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Rainbow trout are considered glucose intolerant because they are poor utilizers of glucose, despite having functional insulin receptors and glucose transporters. Following high carbohydrate meals, rainbow trout are persistently hyperglycemic, which is likely due to low glucose utilization in peripheral tissues including the muscle. Also, rainbow trout myogenic precursor cells (MPCs) treated in vitro with insulin and IGF1 increase glucose uptake and protein synthesis, whereas protein degradation is decreased. Given our understanding of glucose regulation in trout, we sought to understand how glucose concentrations affect protein synthesis, protein degradation; and expression of genes associated with muscle growth and proteolysis in MPCs. We found that following 24 h and 48 h of treatment with low glucose media (5.6 mM), myoblasts had significant decreases in protein synthesis. Also, low glucose treatments affected the expression of both mstn2a and igfbp5. These findings support that glucose is a direct regulator of protein synthesis and growth-related mechanisms in rainbow trout muscle.

Teneurin C-terminal associated peptides (TCAP), bioactive peptides located on the C-terminal end of teneurin proteins, have been shown to regulate stress axis functions due to the high conservation between TCAP and corticotropin releasing factor (CRF). Additionally, recent work demonstrated that TCAP can increase metabolism in rats via glucose metabolism. These metabolic actions are not well described in other organisms, including teleosts. Here we investigated the expression of a tcap isoform, tcap-3, and the potential role of TCAP-3 as a regulator of metabolism across zebrafish life-stages. Using pcr-based analyses, tcap-3 appears to be independently transcribed, in relation to teneurin-3, in muscle tissue of adult zebrafish. Resazurin, respirometry chambers, and mitochondrial metabolism analyses were used to study the metabolic effects of synthetic rainbow trout TCAP-3 (rtTCAP-3) in larval and adult zebrafish. Overall, metabolic activity was enhanced by 48 h of rtTCAP-3 treatment in larvae (bath immersion) and adults (intraperitoneal injections). This metabolic activity increase was due to mitochondrial uncoupling, as mitochondrial respiration increase by rtTCAP-3 was due to proton leak. Additionally, rtTCAP-3 protected larval fish from reduced metabolic activity induced by low temperatures. Subsequently, rtTCAP-3 increased metabolic output in adult zebrafish subjected to accelerated swimming speeds, demonstrating the potent role of rtTCAP-3 in zebrafish metabolism regulation during metabolic challenges. Collectively, these results demonstrate the conserved roles for rtTCAP-3 as a metabolic activator in zebrafish.

Stress is a major factor that contributes to poor production and animal welfare concerns in aquaculture. As such, a thorough understanding of mechanisms involved in the stress response is imperative to developing strategies to mitigate the negative side effects of stressors, including the impact of high stocking densities on growth. The purpose of this study was to determine how the muscle growth inhibitor, myostatin , and the stress-responsive gene HSP90 are regulated in response to crowding stress in rainbow trout ( Oncorhynchus mykiss ), cutthroat trout ( Oncorhynchus clarki ), brook trout ( Salvelinus fontinalis ), and Atlantic salmon ( Salmo salar ). All species exhibited higher cortisol and glucose levels following the handling stress, indicating physiological response to the treatment. Additionally, all species, except rainbow trout, exhibited higher HSP90 levels in muscle after a 48 h crowding stress. Crowding stress resulted in a decrease of myostatin-1a in brook trout white muscle but not red muscle, while, myostatin-1a and -2a levels increased in white muscle and myostatin-1b levels increased in red muscle in Atlantic salmon. In rainbow trout, no significant changes were detected in either muscle type, but myostatin-1a was upregulated in both white and red skeletal muscle in the closely related cutthroat trout. The variation in response to crowding suggests a complex and species-specific interaction between stress and the muscle gene regulation in these salmonids. Only Atlantic salmon and cutthroat trout exhibited increased muscle myostatin transcription, and also exhibited the largest increase in circulating glucose in response to crowding. These results suggest that species-specific farming practices should be carefully examined in order to optimize low stress culture conditions.

Teneurins have well established roles in function and maintenance of the central nervous systems of vertebrates. In addition, teneurin c-terminal associated peptide (TCAP), a bioactive peptide found on the c-terminal portion of teneurins, has been shown to regulate glucose metabolism. Although, the majority of research conducted on the actions of teneurins and TCAPs has strictly focused on neurological systems in rodents, TCAP was first identified in rainbow trout after screening trout hypothalamic cDNA. This suggests a conserved functional role of TCAP across vertebrates, however, the current depth of literature on teneurins and TCAPs in fish is limited. In addition, the overall function of TCAP in regulating metabolism is unclear. This review will highlight work that has been conducted specifically in fish species in relation to the teneurin system and metabolism in order to identify areas of research that are needed for future work.

Glucocorticoids (GCs) strongly regulate myostatin transcript levels in mammals via glucocorticoid response elements (GREs) in the myostatin promoter, and bioinformatics methods suggest that this regulatory mechanism is conserved among many vertebrates. However, the multiple myostatin genes found in some fishes may be an exception. In rainbow trout (Oncorhynchus mykiss), two genome duplication events have produced three putatively functional myostatin genes, myostatin-1a, -1b and -2a, which are ubiquitously and differentially expressed. In addition, in silico promoter analyses of the rainbow trout myostatin promoters have failed to identify putative GREs, suggesting a divergence in myostatin function. Therefore, we hypothesized that myostatin mRNA expression is not regulated by glucocorticoids in rainbow trout. In this study, both juvenile rainbow trout and primary trout myoblasts were treated with cortisol to examine the relationship between this glucocorticoid and myostatin mRNA expression. Results suggest that exogenous cortisol does not regulate myostatin-1a and -1b expression in vivo, as myostatin mRNA levels were not significantly affected by cortisol treatment in either red or white muscle tissue. In red muscle, myostatin-2a levels were significantly elevated in the cortisol treatment group relative to the control, but not the vehicle control, at both 12 h and 24 h post-injection. As such, it is unclear if cortisol was acting alone or in combination with the vehicle. Cortisol increased myostatin-1b expression in a dose-dependent manner in vitro. Further work is needed to determine if this response is the direct result of cortisol acting on the myostatin-1b promoter or through an alternative mechanism. These results suggest that regulation of myostatin by cortisol may not be as highly conserved as previously thought and support previous work that describes potential functional divergence of the multiple myostatin genes in fishes.

Teleosts and other aquatic ectotherms have the ability to withstand prolonged periods of low water temperatures (cold-acclimation) and fasting, and can often respond with phases of accelerated (compensatory) growth when favorable conditions are restored. We assessed whether complete feed restriction prior to (24 °C, days 0–23) and/or during (14 °C, days 24–114) a simulated period of cold-acclimation could elicit episodes of compensatory growth (CG) and catch-up growth upon warm-up to 24 °C and satiation feeding (days 115–148). Control hybrid striped bass (HSB: Morone chrysops × Morone saxatilis ) were fed to satiation throughout the entire experiment under these temperature fluctuations. Compensatory growth and ultimately catch-up growth were achieved in groups of HSB that were deprived of feed during either the initial period at 24 °C (days 0–23), during the cold-acclimation period (14 °C, days 24–114), or during both of these periods (days 0–114). Further, it appears that HSB are better able to compensate for weight loss when skeletal length is not significantly compromised during the treatment period, which occurred in HSB feed restricted during cold-acclimation only. The most dramatic CG responses were defined by specific growth rates (SGRs) up to 4.2 times that of controls and were accompanied by hyperphagia and improvements in temporal and overall feed conversion. Levels of plasma insulin-like growth factor (IGF)-1 and muscle IGF-1 mRNA were significantly correlated to growth rate for all groups throughout the experiment (R 2 = 0.40, 0.23, respectively), with an overcompensation of both observed in HSB with the most elevated SGRs during the CG response. Interestingly, opposing trends were observed between muscle mRNA expression of growth hormone receptor (GHR)-1 and -2, with fasting at 24 °C and 14 °C resulting in depressed levels of GHR-1 and elevated levels of GHR-2 relative to controls. Levels of muscle myostatin (MSTN)-1 mRNA were significantly depressed in HSB fasted at 24 °C and/or 14 °C while MSTN-2 mRNA was lower following initial feed restriction at 24 °C. Likewise, levels of unprocessed pro-MSTN (precursor) and mature MSTN protein were both depressed in fasted fish at 24 °C. This study demonstrates that a previous period of feed restriction and cold-acclimation followed by realimentation at more favorable water temperatures produces a strong CG response and catch-up growth in fish. These studies also suggest that an overcompensation of circulating and local IGF-1 along with changes in MSTN mRNA and protein expression may contribute to accelerated growth rates characteristic of CG. Furthermore, our studies indicate that overall feed conversion can improve by as much as 30% with CG induced through temperature and feeding manipulations with no adverse effects on growth of HSB. This raises the possibility that CG protocols can improve production efficiency of HSB and other temperate teleosts in pond or tank culture.

Zebrafish (Danio rerio) have become an important model organism for developmental biology and human health studies. We recently demonstrated differential growth patterns between the zebrafish and a close relative the giant danio (Danio aequipinnatus), where the giant danio appears to exhibit indeterminate growth similar to most fish species important for commercial production, while zebrafish exhibit determinate growth more similar to mammalian growth. This study focused on evaluating muscle growth regulation differences in adult zebrafish and giant danio utilizing growth hormone treatment as a mode of growth manipulation. Growth hormone treatment resulted in increased overall growth in giant danio, but failed to increase growth in the zebrafish. Growth hormone treatment increased muscle IGF-I and GHrI gene expression in both species, but to a larger degree in the giant danio. In contrast, zebrafish exhibited a larger increase in IrA and IGF-IrB gene expression in muscle in response to GH treatment. In addition muscle myostatin levels were differentially regulated between the two species, with a down-regulation observed in rapidly growing, GH-treated giant danio and an up-regulation in zebrafish not actively growing in response to GH. This is the first report of differential expression of growth-regulating genes in closely related fish species exhibiting opposing growth paradigms. These results further support the role that the zebrafish and giant danio can play important model organisms for determinate and indeterminate growth.

Cortisol, the primary corticosteroid in teleost fishes, is released in response to stressors to elicit local functions, however little is understood regarding muscle-specific responses to cortisol in these fishes. In mammals, glucocorticoids strongly regulate the muscle growth inhibitor, myostatin, via glucocorticoid response elements (GREs) leading to muscle atrophy. Bioinformatics methods suggest that this regulatory mechanism is conserved among vertebrates, however recent evidence suggests some fishes exhibit divergent regulation. Therefore, the aim of this study was to evaluate the conserved actions of cortisol on myostatin and hsp90 expression to determine if variations in cortisol interactions have emerged in salmonid species. Representative salmonids; Chinook salmon (Oncorhynchus tshawytscha), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar); were injected intraperitoneally with a cortisol implant (50μg/g body weight) and muscle gene expression was quantified after 48h. Plasma glucose and cortisol levels were significantly elevated by cortisol in all species, demonstrating physiological effectiveness of the treatment. HSP90 mRNA levels were elevated by cortisol in brook trout, Chinook salmon, and Atlantic salmon, but were decreased in cutthroat trout. Myostatin mRNA levels were affected in a species, tissue (muscle type), and paralog specific manner. Cortisol treatment increased myostatin expression in brook trout (Salvelinus) and Atlantic salmon (Salmo), but not in Chinook salmon (Oncorhynchus) or cutthroat trout (Oncorhynchus). Interestingly, the VC alone increased myostatin mRNA expression in Chinook and Atlantic salmon, while the addition of cortisol blocked the response. Taken together, these results suggest that cortisol affects muscle-specific gene expression in species-specific manners, with unique Oncorhynchus-specific divergence observed, that are not predictive solely based upon mammalian stress responses.

In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-β superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-β superfamily array exists in primitive vertebrates than previously thought.

Recombinant bovine growth hormone (rbGH) increases growth rates in rainbow trout, Oncorhynchus mykiss, and this response is thought to be under the control of the GH–IGF axis, as it is in mammals. However, the mechanisms regulating fish muscle growth are poorly understood. Therefore, an experiment was conducted to examine the effects of rbGH on growthrelated hormones in rainbow trout. Rainbow trout (550F10 g) received an intraperitoneal injection of rbGH (120 A gg 1 BW) or vehicle on days 0 and 21. Blood samples were collected on days 0, 0.5, 1, 3, 7, and 28 and assayed for rainbow trout growth hormone (tGH), rbGH, and IGF-I. As expected, rbGH levels increased (Pb0.05) in circulation 12 h after treatment and continued to increase (Pb0.05) from day 0 to day 7 after treatment. Unexpectedly, levels of rbGH detected at day 28, 7 days after the second injection, were lower than those at day 7. Corresponding to this observation, anti-rbGH antibodies were detectable in serum from treated fish at day 28, but not at day 7. We suggest that the low levels of rbGH detected at day 28 were due to increased clearance of rbGH caused by the anti-rbGH antibodies. Treated fish also exhibited increased serum IGF-I levels (Pb0.01) following rbGH injection, while endogenous tGH did not change (P=0.28). These results suggest that the endogenous negative feedback control loop described in mammals is not activated by rbGH in rainbow trout, as tGH was unaffected by increased circulating rbGH and IGF-I. However, consistent with previous reports, rbGH does increase circulating IGF-I over time and rbGH is detectable throughout the 3-week injection period. To our knowledge, this is the first report demonstrating specific antibody production following an exogenous rbGH injection in fish. D 2005 Elsevier B.V. All rights reserved.

Background: As part of a coordinated effort to expand our research activity at the interface of Aging and Energetics a team of investigators at The University of Alabama at Birmingham systematically assayed and catalogued the top research priorities identified in leading publications in that domain, believing the result would be useful to the scientific community at large.Objective: To identify research priorities and opportunities in the domain of aging and energetics as advocated in the 40 most cited papers related to aging and energetics in the last 4 years.Design: The investigators conducted a search for papers on aging and energetics in Scopus, ranked the resulting papers by number of times they were cited, and selected the ten most-cited papers in each of the four years that include 2010 to 2013, inclusive.Results: Ten research categories were identified from the 40 papers. These included: (1) Calorie restriction (CR) longevity response, (2) role of mTOR (mechanistic target of Rapamycin) and related factors in lifespan extension, (3) nutrient effects beyond energy (especially resveratrol, omega-3 fatty acids, and selected amino acids), 4) autophagy and increased longevity and health, (5) aging-associated predictors of chronic disease, (6) use and effects of mesenchymal stem cells (MSCs), (7) telomeres relative to aging and energetics, (8) accretion and effects of body fat, (9) the aging heart, and (10) mitochondria, reactive oxygen species, and cellular energetics.Conclusion: The field is rich with exciting opportunities to build upon our existing knowledge about the relations among aspects of aging and aspects of energetics and to better understand the mechanisms which connect them.

Muscle growth is an energetically demanding process that is reliant on intramuscular fatty acid depots in most fishes. The complex mechanisms regulating this growth and lipid metabolism are of great interest for human health and aquaculture applications. It is well established that the skeletal muscle chalone, myostatin, plays a role in lipid metabolism and adipogenesis in mammals; however, this function has not been fully assessed in fishes. We therefore examined the interaction between dietary lipid levels and myostatin expression in rainbow trout (Oncorhynchus mykiss). Five weeks of high-fat diet (HFD; 25 % lipid) intake increased white muscle lipid content and decreased circulating glucose levels and hepatosomatic index when compared to low-fat diet (LFD; 10 % lipid) intake. In addition, HFD intake reduced myostatin-1a and myostatin-1b expression in white muscle and myostatin-1b expression in brain tissue. Characterization of the myostatin-1a, myostatin-1b, and myostatin-2a promoters revealed putative binding sites for a subset of transcription factors associated with lipid metabolism. Taken together, these data suggest that HFD may regulate myostatin expression through cis-regulatory elements sensitive to increased lipid intake. Further, these findings provide a framework for future investigations of mechanisms describing the relationships between myostatin and lipid metabolism in fish.

The zebrafish (Danio rerio) has been used extensively as a model system for developmental studies but, unlike most teleost fish, it grows in a determinate-like manner. A close relative, the giant danio (Devario cf. aequipinnatus), grows indeterminately, displaying both hyperplasia and hypertrophy of skeletal myofibers as an adult. To better understand adult muscle hyperplasia, a postlarval/postnatal process that closely resembles secondary myogenesis during development, we characterized the expression of Pax3/7, c-Met, syndecan-4, Myf5, MyoD1, myogenin, and myostatin during in vitro myogenesis, a technique that allows for the complete progression of myogenic precursor cells to myotubes. Pax7 appears to be expressed only in newly activated MPCs while Pax3 is expressed through most of the myogenic program, as are c-Met and syndecan-4. MyoD1 appears important in all stages of myogenesis, while Myf5 is likely expressed at low to background levels, and myogenin expression is enriched in myotubes. Myostatin, like MyoD1, appears to be ubiquitous at all stages. This is the first comprehensive report of key myogenic factor expression patterns in an indeterminate teleost, one that strongly suggests that Pax3 and/or Myf5 may be involved in the regulation of this paradigm. Further, it validates this species as a model organism for studying adult myogenesis in vitro, especially mechanisms underlying nascent myofiber recruitment.

In the last decade, myostatin (MSTN), a member of the TGFβ superfamily, has emerged as a strong inhibitor of muscle growth in mammals. In fish many studies reveal a strong conservation of mstn gene organization, sequence, and protein structures. Because of ancient genome duplication, teleostei may have retained two copies of mstn genes and even up to four copies in salmonids due to additional genome duplication event. In sharp contrast to mammals, the different fish mstn orthologs are widely expressed with a tissue-specific expression pattern. Quantification of mstn mRNA in fish under different physiological conditions, demonstrates that endogenous expression of mstn paralogs is rarely related to fish muscle growth rate. In addition, attempts to inhibit MSTN activity did not consistently enhance muscle growth as in mammals. In vitro, MSTN stimulates myotube atrophy and inhibits proliferation but not differentiation of myogenic cells as in mammals. In conclusion, given the strong mstn expression non-muscle tissues of fish, we propose a new hypothesis stating that fish MSTN functions as a general inhibitors of cell proliferation and cell growth to control tissue mass but is not specialized into a strong muscle regulator.

Although the zebrafish (Danio rerio) has been widely utilized as a model organism for several decades, there is little information available on physiological variation underlying genetic variation among the most commonly used inbred strains. This study evaluated growth performance using physiological and molecular markers of growth in response to fasting in six commonly used zebrafish strains [AB, TU, TL, SJA, WIK, and petstore (PET) zebrafish]. Fasting resulted in a standard decrease in whole blood glucose levels, a typical vertebrate glucose metabolism pattern, in AB, PET, TL, and TU zebrafish strains. Alternatively, fasting did not affect glucose levels in SJA and WIK zebrafish strains. Similarly, fasting had no effect on myostatin mRNA levels in AB, PET, TU, and WIK zebrafish strains, but decreased myostatin-1 and -2 mRNA levels in SJA zebrafish. Consistent with previous work, fasting increased myostatin-2 mRNA levels in TL zebrafish. These data demonstrate that variation is present in growth performance between commonly used inbred strains of zebrafish. These data can help future research endeavors by highlighting the attributes of each strain with regard to growth performance so that the most fitting strain may be utilized.

Gelatinases play a role in adipose and muscle hypertrophy and could be involved in tissue remodeling in response to high-fat diet (HFD) intake. This study tested potential roles of gelatinases (matrix metalloproteinses-2 and -9 [MMP-2 and -9]) in relationship to an antigrowth factor [myostatin (MSTN)] known to be dysregulated in relation to HFD-induced obesity (HFDIO) propensity. In vitro and ex vivo analyses demonstrated that MMP-9 increased mature MSTN levels, indicating a potential role of gelatinases in MSTN activation in vivo. HFD intake resulted in increased body weight and circulating blood glucose values in C57BL/6J and MMP-9 null mice, with no changes observed in SWR/J mice. HFD intake attenuated MMP-9 and MMP-2 mRNA levels in SWR/J mice while elevating MMP-2 levels in skeletal muscle in C57BL/6J mice. In MMP-9 null mice, the effects of HFD intake were muted. Consistent with changes in mRNA levels, HFD intake increased MMP-9 activity in muscle tissue of C57BL/6J mice, demonstrating a strong relationship between HFDIO susceptibility and local MMP regulation. Overall, resistance to HFDIO appears to correspond to low MMP-9 and MSTN levels, suggesting a role of MMP-9 in MSTN activation in local tissue responses to HFD intake.

A strong relationship exists between increased inflammatory cytokines and muscle insulin resistance in obesity. This study focused on identifying a relationship between metabolic propensity and myostatin expression in muscle and spleen cells in response to high-fat diet intake. Using a comparative approach, we analyzed the effects of high-fat diet intake on myostatin and follistatin expression, spleen cell composition, and potential cytokine expression in high-fat diet induced obesity (HFDIO) resistant (SWR/J) and susceptible (C57BL/6) mice models. Results demonstrated overall increased myostatin expression in muscle following high-fat diet intake in HFDIO-susceptible mice, while myostatin expression levels decreased initially in muscle from high-fat diet fed resistant mice. In HFDIO-resistant mice, myostatin expression decreased in spleen, while myostatin increased in spleen tissue from HFDIO-susceptible mice. Proinflammatory cytokine (IL-17, IL-1β, and IFNγ) potential increased in splenocytes from HFDIO-susceptible mice. In comparison, C57BL/6 mice fed a high-fat diet exhibited higher frequencies of CD4(+)/CD44(hi) and CD8(+)/CD44(hi) cells in the spleen compared to control fed mice. Together, these results suggest that susceptibility to high-fat diet induced obesity could be influenced by local myostatin activity in a tissue-specific manner and that splenocytes exhibit differential cytokine production in a strain-dependent manner. This study sets the stage for future investigations into the interactions between growth, inflammation, and metabolism.

Myostatin (MSTN) negatively regulates muscle growth in vertebrates. Salmonids produce two myostatin transcripts from separate genes. Surprisingly, quantitative analyses indicate different regulatory mechanisms for the two myostatin genes in rainbow trout. MSTN1 mRNA levels were elevated 26% following recombinant bovine growth hormone (rbGH) treatment, while MSTN2 mRNA levels were reduced 74% compared to controls. MSTN precursor protein (42 kDa) levels were elevated in rbGH treated fish compared to controls. In addition, circulating cortisol levels were elevated 71% following rbGH treatment compared to controls. In treated and control fish, cortisol levels were elevated 245% at day 0 compared to subsequent days. Treated fish exhibited cortisol levels 207% higher than controls at 0.5 day, and remained at least 50% higher for 7 days following treatment. This pattern of change was positively correlated to MSTN1 mRNA levels. This is the first time a direct relationship has been reported between GH, cortisol, and myostatin. In addition, following rbGH administration, myosin protein concentrations in skeletal muscle samples increased, suggesting that GH regulates expression of the most abundant muscle protein. These results indicate the two myostatin genes are differentially regulated and may possess different functions in rainbow trout muscle, and suggests a possible interaction between GH, cortisol, and muscle growth.