HyperplasiaandCellularityChangesinIGF-1 - OverexpressingSkeletalMuscleofCrucianCarp

GROWTH
FACTORS-CYTOKINES
Hyperplasia and Cellularity Changes in IGF-1Overexpressing Skeletal Muscle of Crucian Carp

Dongliang Li, Qiyong Lou, Gang Zhai, Xuyan Peng, Xiaoxia Cheng, Xiangyan Dai,
Zijian Zhuo, Guohui Shang, Xia Jin, Xiaowen Chen, Dong Han, Jiangyan He,
and Zhan Yin
Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences (D.L., Q.L.,
G.Z., X.P., X.C., X.D., Z.Z., G.S., X.J., X.C., D.H., J.H., Z.Y.), Institute of Hydrobiology, Chinese Academy
of Sciences, Wuhan, Hubei, China; and University of Chinese Academy of Sciences (D.L., G.Z., X.P., X.C.,
X.D., Z.Z., G.S.), Beijing, China
The zebrafish skeletal muscle-specific promoter mylz2 was used to cause crucian carp overexpression of the zebrafish IGF-1 cDNA. In stable transgenic germline F1 progenies, a 5-fold increase in
the level of IGF-1 in skeletal muscle was observed. Evident skeletal muscle hyperplasia was observed
in the transgenic fish through histologic analysis. By analyzing the RNA sequencing transcriptome
of the skeletal muscle of IGF-1 transgenic fish and nontransgenic control fish at 15 months of age,
10 966 transcripts with significant expression levels were identified with definite gene descriptions
based on the corresponding zebrafish genome information. Based on the results of our RNA
sequencing transcriptome profiling analysis and the results of the real-time quantitative PCR analysis performed to confirm the skeletal muscle transcriptomics analysis, several pathways, including
IGF-1 signaling, aerobic metabolism, and protein degradation, were found to be activated in the
IGF-1-overexpressing transgenic fish. Intriguingly, our transcriptional expression and protein assays indicated that the overexpression of IGF-1 stimulated a significant shift in the myofiber type
toward a more oxidative slow muscle type. Although the body weight was surprisingly decreased
by IGF-1 transgenic expression, significantly higher oxygen consumption rates were measured in
IGF-1-overexpressing transgenic fish compared with their nontransgenic control fish. These results
indicate that the sustained overexpression of IGF-1 in crucian carp skeletal muscle promotes myofiber hyperplasia and cellularity changes, which elicit alterations in the body energy metabolism
and skeletal muscle growth. (Endocrinology 155: 2199 2212, 2014)
GF-1, an anabolic peptide growth factor, plays primary

roles in the promotion of organ growth. This growth
factor is a primary mediator of the effects of GH on the
promotion of postnatal development and growth (1). Even
a heterozygous mutation of IGF-1, which is partly compensated by the expression of the other allele, is sufficient
to restrict somatic growth in mice (2, 3). Transgenic mice
overexpressing IGF-1 ubiquitously under the metallothionein promoter exhibit increased body weight and organomegaly (4). Hence, the endocrine and autocrine/paracrine
roles of IGF-1 contribute to postnatal growth. The local
expression of IGF-1 in mouse skeletal muscle results in
significant muscle hypertrophy and induced a shift toward

more oxidative myofibers (5). The use of myogenic cells
and inhibitors of signaling pathways has provided valuable insights into our understanding of the IGF functions
on the regulation of skeletal muscle growth in teleost fish.
It has been reported that IGF-1 in fish has dramatic effects
on muscle growth, protein synthesis, and myoblast proliferation (6 8). However, many of the functional studies
on teleost IGF-1 were performed with myogenic cell cultures (9, 10). Little information on the regulatory roles of
teleost IGF-1 has been derived from studies with in vivo
models.
ISSN Print 0013-7227 ISSN Online 1945-7170

Printed in U.S.A.
Copyright 2014 by the Endocrine Society
Received October 9, 2013. Accepted February 28, 2014.
First Published Online March 10, 2014
Abbreviations: BCAA, branched chain amino acid; CPT1, carnitine palmitoyltransferase-1;

EGFP, enhanced green fluorescent protein; FBP, bisphosphatase; HSL, hormone-sensitive
lipase; IRES, internal ribosome entry site; LPL, lipoprotein lipase; PFK, phosphofructokinase;
RNA-seq, RNA sequencing; RPKM, reads per kilobase of mRNA length per millions of
mapped reads; qRT-PCR, quantitative RT-PCR.
doi: 10.1210/en.2013-1938
Endocrinology, June 2014, 155(6):2199 2212
endo.endojournals.org
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 November 2015. at 19:07 For personal use only. No other uses without permission. . All rights reserved.
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Li et al
Muscle Development in IGF-1 Transgenic Fish
Based on the regulatory function of IGF-1 on body metabolism and muscle, we created transgenic crucian carp
(Carassius auratus) overexpressing the zebrafish IGF-1
under the mylz2 promoter, which is a strong zebrafish
skeletal muscle-specific promoter (11). We observed the
stable and specific overexpression pattern of the zebrafish
IGF-1 in transgenic crucian carp. To our surprise, when
the germline transmission IGF-1 transgenic fish were
raised in the same tank with nontransgenic control fish, we
found a significant loss in skeletal muscle mass compared
with the control fish. In addition, we observed markedly
enhanced levels of muscle hyperplasia and an induced shift
from white muscle toward more oxidative red muscle in
the IGF-1 transgenic fish. Further tests indicated that the
oxygen consumption rates of the IGF-1 transgenic fish
were significantly increased compared with those of control fish. To explore the molecular events underlying the
muscle phenotypes in these transgenic fish, transcriptome
profiling analyses of fish muscle tissue through RNA-seq
were conducted. The global transcriptional survey and the
results of our real-time RT-PCR confirmation tests provide strong evidence indicating elevated levels of protein
synthesis, cell proliferation, energy-dependent oxidation
of complex molecules, and muscle proteolysis in the IGF-1
transgenic fish compared with the control fish. These results provide an excellent in vivo model for globally understanding the critical functions of teleost IGF-1 on myogenic cell differentiation and proliferation and body
energy homeostasis in teleost.
(CLONTECH) as the backbone. The fragment containing

mylz2:igf-1:IRES:egfp was flanked with I-SceI sites (Figure 1A).
The procedures for microinjection, initial enhanced green fluorescent protein (EGFP) screening, and PCR confirmation of the
transgene in the genome were described previously (13). The
transgenic fish were raised with the EGFP-negative control fish
at ratio of 1:1 in the same tank. The data reported here are
derived from F1 generation of crucian carp.
Growth evaluations and muscle histologic analysis

To determine the body weights, all of the fish were weighed
at the ages of 3, 8, 15, and 21 months. The muscle mass was
determined by subtracting the sum of the inner organs, head, and
boiled bone from the whole body weight. The procedures for the
muscle histologic analysis were described previously (13, 14).
For the immunofluorescence assays, the primary antibodies S58
(sc-32733) and F59 (sc-32732) were supplied by Developmental
Studies Hybridoma Bank through Santa Cruz Biotechnology and
were diluted at 1:200. The immunoreaction with the primary
antibody was performed overnight at 4C. The primary antibodies were detected using Alexa-488 (antimouse)- and Alexa555 (antirabbit or goat)-conjugated secondary antibodies (Invitrogen) that were diluted 1:250 in carrier buffer.
Western blot analysis

The protein samples were collected from adult crucian carp
skeletal muscle tissue, liver, and blood as described previously
(14). The primary antibodies of anti-IGF-1 (Abcam Inc; antibody
106836), anti-S58, and anti-F59 were diluted 1:1000. The levels
of -actin in each sample were detected by its specific antibody
(catalog no. 69879; Santa Cruz Biotechnology, Inc) and used as
loading controls.
RNA isolation and preparation for RNA-seq
Sexually mature crucian carp were initially purchased from a

hatchery in Ezhou, Hubei Province. Artificially fertilized eggs
collected from the parent crucian carp were used for microinjection. The experimental crucian carp were maintained following the procedure described previously (12). The fish were fed a
commercial crucian carp pellet (Tianan 906, Haida). All of the
experimental procedures were approved by the Animal Care and
Use Committee at the Institute of Hydrobiology of the Chinese
Academy of Sciences (Approval Protocol no. IHB20100032) and
were in accordance with the National Guiding Principles for the
Care and Use of Laboratory Animals.
The total RNA was isolated from skeletal muscle of 15month-old crucian carp with the Trizol Reagent (Gibco BRL)
following the manufacturers instructions. Each RNA sample
was pooled from 3 females at 8 months of age. The 3 control and
3 transgenic crucian carp were randomly chosen from the same
tank. The RNA samples were then treated with RNase-free
DNase I (QIAGEN). The integrity of the RNA was determined
using an Agilent 2000 analyzer. Poly-A-containing mRNA was
isolated from the total RNA using the PolyATract mRNA Isolation System (Promega Corp). The mRNA fragments were converted into first-strand cDNA using reverse transcriptase and
random primers. The second-strand cDNA synthesis reactions
were conducted using DNA polymerase I and RNase H. After the
end repair process and ligation of the adapters, the products were
subsequently purified and amplified through PCR to create the
final cDNA libraries.
Generation of transgenic crucian carp
Transcriptome analysis
The zebrafish mylz2 promoter was previously described by Ju

et al (11). The zebrafish igf-1 (NCBI accession no. DQ777636.1)
transcript from adult liver was cloned by RT-PCR. The sequences of the transcript and predicted protein were confirmed.
The sequence of the internal ribosomal entry site (IRES) from
encephalomyocarditis virus was cloned from the pIRES2-EGFP
vector (CLONTECH). All of the components were subcloned
and assembled from entry clones using the pEGFP-N1 vector
The 95-base sequences were obtained by sequencing using an

Illumina HiSeq 2000. The sequencing-received raw image data
were transformed by base culling into the sequence data. Before
mapping the reads to the reference database, we filtered all of the
sequences to remove any adaptor and low-quality sequences.
The remaining reads were aligned to the zebrafish genome using
SOAPaligner/soap2, which allows up to 2 base mismatches. All
of the reads were aligned against the zebrafish genome assembly
Materials and Methods

Fish maintenance and husbandry
doi: 10.1210/en.2013-1938
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Figure 1. Generation of transgenic zebrafish. A, Schematic representation of the transgenic construct containing the 5-mylz2:igf-1:IRES:egfp-3fragment flanked by I-SceI sites. B and C, Fluorescence microscopy (B) and normal microscopy (C) images of a germline transmission F1 progeny at
6 days after fertilization. D, Insertion test using gDNA PCR tests. The amplified PCR fragments of the correct size (0.86 kb) are shown in lanes 3
and 4. The DNA ladder marker is shown in lane 5. The -actin fragment was amplified from the gDNA and used as the loading control (0.557 kb)
and is shown in lanes 6 9. The DNA from the control fish without the presence of the inserted fragment is shown in lanes 1 and 2. E, Fish body
weights were determined with 3 females in each group 3, 8, 15, and 21 months of age. The significant differences of body weight between
transgenic fish and control fish were observed at these stages (P .05). F, Fish body length was determined with 3 females in each groups at 3, 8,
15, and 21 months of age. The significant differences of body length between transgenic fish and control fish were observed at these stages (P
.05). G, Representative image of IGF-1 transgenic crucian carp (3 on the right) and their nontransgenic control fish (3 on the left) at 10 months of
age. H, Western blot analysis of IGF-1 expression in skeletal muscle tissue from IGF-1 transgenic crucian carp (upper panel right) and nontransgenic
control fish (upper panel left). The -actin levels in the 2 samples was detected and used as the protein loading controls (lower panel). I, The
Western blot analyses for IGF-1 were quantified by densitometric analysis using Image J. The levels of IGF-1 were normalized to the corresponding
level of -actin in the sample and expressed as the fold change relative to the level of IGF-1 in the control sample. The quantification analyses were
performed 3 times, and the SEs were calculated as indicated. **, P .01. Con, control; Tg, transgenic.
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Li et al
version 9 (Zv9; http://www.sanger.ac.uk) using the SOAPaligner/soap2 software package. The normalized gene expression level was calculated separately as reads per kilobase of
mRNA length per millions of mapped reads (RPKM) for each
library, which facilitates the comparison of transcript levels between samples. The cutoff value for determining the gene transcriptional activity was determined based on a 95% confidence
interval for all RPKM values for each gene. The Gene Ontology
(GO) functional enrichment analysis was conducted using
Blast2GO (version 2.3.5) (http://www.blast2 go.org/). KEGG pathway analyses were performed using the Cytoscape software (version 2.6.2) (http://www.cytoscape.org/) with the ClueGO plug-in
(http://www.ici.upmc.fr/cluego/cluegoDownload.shtml).
RT-PCR and quantitative real-time PCR

To confirm the RNA-seq results, the expression levels of the
selected transcripts in skeletal muscle samples from igf-1-overexpressing transgenic fish and nontransgenic control fish were
determined again through quantitative RT-PCR. The procedure
of the quantitative RT-PCR was described previously (14). The
relative abundance of mRNAs was calculated by the comparative cycle of threshold method using the -actin mRNA as an
internal standard. These measurements were performed in triplicate and exhibited a coefficient of variation of less than 5%. The
expression levels were expressed as fold changes (the mean SE)
compared with the levels of the internal control -actin as the
values. In order to assess the correlation between the fold changes
between transgenic fish and control fish based on the RPKM
values (obtained with RNA-seq) with their corresponding quantitative RT-PCR (qRT-PCR) value of the fold change, the log2
data of the fold changes in the RNA-seq and the qRT-PCR were
plotted along with the linear regression line to examine the relationship between them. The primers that were used for this
analysis are summarized in Supplemental Table 1.
Oxygen consumption measurements

The standard oxygen consumption rates of individual igf-1overexpressing transgenic crucian carp and their control fish at
age of 15 months were measured (15). Briefly, the animals were
starved for 24 hours and then transferred and maintained individually in separate respiration chambers with 3000 mL of fresh
water at 28C. The oxygen concentrations were measured using
a SevenGo pro-SG6 oximeter (Mettler-Toledo AG; Analytical;
CH-8603,) after 6 hours of enclosure within the chamber. The
oxygen concentration in a respiration chamber without any fish
specimen during the same period was treated as the initial oxygen
concentration. The oxygen consumption was expressed as milligrams O2/h/g body weight.
Measurements of free amino acid composition in

muscle
The extraction of free amino acids was performed as described previously (16). The concentrations of free amino acids
in the supernatants were measured by reverse-phase HPLC
(E2695; Waters Corp) equipped with 2475 fluorescence detector
(Waters Corp).
Results
Generation of IGF-1 transgenic crucian carp
The transgenic constructs were linearized and injected
into crucian carp embryos at the one- or two-cell stage at
a concentration of 100 g/mL. One thousand and forty
larvae were screened out from 2100 injected embryos with
the presence of EGFP in the skeletal muscle by fluorescent
microscopy. They were kept in one pond. The remainder
of the F0 fish were kept in another pond. Only 142 F0
crucian carp of the 1040 EGFP-positive larvae survived to
the next breeding season. However, the mature and good
quality eggs could be collected from only 28 F0 females
from the 140 EGFP-positive fish. No mature and good
sperm could be collected from the EGFP-positive males.
The collected eggs were fertilized with the sperm collected
from 3 EGFP-negative F0 male crucian carp. Later, some
of the F1 offspring from these F0 females successfully exhibited the transmission of EGFP-expressing skeletal muscle (Figure 1, B and C). The presence of the mylz2:igf-1:
IRES:egfp fragment in the F1 progenies was confirmed by
PCR screening using genomic DNA samples collected
from the fin of the EGFP-positive fish (Figure 1D). However, poorer growth performance of the IGF-1 transgenic
fish compared with the control fish in the same tank began
to be observed after the age of 3 months (Figure 1, EG).
The cell lysates from the identified igf-1 transgenic crucian
carp were subjected to Western blot analyses. It has been
demonstrated that the protein levels of IGF-1 in muscle,
liver, and blood were markedly increased in the transgenic
fish compared with control fish (Figure 1, H and I; and
Supplemental Figure 1).
Histologic analysis of muscle growth
To determine whether the muscle growth was affected
in the igf-1 transgenic fish, samples from 8-month-old female transgenic and nontransgenic control fish were sectioned to observe the histologic features of the muscle tissues. The surface of the muscle tissue of the transgenic
specimens was not as smooth as that of the nontransgenic
control fish (Figure 2AD). The overall muscle mass was
actually decreased in the igf-1 transgenic crucian carp
compared with control fish raised in the same tank (Supplemental Table 2). Based on the histologic analysis performed on a defined region of the epaxial muscle at the
base of the first pin of the dorsal fin (Figure 2, E and F), the
diameters of the myofibers decreased significantly in
transgenic fish compared with nontransgenic control fish
(Figure 2G). This effect was also evidenced by significant
higher number of myofibers seen within similar cross-sectional area in transgenic fish compared with those in nontransgenic fish (Figure 2H). These results indicate that the
doi: 10.1210/en.2013-1938
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HiSeq System, and the resulting

reads were trimmed to remove any
adapter and low-quality sequences.
All of the reads were assembled
with the Trinity (http://trinityrnaseq.sourceforge.net), TGICL (V2.1),
and Phrap (V23.0) software programs into 68 776 transcriptome
unigenes. Of them, 36 964 unigenes
(53.7%) and 54 952 unigenes
(79.8%) were annotated to a NCBI
nonredundant protein database and
a nonredundant nucleotide database
through matching using BLAST (E
value 1E-5). Crucian carp and zebrafish are both cyprinid fishes. Because the available genomic resources of crucian carp are limited,
more than 80% of these annotated
unigenes were aligned to zebrafish
UniGenes (Figure 3A). When the cutoff for significant expression of the
annotated unigenes was set to 4.0 of
the reads per kb of exon per million
fragments mapped (RPKM) in either
the transgenic or the control fish
skeletal muscle samples, 10 966
transcripts that were aligned to zebrafish UniGenes with definite gene
descriptions (Supplemental Table 3)
were used for further analyses.
The numbers of expressed transcripts in each sample are shown in
the Venn diagram presented in Figure 3B. A total of 4820 common
Figure 2. Enhanced muscle hyperplasia in IGF-1-overexpression transgenic crucian carp. AD,
transcripts are presented in the musImages of sacrificed and skinned nontransgenic control crucian carp (Con) (A and C) and IGF-1
cle tissue samples of both transgenic
transgenic crucian carp (Tg) (B and D) at 8 months of age. E and F, Representative cross-sectional
and control fish (black in Suppleimages of skeletal muscle of control (E) and transgenic (F) crucian carp stained with hematoxylin
mental Table 3). In addition to these
and eosin. G, Diameters of primary muscle fibers measured with Image J software in the
representative regions. H, Numbers of primary muscle fibers measured with Image J software in
common transcripts, 545 transcripts
the representative regions. The quantification analyses were performed with 3 females from each
(4.0 RPKM) are uniquely found in
group. The SEs were calculated as indicated. **, P .01.
the sample from control fish (purple in Supplemental Table 3), and
overexpression of IGF-1 in crucian carp apparently causes
5601 transcripts are only significantly expressed (4.0
hyperplastic muscle growth in the transgenic fish.
RPKM) in the skeletal muscle from IGF-1-overexpressing transgenic crucian carp (Figure 3B; red in SuppleSkeletal muscle transcriptome analysis through
mental Table 3).
RNA-seq
Based on the RNA-seq results, the relative expression
To understand the molecular basis of the effects of
IGF-1 overexpression in crucian carp skeletal muscle, we levels of the total transcripts belonging to muscle contracconducted a comparative transcriptomic study of skeletal tion components, such as myosin, troponin, and tropomuscle samples from transgenic fish and control fish. The modulin, in the total transcript pools were found to be
skeletal muscle samples were sequenced with an Illumina slightly decreased in transgenic fish muscle. Among
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Li et al
Figure 3. Comparative RNA-seq transcriptomics analyses of crucian

carp skeletal muscle. A, Species distribution of the BLAST matches for
each annotated contig from the de novo assembled crucian carp
transcriptome (E-value cut-off 1010). B, Venn diagram of gene
expression profiles related with IGF-1 overexpression in crucian carp
skeletal muscle. Comparison of the datasets from IGF-1 transgenic
crucian carp (Tg) and control fish (Con). The number in the overlapped
region represents the annotated genes that were significantly
expressed (4.0 RPKM) in both samples. The numbers in the
nonoverlapped region are the annotated genes that were significantly
expressed (4.0 RPKM) in only one of the samples. The complete gene
lists are available in Table 1. C, Distribution of various categories of
transcripts in the skeletal muscle transcriptome of IGF-1 transgenic
crucian carp (left) and control fish (right). The pie charts show the
percentage of transcripts in each category relative to the entire set.
The muscle contraction components category is further divided into
slow and fast type for the comparison of the myofiber composition
(small pie charts on the top right).
these transcripts, the relative proportion of slow musclespecific molecules increased markedly in the transgenic
fish muscle. In contrast, the total transcriptional levels of
fast muscle-specific components were decreased signifi-
cantly in the IGF-1-overexpressing muscle tissue (Figure

3C). The total transcriptional levels of mitochondrial proteins were increased slightly in transgenic fish muscle. The
total ribosomal proteins (not including mitochondrial ribosomal proteins) were slightly decreased in the transgenic tissue sample. To our surprise, a 1-fold increase in
the total transcriptional level of the ubiquitin and proteasome transcripts was found in IGF-1 transgenic muscle
compared with control fish muscle (Figure 3C).
To further explore the potential impact on muscle development and growth in IGF-1 transgenic crucian carp,
we queried genes that have been previously shown to be
involved in IGF signaling, muscle cell proliferation, skeletal muscle cellularity, fatty acid metabolism, glucose metabolism, and amino acid metabolism (Table 1). First, the
markedly increased level of igf-1 transcript was confirmed
in the transgenic fish muscle transcriptome. In addition,
the expression levels of IGF-binding protein 5 (igfbp5),
IGF-binding protein 7 precursor (igfbp7), c-Jun protein
(c-jun), early growth response protein 1 (egr1), and Integrin 5 (itga5), which have been reported to be IGF-1inducible transcripts (1720), were found to be increased
in transgenic fish muscle compared with control fish muscle tissue. MyoD, myogenin (myog), and desmin a are
critical genes involved in myogenesis in adult skeletal muscle (2123). Our RNA-seq analyses showed that these key
myogenic regulators are also significantly induced in
IGF-1 transgenic crucian carp muscle. The analysis of the
overexpression of IGF-1 in crucian carp muscle also
showed that many fast muscle-specific molecules, including myosin-binding protein C, fast-type (mybpc2), myosin
heavy chain, fast skeletal muscle (mylz1), fast skeletal myosin light chain 3 (mlc3f), fast muscle troponin I (tnni2),
and troponin T3b, skeletal, fast isoform 2 (tnnt3b), are
down-regulated. Conversely, the expression levels of their
counterparts in slow muscle, such as myosin-binding protein C, slow-type (mybpc1), slow myosin heavy chain 1
(smyhc1), slow myosin heavy chain 2 (smyhc2), slow myosin heavy chain 3 (smyhc3), slow-specific troponin C
(stnnc), troponin I, slow skeletal muscle-like (tnni1al), and
novel slow skeletal troponin T family protein (tnnt2e)
were up-regulated in IGF-1-overexpressing fish muscle.
Red or slow muscle is characterized by its high myoglobin
(mb) content (24), whereas fast-contracting muscle is rich
in parvalbumin (pval) and muscle creatine kinase b (mckb)
(25, 26). As shown by our RNA-seq results, the overexpression of IGF-1 caused a significant elevation in the expression level of mb and decreased levels of pval1b,
pval1d, and pval2 compared with those found in control
fish muscle. Lipoprotein lipase (LPL) is thought to function as a gate keeper for fatty acid uptake into animal
organs (27). Hormone-sensitive lipase (HSL) is considered
doi: 10.1210/en.2013-1938
Table 1.
Selected Relevant Clusters of Annotated Transcripts Regulated in IGF-1 Transgenic Crucian Carp
Symbol
Gene ID
(22200)
Con_RPKM
Tg_RPKM
Activation of IGF signaling and muscle cell proliferation

igf-1
CL3122.Contig1_All 0.113
229.366
igfbp5
Unigene2084_All
3.111
8.40
igfbp7
Unigene11591_All
8.551
27.212
c-jun
9.733
Log2
(Tg_RPKM/
Con_RPKM)
Up/Down
10.987
1.434
1.670
1.240
Up
Up
Up
Up
egr1
CL4798.Contig2_All
1.637
5.331
1.703
Up
itga5
5.403
1.034
Up
myog
CL582.Contig3_All
myod
CL8588.Contig1_All
desmin Unigene21270_All
Skeletal muscle cellularity
mybpc1 CL753.Contig2_All
0.7328
16.735
187.29
4.1992
61.485
1080.709
2.5186
1.8774
2.5286
Up
Up
Up
5.7761
37.0605
2.6817
Up
mybpc2 CL775.Contig1_All
113.05
52.5636
1.1048
Down
smyhc1
smyhc2
smyhc3
CL7051.Contig9_All
CL488.Contig2_All
CL3827.Contig5_All
0.1513
51.361
60.653
17.1448
353.4921
880.5984
6.8242
2.7829
3.8598
Up
Up
Up
myhz1
Unigene12959_All
43.539
9.1499
2.2505
Down
mlc3f
Unigene514_All
23337
9989.161
1.2242
Down
stnnc
tnni1al
CL4691.Contig2_All
CL5730.Contig1_All
351.31
23.808
969.4953
48.4113
1.4645
1.0239
Up
Up
tnni2
tnnt2e
CL6982.Contig4_All
CL1124.Contig3_All
3087.8
135.99
928.7902
934.577
1.7332
2.7809
Down
Up
tnnt3b
CL5279.Contig1_All
3536.5
1726.914
1.0341
Down
mckb
mb1
CL7197.Contig2_All
Unigene17901_All
22129
594.26
10758.97
1933.198
1.0404
1.7018
Down
Up
pval2
Unigene9536_All
pval1d CL3934.Contig1_All
pval1b CL2318.Contig1_All
Fatty acid metabolism
lpl
CL2110.Contig3_All
5957.2
2341.7
4558.2
891.8543
613.4232
929.9067
2.7398
1.9326
2.2933
Down
Down
Down
3.7688
14.3614
1.93
Up
hsll
cpt1a
Unigene37356_All
CL4396.Contig2_All
0.4556
15.933
3.0456
34.1066
2.7409
1.0981
Up
Up
acss2
CL9733.Contig1_All
1.4806
6.0874
2.0396
Up
acat1
CL1269.Contig2_All
10.635
26.3952
1.3115
Up
vlcad
CL4730.Contig3_All
9.5454
30.5286
1.6773
Up
scad
CL4908.Contig1_All
2.8728
7.6587
1.4146
Up
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NCBI Nonredundant
Database Annotation
IGF-1 precursor
IGF-binding protein 5
IGF-binding protein 7 precursor
c-Jun protein [Carassius
auratus]
Early growth response protein
1
Integrin, 5 (fibronectin
receptor, -polypeptide)
Myogenin [Cyprinus carpio]
myoD [Schizothorax prenanti]
Desmin a
Myosin-binding protein C,
slow-type
Myosin-binding protein C, fasttype
Slow myosin heavy chain 1
Myosin heavy chain, fast
skeletal muscle
Fast skeletal myosin light chain
3 [Cyprinus carpio]
Slow-specific troponin C
Troponin I, slow skeletal
muscle-like
Fast muscle troponin I
Novel slow skeletal troponin T
family protein
Troponin T3b, skeletal, fast
isoform 2
Muscle creatine kinase b
Myoglobin 1 [Carassius
auratus]
Parvalbumin-2
Parvalbumin isoform 1d
Parvalbumin isoform 1b
Lipoprotein lipase [Carassius
auratus]
Hormone-sensitive lipase-like
Carnitine Opalmitoyltransferase 1, liver
isoform
Acetyl-coenzyme A synthetase,
cytoplasmic
Acetyl-CoA acetyltransferase,
mitochondrial precursor
Very long-chain specific acylCoA dehydrogenase,
mitochondrial
Short-chain specific acyl-CoA
dehydrogenase,
mitochondrial
(Continued)
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Li et al
Table 1.
Continued
Symbol
Gene ID
(22200)
Con_RPKM
Unigene13021_All
Tg_RPKM
Log2
(Tg_RPKM/
Con_RPKM)
Up/Down
0.7432
6.2537
3.0729
Up
slc25a20 Unigene2515_All
34.345
71.7117
1.0621
Up
pgc-1a
CL5824.Contig4_All
1.2594
3.0171
1.2604
Up
pr285
CL9791.Contig1_All
0.1176
4.4816
5.2521
Up
Glucose metabolism
pfkmb
CL4095.Contig1_All
pfkma
CL561.Contig1_All
aldoa
Unigene17679_All
66.601
89.794
2472.5
27.5277
31.4878
1163.902
1.2746
1.5118
1.087
Down
Down
Down
6.5733
1.8726
1.8116
Down
43.4124
16.0515
1.1228
1.3009
Up
Up
56.5997
1.2675
Up
slc27a1
fbp1
Unigene19259_All
Protein metabolism
aam
aco2
hadhb
CL2928.Contig4_All
23.51
mchm
13.1723
1.0789
Up
dbt
CL3152.Contig3_All
4.2853
11.9778
1.4829
Up
bact2
CL4267.Contig1_All
14.831
34.3267
1.2108
Up
bckdk
CL5179.Contig1_All
1.2468
6.5641
2.3964
Up
Muscle atrophy and apoptosis

foxO1a CL9998.Contig1_All
fbx32
CL8184.Contig2_All
bnip3l
CL4423.Contig3_All
7.114
99.052
13.465
2.3715
22.5917
42.8922
1.585
2.1324
1.6716
Down
Down
Up
casp3b
Unigene39023_All
0.5913
14.3256
4.5986
Up
shfm1
CL8158.Contig2_All
107.75
256.6382
1.252
Up
myst
CL4098.Contig1_All
11.308
49.1251
2.1191
Up
NCBI Nonredundant
Database Annotation
Solute carrier family 27 (fatty
acid transporter), member 1
Mitochondrial
carnitine/acylcarnitine carrier
protein
Peroxisome proliferatoractivated receptor
coactivator 1-
Peroxisomal proliferatoractivated receptor Ainteracting complex 285 kDa
protein-like
Phosphofructokinase, muscle b
Phosphofructokinase, muscle a
Fructose-bisphosphate aldolase
A
Fructose-1,6-bisphosphatase
[Carassius gibelio]
Aspartate aminotransferase 2
Aconitate hydratase,
mitochondrial
Trifunctional enzyme subunit ,
mitochondrial
Methylglutaconyl-CoA
hydratase, mitochondrial
Lipoamide acyltransferase
component of branchedchain -keto acid
dehydrogenase complex,
mitochondrial
BCAA aminotransferase,
mitochondrial
Branched chain -ketoacid
dehydrogenase kinase
Forkhead box O1 a
F-box only protein 32
BCL2/adenovirus E1b 19-kDa
interacting protein 3a like
Caspase 3, apoptosis-related
cysteine protease b
26S proteasome complex
subunit DSS1
Myostatin [Labeo fimbriatus]
CoA, coenzyme A; Con, control; Tg, transgenic.
the rate-limiting enzyme in the hydrolysis of triacylglycerol (28). Carnitine palmitoyltransferase-1 (CPT1) is reported as the gatekeeper for fatty acid entry into the mitochondria (29). The transcriptional levels of lpl, hsll,
cpt1a, and other key enzymes in the fatty acid oxidation
pathway (shown in Table 1) are significantly up-regulated
in IGF-1 transgenic fish muscle compared with the control
samples. The rate of gluconeogenesis is ultimately con-
trolled by the action of a key enzyme, fructose-1,6-bisphosphatase (FBP) (30). In contrast, phosphofructokinase
(PFK) is an important control point in the glycolytic pathway (31). In IGF-1-overexpressing fish muscle, the expression levels of fbp1, pfk muscle a (pfkma), and pfk muscle
b (pfkmb) were significantly down-regulated (as shown in
Table 1). Branched-chain amino acids (BCAAs) can also
be used as metabolic fuels. The mitochondrial form of
doi: 10.1210/en.2013-1938
Figure 4. Real-time RT-PCR confirmation of the representative key

signaling transcripts identified from the comparative crucian carp
skeletal muscle RNA-seq transcriptomics analyses. RNA samples for
quantitative RT-PCR assays were extracted from skeletal muscle of IGF1 female transgenic crucian carp and control fish at 15 months of age
(n 3). The real-time RT-PCR measurements were performed with
specific primers that were designed for desmin a, myoD, smyhc3,
stnnc, mb, mckb, pval2, fbox32, shfm1, mstn, hsll, pfkmb, fbp1,
mchm2, bcat2, casp3b, and bnip3 l. The relative transcript levels are
calculated by real-time RT-PCR using -actin as the internal standard.
The log2 data of the fold changes in the RNA-seq and the qRT-PCR
were plotted along with the linear regression line to examine the
relationship between them. The primers designed for the real-time PCR
analysis are listed in Supplemental Table 1.
BCAA transaminase has been reported to be one of the key

molecules in the BCAA metabolism (32). Our RNA-seq
results indicate that the transcriptional levels of many key
molecules involved the BCAA metabolism are significantly increased in IGF-1 transgenic fish muscle (as shown
in Table 1). Consistent with previous reports, the suppression of the expression of genes encoding ubiquitin ligases,
such as F-box protein 32 (Fbx32) and atrogin-1, was also
observed in IGF-1-overexpressing tissue (as shown in Table 1) (8). However, the IGF-1 overexpression in the transgenic muscle tissue was surprisingly unable to repress the
expression of a large number of skeletal muscle atrophyrelated genes, including BCL2/adenovirus E1b 19-kDa interacting protein 3a like (bnip3l), caspase 3, apoptosisrelated cysteine protease b (casp3b), and 26S proteasome
complex subunit DSS1 (shfm1). In fact, the expression
levels of these genes were even significantly elevated in our
transgenic fish muscle compared with the tissue samples
from nontransgenic control fish (Table 1).
Experimental validation of the RNA-seq analyses
Based on the bioinformatic analyses previously described, another set muscle samples were collected from 3
transgenic female fish and 3 control female fish skeletal at
15 months of age for the experimental verification. We
chose 17 differentially expressed genes, which were se-
2207
lected as representative transcripts in several key pathways. The real-time RT-PCR results confirmed that all of
the 17 transcripts exhibited the similar tendencies in their
expression patterns as was observed in the RNA-seq data.
A least-squares regression analysis provided an R2 value of
0.9715, with a corresponding factor of 1.207, suggesting
a strong inverse relationship between the log2 values of the
fold changes collected from RNA-seq and qRT-PCR results (Figure 4).
The slow MyHC-specific antibodies S58 and F59 have
been widely used as markers for investigations of muscle
fiber type specification in fish (33, 34). Based on crosssectional immune-histologic profiles of the defined lateral
dorsal area (Figure 5A), a markedly larger portion of red
muscle (Figure 5C), higher number of myocytes in the
superficial layers reacted with S58 (Figure 5E) and F59
(Figure 5G) compared with the control fish. Moreover,
Western blot analyses with antibodies S58 and F59 also
demonstrated that higher levels of sMyHC protein were
detected in the muscle samples from transgenic fish compared with the control samples (Figure 5, H and I).
Oxygen consumption rates and muscle free BCAAs
in IGF-1 transgenic crucian carp
Aerobic metabolism consumes dissolved oxygen in the
water. The metabolic rate of fish is ordinarily reflected by
the oxygen consumption rate (35). At 15 months of age, a
group of 6 animals per genotype were employed for the
oxygen consumption measurements. The weight, total
length, and oxygen consumption of each crucian carp
were measured. As shown in Figure 6B, the IGF-1 transgenic crucian carp presented significantly higher oxygen
consumption rates than those of control fish. However,
the weights of the IGF-1 transgenic crucian carp were significantly lower compared with those of the control fish
(Figure 6A).
The BCAAs are the essential amino acids that primarily
metabolized in muscle for energy or protein synthesis (36).
We found that the portion of the free BCAAs in the
muscle of IGF-1 transgenic fish was significantly increased compared with that from the control fish (Supplemental Table 4).
Discussion
In fish, skeletal muscle growth can be normally achieved
by both hypertrophic and hyperplastic muscle growth
throughout the lifecycle. The IGF-1 signal is the main
downstream effective cascade of the GH axis and is also
clearly an anabolic factor for muscle growth. It also has
been reported that the overexpression of IGF-1 promotes
2208
Li et al
ation, and hypertrophy and inhibit

muscle atrophy (6 8). To date, no
study has analyzed an IGF-1 transgenic fish model. Ninety-four percent of the amino acids in the zebrafish and crucian carp IGF-1a
sequences
(NCBI
Nr.
no.
ABG75920) are identical. In fact,
there is only one amino acid difference between the mature IGF-1a
peptides of zebrafish and crucian
carp. When we overexpressed the
zebrafish IGF-1 in crucian carp, we
found a 5-fold increase in IGF-1
protein in the skeletal muscle of the
F1 germline transmission transgenic fish (Figure 1). Evident myocyte hyperplasia was also observed
in the IGF-1-overexpressing skeletal
muscle (Figure 2). At the transcriptional levels, the expression levels of
many known IGF-1-responsive genes
and myogenic regulatory factors, such
as igfbp5, igfbp7, c-jun, egr1, itga5,
myoD, myog, and desmin a, were
found to be significantly increased in
the skeletal muscle of IGF-1 transgenic
crucian carp compared with control
fish (Table 1 and Figure 4). These observations indicate that fish IGF-1 promotes myogenesis through hyperplasia at the postnatal stage in vivo. In
addition, based on transcriptomics
and Western blot analyses, our experimental results demonstrate that
IGF-1 overexpression also promotes a
Figure 5. Histochemistry and immunoblot analyses of myofiber types. A, Diagrammatic image showing
shift toward slow skeletal muscle and
the region selected as the representative areas for sampling. B and C, Representative cross-cutting profiles
a more oxidative myofiber type in
in the defined region showing the overall white and red muscle portions in control crucian carp (B) and
transgenic crucian carp (Figure 3C,
IGF-1 transgenic crucian carp (C). D and E, Representative cross-sectional profiles in the defined region
showing the slow myofibers in control crucian carp (D) and IGF-1 transgenic crucian carp (E) detected by
Table 1, Figure 4, and Figure 5). This
the S58 antibody. F and G, Representative cross-sectional profiles of slow myofibers in the defined region
observation is similar with a previous
in control crucian carp (F) and IGF-1 transgenic crucian carp (G) detected by the F59 antibody. H, Western
report on IGF-1 overexpression in the
blot analyses of the slow myofiber-specific protein expression in skeletal muscle tissue from IGF-1
transgenic mouse (5). Therefore, our
transgenic crucian carp (right) and nontransgenic control crucian carp (left). The myofibers were detected
using the S58 antibody (upper panel) and the F59 antibody (middle panel). The -actin levels in the 2
results clearly indicate the in vivo
samples were detected and used as the protein loading controls (lower panel). I, The Western blot analyses
mitogenic effect and regulatory funcfor S58 and F59 were quantified by densitometric analysis using Image J. The levels of S58 and F59 were
tion on myocyte differentiation in
normalized to the corresponding level of -actin in the sample and expressed as the fold change relative to
the level of markers in the control sample. The quantification analyses were performed 3 times, and the
teleosts.
SEs were calculated as indicated. **, P .01. Con, control; Tg, transgenic.
The myotomal architecture and
muscle fiber organization of fish are
myocyte proliferation in transgenic mammals and cul- intimately related to the particular requirements associtured fish myogenic cells (5, 10). Fish IGF-1 can also ated with flexing the body during swimming and the asdirectly stimulate muscle cell proliferation, differenti- sociated patterns of force generation. Over the lifecycle,
doi: 10.1210/en.2013-1938
2209
olysis for its energy supply (39). The

MyHC and TnC isotypes and the
contents of Pval and mCK are also
major determinants of these contrasting contractile properties of the
different skeletal muscle types (40
42). Therefore, it is usually accepted
that changes in the somatic growth
of fish are mainly attributable to
changes in the total white muscle
growth. In the white muscle of fish, it
has been shown that such changes in
muscle cellularity are linked to
changes in environmental factors,
such as rearing temperature (43), photoperiod (44), endurance exercise
(41), and nutritional conditions (24).
Because a large portion of the body
mass of teleosts is skeletal muscle, an
Figure 6. Body weight and oxygen consumption measurements. A, Body weights of 15-monthalteration in the metabolic rates of
old nontransgenic control crucian carp (con) and IGF-1 transgenic crucian carp (tg). B, Oxygen
consumption rates of 15-month-old nontransgenic control crucian carp (con) and IGF-1
skeletal muscle tissue by IGF-1 would
transgenic crucian carp (tg). The data are presented as the means SE; n 6.
definitely have a great impact on the
metabolism status of the organism.
there are marked changes in the myotomal structure and
The modulation of the muscle mitochondrial content
function associated with an increase in body mass. The begins early in the embryonic development, when the musaxial skeletal muscle mass in most adult teleosts represents cle precursors differentiate and diverge to form distinct
more than 60% of the body mass (37), whereas skeletal fiber types. The metabolic regulation of enzymes allows
muscle only constitutes approximately 30% 40% of the cells to use the existing pool of mitochondria to match
body mass in mammals (38). This large portion of adult their energy production to the energy demand. When
body mass in fish is also an important ancillary metabolic changes in the energy demands persist for long periods,
function in which sarcomeric proteins constitute a reser- most cells can respond by modifying their rates of mitovoir of amino acids that can be mobilized as an energy chondrial biogenesis to induce compensatory changes in
source (37). On the other hand, fast and slow muscles are the mitochondrial capacity. The muscle mitochondrial
2 major types of skeletal muscle in teleosts based on their content remains plastic throughout adulthood, ie, it inshortening velocity. Skeletal muscles in most adult teleosts creases in response to hypermetabolic conditions and deconsist mainly of deep fast white fibers (up to 90%) that creases during periods of reduced activity. Aerobic meare covered by a thin superficial layer of slow red muscle tabolism depends on the capacity of mitochondria to
fibers located under the skin. Fast muscle works mainly in generate ATP at rates that are sufficient to meet the eneranaerobic metabolism and generates a short but strong getic demands of tissue. Because the mitochondrial concontraction force. Slow muscle generates a slow twitch tents of red and white muscles of fish differ by almost
and continuous contraction over a long period. The slow 10-fold and the ratios of mitochondrial enzymes are
red muscle fibers contain abundant mitochondria and de- nearly identical (45), marked changes in the mitochonliver maximum power at the low tail-beat frequencies as- drial content require exquisite coordination of hundreds
sociated with sustained swimming behavior (24). Red or of different genes located in the nucleus and genes encoded
slow muscle is characterized by a high Mb content, a well- by mitochondrial DNA. The synthesis of the appropriate
developed blood supply, a low myofibrillar ATPase activ- amounts of mitochondrial proteins also requires the coity, numerous mitochondria, and high activities of the en- ordination of protein synthesis in both the cytoplasm and
zymes involved in the tricarboxylic acid cycle and the mitochondria. As shown through our transcriptomics
electron transport chain. Red muscle is thought to have an analyses, the total level of the transcripts of mitochondrial
active aerobic metabolism that utilizes carbohydrates, lip- components increases proportionally in IGF-1 transgenic
ids, and even amino acids for fuel. In contrast, white or fast muscle (Figure 3C). The expression level of the master
muscle appears to depend largely on anaerobic glycogen- transcriptional regulator for mitochondrial biogenesis,
2210
Li et al
peroxisomal proliferator-activated receptor coactivator-1 (pgc-1a) (45), is also significantly increased in

IGF-1 transgenic fish muscle (Table 1). In addition, our
experiment also demonstrated elevated oxygen consumption rates in IGF-1 transgenic crucian carp (Figure 6). All
of these differences may be responses to the cellularity
change in skeletal myocytes caused by IGF-1 overexpression in the skeletal muscle of transgenic fish.
The mechanisms involved in the coordinated regulation of the different muscle structural types and metabolic
programs in teleosts are largely unknown. Theoretically,
fast muscle appears to largely depend on anaerobic glycogenolysis for its energy supply, whereas red muscle relies
largely on mitochondrial fuel oxidation for ATP oxidation
(25). In particular, compared with terrestrial animals, the
concentration of protein/amino acids and fats required to
meet the energy requirements of fish is considered high
(46). As shown by our RNA-seq results, the expression
levels of the critical genes involved in glycogenolysis and
gluconeogenesis are down-regulated in IGF-1 transgenic
fish muscle. Moreover, the transcriptional levels of the key
molecules involved in fatty acid and amino acid metabolism were found to be significantly increased (Table 1 and
Figure 4). Because they are raised in the same tank as their
nontransgenic control fish, the IGF-1 transgenic crucian
carp should face a similar daily energy need for swimming
and predation. Therefore, the IGF-1 transgenic fish may
require more amino acids/proteins and fatty acids for fuel
compared with control fish. It is known that skeletal muscle is a major site of metabolic activity and the most abundant tissue in the fish body (60% of the total body mass).
As the largest protein reservoir, fish muscle serves as a
major source of amino acids that are used for energy production by the body during catabolic periods (47). Thus,
the total transcriptional levels of all ubiquitin and proteasome components were significantly increased in IGF-1
transgenic fish muscle (Figure 3C), and the IGF-1 transgenic muscle also exhibited elevated levels of many molecules involved in protein degradation, such as bnip3l,
casp3b, and shfm1 (Table 1 and Figure 4). It was previously reported that IGF-1 is a key molecule in the prevention of muscle atrophy in fish and other animals (8, 48).
We also observed that the expression levels of the typical
molecules associated with muscle atrophy, namely
foxO1a and fbx32, are down-regulated by IGF-1 overexpression (Table 1 and Figure 4). Based on fish myogenic
cell culture, the function of IGF-1 on myostatin suppression has also been suggested (9). However, the level of
myostatin transcripts was increased significantly in our
IGF-1-overexpressing transgenic fish skeletal muscle sample (Table 1 and Figure 4). We hypothesize that all of these
unexpected muscle-wasting phenomena observed in IGF-
1-overexpressing fish may be due to irregular fuel demands from the active hyperplasia and the shifting from
fast to slow muscle in transgenic crucian carp muscle. In
fact, although an enlarged skeletal muscle mass has been
observed in certain body parts of some IGF-1 transgenic
mammals (5, 49), no significant increase in body weight
has been found in IGF-1-overexpressing animals (48, 49).
This finding may be associated with the similar phenomenon resulting from the energy metabolism caused by muscle cellularity changes. Because the skeletal muscle mass in
fish accounts for a higher proportion of their whole body
weight (60%) than other mammalian model animals
(40%) and because fish exhibit a poor carbohydrate
utilization capacity, our IGF-1 transgenic fish may have to
provide a markedly higher amount of energy from amino
acids/proteins and/or fatty acids to meet the requirements
caused by the skeletal muscle cellularity shift. Elevated
expression levels of the transcripts involved in the oxidative metabolism of fatty acids and BCAAs and the protein
degradation process were observed (Figure 3C, Table 1,
and Figure 4). Actually, the portion of BCAAs in the free
amino acid pool of the transgenic fish is also significantly
increased compared with that from the control fish (Supplemental Table 4).
Our current IGF-1-overexpression transgenic crucian
carp provides the first teleost model for the study of the
functions of IGF-1 in vivo. Our results confirmed the mitogenic effects of IGF-1 in fish skeletal muscle growth. We
also demonstrated the role of fish IGF-1 in the regulation
of muscle differentiation. However, somatic growth is
highly dependent on food intake and composition and the
assimilation of nutrients. The in vivo model presented in
this manuscript provides a method for the integration of
all of the effects of IGF-1 on cellular and molecular interactions during growth under traditional physiological
conditions. Our study demonstrated that the overall phenotypes in our IGF-1 transgenic fish may be the result of
the effects of multiple intrinsic factors, such as endocrine/
autocrine signals, the flow of nutrients, the rates of metabolic processes, and energy budgets.
Acknowledgments
Address all correspondence and requests for reprints to: Zhan
Yin, Key Laboratory of Aquatic Biodiversity and Conservation
of the Chinese Academy of Sciences, Institute of Hydrobiology,
Chinese Academy of Sciences, Wuhan, Hubei, China E-mail:
zyin@ihb.ac.cn.
This study was supported by funds obtained from National
Natural Science Foundation of China (no. 30925027) (to Z.Y.),
the National Basic Research Program of China (973 Program;
doi: 10.1210/en.2013-1938
2010CB126302) (to Z.Y.), and the National Basic Research Program of China (973 Program; 2009CB118701) (to J.Y.).
Author Contributions: D.L., Q.L., G.Z., X.P., X.C., X.D.,
Z.Z., and G.S. performed the transgenic work. X.J. performed
the histologic analysis. X.C. performed the transcriptomics analysis. D.H. performed the oxygen consumption measurements.
J.H. performed the data analysis and interpretation. Z.Y. assisted and supervised the research team and wrote the paper.
Disclosure Summary: The authors have nothing to disclose.
16.
17.
18.
19.
References
1. Duan C, Ren H, Gao S. Insulin-like growth factors (IGFs), IGF
receptors, and IGF-binding proteins: Roles in skeletal muscle growth
and differentiation. Gen Comp Endocrinol. 2010;167(3):344 351.
2. Baker J, Liu JP, Robertson EJ, Efstratiadis A. Role of insulin-like
growth-factors in embryonic and postnatal growth. Cell. 1993;
75(1):73 82.
3. Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice
carrying null mutations of the genes encoding insulin-like growth
factor-I (Igf-1) and type-1 IGF receptor (Igf1r). Cell. 1993;75(1):
59 72.
4. Mathews LS, Hammer RE, Behringer RR, et al. Growth enhancement of transgenic mice expressing human insulin-like growth factor
I. Endocrinology. 1988;123(6):28272833.
5. Coleman ME, DeMayo F, Yin KC, et al. Myogenic vector expression
of insulin-like growth factor I stimulates muscle cell differentiation
and myofiber hypertrophy in transgenic mice. J Biol Chem. 1995;
270(20):12109 12116.
6. Castillo J, Codina M, Martnez ML, Navarro I, Gutierrez J. Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of
rainbow trout. Am J Physiol Regul Integr Comp Physiol. 2004;
286(5):R935R941.
7. Daz M, Vraskou Y, Gutierrez J, Planas JV. Expression of rainbow
trout glucose transporters GLUT1 and GLUT4 during in vitro muscle cell differentiation and regulation by insulin and IGF-I. Am J
Physiol Regul Integr Comp Physiol. 2009;296(3):R794 R800.
8. Cleveland BM, Weber GM. Effects of insulin-like growth factor-I,
insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes. Am J Physiol Regul Integr
Comp Physiol. 2010;298(2):R341R350.
9. Garikipati DK, Rodgers BD. Myostatin inhibits myosatellite cell
proliferation and consequently activates differentiation: evidence
for endocrine-regulated transcript processing. J Endocrinol. 2012;
215(1):177187.
10. Bower NI, Johnston IA. Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and
differentiating myogenic cells. Am J Physiol Regul Integr CompPhysiol. 2010;298(6):R1615R1626.
11. Ju BS, Chong SW, He JY, et al. Recapitulation of fast skeletal muscle
development in zebrafish by transgenic expression of GFP under the
mylz2 promoter. Dev Dyn. 2003;227(1):14 26.
12. Yang Y, Xie SQ, Cui YB, Zhu XM, Lei W, Yang YX. Partial and
total replacement of fishmeal with poultry by-product meal in diets
for gibel carp, Carassius auratus gibelio Bloch. Aquaculture Res.
2006;37(1):40 48.
13. Li X, Nie F, Yin Z, He J. Enhanced hyperplasia in muscles of transgenic zebrafish expressing Follistatin1. Sci China Life Sci. 2011;
54(2):159 165.
14. Mei Y, Yin N, Jin X, He J, Yin Z. The regulatory role of the adrenergic agonists phenylephrine and isoproterenol on fetal hemoglobin
expression and erythroid differentiation. Endocrinology. 2013.
15. Nithart M, Alliot E, Salen-Picard C. Production, respiration and
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
2211
ammonia excretion of two polychaete species in a north Norfolk

saltmarsh. J Marine Biol Assoc UK. 1999;79(6):1029 1037.
Wilson GJ, Layman DK, Moulton CJ, et al. Leucine or carbohydrate
supplementation reduces AMPK and eEF2 phosphorylation and extends postprandial muscle protein synthesis in rats. Am J Physiol
Endocrinol Metab. 2011;301(6):E1236 E1242.
Monnier D, Boutillier AL, Giraud P, et al. Insulin-like growth-factor-I stimulates c-fos and c-jun tRanscription in PC12 cells. Mol Cell
Endocrinol. 1994;104(2):139 145.
Jhun BH, Haruta T, Meinkoth JL, et al. Signal transduction pathways leading to insulin-induced early gene induction. Biochemistry.
1995;34(25):7996 8004.
Dupont J, Khan J, Qu BH, Metzler P, Helman L, LeRoith D. Insulin
and IGF-1 induce different patterns of gene expression in mouse
fibroblast NIH-3T3 cells: Identification by cDNA microarray analysis. Endocrinology. 2001;142(11):4969 4975.
Zapf J, Schmid C, Guler HP, et al. Regulation of binding proteins for
insulin-like growth-factors (IGF) in humans. Increased expression of
IGF binding protein 2 during IGF I treatment of healthy adults and
in patients with extrapancreatic tumor hypoglycemia. J Clin Invest.
1990;86(3):952961.
Megeney LA, Kablar B, Garrett K, Anderson JE, Rudnicki MA.
MyoD is required for myogenic stem cell function in adult skeletal
muscle. Genes Dev. 1996;10(10):11731183.
Sabourin LA, Rudnicki MA. The molecular regulation of myogenesis. Clin Genet. 2000;57(1):16 25.
Asakura A, Seale P, Girgis-Gabardo A, Rudnicki MA. Myogenic
specification of side population cells in skeletal muscle. J Cell Biol.
2002;159(1):123134.
Alami-Durante H, Medale F, Cluzeaud M, Kaushik SJ. Skeletal muscle growth dynamics and expression of related genes in white and red
muscles of rainbow trout fed diets with graded levels of a mixture of
plant protein sources as substitutes for fishmeal. Aquaculture. 2010;
303(1 4):50 58.
Fluck M, Hoppeler H. Molecular basis of skeletal muscle plasticity
from gene to form and function. Rev Physiol Biochem Pharmacol.
2003;146:159 216.
van Deursen J, Heerschap A, Oerlemans F, et al. Skeletal muscles of
mice deficient in muscle creatine kinase lack burst activity. Cell.
1993;74(4):621 631.
Greenwood MR. The relationship of enzyme-activity to feedingbehavior in ratslipoprotein-lipase as the metabolic gatekeeper. Int
J Obes. 1985;9(Suppl 1):6770.
Lampidonis AD, Rogdakis E, Voutsinas GE, Stravopodis DJ. The
resurgence of hormone-sensitive lipase (HSL) in mammalian lipolysis. Gene. 2011;477(12):111.
Muoio DM, Neufer PD. Lipid-induced mitochondrial stress and
insulin action in muscle. Cell Metab. 2012;15(5):595 605.
Hopwood NJ, Holzman I, Drash AL. Fructose-1,6-diphosphatase
deficiency. Am J Dis Child. 1977;131(4):418 421.
Sola-Penna M, Da Silva D, Coelho WS, Marinho-Carvalho MM,
Zancan P. Regulation of mammalian muscle type 6-phosphofructo1-kinase and Its implication for the control of the metabolism.
Iubmb Life. 2010;62(11):791796.
Newgard CB. Interplay between lipids and branched-chain amino
acids in development of insulin resistance. Cell Metab. 2012;15(5):
606 614.
Stoiber W, Haslett JR, Wenk R, Steinbacher P, Gollmann HP, Sanger AM. Cellularity changes in developing red and white fish muscle
at different temperatures: simulating natural environmental conditions for a temperate freshwater cyprinid. J Exp Biol. 2002;205
(Pt 16):2349 2364.
Hernandez LP, Patterson SE, Devoto SH. The development of muscle fiber type identity in zebrafish cranial muscles. Anat Embryol.
2005;209(4):323334.
Rosa CE, Figueiredo MA, Lanes CFC, Almeida DV, Monserrat JM,
Marins LF. Metabolic rate and reactive oxygen species production
2212
36.
37.
38.
39.
40.
41.
42.
Li et al
in different genotypes of GH-transgenic zebrafish. Comp BiochemPhysiol B Biochem Mol Biol. 2008;149(1):209 214.
Fried SK, Watford M. Leucing weight with a futile cycle. Cell Metab.
2007;6(3):155156.
Johnston IA, Bower NI, Macqueen DJ. Growth and the regulation
of myotomal muscle mass in teleost fish. J ExpBiol. 2011;214
(Pt 10):16171628.
Velloso CP. Regulation of muscle mass by growth hormone and
IGF-I. Br J Pharmacol. 2008;154(3):557568.
Johnston IA, Davison W, Goldspink G. Energy-metabolism of carp
swimming muscles. J Comp Physiol. 1977;114(2):203216.
Elworthy S, Hargrave M, Knight R, Mebus K, Ingham PW. Expression of multiple slow myosin heavy chain genes reveals a diversity of
zebrafish slow twitch muscle fibres with differing requirements for
Hedgehog and Prdm1 activity. Development. 2008;135(12):2115
2126.
van der Meulen T, Schipper H, van den Boogaart JG, Huising MO,
Kranenbarg S, van Leeuwen JL. Endurance exercise differentially
stimulates heart and axial muscle development in zebrafish (Danio
rerio). Am J Physiol Regul Integr Comp Physiol. 2006;291(4):
R1040 R1048.
Seebacher F, Walter I. Differences in locomotor performance between individuals: importance of parvalbumin, calcium handling
and metabolism. J Exp Biol. 2012;215(Pt 4):663 670.
43. Alami-Durante H, Olive N, Rouel M. Early thermal history significantly affects the seasonal hyperplastic process occurring in the
myotomal white muscle of Dicentrarchus labrax juveniles. Cell Tissue Res. 2007;327(3):553570.
44. Johnston IA, Manthri S, Smart A, Campbell P, Nickell D, Alderson
R. Plasticity of muscle fibre number in seawater stages of Atlantic
salmon in response to photoperiod manipulation. J ExpBiol. 2003;
206(Pt 19):34253435.
45. Leary SC, Lyons CN, Rosenberger AG, Ballantyne JS, Stillman J,
Moyes CD. Fiber-type differences in muscle mitochondrial profiles.
Am J Physiol Regul Integr Comp Physiol. 2003;285(4):R817
R826.
46. Kaushik SJ, Seiliez I. Protein and amino acid nutrition and metabolism in fish: current knowledge and future needs. Aquaculture Res.
2010;41(3):322332.
47. Wang J, Salem M, Qi N, Kenney PB, Rexroad CE 3rd, Yao J. Molecular characterization of the MuRF genes in rainbow trout: Potential role in muscle degradation. Comp Biochem Physiol B
Biochem Mol Biol. 2011;158(3):208 215.
48. Shavlakadze T, White JD, Davies M, Hoh JF, Grounds MD. Insulinlike growth factor I slows the rate of denervation induced skeletal
muscle atrophy. Neuromusc Disord. 2005;15(2):139 146.
49. Musaro` A, McCullagh K, Paul A, et al. Localized Igf-1 transgene
expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet. 2001;27(2):195200.
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GROWTH

Hyperplasia and Cellularity Changes in IGF-1Overexpressing Skeletal Muscle of Crucian Carp

GF-1, an anabolic peptide growth factor, plays primary

significant muscle hypertrophy and induced a shift toward

ISSN Print 0013-7227 ISSN Online 1945-7170

Abbreviations: BCAA, branched chain amino acid; CPT1, carnitine palmitoyltransferase-1;

Endocrinology, June 2014, 155(6):2199 2212

Muscle Development in IGF-1 Transgenic Fish

Endocrinology, June 2014, 155(6):2199 2212

(CLONTECH) as the backbone. The fragment containing

Growth evaluations and muscle histologic analysis

Western blot analysis

RNA isolation and preparation for RNA-seq

Sexually mature crucian carp were initially purchased from a

Generation of transgenic crucian carp

The zebrafish mylz2 promoter was previously described by Ju

The 95-base sequences were obtained by sequencing using an

Materials and Methods

Muscle Development in IGF-1 Transgenic Fish

RT-PCR and quantitative real-time PCR

Oxygen consumption measurements

Measurements of free amino acid composition in

Endocrinology, June 2014, 155(6):2199 2212

HiSeq System, and the resulting

Muscle Development in IGF-1 Transgenic Fish

Figure 3. Comparative RNA-seq transcriptomics analyses of crucian

Endocrinology, June 2014, 155(6):2199 2212

cantly in the IGF-1-overexpressing muscle tissue (Figure

Activation of IGF signaling and muscle cell proliferation

Muscle Development in IGF-1 Transgenic Fish

Endocrinology, June 2014, 155(6):2199 2212

Muscle atrophy and apoptosis

CoA, coenzyme A; Con, control; Tg, transgenic.

Figure 4. Real-time RT-PCR confirmation of the representative key

BCAA transaminase has been reported to be one of the key

Muscle Development in IGF-1 Transgenic Fish

Endocrinology, June 2014, 155(6):2199 2212

ation, and hypertrophy and inhibit

olysis for its energy supply (39). The

Muscle Development in IGF-1 Transgenic Fish

peroxisomal proliferator-activated receptor coactivator-1 (pgc-1a) (45), is also significantly increased in

Endocrinology, June 2014, 155(6):2199 2212

ammonia excretion of two polychaete species in a north Norfolk

Muscle Development in IGF-1 Transgenic Fish

Endocrinology, June 2014, 155(6):2199 2212

Members receive free electronic delivery of

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