation through multiple pathways that culminate in a net increase of protein synthesis, mitochondrial biogenesis, and improved metabolic efficiency. This review focuses on regulation, function, and transcriptional targets of CREB (cAMP response element binding protein) and CRTCs (CREB regulated transcriptional coactivators) in skeletal muscle and the potential for targeting this pathway to sustain muscle mass and metabolic function in type 2 diabetes and cancer. Although the muscle-autonomous roles of these proteins might render them excellent targets for both conditions, pharmacologic targeting must be approached with caution. Gain of CREB-CRTC function is associated with excess liver glucose output in type 2 diabetes, and growing evidence implicates CREB-CRTC activation in proliferation and invasion of different types of cancer cells. We conclude that deeper investigation to identify skeletal muscle specific regulatory mechanisms that govern CREB-CRTC transcriptional activity is needed to safely take advantage of their potent effects to invigorate skeletal muscle to potentially improve health in people with type 2 diabetes and cancer.
... It has recently been suggested that even angiotensin-converting enzyme (ACE) inhibitors could have beneficial effects on muscle, increasing the number of mitochondria and serum IGF-1 (111), but further studies are needed to better understand the effects of these molecules on skeletal muscle. The urocortin 2 (Ucn2) is a peptide, commonly expressed in the central nervous system and other tissues, recently considered as a potential therapeutic option for sarcopenia, because of its relationship with muscle wasting, insulin resistance, obesity, and diabetes (112). Considering that glucose is an important energy source for skeletal muscle, the insulin resistance could have systemic effects for this tissue (113). ...
Sarcopenia, defined as the loss of muscle mass and function, has important consequences in terms of increasing frailty, disability, and social and healthcare costs. The diagnosis of sarcopenia should be considered in all patients presenting a decline in physical function , muscle strength and general health conditions. Given that the progressive reduction of muscle mass and strength occurs also in aging, the switch towards a pathological condition has been established by combining diagnostic cutoffs and risk factors for reduced mobility , poor quality of life, and increased morbidity and mortality. On the other hand, the introduction of different criteria for the diagnosis of sarcopenia has hindered the development of guidelines for the management of this disorder. The objective of the treatment of muscle wasting is to maintain or improve muscle mass, and both mechanical and metabolic muscle functions. The management of sarcopenia should be multifactorial and interdisciplinary, including exercise, particularly muscle strengthening training, intake of proteins and vitamin D, and treatment of diseases causing muscle loss. To date there are no drugs that have been specifically approved for the treatment of sarcopenia (or other conditions causing reduced muscle function) although many substances are commonly used for this purpose. Several drugs have been studied to improve muscle mass and function, such as testosterone, estrogens, selective modulators of the androgen receptor (SARMs), Clinical Cases in Mineral and Bone Metabolism 2018; 15(3):407-415 407 ghrelin, anti-cytokines (IL-1, IL-6,TNF-α), and myostatin inhibitors. Identification of novel molecules targeting specific biological pathways whose stimulation or inhibition produces net anabolic effects on skeletal muscle might be a significant step forward for the treatment of muscle disorders.
... Recently, it has been shown that a particular peptide expressed in the central nervous system and peripheral tissues, urocortin II, has potential as a therapeutic agent for muscle loss due to sarcopenia [67] . The link between urocortin II and muscle loss due to aging involves the relationship between sarcopenia, insulin resistance, obesity, and diabetes. ...
Sarcopenia is a complex and multifactorial disease that includes a decrease in the number, structure and physiology of muscle fibers, and age-related muscle mass loss, and is associated with loss of strength, increased frailty, and increased risk for fractures and falls. Treatment options are suboptimal and consist of exercise and nutrition as the cornerstone of therapy. Current treatment principles involve identification and modification of risk factors to prevent the disease, but these efforts are of limited value to the elderly individuals currently affected by sarcopenia. The development of new and effective therapies for sarcopenia is challenging. Potential therapies can target one or more of the proposed multiple etiologies such as the loss of regenerative capacity of muscle, age-related changes in the expression of signaling molecules such as growth hormone, IGF-1, myostatin, and other endocrine signaling molecules, and age-related changes in muscle physiology like denervation and mitochondrial dysfunction. The present paper reviews regenerative medicine strategies that seek to restore adequate skeletal muscle structure and function including exogenous delivery of cells and pharmacological therapies to induce myogenesis or reverse the physiologic changes that result in the disease. Approaches that modify the microenvironment to provide an environment conducive to reversal and mitigation of the disease represent a potential regenerative medicine approach that is discussed herein.
... In addition to functional adaptation cAMP signaling participates in muscle precursor cell differentiation, migration, and fusion. In adult muscle, stimulation of cAMP production showed slow degeneration or promoted regeneration in rodent models of necrotic muscle injury and Duchenne's muscular dystrophy [45][46][47][48]. Reports suggested that cAMP may not only upregulated the expression of growth-promoting genes, but also limited the expression of genes that negatively impact axonal regeneration [49]. ...
Background: Extracts of the plant Eurycoma longifolia Jack (EL) have been shown to possess cytotoxic, antimalarial, anti-ulcer, antipyretic activities. The roots have been frequently prescribed for the treatment of persistent fever, tertian malaria, sexual insufficiency, dysentery, glandular swelling, antipyretic, complications after childbirth. However, there is little data concerning the ability of Eurycoma longifolia Jack on antinociception. A ethno-medicinal report strongly indicated its potential therapeutic effects in facial and spinal pain modulation. The functional neuroanatomical map of EL in spinal cord nuclei is not available. Objective: The aim of this study was to investigate the neuroanatomical functional site of the EL extracts (root) in rat spinal nucleus by means of c-Fos immunohistochemistry. Results: Our present study demonstrated that i.p., administration of EL (Tongkat Ali, 8mg/kg) significantly (p<0.01) increase in the number of c-Fos immunoreactive neurons in the dorsal and ventral nuclei of the rat. Conclusion: The result of the present study strongly indicates that Tongkat Ali has neuromodulatory effects on sensory and motor neurons. To the best of our knowledge, it is the first report which indicates that EL might play crucial roles in motor and sensory neuromodulation. Our findings warrant further study to reveal the underlying molecular mechanisms of EL induced sensory and motor neuromodulation. Background: write background about topic of paper. Objective:
... atrophy and promote hypertrophy (Chanalaris, et al. 2005; Hinkle, et al. 2003; Reutenauer-Patte, et al. 67 2012). Ucn2 knockout mice show increased whole body insulin sensitivity and resist the effects of a 68 HFD, due to CRFR2-mediated activation of Akt and ERK1/2 signalling in skeletal muscle (Chen, et 69 al. 2006). ...
Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes (T2D) and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (Ucns) are well-established as neuropeptides but also have roles in metabolism in peripheral tissues. We showed recently that global overexpression of Ucn3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high fat diet. Here, we aimed to establish whether short-term local Ucn3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. Ucn3 was expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied versus the contralateral muscles after one week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (p=0.030), associated with increased insulin-like growth factor-1 (IGF1) and IGF1 receptor mRNA and increased Ser256 phosphorylation of forkhead transcription factor. Glucose clearance into test muscles after an intraperitoneal glucose load was increased by 23% (p=0.018) per unit mass, associated with increased GLUT1 (34% increase; p=0.026) and GLUT4 (48% increase; p=0.0009) protein and significantly increased phosphorylation of insulin receptor substrate-1, Akt, Akt substrate of 160kDa, glycogen synthase kinase-3β, AMP-activated protein kinase (AMPK) and its substrate acetyl coA carboxylase. Thus, Ucn3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promise for treatment of IR syndromes including sarcopaenic obesity.
... The Ucn2-null mice exhibit increased insulin sensitivity and are protected from fat-induced insulin resistance (8). In addition, CRFR2 activation was demonstrated to increase SM mass (24), reduce SM mass loss in atrophying SM due to denervation or casting, and to increase nonatrophying SM mass (25). ...
Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays and calcium mobilization measurements, we further demonstrate the functionality of these differentially expressed receptors. Using luciferase reporter assays we show a differential activation of CRFR promoters during myogenic differentiation. Transfections with different fragments of the 5'-flanking region of the mCRFR2β gene fused to a luciferase reporter gene show a promoter-dependent expression of the reporter gene and reveal the importance of the myocyte enhancer factor 2 consensus sequence located at the 3'-proximal region of CRFR2β promoter. Furthermore, we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat diet and chronic variable stress conditions. Performing a whole-genome expression microarray analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are elevated in insulin-resistant states. In correlation with the SM CRFR2β levels, the SM retinol-binding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable stress conditions. The current findings further position the SM CRFR2 pathways as a relevant physiological system that may affect the known reciprocal relationship between psychological and physiological challenges and the metabolic syndrome.
... For example, this was observed in animals with experimentally induced nerve damage. The agonist has also been reported to induce skeletal muscle hypertrophy [46]. ...
G-protein coupled receptors (GPCRs) represent a large class of cell surface receptors that mediate a multitude of functions. Over the years, a number of GPCRs and ancillary proteins have been shown to be expressed in skeletal muscle. Unlike the case with other muscle tissues like cardiac and vascular smooth muscle cells, there has been little attempt at systematically analyzing GPCRs in skeletal muscle. Here we have compiled all the GPCRs that are expressed in skeletal muscle. In addition, we review the known function of these receptors in both skeletal muscle tissue and in cultured skeletal muscle cells.
The clinical manifestation of osteopenia/osteoporosis is the fragility fracture, which is associated with significant morbidity and mortality. It also causes a high risk of disability. Fragility fractures may occur spontaneously but most frequently they are the consequence of falls. Sarcopenia, the age-related loss of muscle mass and physical performance, is becoming an increasing medical and financial concern in aging societies. Sarcopenia is evident in around 20% of over 70-year-olds; the figure rises to 50% for those over the age of 80. Those affected by this syndrome exhibit impaired mobility, a higher disability rate, and also a higher risk for falls and fractures. There is an intensive and complex interaction, between bones and muscles, with a magnified negative impact on the individual health. Individuals suffering from both, sarcopenia and osteopenia, are identified as osteosarcopenic or sarcoosteopenic. Fracture risk is increased 3.5-fold in male osteosarcopenia patients and herewith significantly higher than in sarcopenia and osteopenia alone. The importance of osteosarcopenia is not just linked to such potential additive risk of negative functional outcome, but also the suggestion that osteosarcopenia results from progression of osteopenia and/or sarcopenia. Consequently, therapeutic interventions (nutrition, exercise, vitamin D) should be started early in the development of this “hazardous duet” in a trial to prevent or at least stop such negative synergistic effects of osteosarcopenia on physical performance and bone turnover. This chapter starts by discussing osteosarcopenia and the impact of aging on the human body and the potential mechanisms of age-related sarcopenia. It then highlights the biochemical communication between muscle and bone and how muscle and bone act as an endocrine organ. The chapter then discusses osteosarcopenia is standard practice, presenting a case finding practical algorithm as well as categories of sarcopenia and sarcopenia-like conditions. This is followed by tools of diagnosis and treatment protocols of osteosarcopenia. The chapter concludes by presenting patient-centered care approach for osteosarcopenia patients.
Activation of corticotropin-releasing factor receptor 2β (CRFR2β) results in increased skeletal muscle mass and the prevention of muscle atrophy. Using a luciferase reporter assay, we screened 357 functional food factors that activate CRFR2β and, subsequently, confirmed that nobiletin (NBT) increases CRFR2β activity. Additionally, we found that NBT augments the activity of the endogenous peptide ligand urocortin 2 (Ucn2) in a concentration-dependent manner. Computational simulation of CRFR2β confirmed that transmembrane domains (TMs) 1 and 2 are important for the synergistic activity of NBT and also identified important amino acids in these domains. Finally, we demonstrated that a co-administration of Ucn2 and NBT increases the hypertrophic signal in mouse skeletal muscle. These observations demonstrate that NBT can activate CRFR2β and amplify the agonistic activity of Ucn2 and that such food-derived molecules have the potential to enhance endogenous G protein-coupled receptor ligand activities and contribute to the maintenance of skeletal muscle mass and function.
Aging is a one-way process associated with profound structural and functional changes in the organism. Indeed, the neuromuscular system undergoes a wide remodeling, which involves muscles, fascia, and the central and peripheral nervous systems. As a result, intrinsic features of tissues, as well as their functional and structural coupling, are affected and a decline in overall physical performance occurs. Evidence from the scientific literature demonstrates that senescence is associated with increased stiffness and reduced elasticity of fascia, as well as loss of skeletal muscle mass, strength, and regenerative potential. The interaction between muscular and fascial structures is also weakened. As for the nervous system, aging leads to motor cortex atrophy, reduced motor cortical excitability, and plasticity, thus leading to accumulation of denervated muscle fibers. As a result, the magnitude of force generated by the neuromuscular apparatus, its transmission along the myofascial chain, joint mobility, and movement coordination are impaired. In this review, we summarize the evidence about the deleterious effect of aging on skeletal muscle, fascial tissue, and the nervous system. In particular, we address the structural and functional changes occurring within and between these tissues and discuss the effect of inflammation in aging. From the clinical perspective, this article outlines promising approaches for analyzing the composition and the viscoelastic properties of skeletal muscle, such as ultrasonography and elastography, which could be applied for a better understanding of musculoskeletal modifications occurring with aging. Moreover, we describe the use of tissue manipulation techniques, such as massage, traction, mobilization as well as acupuncture, dry needling, and nerve block, to enhance fascial repair.
Sarcopenia in cirrhosis is a multifaceted process. As our understanding of the complex biology of skeletal muscle improves, we are getting closer to identifying specific therapies to treat muscle depletion in cirrhotic and non-cirrhotic populations. Anabolic hormones including testosterone and growth factor are often reduced in cirrhosis; pharmacological therapies that replenish these deficiencies may assist in preventing or reversing muscle depletion. In cirrhosis, hyperammonemia and portal hypertension both play important roles in the pathogenesis of sarcopenia. Specific treatments and interventions targeting these physiological abnormalities may also have added anabolic effects on muscle. Myostatin has emerged as key negative regulator of skeletal muscle in many chronic disease states including cirrhosis. Novel therapies that target different components of the extracellular myostatin signaling pathway are currently in various stages of development. Other drugs in clinical development include selective androgen receptor modulators, ghrelin agonists, and urocortin II agonists. However, while mechanistically these interventions offer possible treatments for sarcopenia in cirrhosis, there is a lack of large randomized trials to support any pharmacological intervention in this population. Therapeutic clinical trials in sarcopenia face significant challenges including a lack of consensus in the definition and assessment methodology for sarcopenia and the heterogenous population who each likely has differing underlying contributors to muscle wasting. Importantly, it is yet to be determined whether increasing muscle mass translates to improved patient outcomes.
Key points:
Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy (DMD), with strong evidence of attendant systemic and muscle inflammation. We performed a two-week intervention in young wild-type and mdx mice, consisting of either injection of saline or co-administration of a neutralizing interleukin-6 receptor antibody (xIL-6R) and Urocortin-2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL-6R and Ucn2 co-treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro-inflammatory cytokine interleukin-1β was reduced and the concentration of the anti-inflammatory cytokine interleukin-10 was increased in mdx diaphragm following drug co-treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing.
Abstract:
The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six-week-old male mdx (n = 32) and wild-type (WT; n = 32) mice received either saline (0.9% w/v) or a co-administration of neutralizing interleukin-6 receptor antibodies (xIL-6R; 0.2 mg/kg) and corticotrophin-releasing factor receptor 2 agonist (Urocortin-2; 30 μg/kg), subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force- and power-generating capacity. The number of centrally-nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co-treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co-treatment preserved MyHC type IIx complement in mdx muscle. Drug co-treatment increased the cross-sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL-1β, IL-6, KC/GRO and TNF-α were significantly increased in mdx diaphragm compared with WT. Drug co-treatment significantly decreased IL-1β and increased IL-10 in mdx diaphragm. Drug co-treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies. This article is protected by copyright. All rights reserved.
New Findings
What is the central question of this study?
We previously reported impaired upper airway dilator muscle function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Our aim was to assess the effect of blocking interleukin‐6 receptor signalling and stimulating corticotrophin‐releasing factor receptor 2 signalling on mdx sternohyoid muscle structure and function.
What is the main finding and its importance?
The interventional treatment had a positive inotropic effect on sternohyoid muscle force, restoring mechanical work and power to wild‐type values, reduced myofibre central nucleation and preserved the myosin heavy chain type IIb fibre complement of mdx sternohyoid muscle. These data might have implications for development of pharmacotherapies for DMD with relevance to respiratory muscle performance.
The mdx mouse model of Duchenne muscular dystrophy shows evidence of impaired pharyngeal dilator muscle function. We hypothesized that inflammatory and stress‐related factors are implicated in airway dilator muscle dysfunction. Six‐week‐old mdx ( n = 26) and wild‐type (WT; n = 26) mice received either saline (0.9% w/v) or a co‐administration of neutralizing interleukin‐6 receptor antibodies (0.2 mg kg ⁻¹ ) and corticotrophin‐releasing factor receptor 2 agonist (urocortin 2; 30 μg kg ⁻¹ ) over 2 weeks. Sternohyoid muscle isometric and isotonic contractile function was examined ex vivo . Muscle fibre centronucleation and muscle cellular infiltration, collagen content, fibre‐type distribution and fibre cross‐sectional area were determined by histology and immunofluorescence. Muscle chemokine content was examined by use of a multiplex assay. Sternohyoid peak specific force at 100 Hz was significantly reduced in mdx compared with WT. Drug treatment completely restored force in mdx sternohyoid to WT levels. The percentage of centrally nucleated muscle fibres was significantly increased in mdx , and this was partly ameliorated after drug treatment. The areal density of infiltrates and collagen content were significantly increased in mdx sternohyoid; both indices were unaffected by drug treatment. The abundance of myosin heavy chain type IIb fibres was significantly decreased in mdx sternohyoid; drug treatment preserved myosin heavy chain type IIb complement in mdx muscle. The chemokines macrophage inflammatory protein 2, interferon‐γ‐induced protein 10 and macrophage inflammatory protein 3α were significantly increased in mdx sternohyoid compared with WT. Drug treatment significantly increased chemokine expression in mdx but not WT sternohyoid. Recovery of contractile function was impressive in our study, with implications for Duchenne muscular dystrophy. The precise molecular mechanisms by which the drug treatment exerts an inotropic effect on mdx sternohyoid muscle remain to be elucidated.
Treatment of rats after burn-injury with the cyclic AMP phosphodiesterase (PDE) inhibitor, torbafylline (also known as HWA 448) significantly reversed changes in rat skeletal muscle proteolysis, PDE4 activity, cAMP concentrations and mRNA expression of TNFα, IL-6, ubiquitin and E3 ligases. Torbafylline also attenuated muscle proteolysis during in vitro incubation, and this effect was blocked by the inhibitor Rp-cAMPS. Moreover, torbafylline significantly increased phopho-Akt levels, and normalized downregulated phospho-FOXO1 and phospho-4E-BP1 in muscle of burn rats. Similarly, torbafylline also normalized phosphorylation levels of Akt and its downstream elements in TNFα+IFNγ treated C2C12 myotubes. Torbafylline enhanced protein levels of exchange protein directly activated by cAMP (Epac) both in skeletal muscle of burn rats and in TNFα+IFNγ treated C2C12 myotubes. Pretreatment with a specific antagonist of PI3K or Epac significantly reversed the inhibitory effects of torbafylline on TNFα+IFNγ-induced MAFbx mRNA expression and protein breakdown in C2C12 myotubes. Torbafylline inhibits burn-induced muscle proteolysis by activating multiple pathways through PDE4/cAMP/Epac/ PI3K/Akt.
The main aim of this thesis was the functional characterization of the just recently identified neuropeptide Urocortin 2 (UCN2) by means of a
conditional gain-of-function mouse model. A combination of the Cre/loxP system and the ubiquitously expressed Rosa26 locus allowed the
dose dependent and cell type specific expression of UCN2. UCN2 was shown to be specifically overexpressed in the CNS, and the resulting effects on the expression patterns of CRH related genes, the balance of stress hormones and furthermore the behaviour of the mice were analyzed. The results substantially contribute to the elucidation of the physiological function of UCN2 and show its potential role on fear- and depression related behaviour.
In conscious male Sprague-Dawley rats, we compared regional hemodynamic actions of the selective corticotropin-releasing factor type 2 (CRF(2)) receptor ligands human and mouse urocortin 2 (hUCN2 and mUCN2, respectively) with those of CRF. Bolus i.v. doses of 3 and 30 pmol kg(-1) hUCN2, mUCN2, or CRF had no significant hemodynamic actions, but at doses of 300 and 3000 pmol kg(-1), all three peptides caused dose-dependent tachycardia and hypotension, with rapid-onset, short-duration, mesenteric vasodilatation and slower-onset, more prolonged hindquarters vasodilatation but little or no change in renal vascular conductance. Pretreatment with the nonselective CRF receptor antagonist astressin or the selective CRF(2) receptor antagonist antisauvagine 30 abolished all the cardiovascular actions of all three peptides. Indomethacin had no effect on responses to hUCN2, and there was no evidence for any involvement of nitric oxide (NO) in the vasodilator actions of hUCN2. There was no evidence that recruitment of angiotensin- and endothelin-mediated vasoconstrictor mechanisms counteracted the vascular actions of hUCN2. The results indicate that the hemodynamic effects of i.v. hUCN2, mUCN2, and CRF depend on activation of CRF(2) receptors and do not involve NO or prostanoids.
Urocortin 1, a human homologue of fish urotensin I, together with its related-compounds (urocortins 2 and 3), comprises a distinct family of stress peptides. Urocortin 1 has a high affinity for both corticotropin-releasing factor (CRF) type 1 receptor (CRF1) and CRF type 2 receptor (CRF2), and urocortins 2 and 3 have a high affinity for CRF2, while CRF has a low affinity for CRF2 and a high affinity for CRF1. These differences of the binding affinity with receptors make the biological actions of these peptides. Besides the binding affinity with receptors, the limited overlap of the distribution of CRF and urocortins may also contribute to the differences of physiological roles of each peptide. Urocortins show 'stress-coping' responses such as anxiolysis and dearousal in the brain. In the periphery, recent studies show the potent effects of urocortins on the cardiovascular and immune systems. In this review article, we take a look over the series of peptides included in this family, especially in terms of the versatility of biological actions, along with the various characters of the receptors.
Of the two corticotropin releasing factor receptors known, corticotrophin releasing factor 2 receptor (CRF2R) is expressed in skeletal muscle. The function of this receptor in skeletal muscle is at present unknown. In order to better understand the role of the CRF2R in skeletal muscle, we treated rats with CRF2R agonists and evaluated the effect of these agents on normal and denervated muscle mass. Rats treated with the non-selective CRFR agonist, sauvagine, did not demonstrate any significant and consistent change in non-denervated and denervated fast twitch [tibialis anterior (TA) or extensor digitorum longus (EDL)] or slow/mixed twitch [medial gastrocnemius (MG) or soleus] fiber muscle mass. In adrenalectomized rats, sauvagine treatment resulted in no significant and consistent change in non-denervated fast or slow/mixed twitch fiber muscles but did cause a significant and consistent increase in denervated fast twitch (TA and EDL) but not slow/mixed twitch muscle mass. Interestingly adrenalectomy had no effect on the degree of muscle atrophy. Rats treated with the CRF2R selective agonist urocortin 2 demonstrated an increase in non-denervated and denervated fast and slow/mix twitch fiber muscle mass. The urocortin 2 induced increase in muscle mass was accompanied by an increase in muscle fiber cross-sectional area and muscle absolute force. These studies demonstrated that activation of the CRF2R decreased the level of skeletal muscle mass, force, and myocyte cross-sectional area loss resulting from sciatic nerve damage and increased the mass, force and myocyte cross-sectional area of normal (non-atrophying) skeletal muscle. In addition, we also observed that removal of the adrenals increased the effectiveness of the non-selective CRFR agonists sauvagine, presumably via the removal of the pro-atrophy influence of adrenal produced corticosteroids. These results demonstrate that pharmacological modulation of the CRF2R may be a viable method to treat skeletal muscle atrophy.
Corticotropin releasing factor 2 receptor selective analogs of the amphibian peptide sauvagine, a member of the corticotropin releasing factor (CRF) peptide family, have therapeutic potential for the treatment of skeletal muscle atrophy. Previously, we demonstrated that [P11X12X13]Svg peptides have improved CRF2R selectivity, although not to the level of CRF2R selective hormones such as urocortin 2 and urocortin 3. Since we also demonstrated a potential for improvement in selectivity of sauvagine by modifications of residues 35 and 39, we investigated substitutions of these amino acids in selected [P11X12X13]Svg peptides. We have observed that substitution of Arg35 in sauvagine to Ala35 (the amino acid found in all CRF2R selective agonists), increased the selectivity of [P11, X12, X13]Svg analogs. In contrast, substitution of Asp39 in sauvagine to Ala39 (also the amino acid found in all CRF2R selective agonists) did not further increase the selectivity of [P11, X12, X13, A35]Svg analogs. Thus, the residues 35 along with 11, 12, and 13 in sauvagine represent important sites for improving CRF2R selectivity.
Urocortin 3 (Ucn 3)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. In the present study, we studied expression of Ucn3/SCP in the normal adrenal and adrenal tumors by radioimmunoassay and reverse transcriptase-polymerase chain reaction (RT-PCR). High concentrations of immunoreactive (IR)-Ucn3 were present in the normal portions of adrenal glands (4.2+/-0.51 pmol/g wet weight, mean+/-S.E.M., n = 14), and the levels were higher than those in the brain. IR-Ucn3 was also detected in the tumor tissues of aldosterone-secreting adenomas (6.2+/-0.6 pmol/g wet weight, n = 10), cortisol-secreting adenomas (5.0+/-1.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.9+/-0.4 pmol/g wet weight, n = 7). Reverse phase high performance liquid chromatography showed that IR-Ucn3 in normal portions of adrenal glands and aldosterone-secreting adenomas was eluted mainly in the positions of Ucn3 and SCP with several minor peaks eluting earlier. The RT-PCR showed expression of Ucn3 mRNA in normal portions of adrenal gland (positive ratio; 4/4), aldosterone-secreting adenomas (3/4), cortisol-secreting adenomas (1/3) and pheochromocytomas (6/7). These findings indicate that Ucn3 is produced in normal adrenal and adrenal tumors (both adrenocortical tumors and pheochromocytomas), and suggest that Ucn3 acts as an autocrine or paracrine regulator in normal adrenal and adrenal tumors.
Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.
Corticotrophin-releasing factor 2 receptor (CRF2R) agonists prevent muscle atrophy due to immobilization, denervation, and corticosteroid-induced muscle atrophy in wildtype mice. We hypothesized that a CRF2R agonist will increase skeletal muscle mass in mdx mice. Mdx (C57BL/10ScSn-Dmd(mdx)) and wildtype (C57BL/6) mice were divided into four groups: sedentary placebo, sedentary CRF2R agonist, exercised placebo, and exercised CRF2R agonist. Mice exercised on a treadmill twice weekly for 30 min (8-12 m/min, 8 weeks). Muscle and heart weights, serum creatine kinase, and gamma-glutamyltransferase activities were measured. The CRF2R agonist increased extensor digitorum longus and soleus muscle weights (P < 0.05) in wildtype and mdx mice. Sedentary mdx CRF2R and exercised mdx placebo mice had lower serum creatine kinase activity than sedentary mdx placebo mice. CRF2R-treated mice had decreased heart weights compared to placebo-treated mice. We conclude that CRF2R agonists should be further evaluated as a potential therapy for dystrophinopathies.
Previous studies have demonstrated an effect of corticotropin-releasing factor 2 receptor (CRF2R) agonists in the maintenance of skeletal muscle mass. The aim of this study was to evaluate the effects of a CRF2R agonist in preserving skeletal muscle in a mouse cachexia model. Implantation of a fast-growing tumor to mice (Lewis lung carcinoma) resulted in a clear cachectic state characterized by a profound muscle wasting. We found that administration of a CRF2R agonist (PG-873637) at 100 microg/kg/day by means of osmotic minipumps to tumor-bearing mice resulted in beneficial effects on muscle weight loss. Thus, muscle loss was partially reversed by the CRF2R agonist at different stages of tumor growth (at day 14 after tumor inoculation: 12% in tibialis posterior; 9% in gastrocnemius; and 48% in soleus). Moreover, the CRF2R agonist significantly reduced both the number of metastases and their mass (at day 19 after tumor inoculation: 66% and 61%, respectively). These data suggest a potentially beneficial effect of the CRF2R agonist in preserving skeletal muscle during cancer cachexia and open a line of research for the development of new therapeutic approaches for the treatment of muscle wasting associated with cancer.
Corticotropin-releasing factor (CRF) mediates many critical aspects of the physiological response to stress. These effects are elicited by binding to specific high-affinity receptors, which are coupled to guanine nucleotide stimulatory factor (Gs)-response pathways. Recently, a gene encoding a receptor for CRF, expressed in pituitary and the central nervous system (PC-CRF receptor), was isolated and characterized. Here we report the identification and characterization of a second, distinct CRF receptor that is expressed primarily in heart and skeletal muscle and exhibits a specific ligand preference and antagonist sensitivity compared with the PC-CRF receptor. We refer to this second receptor as the heart/muscle (HM)-CRF receptor.
Corticotropin-releasing factor (CRF), urocortin, sauvagine and urotensin I form the CRF family. These peptides bind with different affinities to two subtypes of CRF receptor (CRFR), CRFR1 and CRFR2. The latter exists as two splice variants, the neuronal CRFR2a and the peripheral CRFR2b. CRFR is a G protein-dependent receptor which acts mainly through Gs enhancing cAMP production. However, CRFR1 expressed in neutrophils of the spleen in response to immunologic stimulation and psychological stress does not seem to function through Gs, as indicated by the inability of CRF to stimulate the cAMP production of CRFR1+ neutrophils. Besides the two receptors, a 37 kD CRF binding protein (CRF-BP) binds several CRF peptides with high affinity. CRFR and CRF-BP do not share a common amino acid sequence representing the ligand binding site. In view of the unusually slow offrate of CRF-BP, it is proposed that CRF-BP provides an efficient uptake of free extracellular CRF. Thus, the time of exposure of CRFR to CRF or urocortin can be limited. At this time, the fate of the ligand CRF-BP complex is unclear. CRFR1 is not only involved in the hypophyseal stimulation of corticotropin release, but hippocampal CRFR1 mediates enhancement of stress-induced learning. CRFR1 may also be involved in basic anxiety. In contrast, at least in the mouse, CRFR2 of the lateral intermediate septum mediates tonic impairment of learning. In response to stressful stimuli or after local injection of high CRF doses, CRFR2 mediates anxiety. Effects requiring CRFR2 can be blocked specifically by the recently developed peptidic antagonist antisauvagine-30.
Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G-protein-coupled receptors, Crhr1 and Crhr2, causing (among other transductional events) phosphorylation of the transcription factor Creb. The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1. The enhanced anxiety of Crhr2-deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2-/- mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2-deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender.
The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2. These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2-/- mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2-/- mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2-/- mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2-/- mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2-/- mice following Ucn, but Crhr2-/- mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2-/- mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2-/- mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.
Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.
The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.
Corticotropin-releasing factor (CRF) injected peripherally induces clustered spike-burst activity in the proximal colon through CRF(1) receptors in rats. We investigated the effect of intraperitoneal CRF on proximal colon ganglionic myenteric cell activity in conscious rats using Fos immunohistochemistry on the colonic longitudinal muscle/myenteric plexus whole mount preparation. In vehicle-pretreated rats, there were only a few Fos immunoreactive (IR) cells per ganglion (1.2 +/- 0.6). CRF (10 microg/kg ip) induced Fos expression in 19.6 +/- 2.1 cells/ganglion. The CRF(1)/CRF(2) antagonist astressin (33 microg/kg ip) and the selective CRF(1) antagonist CP-154,526 (20 mg/kg sc) prevented intraperitoneal CRF-induced Fos expression in the proximal colon (number of Fos-IR cells/ganglion: 2.7 +/- 1.2 and 1.0 +/- 1.0, respectively), whereas atropine (1 mg/kg sc) had no effect. Double labeling of Fos with protein gene product 9.5 revealed the neuronal identity of activated cells that were encircled by varicose fibers immunoreactive to vesicular acetylcholine transporter. Fos immunoreactivity was mainly present in choline acetyltransferase-IR nerve cell bodies but not in the NADPH-diaphorase-positive cells. These results indicate that peripheral CRF activates myenteric cholinergic neurons in the proximal colon through CRF(1) receptor.
Angiogenesis is regulated by means of a balance between activators and inhibitors. However, little is known regarding the regulation of the quiescent state of adult vessels. Corticotropin-releasing factor receptor 2 (CRFR2) is found in both endothelial and smooth muscle cells (SMCs) in the vasculature, where its function has remained elusive. We have investigated the role of CRFR2 as a determinant of tissue vascularization by comparing control and CRFR2-deficient mice with immunohistological and morphometric techniques. To define the mechanisms responsible for CRFR2 inhibition of angiogenesis, we have also examined in vitro the effect of ligand activation on cell proliferation, cell cycle protein phosphorylation, and capillary tube formation. Our results demonstrate that mice deficient for CRFR2 become hypervascularized postnatally. Activation of this receptor in vitro results in reduced vascular endothelial growth factor (VEGF) release from SMCs, an inhibition of SMC proliferation, and inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibits growth factor-induced vascularization. Western blots show that cell cycle retinoblastoma protein, which is essential for cell cycle progression, is decreased by CRFR2 agonist treatment in SMCs. These results suggest that CRFR2 is a critical component of a pathway necessary for tonic inhibition of adult neovascularization. CRFR2 may be a potential target for therapeutic modulation of angiogenesis in cancer and ischemic cardiovascular disease.
Peptides encoded by the urocortin II (Ucn II) gene were recently identified as new members of the corticotropin-releasing factor (CRF) family. Ucn II is a specific ligand for the type 2 CRF receptor. Using RT-PCR, DNA sequencing, and immunofluorescence staining, we report the expression of Ucn II mRNA in several human and mouse (m) neuronal cell lines. Using these neuronal cell lines, we provide evidence that exposure to glucocorticoid hormones increases mUcn II mRNA expression and promoter activation. The effect of glucocorticoids on mUcn II mRNA expression was tested in the Ucn II/glucocorticoid receptor-positive cell line NG108-15. The results demonstrate that mUcn II mRNA expression is up-regulated by dexamethasone in a dose- and time-dependent fashion. Computer analysis revealed the presence of 14 putative half-palindrome glucocorticoid response element sequences within 1.2 kb of the mUcn II 5' flanking region. Transfections with different fragments of the 5'-flanking region of the mUcn II gene fused to a luciferase reporter gene showed a promoter-dependent expression of the reporter gene and regulation by dexamethasone. Promoter deletion studies clarify the sufficient putative glucocorticoid response element site mediating this effect. The steroid hormone antagonist RU486 blocked the effect of dexamethasone on mUcn II mRNA expression and promoter activation, suggesting a direct glucocorticoid receptor-mediated effect of dexamethasone on mUcn II mRNA expression. Ucn II is expressed in vivo in the hypothalamus, brainstem, olfactory bulb, and pituitary. Low levels were also detected in the mouse cortex, hippocampus, and spinal cord. We demonstrated that mUcn II gene transcription was stimulated by glucocorticoid administration in vivo and inhibited by removal of glucocorticoids by adrenalectomy. Administration of dexamethasone to mice resulted in an increase of mUcn II levels in the hypothalamus and brainstem but not in the olfactory bulb region 12 h following ip injection. In light of our present data and the current literature, we propose a putative link between the CRF receptor 1 and CRF receptor 2 pathways.
Two receptors activated by the corticotropin-releasing factor (CRF) family of peptides have been identified, the CRF 1 receptor (CRF1R) and the CRF 2 receptor (CRF2R). Of these, the CRF2R is expressed in skeletal muscle. To understand the role of the CRF2R in skeletal muscle, we utilized CRFR knockout mice and CRF2R-selective agonists to modulate nerve damage and corticosteroid- and disuse-induced skeletal muscle atrophy in mice. These analyses demonstrated that activation of the CRF2R decreased nerve damage and corticosteroid- and disuse-induced skeletal muscle mass and function loss. In addition, selective activation of the CRF2R increased nonatrophy skeletal muscle mass. Thus we describe for the first time a novel activity of the CRF2R, modulation of skeletal muscle mass.
Corticotropin-releasing factor (CRF), urocortin, sauvagine and urotensin 1 form the CRF family. These peptides bind with different affinities to two subtypes of CRF receptor (CRFR), CRFR1 and CRFR2. The latter exists as two splice variants, the neuronal CRFR2 alpha and the peripheral CRFR2 beta. CRFR is a G protein-dependent receptor which acts mainly through G(s) enhancing cAMP production. However, CRFR1 expressed in neutrophils of the spleen in response to immunologic stimulation and psychological stress does not seem to function through G(s) as indicated by the inability of CRF to stimulate the cAMP production of CRFR1(+) neutrophils. Besides the two receptors, a 37 kD CRF binding protein (CRF-BP) binds several CRF peptides with high affinity. CRFR and CRF-BP do not share a common amino acid sequence representing the ligand binding site. In view of the unusually slow off rate of CRF-BP, it is proposed that CRF-BP provides an efficient uptake of free extracellular CRF. Thus, the time of exposure of CRFR to GRF or urocortin can be limited. At this time, the fate of the ligand CRF-BP complex is unclear. CRFR1 is not only involved in the hypophyseal stimulation of corticotropin release, but hippocampal CRFR1 mediates enhancement of stress-induced learning. CRFR1 may also be involved in basic anxiety. In contrast, at least in the mouse, CRFR2 of the lateral intermediate septum mediates tonic impairment of learning. In response to stressful stimuli or after local injection of high CRF doses, CRFR2 mediates anxiety. Effects requiring CRFR2 can be blocked specifically by the recently developed peptidic antagonist antisauvagine-30.
We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.
: Corticotropin-releasing factor (CRF) was recognized biologically in the 1950s and was first isolated from ovine hypothalamus and characterized as a 41 amino acid peptide in 1981. Subsequently, rat and human CRF were identified and found to be identical to one another, while differing from ovine CRF by seven residues. A variety of experimental observations indicate that CRF is the key neuroregulator of the hypothalamic-pituitary-adrenal cortical (HPA) axis. The actions of CRF on adrenocorticotropic hormone (ACTH) secretion are potentiated by vasopressin and blunted by glucocorticoid negative feedback. The broad central and peripheral distribution of the peptide and its receptors supports the notion that CRF is an important local regulator within the central nervous, immune, and other systems. Further, CRF mediates numerous complementary stress-related endocrine, immune, autonomic, and behavioral responses.
Antagonists of CRF, such as [alpha]-helical CRF or astressin, block many stress-induced physiologic and pathophysiologic responses in experimental animals, and perturbations of the CRF system or the HPA have been reported in human affective disorders. The effects of CRF within the central nervous system may be anatomically and temporally limited by a high-affinity binding protein (CRF-BP). The actions of CRF are mediated by seven transmembrane-domain G-protein-coupled receptors (CRF-R) derived from two genes (R1 and R2), each of which has alternative splice variants. We have identified a novel urotensin and CRF-like peptide, urocortin, in the rat brain and the human genome. Urocortin has a high affinity for CRF-R1 and R2 as well as for CRF-BP. Synthetic urocortin has potent biological actions on both CRF-R1 (pituitary ACTH release) as well as CRF-R2 (vasodilation, reduction of vascular permeability) mediated events. This novel peptide appears to be an endogenous ligand for CRF-type 2 receptors.
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Corticotropin-releasing factor (CRF) and CRF-related neuropeptides have an important role in the central nervous system to mediate behavioral responses to stressors. CRF receptor antagonists are very effective in reversing stress-induced suppression and activation in behavior. An additional CRF-like neuropeptide, urocortin, has been identified in the brain and has a high affinity for the CRF-2 receptor in addition to the CRF-1 receptor. Urocortin has many of the effects of CRF but also is significantly more potent than CRF in decreasing feeding in both meal-deprived and free-feeding rats. In mouse genetic models, mice over-expressing CRF show anxiogenic-like responses compared to wild-type mice, and mice lacking the CRF-1 receptor showed an anxiolytic-like behavioral profile compared to wild-type mice. Results to date have led to the hypothesis that CRF-1 receptors may mediate CRF-like neuropeptide effects on behavioral responses to stressors, but CRF-2 receptors may mediate the suppression of feeding produced by CRF-like neuropeptides. Brain sites for the behavioral effects of CRF include the locus coeruleus (LC), paraventricular nucleus (PVN) of the hypothalamus, the bed nucleus of the stria terminalis (BNST), and the central nucleus of the amygdala. CRF may also be activated during acute withdrawal from all major drugs of abuse, and recent data suggest that CRF may contribute to the dependence and vulnerability to relapse associated with chronic administration of drugs of abuse. These data suggest that CRF systems in the brain have a unique role in mediating behavioral responses to diverse stressors. These systems may be particularly important in situations were an organism must mobilize not only the pituitary adrenal system, but also the central nervous system in response to environmental challenge. Clearly, dysfunction in such a fundamental brain-activating system may be the key to a variety of pathophysiological conditions involving abnormal responses to stressors such as anxiety disorders, affective disorders, and anorexia nervosa.
The effect of CRF microinjected into the dorsal vagal complex (DVC) on centrally-stimulated gastric contractility was investigated in fasted, urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and contractility was analyzed by computer. Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, (26 pmol) into the DVC induced a 12.2-fold stimulation of gastric contractility within 30 min. Corticotropin-releasing factor (CRF) (63-210 pmol) microinjected into the DVC concomitantly with RX 77368 (26 pmol) induced a dose-related inhibition of stimulated gastric contractility. Neither CRF alone (210 pmol) nor vehicle modified basal gastric contractility. Microinjection of kainic acid (141 pmol) into the raphe pallidus nucleus induced a 3.6-fold stimulation of gastric contractility after 45 min. This stimulation was suppressed by bilateral microinjection of CRF (105 pmol/site) into the DVC. These results demonstrate that CRF acts in the DVC to inhibit centrally-stimulated gastric contractility and suggest that TRH and CRF may interact in the DVC to regulate gastric motor function.
A variety of peptides (corticotropin releasing factor (= CRF), cholecystokinin-derived peptides, neurotensin, bombesin, angiotensin II, bradykinin and substance P) induce a contraction of the myenteric plexus/longitudinal muscle preparation of the guinea-pig ileum. This excitatory effect is rapid in onset and disappears within a few minutes in the continued presence of the peptide. A part of the contractile response is antagonized by atropine indicating that acetylcholine (ACh) is involved in this effect. Following cessation of the peptide-induced contraction, a second contractile response can be elicited by the opioid antagonist naloxone. The magnitude of this naloxone induced contraction is related to the "atropine-sensitive" component of the initial contractory effect of the peptides. It appears that the peptidergic excitatory action on the plexus which is associated with release of ACh, initiates the release of opioid peptides in this tissue.
We studied the effect of corticotropin-releasing factor (CRF) on airway smooth muscle functions in isolated rabbit tracheal segments under isometric conditions in vitro. The addition of ovine CRF synthesized by solid-phase methods did not cause muscle contraction, but it potentiated the contractile response to electrical field stimulation (EFS) at 5 Hz in a dose-dependent fashion, the maximal increase from the baseline response being 43.9 +/- 6.1% (mean +/- SE, p less than 0.001). This effect was not influenced by propranolol, phentolamine, indomethacin, pyrilamine, or (D-Pro2,D-Trp7.9)-substance P, but was completely inhibited by alpha-helical CRF, a CRF receptor antagonist. CRF (10(-7) M) also increased the responses to EFS at all frequencies of stimulation (1 to 40 Hz), so that the stimulus frequency required to produce a half-maximal contraction (ES50) decreased from 7.7 +/- 1.0 to 3.8 +/- 0.6 Hz (p less than 0.01). In contrast, contractile response to administered acetylcholine was not affected by CRF. CRF-induced potentiation of the response to EFS (5 Hz) was further increased from 44.5 +/- 5.4 to 144.6 +/- 11.1% in the presence of physostigmine and was abolished by atropine. These results suggest that CRF prejunctionally potentiates the vagally mediated contraction of airway smooth muscle through activation of CRF receptors on the cholinergic nerve terminals, likely involving the accelerated release of acetylcholine.
We have recently described the cloning and characterization of a novel corticotropin-releasing factor receptor subtype (CRF2) from rat brain that exists in two alternatively spliced forms, CRF2 alpha and CRF2 beta. These forms differ in their N-terminal coding sequence which results in the production of two distinct receptors of 411 and 431 amino acids, respectively. To assess whether these two forms might represent distinct targets for CRF action, RNase protection and in situ hybridization studies were performed using specific N-terminal cRNA probes. The results showed a differential distribution of the mRNAs for these two receptor forms in the rat. The mRNA for CRF2 alpha is found almost exclusively in the brain, particularly in the hypothalamus, lateral septum, and olfactory bulb, whereas the mRNA for CRF2 beta appears to be both in the brain and in the periphery, with the greatest abundance in the heart and skeletal muscle. Thus, the data suggest that these alternatively spliced forms of the CRF2 receptor may represent functionally distinct CRF receptors. In addition, it highlights the importance of probe specificity for in situ hybridization studies.
Corticotropin-releasing factor (CRF) plays a major role in coordinating the endocrine, autonomic, behavioral and immune responses to stress through actions in the brain and the periphery. CRF receptors identified in brain, pituitary and spleen have comparable kinetic and pharmacological characteristics, guanine nucleotide sensitivity and adenylate cyclase-stimulating activity. Differences were observed in the molecular mass of the CRF receptor complex between the brain (58,000 Da) and the pituitary and spleen (75,000 Da), which appeared to be due to differential glycosylation of the receptor proteins. The recently cloned CRF receptor in the pituitary and the brain (designated as CRF1) encodes a 415 amino acid protein comprising seven putative membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal peptide/growth hormone-releasing hormone subfamily of G-protein-coupled receptors. A second member of the CRF receptor family encoding a 411 amino acid rat brain protein with approximately 70% homology to CRF1 has recently been identified (designated as CRF2); there exists an additional splice variant of the CRF2 receptor with a different N-terminal domain encoding a protein of 431 amino acids. In autoradiographic studies, CRF receptors were localized in highest densities in the anterior and intermediate lobes of the pituitary gland, olfactory bulb, cerebral cortex, amygdala, cerebellum and the macrophage-enriched zones and red pulp regions of the spleen. CRF can modulate the number of CRF receptors in a reciprocal manner. For example, stress and adrenalectomy increase hypothalamic CRF secretion which, in turn, down-regulates CRF receptors in the anterior pituitary. CRF receptors in the brain and pituitary are also altered as a consequence of the development and aging processes. In addition to a physiological role for CRF in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli, recent clinical data implicate CRF in the etiology and pathophysiology of various endocrine, psychiatric, neurologic and inflammatory illnesses. Hypersecretion of CRF in the brain may contribute to the symptomatology seen in neuropsychiatric disorders, such as depression, anxiety-related disorders and anorexia nervosa. Furthermore, overproduction of CRF at peripheral inflammatory sites, such as synovial joints may contribute to autoimmune diseases such as rheumatoid arthritis. In contrast, deficits in brain CRF are apparent in neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, as they relate to dysfunction of CRF neurons in the brain areas affected in the particular disorder. Strategies directed at developing CRF-related agents may hold promise for novel therapies for the treatment of these various disorders.
Corticotropin-releasing factor (CRF) is a 41-amino acid neuropeptide, which is recognized as a critical mediator of complimentary, stress-related endocrine, autonomic, and behavioral responses in mammalian species. CRF belongs to a family of structurally related peptides including frogskin sauvagine and fish urotensin I. The effects of CRF and related peptides are mediated by two distinct receptors, which differ in their anatomical distribution, as well as in their pharmacological characteristics. In addition, CRF is bound with high affinity by a CRF binding protein (CRF-BP), which is a putative inhibitor of CRF action. CRF is probably not the sole endogenous ligand for CRF receptors or the CRF-BP, since a second mammalian member of the CRF family, urocortin, has recently been identified. This article describes recent findings with respect to CRF, its receptors, binding protein, and CRF-related peptides, which provide further insights into the role and mechanisms of CRF action in stress responses.
We tested the effect of endotoxin on the peripheral corticotropin-releasing hormone receptor (CRH-R2), which is highly expressed in the heart. Systemic injection of LPS markedly downregulated CRH-R2 mRNA levels in the heart in a dose and time dependent manner. In contrast, CRH-R2 levels in skeletal muscle increased following exposure to endotoxin. These results suggest that CRH-R2 may be differentially regulated in cardiac tissue and skeletal muscle. Finding that CRH-R2 expression in the heart is modulated by endotoxin, a potent inducer of cardiovascular dysregulation, suggests a possible link between CRH and the cardiovascular response to stress.
The effect of corticotropin-releasing factor (CRF) on contractions of guinea pig isolated airways in response to electrical or chemical stimulation of tachykinergic nerve fibers was studied. CRF (1 microgram/ml) caused a 70% enhancement of the peak magnitude of the response to electrical field stimulation (EFS). CRF had a similar effect on contractions of the isolated bronchus evoked by capsaicin. CRF also potentiated contractions evoked by exogenously applied substance P. This effect was selective, as CRF has no effect on contractions evoked by neurokinin A or the substance P analog ASMSP. The potentiation of the substance P-induced contractions of airway smooth muscle was blocked by the CRF receptor antagonist alpha helical (9-41) CRF. These data support the hypothesis that CRF enhances the airway smooth muscle response to stimulation of tachykinin-containing nerve fibers and that this effect is due to a post-junctional mechanism of action.
Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioural and immune responses to stress, and has been implicated in the stress-like and other aversive consequences of drug abuse, such as withdrawal from alcohol. Two CRH receptors, Crhr1 and Crhr2, have been identified in the mouse. Crhr1 is highly expressed in the anterior pituitary, neocortex, hippocampus, amygdala and cerebellum, and activation of this receptor stimulates adenylate cyclase. Here we show that in mice lacking Crhr1, the medulla of the adrenal gland is atrophied and stress-induced release of adrenocorticotropic hormone (ACTH) and corticosterone is reduced. The homozygous mutants exhibit increased exploratory activity and reduced anxiety-related behaviour under both basal conditions and following alcohol withdrawal. Our results demonstrate a key role of the Crhr1 receptor in mediating the stress response and anxiety-related behaviour.
Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.
Corticotropin-releasing factor (CRF) has been widely implicated as playing a major role in modulating the endocrine, autonomic, behavioral and immune responses to stress. The recent cloning of multiple receptors for CRF as well as the discovery of non-peptide receptor antagonists for CRF receptors have begun a new era of CRF study. Presently, there are five distinct targets for CRF with unique cDNA sequences, pharmacology and localization. These fall into three distinct classes, encoded by three different genes and have been termed the CRF1 and CRF2 receptors (belonging to the superfamily of G-protein coupled receptors) and the CRF-binding protein. The CRF2 receptor exists as three splice variants of the same gene and have been designated CRF2a CRF2b and CRF2g. The pharmacology and localization of all of these proteins in brain has been well established. The CRF1 receptor subtype is localized primarily to cortical and cerebellar regions while the CRF2a receptor is localized to subcortical regions including the lateral septum, and paraventricular and ventromedial nuclei of the hypothalamus. The CRF2b receptor is primarily localized to heart, skeletal muscle and in the brain, to cerebral arterioles and choroid plexus. The CRF2g receptor has most recently been identified in human amygdala. Expression of these receptors in mammalian cell lines has made possible the identification of non-peptide, high affinity, selective receptor antagonists. While the natural mammalian ligands oCRF and r/hCRF have high affinity for the CRF1 receptor subtype, they have lower affinity for the CRF2 receptor family making them ineffective labels for CRF2 receptors. [125I]Sauvagine has been characterized as a high affinity ligand for both the CRF1 and the CRF2 receptor subtypes and has been used in both radioligand binding and receptor autoradiographic studies as a tool to aid in the discovery of selective small molecule receptor antagonists. A number of non-peptide CRF1 receptor antagonists that can specifically and selectively block the CRF1 receptor subtype have recently been identified. Compounds such as CP 154,526 (12), NBI 27914 (129) and Antalarmin (154) inhibit CRF-stimulation of cAMP or CRF-stimulated ACTH release from cultured rat anterior pituitary cells. Furthermore, when administered peripherally, these compounds compete for ex vivo [125I]sauvagine binding to CRF1 receptors in brain sections demonstrating their ability to cross the blood-brain-barrier. In in vivo studies, peripheral administration of these compounds attenuate stress-induced elevations in plasma ACTH levels in rats demonstrating that CRF1 receptors can be blocked in the periphery. Furthermore, peripherally administered CRF1 receptor antagonists have also been demonstrated to inhibit CRF-induced seizure activity. These data clearly demonstrate that non-peptide CRF1 receptor antagonists, when administered systemically, can specifically block central CRF1 receptors and provide tools that can be used to determine the role of CRF1 receptors in various neuropsychiatric and neurodegenerative disorders. In addition, these molecules will prove useful in the discovery and development of potential orally active therapeutics for these disorders.
CRF and urocortin, administrated systemically, exert peripheral biological actions which may be mediated by brain pathways. We identified brain neuronal activation induced by intravenous (i.v.) injection of CRF and urocortin in conscious rats by monitoring Fos expression 60 min later. Both peptides (850 pmol/kg, i.v.) increased the number of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala, nucleus tractus solitarius and area postrema compared with vehicle injection. Urocortin induced a 4-fold increase in the number of Fos-positive cells in the supraoptic nucleus and a 3.4-fold increase in the lateral magnocellular part of the paraventricular nucleus compared with CRF. Urocortin also elicited Fos expression in the accessory hypothalamic neurosecretory nuclei, ependyma lining the ventricles and choroid plexus which was not observed after CRF. The intensity and pattern of the Fos response were dose-related (85, 255 and 850 pmol/kg, i.v.) and urocortin was more potent than CRF. Neither CRF nor urocortin induced Fos expression in the lateral septal nucleus, Edinger-Westphal nucleus, dorsal raphe nucleus, locus coeruleus, or hypoglossal nucleus. These results show that urocortin, and less potently CRF, injected into the circulation at picomolar doses activate selective brain nuclei involved in the modulation of autonomic/endocrine function; in addition, urocortin induces a distinct activation of hypothalamic neuroendocrine neurons.
The CRF receptors belong to the VIP/GRF/PTH family of G-protein coupled receptors whose actions are mediated through activation of adenylate cyclase. Two CRF receptors, encoded by distinct genes, CRF-R1 and CRF-R2, and that can exist in two alternatively spliced forms, have been cloned. The type-1 receptor is expressed in many areas of the rodent brain, as well as in the pituitary, gonads, and skin. In the rodent, one splice variant of the type-2 receptor, CRF-R2 alpha, is expressed mainly in the brain, whereas the other variant, CRF-R2 beta, is found not only in the CNS, but also in cardiac and skeletal muscle, epididymis, and the gastrointestinal tract. The poor correlation between the sites of expression of CRF-R2 and CRF, as well as the relatively low affinity of CRF for CRF-R2, suggested the presence of another ligand, whose existence was confirmed in our cloning of urocortin. This CRF-like peptide is found not only in brain, but also in peripheral sites, such as lymphocytes. The broad tissue distribution of CRF receptors and their ligands underscores the important role of this system in maintenance of homeostasis. Functional studies of the two receptor types reveal differences in the specificity for CRF and related ligands. On the basis of its greater affinity for urocortin, in comparison with CRF, as well as its brain distribution, CRF-R2 may be the cognate receptor for urocortin. Mutagenesis studies of CRF receptors directed toward understanding the basis for their specificity, provide insight into the structural determinants for hormone-receptor recognition and signal transduction.
Research on the contribution of CRH receptor stimulation to energy homeostasis has focused on forebrain substrates. In this study, we explored the effects of caudal brainstem administration of the CRH receptor agonist, urocortin, on food intake and body weight, and on plasma glucose and corticosterone (CORT) in non-deprived rats. Urocortin (0, 0.3, 1, 3 microg) delivered, respectively, to the fourth and lateral ventricles yielded substantial suppression of food intake measured 2, 4 and 24 h later. A significant but more modest anorexia was observed between 24 and 48 h after injection. Intake responses did not differ between the injection sites, but body weight loss measured 24 h after lateral-i.c.v. injection was substantially greater than that after fourth-i.c.v. injection. Fourth-i.c.v. urocortin administration (3 microg) produced substantial elevations in plasma glucose and CORT that were not distinguishable in magnitude and duration from responses to lateral-i.c.v. delivery. Unilateral microinjection of urocortin into the dorsal vagal complex significantly reduced 24-h food intake at a dose (0.1 microg) that was subthreshold for the response to ventricular administration, suggesting that fourth-i.c.v. effects are mediated in part by stimulation of CRH receptors in this region of the caudal brainstem. The results indicate that similar effects can be obtained from stimulation of anatomically disparate populations of CRH receptors, and that interactions between forebrain and hindbrain structures should be considered in the evaluation of CRH contributions to food intake and body weight control.
Since few previous studies have examined the effects of urocortin on physiological fed and fasted gastrointestinal motility, we administered urocortin intracerebroventricularly (icv) or intravenously (iv) in freely moving conscious rats and examined the changes in antral and duodenal motility. Icv and iv injection of urocortin disrupted fasted motor patterns of gastroduodenal motility, which were replaced by fed-like motor patterns. When urocortin was given icv and iv in the fed state, the motor activity remained like the fed patterns but % motor index (%MI) was decreased in the antrum and increased in the duodenum. Increase in the %MI in the duodenum induced by urocortin was shown as a nonpropagated event, since the transit of nonnutrient contents in the duodenum was decreased by icv and iv injection of urocortin. Changes in the gastroduodenal motility induced by icv injection of urocortin were abolished in animals with truncal vagotomy but not altered in animals with mechanical sympathectomy, suggesting that the vagal pathway may mediate the central action of urocortin. Neither urocortin antiserum nor alpha-helical CRF-(9-41) affected fed and fasted gastroduodenal motility, suggesting that endogenous urocortin is not involved in regulation of basal gastroduodenal motility.
Adaptive stress responses mediated by the endocrine, autonomic, cardiovascular and immune systems are essential for the survival of the individual. Initial stress-induced responses provide a vital short-term metabolic lift, but prolonged or inappropriate exposure to stress can compromise homeostasis thereby leading to disease. This 'fight-or-flight' response is characterized by the activation of the corticotropin-releasing hormone (CRH)-adrenocorticotropin-glucocorticoid axis, mediated by the type 1 CRH receptor. In contrast, the type 2 CRH receptor mediates the stress-coping responses during the recovery phase of stress. We identified human stresscopin (SCP) and stresscopin-related peptide (SRP) as specific ligands for the type 2 CRH receptor. The genes encoding these peptides were expressed in diverse peripheral tissues as well as in the central nervous system. Treatment with SCP or SRP suppressed food intake, delayed gastric emptying and decreased heat-induced edema. Thus SCP and SRP might represent endogenous ligands for maintaining homeostasis after stress, and could allow the design of drugs to ameliorate stress-related diseases.
Abnormal signaling at corticotropin-releasing factor CRF1 and CRF2 receptors might contribute to the pathophysiology of stress-related disorders such as anxiety, depression and eating disorders, in addition to cardiac and inflammatory disorders. Recently, molecular characterization of CRF1 and CRF2 receptors and the cloning of novel ligands--urocortin, stresscopin-related peptide/urocortin II, and stresscopin/urocortin III--have revealed a far-reaching physiological importance for the family of CRF peptides. Although the physiological roles of the CRF2 receptor remain to be defined, the preclinical and clinical development of specific small-molecule antagonists of the CRF1 receptor opens new avenues for the treatment of psychiatric and neurological disorders.
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