The study was supported by the Marine Alliance for Science and Technology for Scotland (Scottish Funding Council grant HR09011) and by the Canadian Biotechnology Strategy (to R.H.D.). Deposited in PMC for immediate releaseCoho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.Publisher PDFPeer reviewe

Devlin, R.H.

Garcia de la Serrana Castillo, Daniel

Johnston, I.A.

Journal of Experimental Biology

English

PubMed

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity

University of St Andrews Research Portal

Muscle fibre size optimisation provides flexibility for energy budgeting in calorie-restricted coho salmon transgenic for growth hormone

Abstract
               Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1-year) (TF) or the same smaller ration of wild-type fish (2-years) (TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating 2-fold higher rate of fibre recruitment in the GH-transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 21% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were down-regulated in TR relative to TF and WT fish. We suggest muscle fibre size optimisation allows the relocation of energy from maintenance to locomotion explaining the observation that calorie-restricted transgenics grow at the same rate as WT whilst exhibiting markedly higher foraging activity.</jats:p

Ian A. Johnston

Daniel Garcia de la serrana

Robert H. Devlin

Crossref

The Journal of Experimental Biology3392© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 3392-3395 doi:10.1242/jeb.107664ABSTRACTCoho salmon (Oncorhynchus kisutch) transgenic for growth hormone(GH) show substantially faster growth than wild-type (WT) fish. Wefed GH-transgenic salmon either to satiation (1 year; TF) or the samesmaller ration of wild-type fish (2 years; TR), resulting in groupsmatched for body size to WT salmon. The myotomes of TF and WTfish had the same number and size distribution of muscle fibres,indicating a twofold higher rate of fibre recruitment in the GHtransgenics. Unexpectedly, calorie restriction was found to decreasethe rate of fibre production in transgenics, resulting in a 20% increasein average fibre size and reduced costs of ionic homeostasis. Genesfor myotube formation were downregulated in TR relative to TF andWT fish. We suggest that muscle fibre size optimisation allows thereallocation of energy from maintenance to locomotion, explaining theobservation that calorie-restricted transgenics grow at the same rateas WT fish whilst exhibiting markedly higher foraging activity.KEY WORDS: Growth, Myotube formation, Transgenesis, Optimalfibre size hypothesisINTRODUCTIONGrowth hormone (GH) has pervasive effects on growth rate,behaviour, feeding, metabolism, osmoregulation, smoltification andimmunity in fish (Björnsson, 1997). Growth rate is substantiallyhigher in GH-transgenic salmonid fish than in wild-type (WT) fish,although the effects vary with promoter type and family origin(Leggatt et al., 2012). Fast growth in transgenics is associated withmarkedly higher appetite and feeding motivation than WT fish(Sundstrom et al., 2003).GH binds to receptors in the liver and induces insulin-like growthfactor-1 (IGF1) synthesis, which is secreted into the generalcirculation, promoting growth in peripheral tissues such as skeletalmuscle (Johnston et al., 2011). Binding of IGF1 to its receptoractivates several downstream signaling cascades including the PI3K-Akt-TOR pathway to stimulate protein synthesis and inhibit proteindegradation by the ubiquitin proteasome pathway (Johnston et al.,2011).Primary effects of GH transgenesis can be distinguished fromsecondary effects linked to appetite by comparing transgenics fedSHORT COMMUNICATION1Scottish Oceans Institute, School of  Biology, University of  St Andrews, StAndrews, KY16 8LB, UK. 2Fisheries and Oceans Canada, 4160 Marine Drive,West Vancouver, BC V7V 1N6, Canada.*Author for correspondence (iaj@standrews.ac.uk)This is an Open Access article distributed under the terms of  the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricteduse, distribution and reproduction in any medium provided that the original work is properlyattributed. Received 12 May 2014; Accepted 23 July 2014either to satiation (TF) or with the reduced ration (TR) of WT fish,producing groups of the same body size. GH-transgenic cohosalmon [Oncorhyncus kisutch (Walbaum 1792)] fed to satiation hadhigher plasma GH and increased levels of tissue GH relative to WTfish (Raven et al., 2008). Plasma GH levels were higher still in TRfish, whereas plasma and tissue IGF1 concentrations were similar inTR and WT groups, reflecting similar feeding and growth rates(Raven et al., 2008). TR fish also maintain a much higher feedingmotivation and are more active than WT fish (Sundstrom et al.,2003).Teleosts recruit muscle fibres until they reach approximately40–50% of the maximum adult size (Johnston et al., 2011). Onereport suggested that muscle in GH-transgenic coho salmon grewmore by hyperplasia than hypertrophy than in WT fish (Hill et al.,2000), although in this study fibre number was not estimated. Inorder to test the hypothesis that GH transgenesis enhanceshyperplastic muscle growth, we compared fibre number anddiameter in WT, TF and TR groups of the same body length andmeasured expression of gh, igf1 and genes required for myotubeformation.RESULTS AND DISCUSSIONTF coho salmon had a higher appetite and grew faster than WT fish,recruiting fast muscle fibres at twice the rate, but showed a similarcontribution of hyperplasia and hypertrophy to reach a given bodylength, resulting in a similar average muscle fibre size (Fig. 1A,B;supplementary material Table S1); i.e. the hypothesis of an increasedimportance of hyperplasia in these transgenics was not supported(Hill et al., 2000). Unexpectedly, TR fish recruited 49% fewer fibresthan WT fish on the same feeding regime and 59% fewer fibres thanTF fish (P<0.05; Fig. 1B; supplementary material Table S1).Because there were only relatively small and not statisticallysignificant differences in total muscle cross-sectional area betweengroups (supplementary material Table S1), this result reflects agreater contribution of fibre hypertrophy to growth in TR fish. Theaverage fibre diameter was 20% greater in TR fish (49 μm) than inthe TF and WT groups (41 μm; supplementary material Table S1).We fitted probability density functions (PDFs) to the distributionsof fibre diameter and compared specific percentiles using multi-Wilcoxon tests. The average PDF of the TR group was significantlydifferent from that of the TF and WT groups (Kolmogorov–Smirnoff; P<0.05). TR fish had larger diameter fibres across thewhole range of fibre sizes, i.e. increased hypertrophy was evidentfor cohorts of fibres with different birthdays (Fig. 1C,D). Growthhormone (gh) mRNA levels (Fig. 2A) were similar in TF and TRfish and much higher than in WT fish, as expected (P<0.01). Incontrast, igf1 expression in muscle was significantly higher in TFthan in WT fish (P<0.05), reflecting higher food intake and growth(Sundstrom et al., 2003), whereas WT and TR fish were notMuscle fibre size optimisation provides flexibility for energybudgeting in calorie-restricted coho salmon transgenic for growth hormoneIan A. Johnston1,*, Daniel Garcia de la serrana1 and Robert H. Devlin2The Journal of Experimental Biology3393SHORT COMMUNICATION The Journal of Experimental Biology (2014) doi:10.1242/jeb.107664significantly different from each other. The expression of genesrequired for myoblast fusion during hyperplastic growth, includingdock1, dock5, crkl, itgb1, cadh15 and tmem8c, were twofold tofourfold downregulated in TR relative to the other groups (P<0.05;gene names are given in full in supplementary material Table S2).Muscle fibre size is under strong evolutionary selection in fishand can be adjusted by altering the lifetime production of musclefibres (Johnston et al., 2003). ATP-dependent ion pumpingcounteracts passive ion leak across the sarcolemmal membrane sothat the energy cost of maintaining a negative resting membranepotential is proportional to fibre surface/volume ratio. Thus largefibres are cheaper to maintain than smaller ones (Johnston et al.,2012; Jimenez et al., 2013). The optimal muscle fibre sizehypothesis envisages a trade-off between minimising surface/volume ratio on the one hand and avoiding diffusional constraintsfor diffusion of gas and metabolites on the other (Johnston et al.,2012, Johnston et al., 2003). Examples of fibre size optimisationover evolutionary time scales include the radiation of Antarcticnotothenioid fishes following climatic cooling, resulting in arelaxation of diffusional constraints and ‘giant muscle fibres’through a dramatic reduction in body-size-corrected fibre number(Johnston et al., 2003). Similarly, dwarfism in land-locked salmonidand stickleback populations was associated with a large reduction infibre number relative to the large-bodied ancestral anadromous state,enabling a similar scaling of fibre diameter to body size (Johnstonet al., 2012). The results of the present study indicate that, at leastunder certain circumstances, fibre size optimization may alsooperate within the lifetime of an individual.The GH-axis is tightly coupled to the energy status of the fish andlinked to feed intake via the expression of GH, IGF1 and theirreceptors (Raven et al., 2008). TR fish maintained a high GH outputwhilst experiencing a limitation in the supply of the amino acids andother nutrients required for growth. Our working hypothesis is thatthe uncoupling of the GH-axis from energy status directly affectedsome as-yet-unknown component of the signalling pathwaysregulating myotube formation and hypertrophic growth, providingthe stimulus for muscle fibre size optimization. GH transgenicsdriven by the metallothionein-B promoter showed increased GHexpression in all non-pituitary cell types including muscle and werenot sensitive to the metabolic signals normally influencingendogenous GH regulation, e.g. GH receptor expression was notincreased with calorie restriction in TR fish as it was in WT fish(Raven et al., 2008). It is therefore possible that the effects of calorierestriction on fibre production differ between TR and WT fish, butthis remains to be investigated. We found that the median fibrediameter was 20% higher in the TR than the WT fish (Fig. 1B),which is expected to produce proportional energy savings in costsof ionic homeostasis. The energy saved could theoretically bereallocated to other aspects of the energy budget such as routineswimming activity, and may provide an explanation as to why TRfish were observed to exhibit much higher foraging activity whilstgrowing at a rate similar to that of WT fish fed the same diet.MATERIALS AND METHODSFishCoho salmon (Oncorhynchus kisutch) were reared in a containment facilityat Fisheries and Oceans Canada, West Vancouver. WT fish were from the2010 brood of Chehalis River strain (BC, Canada). The strain M77transgenic coho salmon were derived from the Chehalis River strainproduced using the OnMTGH1 construct as previously described (LeggattFork length (cm)16.0Fibre number6.01048.01041.01051.21051.41051.61051.8105Percentile0Fibre diameter (µm)020406080100120140 2 cm   WTTFTR19.018.518.017.517.016.510080604020Fibre diameter (µm)Probability density function0.030.020.0100 50 100 150A BC DWTTFTRFig. 1. Analysis of muscle growth patterns incoho salmon (Oncorhynchus kisutch).(A) Representative images of fish in the differenttreatment groups. WT, wild type; TF, growthhormone (GH) transgenics fed to satiation; TR,calorie-restricted GH transgenics growing at theWT rate. (B) Relationship between fibre numberand fish length. Filled triangles, WT; filledcircles, TF; open circles, TR. Lines were fittedby least squares regression. (C) Probabilitydensity functions (PDFs) of muscle fibrediameter. The dashed lines represent theaverage PDFs of groups and the solid line thePDF of the combined groups. The shaded arearepresents the 1000 bootstraps of the combinedgroup. (D) Percentiles of fibre diameter for WT,TF and TR groups. Values represent means ±s.e.m.The Journal of Experimental Biology3394SHORT COMMUNICATION The Journal of Experimental Biology (2014) doi:10.1242/jeb.107664et al., 2012). Fish were reared in freshwater at 10±1°C with a naturalphotoperiod, and fed a commercial diet (Skretting, Vancouver, Canada). The2011 brood of GH transgenics were fed to satiation thrice daily (TF group).The 2010 GH transgenics (TR group) were fed the same ration as the WTgroup, to produce similar body masses (supplementary material Table S1).Experiments were conducted meeting Canadian Council for Animal Careguidelines, and were approved by the Department of Fisheries and OceansPacific Region Animal Care Committee under Animal Use Protocol no. 10-016. TF, TR and WT groups showed no significant differences in fork length(Lf); however, body mass (M) was significantly higher in the TF group thanin the WT group (P<0.05; supplementary material Table S1) and conditionfactor (M/Lf3×100) was higher in the TF group than either the TR or WTgroups, reflecting differences in body shape (Fig. 1A). The total cross-sectional area of fast muscle at the position of the anal vent was notstatistically different between groups (supplementary material Table S1).Muscle morphometryA steak ~5 mm thick was made at the level of the anal vent. Three blockswere prepared to sample one entire half of the myotomal cross-section, andthese were frozen in isopentane (2-methyl butane) cooled to its freezingpoint in liquid nitrogen. Sections were stained with myosin ATPase todistinguish between fibre types and hematoxylin and eosin for morphometricanalysis (Johnston et al., 2012). Photographs of tissue sections were takenat a magnification of ×200, and four fields, selected at random, werephotographed per block per fish. The cross-sectional areas of the entire fastmuscle portion of the myotome and approximately 300 fast muscle fibresper block were digitised. In total, 800 fast muscle fibres were randomlyselected per fish using a program written in R+ (http://www.r-project.org/)and smooth PDFs were fitted using a kernel function. The averagesmoothing parameter used (0.284) was similar between groups. Bootstraptechniques (n=1000) were used to distinguish underlying structure in thedistributions from random variation. A non-parametric Kolmogorov–Smirnoff two-sample test was used to test the null hypothesis that the PDFsof groups were equal over all diameters (see Johnston et al., 2012). Data onfish size and muscle cellularity parameters were tested for normality(Shapiro–Wilk test) and equal variance and analysed using a one-wayANOVA with pairwise multiple comparisons by the Holm–Sidak method(overall significance level equal to 0.05).Gene expression analysisPure fast muscle was dissected from dorsal epaxial myotomes. Total RNAextraction, quality analysis and concentration protocols were as describedpreviously (Macqueen et al., 2013). Tissues were sampled at a similar timeof day for six fish per group and the RNA was stored at −80°C. A detaileddescription of the cDNA synthesis, primer design, qPCR reaction setup anddata analysis is provided elsewhere (Macqueen et al., 2013) and insupplementary material Table S2. Briefly, a total of 1 μg of RNA from sixindividuals for each of the treatments (WT, TF, TR) was reverse transcribedto cDNA using the Quantitec reverse transcription kit (QIAGEN,Manchester, UK) including a gDNA removal step. Minus reversetranscriptase was performed using 1 μg of RNA from a pool created withRNA from all samples. Six microlitres per sample were mixed with 7.5 μlof SensiFAST SYBR Lo-ROX 2X master mix (Bioline, London, UK)containing 400 nmol l−1 of sense/antisense primers. Reactions wereperformed in duplicate in a Mx3005P Thermocycler (Agilent, Berkshire,UK), with one cycle of 2 min at 95°C and 40 cycles of 5 s at 95°C and 20 sdock10246Relative expression (a.u.)dock50246crkl012345itgb1012345cadh15246tmem8c1234igf100.20.40.60.81.0gh00.10.20.3ABWT     TF     TRWT     TF     TRWT     TF     TR WT     TF     TR WT     TF     TRWT     TF     TR WT     TF      TR WT     TF      TRa,bFig. 2. Gene expression analysis in coho salmon(Oncorhynchus kisutch) fast skeletal muscle. Geneexpression in fast skeletal muscle measured by qPCR forgh, igf1, dock5, dock1, cadherin-15, tmem8c, itgb1 and crkl(see supplementary material Table S2 for abbreviations).(A) GH axis genes. (B) Myotube formation genes. Resultsrepresent means ± s.e.m., 6 fish per group. Different lettersindicate significant differences between means (P<0.05).The Journal of Experimental Biology3395SHORT COMMUNICATION The Journal of Experimental Biology (2014) doi:10.1242/jeb.107664at 65°C, followed by a dissociation curve analysis, which resulted in a singlepeak in all cases. The stability of the four housekeeping genes rpl27, rpl13,ef1a and β-actin was analysed as previously described (Macqueen et al.,2013). Rpl13 and ef1a were found to be the most stable reference genes(average expression stability value M=0.058). Normalization of geneexpression was performed using the geometric average of rpl13 and ef1a.All expression values are expressed as arbitrary units. Expression betweengroups was compared using a one-way ANOVA with a Bonferroni post hoccorrection using the SPSS21 statistical package (IBM).Competing interestsThe authors declare no competing financial interests. Author contributionsI.A.J. and R.H.D. conceived the study. R.H.D. was responsible for fish husbandryand generation of the experimental groups. I.A.J. was responsible for thehistological analysis. D.G.d.l.s. was responsible for the RNA extraction and RT-qPCR analysis. I.A.J. and D.G.d.l.s. carried out the statistical analysis. I.A.J. wrotethe paper with input from the other authors.FundingThe study was supported by the Marine Alliance for Science and Technology forScotland (Scottish Funding Council grant HR09011) and by the CanadianBiotechnology Strategy (to R.H.D.). Deposited in PMC for immediate release.Supplementary materialSupplementary material available online athttp://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.107664/-/DC1ReferencesBjörnsson, B. T. (1997). The biology of salmon growth hormone: from daylight todominance. Fish Physiol. 17, 9-24.Hill, J. A., Kiessling, A. and Devlin, R. H. (2000). Coho salmon (Oncorhynchuskisutch) transgenic for a growth hormone gene construct exhibit increased rates ofhyperplasia and detectable levels of differential gene expression. Can. J. Aquat. Sci57, 939-950. Jimenez, A. G., Dillaman, R. M. and Kinsey, S. T. (2013). Large fibre size in skeletalmuscle is metabolically advantageous. Nat. Commun. 4, 2150.Johnston, I. A., Fernández, D. A., Calvo, J., Vieira, V. L. A., North, A. W.,Abercromby, M. and Garland, T., Jr (2003). Reduction in muscle fibre numberduring the adaptive radiation of notothenioid fishes: a phylogenetic perspective. J.Exp. Biol. 206, 2595-2609. Johnston, I. A., Bower, N. I. and Macqueen, D. J. (2011). Growth and the regulationof myotomal muscle mass in teleost fish. J. Exp. Biol. 214, 1617-1628. Johnston, I. A., Kristjánsson, B. K., Paxton, C. G., Vieira, V. L. A., Macqueen, D. J. and Bell, M. A. (2012). Universal scaling rules predict evolutionary patterns of myogenesis in species with indeterminate growth. Proc. Biol. Sci. 279, 2255-2261. Leggatt, R., Biagi, C. A., Smith, J. L. and Devlin, R. H. (2012). Growth of growthhormone transgenic coho salmon Oncorhynchus kisutch is influenced by constructpromoter type and family line. Aquaculture 356-357, 193-199. Macqueen, D. J., Garcia de la Serrana, D. and Johnston, I. A. (2013). Evolution ofancient functions in the vertebrate insulin-like growth factor system uncovered bystudy of duplicated salmonid fish genomes. Mol. Biol. Evol. 30, 1060-1076. Raven, P. A., Uh, M., Sakhrani, D., Beckman, B. R., Cooper, K., Pinter, J., Leder,E. H., Silverstein, J. and Devlin, R. H. (2008). Endocrine effects of growth hormone overexpression in transgenic coho salmon. Gen. Comp. Endocrinol. 159,26-37. Sundstrom, L. F., Devlin, R. H., Johnsson, J. I. and Biagi, C. A. (2003). Verticalposition reflects increased feeding motivation in growth hormone transgenic cohosalmon (Oncorhynchus kisutch). Ethology 109, 701-712. 

Muscle fibre size optimisation provides flexibility to energy budgeting in calorie-restricted Coho salmon transgenic for growth hormone

University of St. Andrews - Pure

The study was supported by the Marine Alliance for Science and Technology for Scotland (Scottish Funding Council grant HR09011) and by the Canadian Biotechnology Strategy (to R.H.D.). Deposited in PMC for immediate releaseCoho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.Peer reviewe

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Muscle fibre size optimisation provides flexibility for energy budgeting in calorie-restricted coho salmon transgenic for growth hormone

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University of St. Andrews - Pure

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