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In this study, we used juvenile rainbow trout to examine the direct effects of selected environmental estrogens (EE), specifically, 17 β-estradiol (E2), β-sitosterol (βS), and 4-n-nonylphenol (NP), on target tissue sensitivity to insulin-like growth factor (IGF) as assessed by expression of IGF receptor type 1 (IGFR1) mRNAs and IGF-1 binding capacity, as well as on the cell signaling pathways through which EE exert their effects. E2 and NP inhibited IGFR1A and IGFR1B mRNA expression in a time- and concentration-related manner in gill and muscle; however, βS had no effect on expression of IGFR1 mRNAs in either tissue. NP reduced

Fish in aquatic habitats are exposed to increasing concentrations and types of environmental contaminants, including environmental estrogens (EE). While there is growing evidence to support the observation that endocrine-disrupting compounds (EDCs) possess growth-inhibiting effects, the mechanisms by which these physiological effects occur are poorly understood. In this study, we examined the direct effects of EE, specifically 17β-estradiol (E2), β-sitosterol (βS), and 4-n-nonylphenol (NP), on GH sensitivity as assessed by mRNA expression and functional expression of growth hormone receptor in hepatocytes, gill filaments, and muscle in rainbow trout (Oncorhynchus mykiss). Additionally, we examined the effects of EE on signaling cascades related to growth hormone signal transduction (i.e., JAK-STAT, MAPK, and PI3K-Akt). Environmental estrogens directly suppressed the expression of GHRs in a tissue- and compound-related manner. The potency and efficacy varied with EE; effects were most pronounced with E2 in liver. EE treatment deactivated the JAK-STAT, MAPK, and PI3K-Akt pathways in liver a time-, EE- and concentration-dependent manner. Generally, E2 and NP were most effective in deactivating pathway elements; maximum suppression for each pathway was rapid, typically occurring at 10-30min. The observed effects occurred via an estrogen-dependent pathway, as indicated by treatment with an ER antagonist, ICI 182,780. These findings suggest that EEs suppress growth by reducing GH sensitivity in terms of reduced GHR synthesis and reduced surface GHR expression and by repressing GH signaling pathways.

Environmental estrogens (EE) have been found to disrupt a host of developmental, reproductive, metabolic, and osmoregulatory process in a wide-range of animals, particularly those in aquatic ecosystems where such compounds concentrate. Previously, we showed that EE inhibited post-embryonic organismal growth of rainbow trout in vivo, but the precise mechanism(s) through which EE exert their growth inhibiting effects remain unknown. In this study, we used rainbow trout (Oncorhynchus mykiss) as a model to investigate the direct effects of 17β-estradiol (E2), β-sitosterol (βS), and 4-n-nonylphenol (NP) on the synthesis of insulin-like growth factors (IGFs) and to elucidate the mechanism(s) by which EEs exert such effects. E2, βS, and NP significantly inhibited the expression of both IGF-1 and IGF-2 mRNAs in liver and gill in a time- and concentration-related manner. Although the response evoked by each EEs on the expression of IGF mRNAs was similar, the potency and efficacy varied with EE; the rank order potency/ efficacy was as follows: E2> NP> βS. The effects of EEs on the expression of IGF mRNAs was blocked by the estrogen receptor (ER) antagonist, ICI 182,780. The mechanism(s) through which EEs inhibit IGF mRNA expression were investigated in isolated liver cells in vitro. EE treatment deactivated JAK, STAT, ERK, and AKT. Moreover, blockade of growth hormone (GH)-stimulated IGF expression by EE was accompanied by deactivation of JAK, STAT, ERK, and AKT. EEs also increased the expression of suppressor of cytokine signaling 2 (SOCS-2), a known inhibitor of JAK-2--an action that also was blocked by ICI 182,780. These results indicate that EEs directly inhibit the expression of IGF mRNAs by disrupting GH post-receptor signaling pathways (e.g., JAK, STAT, ERK, and AKT) in an ER-dependent manner.

Introduction Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy. Methods Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS). Results Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14. Conclusions Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy. Muscle Nerve 48: 393–402, 2013

Previous studies show that successful adaptation of euryhaline teleost fish to seawater (SW) involves the GH-IGF system. The increasing occurrence, distribution, and concentration of environmental contaminants, including environmental estrogens (EE), in aquatic habit over recent time may compromise the hypoosmoreegulatory ability of fish. In this study, we used rainbow trout (Oncorhynchus mykiss) to assess the effects of EE on the GH-IGF system and adaptation to increased salinity. Juvenile trout (ca. 30 g) were exposed to either low (10 μg/l) or high (100 μg/l) concentrations of β-sitosterol, 4-n-nonylphenol (NP), or 17β-estradiol (E2) for 28 days in fresh water (FW); after which, fish were exposed to 20‰ SW. Plasma chloride levels in control fish rose initially, and then declined to initial levels after 48 h. By contrast, plasma chloride levels in all EE-treated groups except β-sitosterol low increased and remained elevated over initial levels after 48 h. Levels GH receptor 1 (GHR 1), GHR 2, insulin-like growth factor-1 (IGF-1), and IGF-2mRNAs in liver of control fish increased 6–12 h after SW exposure. In gill, levels of GHR 1, GHR 2, IGF-1, IGF-2, IGF receptor 1A (IGFR1A), and IGFR1B mRNAs increased in control fish 6–12 h after 20‰ SW exposure. Levels of IGFR1A and IGFR1B mRNAs in white muscle and of IGFR1A mRNA in red muscle increased in control fish 6–12 h after 20‰ SW exposure. Expression of all mRNAs in liver, gill, and red and white muscle declined from peak levels in control fish by 48 h after transfer. Exposure of fish to β-sitosterol, NP, and E2 abolished or attenuated normal salinity-induced changes in the expression of GHR, IGF, and IGFR1 mRNAs in all tissues. These results indicate that EE reduces salinity adaptation by inhibiting components of the GH-IGF system.

Introduction:In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles. Methods: An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short- and long-latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice. Results: PF did not change with one- and three-times-per-week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT. Conclusions: The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models. Muscle Nerve, 2012

Although environmental estrogens (EE) have been found to disrupt a wide variety of developmental and reproductive processes in vertebrates, there is a paucity of information concerning their effects on organismal growth, particularly postembryonic growth. In this study, we exposed juvenile rainbow trout (Oncorhynchus mykiss) to 17β-estradiol (E2) β-sitosterol (βS), or 4-n-nonylphenol (NP) to assess the effects of EE on overall organismal growth and on the growth hormone-insulin-like-growth factor (GH-IGF) system. EE treatment significantly reduced food conversion, body condition, and body growth. EE-inhibited growth resulted from alterations in peripheral elements of the GH-IGF system, which includes multiple GH receptors (GHRs), IGFs, and IGF receptors (IGFRs). In general, E2, βS, and NP reduced the expression of GHRs, IGFs, and IGFRs; however, the effects varied in an EE-, tissue-, element type-specific manner. For example, in liver, E2 was more efficacious than either βS, and NP in reducing GHR expression, and the effect of E2 was greater on GHR 1 than GHR2 mRNA. By contrast, in gill, all EEs affected GHR expression in a similar manner and there was no difference in the effect on GHR1 and GHR 2 mRNA. With regard to IGF expression, all EEs reduced hepatic IGF1 and IGF2 mRNA levels, whereas as in gill, only E2 and NP significantly reduced IGF1 and IGF2 expression. Lastly, E2 and NP reduced the expression of IGFR1A and IGFR1B mRNA expression similarly in gill and red and white muscle, whereas βS had no effect on expression of IGFR mRNAs. These findings indicate that EEs disrupt post-embryonic growth by reducing GH sensitivity, IGF production, and IGF sensitivity.

Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy.Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS).Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14.Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy.

In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles.An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short- and long-latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice.PF did not change with one- and three-times-per-week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT.The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models.

Members of the bone morphogenetic protein-1/mammalian tolloid (BMP-1/mTLD) family of proteases cleave diverse extracellular proteins, including the growth inhibitor myostatin. The purpose of this work was to examine the expression of BMP-1/mTLD, tolloid-like-1 and -2 (TLL1 and TLL2) in hindlimb muscles of the mouse in vivo and in C(2)C(12) muscle cells in vitro. Quantitative real-time polymerase chain reaction revealed that neither BMP-1/mTLD nor TLL1 mRNA levels differed between the predominantly fast-twitch tibialis anterior (TA) and gastrocnemius (GAST) muscles and the more slow-twitch soleus (SOL) muscle; TLL2 mRNA levels were not detectable in any of the muscles examined. Interestingly, however, immunohistochemical analysis revealed that BMP-1 protein was expressed in type I and IIa but not in IIb fibers. TLL1 mRNA levels significantly increased in the TA but not the SOL with 3 days of hindlimb suspension and significantly decreased in both TA and SOL in response to 2 days of food deprivation. In contrast, BMP-1/mTLD mRNA levels were unaffected in either muscle by either condition. In addition, BMP-1/mTLD and TLL1 mRNA levels significantly decreased during C(2)C(12) myoblast differentiation in vitro, and activity of a 1,200-bp mouse TLL1 promoter construct was significantly decreased in C(2)C(12) myotubes by differentiation, by mutation of an nuclear factor kappa-beta (NF-kappaB) site, or deletion of a sma/mothers against decapentaplegic (SMAD) site. Together, these data demonstrate that TLL1 mRNA levels are altered by loading, energy status, and differentiation, and thus its expression may be regulated so as to modulate activity of myostatin or other extracellular substrates during these adaptive states.

In this study, we examined the effects of 2-week hindlimb un-loading in mice followed by re-ambulation with voluntary access to running wheels. The recovery period was terminated at a time point when physical performance--defined by velocity, time, and distance ran per day--of the suspended group matched that of an unsuspended group. Mice were assigned to one of four groups: unsuspended non-exercise (Control), 14 days of hindlimb suspension (HS), 7 days of access to running wheels (E7), or 14 days of HS plus 7 days access to running wheels (HSE7). HS resulted in significant decreases in body and muscle mass, hindlimb strength, soleus force, soleus specific force, fatigue resistance, and fiber cross sectional area (CSA). Seven days of re-ambulation with access to running wheels following HS recovered masses to Control values, increased fiber CSA, increased resistance to fatigue and improved recovery from fatigue in the soleus. HS resulted in a myosin heavy chain (MHC) phenotype shift from slow toward fast-twitch fibers, though running alone did not influence the expression of MHC fibers. Compared to the Control group, HSE7 mice did not recover functional hindlimb strength as assessed through measurements either in vivo or ex vivo. Results from this study demonstrate that 7 days of muscle re-loading with access to wheel-running following HS can stimulate muscle to regain mass and fiber CSA and exhibit improved metrics of fatigue resistance and recovery, yet muscles remain impaired in regard to strength. Understanding this mismatch between muscle morphology and strength may prove of value in designing effective exercise protocols for disuse muscle atrophy rehabilitation.

Both spaceflight and bed rest studies are known to cause substantial muscle loss in humans. Of particular interest in atrophy studies is the postural, slow-twitch fiber soleus muscle. It has been demonstrated that during periods of disuse, slow-twitch fibers undergo fiber type switching and morph into fast-twitch fibers [1]. Alternatively, when exercise is prescribed, there is switching of fast-to slow-twitch fibers [2], indicating a profound adaptability of the muscle to exercise.Copyright © 2007 by ASME