AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus GmbHGöttingen, Germany10.5194/aab-58-33-2015Breed-specific lipid-related gene expression in the subcutaneous fat of
Large White and Erhualian pigs at weaningZhengY.PanS.HuangY.CiL.ZhaoR.YangX.yangxj@njau.edu.cnKey Laboratory of Animal Physiology and Biochemistry, Nanjing Agricultural
University, Nanjing, ChinaX. Yang (yangxj@njau.edu.cn)4March2015581334122November201324September2014This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/58/33/2015/aab-58-33-2015.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/58/33/2015/aab-58-33-2015.pdf
The Erhualian (EHL) pig possesses significantly lower growth rates and
higher adipose deposition compared with the Large White (LW) pig. To further
understand the mechanism of breed lipid deposition difference at the early
postnatal age, we employed an animal model of EHL and LW pigs at weaning age to
compare the lipid metabolism differences in subcutaneous fat. The result
showed that serum triglyceride in EHL was significantly higher
(P < 0.05) than that of LW. Peroxisome proliferator-activated receptor-γ
protein level in EHL was significantly higher (P < 0.01) though CCTTA
enhancer-binding protein level demonstrated no change compared with LW pigs.
Hormone sensitive lipase, adipose tissue triglyceride lipase mRNA expression
and the lipase activity were significantly lower (P < 0.05) in EHL.
Uncoupling protein-2 protein content was significantly lower (P < 0.01) in EHL than that in LW pigs. We first cloned the nucleotide sequence
of Zinc-α2-glycoprotein (ZAG) with 1090 bp and found that both ZAG
mRNA expression and protein level in EHL pigs was significantly lower
(P < 0.01) than that of LW pigs. β3 adrenergic receptor mRNA
expression in EHL pigs was significantly higher (P < 0.01) than that of LW
pigs, though tumour necrosis factor α gene expression demonstrated
no significant difference. Therefore, the significant breed lipid metabolism
difference in subcutaneous fat exists at an early postnatal age
between EHL and LW pigs, and this difference may originate from two causes
including the increased lipid synthesis and reduced lipid mobilization in
EHL pigs compared with LW pigs.
Introduction
In recent years, an epidemic of obesity has been observed around the world.
Adipose metabolism has received much attention because substantial adipose
deposition is related to many metabolic diseases such as diabetes,
hypertension, etc. Furthermore, it was estimated that the genetic basis for
obesity could approximately range from 40 to 70 % (Singla et al., 2010).
The pig is one of the most important domestic species for meat production
and is also an ideal model for metabolic studies including obesity (Spurlock
et al., 2008). Concerning the breed specific adipose metabolism, though there have
been some reports demonstrating the breed differences (Quintanilla et al.,
2002;
Li et al., 2008a, b; Nakajima et al., 2011; Wei et al., 2011), most of them were
interested in the pigs' metabolism at the finishing period. Actually, early
life is a critical period for body lipid deposition (Wells, 2007). After
birth, the adipose tissue expansion takes place rapidly as a result of
increased fat cell size as well as an increase in fat cell number (Gregoire
et al., 1998). Therefore, understanding the adipose metabolism at the early
postnatal stage is also decisive for clarifying the breed difference of lipid
deposition in pigs.
Fat metabolism mainly includes adipogenesis and lipolysis. CCTTA
enhancer-binding protein (C/EBPβ) and peroxisome
proliferator-activated receptor-γ (PPARγ) are
well-established key transcription factors for adipogenesis. PPARγ
is highly expressed and essentially required for the formation,
differentiation and survival of adipose tissue in vivo (Koh et al., 2009).
C/EBPβ is an important factor that causes adipogenesis and plays a
dual role as a stimulator of cell determination and differentiation
(Darlington et al., 1998). Lipolysis depends most on adipose tissue triglyceride
lipase (ATGL) and hormone sensitive lipase (HSL). ATGL could catalyse the
initial step of the triglyceride's hydrolysis (Schweiger et al., 2006). HSL is
highly expressed in adipocytes, and lipolysis is influenced by its
protein content, activity and the distribution in the cells (Granneman et al., 2007). For the fat utilization, carnitine palmitoyl transferase-I (CPT1) is
a limited enzyme of fatty acid β-oxidation and uncoupling protein-2
(UCP2) plays a critical role in the biological traits of animal body weight,
basal metabolic rate and energy conversion (Li et al., 2007). Whether these lipid
metabolism genes play a role in breed differences at early postnatal age in
pigs is not clear.
In the past 20 years, it has been recognized that adipose tissue produces a large
number of cytokines which are termed “adipokines”. These adipokines have
an autocrine function on adipose tissue homeostasis and they are secreted
into the peripheral circulation with a systemic effect on metabolic events
(Guerre-Millo, 2004; Fantuzzi et al., 2005). Recently, observations demonstrated
that Zinc-α2-glycoprotein (ZAG) exists in both white and brown
adipose tissue (Bing et al., 2004; Mracek et al., 2010) and the expression of ZAG is
inversely related to the mass of white adipose tissue (Dahlman et al., 2005; Rolli
et al., 2007). It has been found that ZAG could bound to β3 adrenergic receptors
(β3-AR) and could activate GTP-dependent adenylate cyclase to elevate
intracellular cAMP level and HSL activity (Russell et al., 2002, 2004).
Additionally, tumour necrosis factor α (TNFα) causes a
dose-dependent decrease of ZAG expression (Bao et al., 2005). However, until
now,
studies on ZAG have been restricted to humans and rats, and no results have been observed
in pigs. Whether it is expressed in adipose tissue of pigs and whether it is
differently expressed in the two breeds is unknown.
Erhualian (EHL), a famous Chinese indigenous pig breed, belongs to the same
family as Meishan, which is characterized by high reproductive rates and
superior meat quality. Compared with Large White pigs (LW), EHL pigs
possess significantly lower growth rate and higher adipose deposition. Though
previous studies have shown the cellular difference of adipocyte in
subcutaneous fat (Nakajima et al., 2011) and the quantitative trait locus (QTL)
analysis of growth and fatness data between growing Landrace and Meishan
pigs (Quintanilla et al., 2002; Wei et al., 2011), the adipose metabolism difference at
the early postnatal stage is still largely unclear between these two breeds.
Therefore in the present study we employed LW and EHL pigs as animal models
and detected the expression of lipogenesis-related genes, including
PPARγ and C/EBPβ, lipolysis-related genes, including HSL and
ATGL, fatty acid oxidation-related genes, including CPT1 and UCP2 and the
adipokine ZAG, to compare the difference in subcutaneous fat between LW
and EHL pigs at weaning age. The results will shed new light on the difference in lipid metabolism of different pig breeds.
Material and methods
Six male piglets from three litters (two from each litter) of purebred LW
and EHL sows from two neighbouring pig breeding farms in Changzhou, Jiangsu
Province, China, were slaughtered at 25 days of age just before weaning. The
subcutaneous fat was taken and immediately placed into liquid nitrogen and
stored at -80 ∘C. The slaughter and sampling procedures
complied with the “Regulation regarding the Management and Treatment of
Experimental Animals” (2008) no. 45 set by Jiangsu Provincial People's
Government. The experiment protocol was specifically approved by the Animal
Ethics Committee of Nanjing Agricultural University.
RNA extraction and reverse transcription (RT)
Total RNA was extracted using Trizol Plus RNA Purification Kit (Invitrogen,
Thermo Fisher Scientific Inc., Waltham, MA, USA). Concentration of the
extracted RNA was measured using NanoDrop 1000 Spectrophotometer. Ratios of
absorption (260/280 nm) of all preparations were between 1.8 and 2.0. RNA
integrity was confirmed by agarose electrophoresis and DNA contamination was
examined by PCR. Reverse transcription was performed using the total RNA (2 µg)
in a final volume of 25 µL containing 1 × RT-buffer,
100 U Moloney Murine Leukemia Virus reverse transcriptase (M-MLV) (Promega
Corp., Fitchburg, WI, USA), 8 U RNase inhibitor (Promega), 5.3 µmol L-1
random hexamer primers and 0.8 µmol L-1 dNTP (TaKaRa Bio Inc., Otsu,
Shiga, Japan).
PCR
PCR was performed in Gene Amp PCR system (PerkinElmer Inc., Waltham, MA,
USA). 2 µL RT product was used for general PCR in a final volume of
50 µL containing 2.5 U LA Taq (TaKaRa, Japan), 5 µL 10 × LA
PCR Buffer, 400 µM dNTPs, 36.5 µL ddH2O and 0.2 µM
forward and reverse primers for ZAG (forward primes:
5'-CCCAGCCATTCCCATAACTA-3', reverse primers: 5'-CCCAGTGCTGGCACTCTTCT-3'),
using a program of 35 cycles (30 s at 95 ∘C, 30 s at 58 ∘C, 30 s at 72 ∘C)
and a final step of 10 min at 72 ∘C. The obtained PCR products were analysed on 2 % agarose gel
and purified with an AxyPrep DNA Gel Extraction Kit (Axygen Scientific,
Inc., Hangzhou, China) and then sequenced.
Quantitative PCR
Quantitative PCR was performed with Mx3000P (Stratagene, Agilent
Technologies, Inc., Santa Clara, CA, USA). Mock RT and no template controls
(NTC) were set to monitor the possible contamination of genomic DNA both at
RT and PCR. The pooled sample made by mixing equal quantities of total RT
products (cDNA) from all samples was used to optimize PCR conditions and
tailor the standard curves for each target gene. Melting curves were
performed to ensure a single specific PCR product for each gene. 2 µL
of the eightfold dilution of RT product were used for RT-PCR in a final
volume of 25 µL containing 12.5 µL SYBR Green Real-time PCR Master
Mix (TaKaRa, Japan) and 0.4 µM of each forward and reverse primers for
ZAG, HSL, PPARγ, C/EBPβ, TNFα, β3-AR, ATGL,
UCP2 and CPT1 genes (sequences of primers were shown in Table 1). All samples
were normalized with the reference gene 18S rRNA. The PCR products for each
gene were sent for sequencing to verify accuracy (Haojia Biotech,
Ltd., Shanghai, China). The reported sequences exactly matched those
published in GenBank. The method of 2-Ct was used to analyse the
real-time PCR data. All samples were included in the same run of RT-PCR and
repeated in triplicates.
Protein extraction and western blot analysis
Total protein was extracted from 500 mg of frozen subcutaneous fat in 1 mL
lysis buffer (15 0 mmol L-1 NaCl,
10 mmol L-1 Tris-HCl, 5 mmol L-1 EDTA, 1 %
Triton X-100 and 0.1 % SDS (Sinopharm Chemical Reagent Co., Ltd.,
Shanghai, China). The protease inhibitor cocktail (Roche Applied Science)
was added according to the manufacturer's instructions. The protein
concentration was measured with the BCA Protein Assay Kit (Pierce, Rockford,
IL, USA) according to the manufacturer's instructions. Thirty micrograms of
protein extract were used for electrophoresis on a 12 % SDS-PAGE gel.
western blot analyses for ZAG (13399-1-AP, Proteintech Group, USA, diluted
1:5000), C/EBPβ (sc-150x, Santa Cruz, USA, diluted 1:2000), UCP-2
(11081-1-AP, Proteintech group, USA, diluted 1:5000) and PPARγ
(BS1587, Bioworld, China, diluted 1:1000) were carried out according to the
recommended protocols provided by the manufacturers. β-actin (AP0060,
Bioworld, USA, diluted 1:6000) was used as a reference in the western blot
analyses.
Lipase (including HSL and ATGL) activity assay
500 mg frozen subcutaneous fat in 1 mL homogenization buffer (0.1 mmol L-1
K+-PBS containing 1 mmol L-1 MgCl2, 1 mmol L-1 DTT and 1 mmol L-1 EDTA)
was homogenized in ice for 30 min and then centrifuged for 10 min at
12 000 ×g at 4 ∘C. The protein concentration of
supernatants was measured with the BCA Protein Assay Kit (Pierce Chemical
Corp., Rockford, USA). Triolein without glycerin was used as substrate which
can be hydrolysed to glycerol by the two enzymes, HSL and ATGL. The
supernatants together with prepared triolein were incubated for 1 h at 37 ∘C.
The lipases in supernatants activate the lipolytic
degradation of the triolein emulsion. The released glycerol was measured
with a commercial kit (Applygen, China). To fit the activities of the
enzymes to the linear range of standard curves constructed with pure
enzymes, all samples were measured in duplicates at appropriate dilutions.
The activity of lipases is expressed as nanomoles released glycerol per
milligram protein per hour.
All data are presented as mean ± standard error. Statistical analyses were
carried out with SPSS 13.0 for Windows. The differences were analysed using
a t test for independent samples. A P value of less than 0.05 was considered
to be significant.
mRNA and protein expression of genes involved in adipogenesis in the
subcutaneous fat of LW and EHL pigs.
(a) PPARγ mRNA expression, (b) C/EBPβ mRNA expression, (c)
PPARγ protein expression, (d) C/EBPβ protein expression.
The values shown represent the mean ± standard error, n=6; **P < 0.01
vs. LW.
ResultsBody weight and serum triglyceride concentration between LW and
EHL pigs
The body weight of LW and EHL pigs at weaning age was 8.10 ± 0.34
and 4.08 ± 0.25 kg, respectively. The serum triglyceride content of LW
and EHL pigs was 0.29 ± 0.03 and 0.62 ± 0.12 mmol L-1,
respectively. Both the body weight and serum triglyceride concentration
demonstrated a significant breed difference (P < 0.05).
Lipogenesis- and lipolysis-related gene expression in the
subcutaneous fat between LW and EHL pigs
Though C/EBPβ mRNA expression was significantly lower (P < 0.01) in the subcutaneous fat of EHL than in that of LW pigs; the protein
expression demonstrated no difference. PPARγ mRNA expression showed
no difference between the two breeds, while its protein level was
significantly higher (P < 0.01) in the subcutaneous fat of EHL than in
that of LW pigs (Fig. 1).
The mRNA expression of HSL and ATGL was significantly lower (P < 0.01)
in the subcutaneous fat of EHL than in that of LW pigs (Fig. 2). The
lipases (including HSL and ATGL) activity was also significantly lower
(P < 0.05) in the subcutaneous fat of EHL than in that LW pigs
(Fig. 2). mRNA expression of CPT1 and UCP2, two key genes involved in fatty acid
oxidation, was significantly lower (P < 0.05) in the subcutaneous
fat tissue of EHL than that of LW pigs. Furthermore, UCP2 protein level was
also significantly lower (P < 0.01) in the subcutaneous fat of EHL
than that of LW pigs (Fig. 3).
mRNA and protein expression of genes involved in lipolysis and lipases
activity in the subcutaneous fat of LW and EHL pigs
The values shown represent the mean ± standard error, n=6; *P < 0.05 vs.
LW; **P < 0.01 vs. LW.
(a) HSL mRNA expression, (b) ATGL mRNA expression and (c) Lipases activity (HSL
and ATGL).
mRNA and protein expression of genes related to energy metabolism in the
subcutaneous fat of LW and EHL pigs.
(a) CPT1 mRNA expression, (b) UCP2 mRNA expression and (c) UCP2 protein expression.
The values shown represent the mean ± standard error, n=6; *P < 0.05
vs. LW; **P < 0.01 vs. LW.
Porcine ZAG gene sequencing and its mRNA and protein expression between LW
and EHL pigs.
(a) Homology analysis, (b) porcine ZAG cDNA sequence, (c) ZAG mRNA
expression and (d) ZAG protein expression.
The values shown represent the mean ± standard error, n=6; **P < 0.01
vs. LW.
mRNA expression of TNFα and β3-AR in subcutaneous fat of LW
and EHL pigs.
(a) TNFα mRNA expression and (b)β3-AR mRNA
expression.
The values shown represent the mean ± standard error, n=6; **P < 0.01
vs. LW.
Porcine ZAG gene sequencing and expression
In the present study, we cloned the nucleotide sequence containing the
complete open reading frame (ORF) of porcine ZAG with 1090 bp. The ORF
sequences are conservative between species. MegAlign software (DNASTAR,
Inc., Madison, WI, USA) was used to compare the homology of ZAG. The results
showed that porcine ZAG gene nucleotide sequence possesses 82.2 %
homology with cattle and 69.5 % homology with mice, respectively. Both
the mRNA and protein expression of ZAG in the subcutaneous fat of EHL was
significantly lower (P < 0.01) than that of LW pigs (Fig. 4).
TNFα, β3-AR mRNA expression
in the subcutaneous fat between LW and EHL pigs
The expression of β3-AR mRNA in subcutaneous fat of EHL pigs was
significantly higher (P < 0.01) than that of LW pigs. No difference was
observed in the expression of TNFα between the two breeds of pigs
(Fig. 5).
Discussion
The EHL pig is a famous Chinese indigenous pig breed with high reproductive traits
and meat quality, similar to its close relative the Meishan (MS) pig,
whereas the LW pig is a lean type of pig. Concerning the fat deposition
between these two breeds, previous studies suggested that adipose tissue
development occurs much earlier in MS than in LW pigs (Mourot et al., 1996), and
preadipocyte proliferation also demonstrated the breed difference in vitro
(Gerfault et al., 1999). Recent studies have shown that the quantitative trait locus
(QTL) for the lipid metabolic characteristics is in both two breeds located
on chromosome 7 (Demars et al., 2007; Gondret et al., 2012). In the present study, we
demonstrated that though the body weight of the EHL pig is only half of LW
pigs, the serum triglyceride level in EHL pigs is significantly higher
compared with LW pigs. Additionally, a significant difference was also observed
in both adipogenesis- and lipolysis-related gene expressions between EHL and
LW piglets at weaning age.
Regarding the adipogenesis, C/EBPβ and PPARγ are
well-established key transcription factors. PPARγ could stimulate
the differentiation from preadipocytes into mature adipocytes and is closely
associated with fat formation. PPARγ-deficient mice exhibited
smaller adipocytes and decreased fat mass compared with wild mice
(Kubota et al., 1994). C/EBPβ is also known to directly influence
adipocytes' development (Rosen et al., 2000). It could lead preadipocytes
to the
proliferative stage and activates PPARγ to promote the
differentiation of adipocytes (Devine et al., 1999). C/EBPβ also could make
other cells turn into adipocytes (Wu et al., 1995). In the present study, though
the mRNA expression of C/EBPβ decreased, the protein concentration
demonstrated no significant difference. The protein level of PPARγ
was significantly higher in EHL than in LW pigs. PPARγ could regulate
the expression of lipid metabolism-related genes to promote the synthesis of
triglycerides in adipocytes and could cause the cell volume increase
(Vidal-Puig et al., 1997). Additionally, PPARγ could also inhibit the
expression of genes involved in lipolysis and fatty acid release (Prusty et al., 2002). From our study we infer that EHL pigs may possess greater
capability for adipogenesis.
For the lipolysis, the mobilization of triglyceride in fat cells is mainly
dependent on HSL and ATGL. ATGL could hydrolyse triglycerides and HSL could
hydrolyse both triglycerides and diglycerides. Both of them are the limited
enzymes of triglyceride hydrolysis process. The present study showed that
EHL pigs had significantly lower HSL and ATGL mRNA expression than LW pigs, and
the lipase activity of the EHL pig was also lower than that of the LW pigs.
The results showed that EHL pigs lesser capability for lipolysis than LW
pigs. CPT is a limited enzyme of fatty acid β-oxidation. Intensive
research has revealed a critical role for UCP in the control of mitochondrial
reactive oxygen species production and in various physiological events such
as obesity and diabetes (Rosen et al., 2000; Zhang et al., 2001). Elevated UCP
expression could promote the utilization of the free fatty acids and could
translate their energy into heat production (Ceperuelo-Mallafré et al., 2009).
In the present study, the mRNA expressions of CPT1 and UCP2 were significantly lower in the subcutaneous fat of EHL than in that of LW pigs. The level of
UCP2 protein decreased significantly in EHL compared with LW pigs. It seems
that the breed difference of lipid deposition has existed at early postnatal
age. Similar to breed lipid metabolism differences at the finishing stage (Li
et al., 2008a, b), the two breeds demonstrated a big difference in lipid
metabolism at weaning age. EHL pigs possess significantly greater
capability for adipogenesis and the lesser capability for lipolysis and utilization
compared with LW pigs.
ZAG is newly defined as a kind of adipokine. A previous study has
demonstrated that elevated ZAG could decrease blood glucose, triglyceride
and free fatty acid levels in mice compared with the control group
(Ceperuelo-Mallafré et al., 2009). Furthermore, there is a clear correlation
between ZAG content and body weight reduction (Todorov et al., 1998). Both body
weight and epididymal fat were reduced in ZAG overexpressive mice (Gong et al., 2009). An injection of human ZAG could increase over-fed mice's utilization
of sugar intake, the rate of glucose metabolism and lipid oxidation in
heart, brain and brown fat tissue (Russell and Tisdale, 2002). There is
growing evidence pointing to ZAG as a potential therapeutic target for
obesity (Gong et al., 2009; Gao et al., 2010). However, until now, there has been no report on
ZAG in pigs. In the present study, we first cloned a part of porcine ZAG
cDNA sequence including ORF, and the nucleotide sequence homology analysis
showed that porcine ZAG gene possesses the highest homology with cattle
(82.2 %). Furthermore, both mRNA expression (almost 10 times higher) and
protein level (2 times higher) of ZAG were significantly lower in EHL
compared with LW pigs. The present study suggests that ZAG expressed in
porcine adipose tissue, and its expression existed great breed difference at
the early postnatal age. It may directly correlate with the fat deposition
in pigs.
It has been demonstrated that ZAG bounds to β3-AR and activated
GTP-dependent adenylate cyclase in the membrane as well as intracellular
cAMP level and HSL activity (Russell et al., 2002, 2004). The effect of
ZAG can be blocked by β3-AR antagonist SR59230 (Russell et al., 2002). In
the present study, β3-AR mRNA expression in white adipose tissue of
EHL was significantly higher than that of LW pigs. This may be due to the
low content of ZAG. It has been shown that the proinflammatory cytokine
TNFα decreased in a dose-dependent manner after ZAG expression (Bao
et al., 2005), whereas in the present study TNFα gene expression
demonstrated no significant difference between the two breeds. Regardless, it
needs further study to better understand the pathway of ZAG in porcine lipid
metabolism.
In conclusion, EHL and LW pigs demonstrated a significant breed difference
in lipid metabolism at early postnatal age. EHL pigs showed greater
capability for lipid synthesis and lower lipid mobilization compared with LW
pigs. ZAG may be an important adipokine involved in the formation of
breed-specific fat deposition.
Acknowledgements
This study was supported by the National Basic Research Program of China
(2012CB124703), the Special Fund for Agro-scientific Research in the Public
Interest (201003011), the Program for New Century Excellent Talents in
University (NCET-12-0889) and the Priority Academic Program Development of
Jiangsu Higher Education Institutions.
Edited by: K. Wimmers
Reviewed by: two anonymous referees
ReferencesBao, Y., Bing, C., Hunter, L., Jenkins, J. R., Wabitsch, M., and Trayhurn,
P.:
Zinc-α2-glycoprotein, a lipid mobilizing factor, is expressed and
secreted by human (SGBS) adipocytes, FEBS Lett., 579, 41–47, 2005.Bing, C., Bao, Y., Jenkins, J., Sanders, P., Manieri, M., Cinti, S., Tisdale, M. J.,
and Trayhurn, P.: Zinc-α2-glycoprotein, a lipid mobilizing factor,
is expressed in adipocytes and is up-regulated in mice with cancer cachexia,
P. Natl. Acad. Sci. USA, 101, 2500–2505, 2004.Ceperuelo-Mallafré, V., Näf, S., Escoté, X., Caubet, E., Gomez, J. M.,
Miranda, M., Chacon, M. R., Gonzalez-Clemente, J. M., Gallart, L., Gutierrez, C.,
and Vendrell,
J.: Circulating and Adipose Tissue Gene Expression of Zinc-α2-Glycoprotein in Obesity: Its Relationship with Adipokine and Lipolytic
Gene Markers in Subcutaneous and Visceral Fat, J. Clin. Endocrinol. Metab., 94,
5062–5069, 2009.
Dahlman, I., Kaaman, M., Olsson, T., Tan, G. D., Bickerton, A. S. T., Wåhlén, K.,
Andersson, J., Nordström, E. A., Blomqvist, L., Sjögren, A., Forsgren, M.,
Attersand, A., and Arner, P.: A Unique Role of Monocyte Chemoattractant
Protein 1 among Chemokines in Adipose Tissue of Obese Subjects, J. Clin. Endocrinol. Metab., 90,
5834–5840, 2005.
Darlington, G. J., Ross, S. E., and MacDougald, O. A.: The role of C/EBP genes in
adipocyte differentiation, J. Biol. Chem., 273, 30057–30060, 1998.
Demars, J., Riquet, J., Sanchez, M. P., Billon, Y., Hocquette, J. F., Lebret, B.,
Iannuccelli, N., Bidanel, J. P., Milan, D., and Gondret, F.: Metabolic and
histochemical characteristics of fat and muscle tissues in homozygous or
heterozygous pigs for the body composition QTL located on chromosome 7,
Physiol. Genomics, 30, 232–241, 2007.Devine, J. H., Eubank, D. W., Clouthier, D. E., Tontonoz, P., Spiegelman, B. M., Hammer, R.
E., and
Beale, E. G.: Adipose Expression of the Phosphoenolpyruvate Carboxykinase
Promoter Requires Peroxisome Proliferator-activated Receptor γ and
9-cis-Retinoic Acid Receptor Binding to an Adipocyte-specific Enhancer in Vivo, J. Biol. Chem., 274,
13604–13612, 1999.
Fantuzzi, G.: Adipose tissue, adipokines, and inflammation, J. Allergy
Clin. Immunol.,
115, 911–919, 2005.
Gao, D., Trayhurn, P., and Bing, C.: Macrophage-secreted factors inhibit ZAG
expression and secretion by human adipocytes, Mol. Cell Endocrinol., 325,
135–142, 2010.
Gerfault, V., Louveau, I., and Mourot, J.: The Effect of GH and IGF-I on
Preadipocytes from Large White and Meishan Pigs in Primary Culture, Gen.
Comp.
Endocr., 114, 396–404, 1999.
Gondret, F., Riquet, J., Tacher, S., Demars, J., Sanchez, M. P., Billon, Y., Robic, A.,
Bidanel, J. P., and Milan, D.: Towards candidate genes affecting body fatness at
the SSC7 QTL by expression analyses, J. Anim. Breed Genet., 129, 316–324, 2012.Gong, F. Y., Zhang, S. J., Deng, J. Y., Zhu, H. J., Pan, H., Li, N. S., and Shi, Y. F.: Zinc-α2-glycoprotein is involved in regulation of body weight through inhibition
of lipogenic enzymes in adipose tissue, Int. J. Obes., 33, 1023–1030, 2009.
Granneman, J. G., Moore, H. P. H., Granneman, R. L., Greenberg, A. S., Obin, M. S., and Zhu,
Z.:
Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes, J. Biol. Chem., 282,
5726–5735, 2007.
Gregoire, F. M., Smas, C. M., and Sul, H. S.: Understanding Adipocyte
Differentiation,
Physiol. Rev., 78, 783–809, 1998.
Guerre-Millo, M.: Adipose tissue and adipokines: for better or worse,
Diabetes Metab., 30, 13–19, 2004.Koh, Y. J., Park, B. H., Park, J. H., Han, J., Lee, I. K., Park, J. W., and Koh, G. Y.: Activation
of PPARγ induces profound multilocularization of adipocytes in adult
mouse white adipose tissues, Exp. Mol. Med., 41, 880–895, 2009.Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S.,
Nakano, R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, Y., Okuno, A., Murakami, K.,
Sekihara, H., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K.,
Kitamura,
T., Fujita, T., Ezaki, O., Aizawa, S., Nagai, R., Tobe, K., Kimura, S., and Kadowaki,
T.:
PPARγ Mediates High-Fat Diet-Induced Adipocyte Hypertrophy and
Insulin Resistance, Mol. Cell, 4, 597–609, 1994.
Li, M., Li, X., Zhu, L., Jiang, Y., and Tang, G.: [Developmental expression changes
of specific genes in adipose tissue for different pig breeds by using
pathway-focused microarray], Chinese J. Biotech., 24, 665–672, 2008a (in
Chinese).
Li, M., Zhu, L., Li, X., Shuai, S., Teng, X., Xiao, H., Li, Q., Chen, L., Guo, Y., and Wang,
J.: Expression profiling analysis for genes related to meat quality and
carcass traits during postnatal development of backfat in two pig breeds,
Sci. China Ser. C, 51, 718–733, 2008b.
Li, Y., Li, H., Zhao, X., Li, N., and Wu, C.: UCP2 and 3 Deletion Screening and
Distribution in 15 Pig Breeds, Biochem. Genet., 45, 103–111, 2007.Mourot, J., Kouba, M., and Bonneau, M.: Comparative study of in vitro lipogenesis in
various adipose tissue in the growing meishan pig: Comparison with the large
white pig (Sus domesticus), Comp. Biochem. Phys. B, 115, 383–388, 1996.Mracek, T., Ding, Q., Tzanavari, T., Kos, K., Pinkney, J., Wilding, J., Trayhurn, P.,
and Bing,
C.: The adipokine zinc-α2-glycoprotein (ZAG) is downregulated
with fat mass expansion in obesity, Clin. Endocrinol., 72, 334–341, 2010.
Nakajima, I., Oe, M., Ojima, K., Muroya, S., Shibata, M., and Chikuni, K.: Cellularity
of developing subcutaneous adipose tissue in Landrace and Meishan pigs:
Adipocyte size differences between two breeds, Anim. Sci. J., 82, 144–149, 2011.Prusty, D., Park, B. H., Davis, K. E., and Farmer, S. R.: Activation of MEK/ERK
Signaling Promotes Adipogenesis by Enhancing Peroxisome
Proliferator-activated Receptor γ (PPARγ) and C/EBPα
Gene Expression during the Differentiation of 3T3-L1 Preadipocytes, J. Biol. Chem., 277,
46226–46232, 2002.
Quintanilla, R., Milan, D., and Bidanel, J. P.: A further look at quantitative
trait loci affecting growth and fatness in a cross between Meishan and Large
White pig populations, Genet. Sel. Evol., 34, 193–210, 2002.Rolli, V., Radosavljevc, M., Astier, V., Macquin, C., Castan-Laurell, I., Visentin, V.,
Guigné, C., Carpéné, C., Valet, P., Gilfillan, S., and Bahram,
S.:
Lipolysis is altered in MHC class I zinc-α2-glycoprotein deficient
mice, FEBS Lett., 581, 394–400, 2007.
Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M.: Transcriptional
regulation of adipogenesis, Genes. Dev., 14, 1293–1307, 2000.Russell, S. T. and Tisdale, M. J.: Effect of a tumour-derived lipid-mobilising
factor on glucose and lipid metabolism in vivo, Br. J. Cancer., 87,
580–584, 2002.Russell, S. T., Hirai, K., and Tisdale, M. J.: Role of β3-adrenergic
receptors in the action of a tumour lipid mobilizing factor, Br. J. Cancer., 86,
424–428, 2002.Russell, S. T., Zimmerman, T. P., Domin, B. A., and Tisdale, M. J.: Induction of lipolysis
in vitro and loss of body fat in vivo by zinc-α2-glycoprotein, BBA
Mol. Cell. Biol. L., 1636, 59–68, 2004.Schweiger, M., Schreiber, R., Haemmerle, G., Lass, A., Fledelius, C., Jacobsen, P.,
Tornqvist, H., Zechner, R., and Zimmermann, R.: Adipose Triglyceride Lipase and
Hormone-sensitive Lipase Are the Major Enzymes in Adipose Tissue
Triacylglycerol Catabolism, J. Biol. Chem., 281, 40236–40241, 2006.
Singla, P., Bardoloi, A., and Parkash, A. A.: Metabolic effects of obesity: A
review, World J. Diabetes, 1, 76–88, 2010.
Spurlock, M. E. and Gabler, N. K.: The Development of Porcine Models of Obesity
and the Metabolic Syndrome, J. Nutr., 138, 397–402, 2008.
Todorov, P. T., McDevitt, T. M., Meyer, D. J., Ueyama, H., Ohkubo, I., and Tisdale, M.
J.:
Purification and Characterization of a Tumor Lipid-mobilizing Factor, Cancer
Res., 58, 2353–2358, 1998.
Vidal-Puig, A. J., Considine, R. V., Jimenez-Liñan, M., Werman, A., Pories, W. J.,
Caro,
J. F., and Flier, J. S.: Peroxisome proliferator-activated receptor gene
expression in human tissues. Effects of obesity, weight loss, and regulation
by insulin and glucocorticoids, J. Clin. Invest., 99, 2416–2422, 1997.
Wei, W. H., Skinner, T. M., Anderson, J. A., Southwood, O. I., Plastow, G., Archibald, A. L.,
and Haley, C. S.: Mapping QTL in the porcine MHC region affecting fatness and
growth traits in a Meishan/Large White composite population, Anim. Genet., 42,
83–85, 2011.
Wells, J. C. K.: The programming effects of early growth, Early Hum. Dev., 83,
743–748, 2007.
Wu, Z., Xie, Y., Bucher, N. L., and Farmer, S. R.: Conditional ectopic expression of
C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis,
Genes. Dev. Oct., 9, 2350–2363, 1995.Zhang, C. Y., Baffy, G., Perret, P., Krauss, S., Peroni, O., Grujic, D., Hagen, T.,
Vidal-Puig, A. J., Boss, O., Kim, Y. B., Zheng, X. X., Wheeler, M. B., Shulman, G. I., Chan, C. B.,
and Lowell, B. B.: Uncoupling Protein-2 Negatively Regulates Insulin Secretion
and Is a Major Link between Obesity, β Cell Dysfunction, and Type 2
Diabetes, Cell, 105, 745–755, 2001.