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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Key Laboratory of Animal Physiology and Biochemistry, Nanjing Agricultural University, Nanjing 210095, China;
and
Research Institute for the Biology of Farm Animals, D-18196 Dummerstorf, Germany; and
Department of Animal Sciences, Washington State University, Pullman 99164-6351
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: association • intramuscular fat content • mutation • messenger ribonucleic acid expression • pig • sterol regulatory element binding transcription factor 1
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Therefore, a major challenge to the pig industry is to produce lean pork with a reasonable IMF content, without significantly increasing other fat depots. Although IMF content is associated with total carcass fat in general, studies have shown that the developmental profiles of each fat depot can be different (Kouba et al., 1999; Hausman and Poulos, 2004). This suggests that it is possible to identify the key factors or genes involved in the regulation of IMF deposition without affecting other fat depots (Gerbens et al., 2000).
Sterol regulatory element binding transcription factor 1 (SREBF1) is a transcription factor involved in adipocyte differentiation (Briggs et al., 1993; Yokoyama et al., 1993) as well as in the biosynthesis of cholesterol and fatty acids (Brown and Goldstein, 1997). Although SREBF1 is predominantly expressed in adipose tissue and liver (Tontonoz et al., 1993; Kim and Spiegelman, 1996; Shimano et al., 1997), its expression in muscle tissue has also been reported in human and mouse (Shimomura et al., 1997). Furthermore, SREBF1 regulates glucose and lipid metabolism in myocytes (Guillet-Deniau et al., 2002; Gosmain et al., 2004; Guillet-Deniau et al., 2004). In humans, SREBF1 gene polymorphisms are associated with obesity and type 2 diabetes (Eberle et al., 2004). Overall, SREBF1 plays a central role in energy homeostasis by promoting glycolysis, lipogenesis, and adipogenesis. Intramuscular fat deposition depends on i.m. adipogenesis and lipogenesis in both adipocytes and myofibers. Therefore, the objectives of the current study were to elucidate the relationship between SREBF1 mRNA expression and IMF deposition in muscle and to identify the genetic polymorphisms in the gene that can be used to improve IMF content in pigs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The animal experiments and protocols used in this study were approved by the Nanjing Agricultural University Institutional Animal Care and Use Committee.
Purebred Erhualian and Sutai pigs were used in the current study. Like Meishan, Erhualian is 1 of the 7 strains of the Taihu breed, which is known for high prolificacy. These pigs reach puberty at 2.5 to 3 mo of age, achieve high embryo survival rates, and average 15 to 16 pigs per litter (Wang, 1988). However, they are slow-growing and fat, but meat from these pigs is palatable due to its high IMF content (David, 1989). Sutai is a newly developed line of pig containing 50% Erhualian and 50% Duroc. These pigs have improved lean production as well as high IMF content.
The Erhualian pigs used in Exp. 1 were produced in the Nanjing Agricultural University Animal Research Center. Four litters of newborn Erhualian piglets were randomly chosen from sows in their second or third parities and raised until weaning at 45 d of age. The lactating sows had access to feed and water ad libitum. Diets were formulated to meet the requirements of Erhualian sows (Gao et al., 2004). At 3, 20, and 45 d of age, 1 male and 1 female Erhualian piglet from each litter (n = 4 for each sex at each age) were randomly selected and slaughtered for tissue sampling. Samples were taken immediately (within 5 to 10 min of death) from the LM at the last rib. A portion of the sample was put immediately into liquid N and stored at –80°C, whereas the remainder was used to determine IMF content. The IMF content was measured in triplicate via the Soxhlet extraction method using petroleum ether as the solvent (AOAC, 2000).
Sutai pigs used in Exp. 2 were raised in the Sutai Breeding Center located in Suzhou, Jiangsu Province. Sutai fattening pigs (n = 100; 68 castrated males, 32 females) were slaughtered at approximately 180 d of age. The average BW was 87.6 ± 5.2 kg. Muscle sampling and the measurement of IMF content were completed using the same procedures described above. Backfat thickness (BFT) was measured by vernier caliper (Mitutoyo Co., Kawasaki, Japan) between the third and fourth from the last ribs. Based on these measurements, 3 groups (n = 20 per group) with high, intermediate, and low IMF content were selected from these 100 animals.
Expression of the SREBF1 Gene in the LM
Total RNA was extracted from the tissue samples using a single-step method with the acid guanidinium thiocyanate-phenol-chloroform (Chomczynski and Sacchi, 1987). Total RNA concentration was then quantified by measuring the absorbance at 260 nm in an Eppendorf Biophotometer (Eppendorf AG, Hamburg, Germany). Ratios of absorption (260/280 nm) were from 1.8 to 2.0 for all samples prepared. The integrity of the RNA was verified by electrophoresis through a 1.4% agarose-formaldehyde. Total RNA (2 µg) was then reverse-transcribed by incubation at 42°C for 1 h in a 25-µL mixture consisting of 10 U of avian myeloblastosis virus reverse transcriptase (Promega Co., Shanghai, China), 10 U of RNase inhibitor (Promega), 12 µmol random primers, 50 mmol Tris-HCl (pH 8.3), 10 mmol MgCl2, 50 mmol KCl, 10 mmol DL-dithiothreitol, 0.5 mmol spermidine, and 0.8 mmol each deoxynucleoside triphosphate. The reaction was terminated by heating at 95°C for 5 min and quickly cooling on ice.
The reverse transcription (RT) reaction mix (2 µL) was used for PCR in a final volume of 25 µL containing 0.5 U of Taq DNA polymerase, 5 mmol Tris-HCl (pH 9.0), 10 mmol NaCl, 0.1 mmol DL-dithiothreitol, 0.01 mmol EDTA, 5% (wt/vol) glycerol, 0.1% (wt/vol) Triton X-100, 0.2 mmol each deoxynucleoside triphosphate, 1.0 mmol MgCl2, 1.0 µL of 18S primer pair, 1.0 µL of 18S competimer, and 0.5 µmol of each primer, which were designed based on the porcine SREBF1 mRNA sequence (GenBank Accession No. AF102873): forward, 5'-GCGACGGTGCCTCTGGTAGT-3' and reverse, 5'-CGCAAGACGGCGGATTTA-3'. The amplified PCR product was 218 bp in size. The Quantum RNA 18S Internal Standards Kit (1716, Ambion Inc., Austin, TX) containing primers and competimers was used as an internal control.
To eliminate the possible contamination of genomic DNA and environmental DNA in the RT-PCR, pooled samples were formed by mixing equal quantities of total RNA from all samples and were used to optimize PCR conditions and normalize for intraassay variations. Optimal amplification of SREBF1 cDNA within its linear range was achieved with 5 min at 94°C, 28 cycles of 30 s at 94°C, 30 s at 58°C, 40 s at 72°C, and a final extension at 72°C for 10 min. All samples were included in the same run of RT-PCR and repeated at least 3 times.
An aliquot of PCR products was analyzed by electrophoresis on 8.0% PAGE gels. The gels were stained with ethidium bromide and photographed with a digital camera. The net intensities of individual bands were measured using Kodak Digital Science 1D software (Eastman Kodak Company, Rochester, NY). The SREBF1 bands were then normalized to the 18S ribosomal RNA bands.
Detection of Genetic Polymorphisms in the Porcine SREBF1 Gene
The PCR-single-strand conformation polymorphism (SSCP) technique (Orita et al., 1989) was used to identify genetic polymorphisms in the porcine SREBF1 gene. The PCR products were obtained using the same primer pairs and reaction conditions as described above. After the PCR reaction, products were denatured at 98°C for 10 min and then immediately cooled on ice. An aliquot of denatured PCR product was analyzed by electrophoresis on 12% PAGE gels. The polymorphic bands (the bands with different electrophoretic mobility stand for various sequences) were visualized with silver stain (Hoshino et al., 1992). Three samples of each homozygote were chosen and sequenced. The Mfold web server (http://www.bioinfo.rpi.edu/~zukerm/rna/) was used to predict mRNA secondary structure changes caused by coding polymorphisms.
Statistical Analysis
The statistical analysis was performed using the GLM procedure (SPSS Inc., Chicago, IL). For Exp. 1, data were analyzed using the following model: Yij = µ+ Ai + Bj + eij, where Yij = the observation made for a given dependent variable; µ= the overall mean; Ai = the fixed effect of the ith age; Bj = the fixed effect of the jth sex; and eij = the random error. Sex differences at the same age were analyzed by independent samples t test of SPSS 11.0. For Exp. 2, the data were analyzed according to the following model: Yij = µ+ Di + Ej + eij, where Yij = the value of the ijth dependent variable; µ= the overall mean; Di = the fixed effect of the ith group; Ej = the fixed effect of the jth sex; and eij = the random error. The analysis of the genotypes effect was performed with the same model, except that Di represented the fixed effect of the ith genotype.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sex had a significant effect on IMF content and SREBF1 gene expression in Erhualian pigs. Regardless of age, the IMF content in female piglets was greater than in males (2.7 ± 0.9% vs. 1.9 ± 0.6%, P < 0.05; Figure 1a). The same trend was also observed in the SREBF1 mRNA expression between female and male piglets (1.01 ± 0.11 vs. 0.79 ± 0.13, P < 0.05; Figure 1b).
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shows the average IMF content of each group, which was significantly different among groups (P < 0.05). Likewise, SREBF1 mRNA level increased as IMF content increased (P < 0.05) among groups (Figure 2b). Additionally, SREBF1 mRNA expression was positively correlated with IMF content (r = 0.67, P < 0.01, n = 60).
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The amplified PCR product of the SREBF1 gene was 218 bp in size. Two SSCP alleles were identified after PAGE analysis, indicating that SREBP1 is polymorphic. Each homozygote produced 2 bands. The genotype of the homozygote with 2 relatively far-separated bands was designated as AA and the other with the less-separated bands as BB. The AB heterozygote contained 3 bands (Figure 3). The frequencies of AA, AB, and BB genotypes were 50, 36, and 14%, respectively.
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As shown in Figure 4a, animals with the AA genotype had the lowest IMF content compared with those animals with AB and BB genotypes (P < 0.05). Similarly, level of SREBP1 mRNA expression was least (P < 0.05) in pigs with the AA genotype (Figure 4b). However, no difference in the BFT existed among the 3 genotypes, being 2.63 ± 0.49 cm for AA, 2.61 ± 0.5 cm for AB, and 2.65 ± 0.48 cm for BB, respectively (Figure 4c).
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Sequencing PCR products for homozygotes AA and BB revealed 2 SNP, T1006C and C1033T, in the porcine SREBF1 gene (NM_214157). Both polymorphic sites correspond to the third nucleotides of triplet codons, which code 332I and 341N, respectively. Neither nucleotide substitutions changed AA sequence, but they affected the secondary structure of SREBF1 mRNA (Figure 5). Only 86 bp of sequence surrounding these 2 SNP were selected for a more localized structure analysis. Among them, 25 were single-strand bases for the haplotype T-C and 26 for C-T, respectively (Figure 5). At position T1006C, allele T produced an interior loop (Figure 5a), whereas allele C formed a multiloop from G13 to C43 (Figure 5b). At position C1033T, both alleles created a hairpin loop, but the former with a closing pair of A60 – T69 plus 2 stacks (C58 – G71 and G59 – C70) and the latter with a closing pair of A60 – T66 plus 3 stacks (A57 – T69, T58 – A68, and G59 – C67; Figure 5a and 5b).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The sterol regulatory element 1 is a key regulatory element in the promoter of several genes involved in cholesterol homeostasis (Yokoyama et al., 1993; Wang et al., 1994; Weber et al., 2004). The consensus E-box site is similar to a carbohydrate response element, which regulates transcription in genes involved in fatty acid and triglyceride metabolism in liver and fat in response to carbohydrate. The SREBF1 is also a major mediator of insulin action on the expression of glycolytic and lipogenic genes in both adipocytes and myocytes (Kim et al,. 1998; Foretz et al., 1999; Guillet-Deniau et al., 2002).
Our study demonstrated the SREBF1 gene expression is significantly correlated with IMF content in LM of pigs. There was a sex difference in IMF content during the early postnatal stage, being greater in females than males at 3 and 45 d of age. The results were in agreement with previous reports in both pigs and goats in later developmental stages (Gallo et al., 1997; Rauw et al., 2003). Accordingly, the mRNA expression of SREBF1 in LM was also greater in female than male piglets during the suckling period. This is the first report on sex differences in SREBF1 expression in young pigs. A positive correlation between IMF content and expression of SREBF1 mRNA in muscle was further observed in fattening pigs in Exp. 2. The positive correlation between IMF content and the muscle expression of SREBF1 in both suckling piglets and adult fattening pigs imply that SREBF1 plays an important role in lipid accumulation of pig muscle.
Analysis using PCR-SSCP revealed that porcine SREBF1 is polymorphic. Sequencing confirmed that 2 SNP in the coding region [i.e., T/C and C/T substitutions at T1006C and C1033T (NM_214157)] were responsible for the different PCR-SSCP patterns. Theoretically, there should be 4 haplotypes for 2 sites: T-C, C-T, T-T, C-C, respectively. However, in this study, only T-C and C-T haplotypes were observed, indicating that these 2 polymorphic sites are completely linked. This was further confirmed by comparing the SREBF1 sequences available in the GenBank database: only 2 haplotypes, T-C (AY272051) and C-T (DQ464433, NM_214157, AY496867, AY307771, AF102873), have been reported in pigs.
Significant differences in both IMF content and SREBP1 mRNA expression level were found between the AA genotype and the other 2 genotypes, AB and BB. As discussed above, these genotypes result from 2 mutations at T1006C and C1033T (NM_214157). Although these coding SNP are silent, they do affect the secondary structure of mRNA (Figure 5
). The change of mRNA secondary structure may affect translation (Baim et al., 1985). Therefore, our study provides evidence that the different haplotypes might affect SREBF1 mRNA stability and result in different phenotypes, such as IMF, in the current study.
An ideal genetic marker for improving pork quality should have a positive effect on IMF, without affecting other body fat contents. This study revealed 2 alleles of the SREBF1 gene, which had significant effect on IMF content but not BFT. Therefore, the SREBF1 gene is a promising genetic marker for IMF content selection in pigs. However, the mechanism underlying the tissue-specific effect of SREBF1 remains to be elucidated. We presume that the effects of SREBF1 on adipogenesis and lipid metabolism may differ between fat and muscle tissue, which is supported by the observation that constitutive overexpression of the nuclear form of SREBF1 in transgenic mice induced divergent effects on white fat depots and skeletal muscles (Shimomura et al., 1998).
In conclusion, we demonstrated a close relationship between IMF deposition and SREBF1 mRNA expression in LM of both suckling and fattening pigs. A mutation in the SREBF1 gene was described and found to have a significant effect on IMF deposition, as well as SREBF1 mRNA expression. The results suggest that SNP of SREBF1 could be used as genetic markers to improve IMF content in pigs.
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Footnotes |
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2 Corresponding author: zhao.ruqian{at}gmail.com
Received for publication January 25, 2007. Accepted for publication September 3, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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