Test for positional candidate genes for body composition on pig chromosome 6

One QTL affecting backfat thickness (BF), intramuscular fat content (IMF) and eye muscle area (MA) was previously localized on porcine chromosome 6 in an F₂ cross between Iberian and Landrace pigs. This work was done to study the effect of two positional candidate genes on these traits: H-FABP and LEPR genes. The QTL mapping analysis was repeated with a regression method using genotypes for seven microsatellites and two PCR-RFLPs in the H-FABP and LEPR genes. H-FABP and LEPR genes were located at 85.4 and 107 cM respectively, by linkage analysis. The effects of the candidate gene polymorphisms were analyzed in two ways. When an animal model was fitted, both genes showed significant effects on fatness traits, the H-FABP polymorphism showed significant effects on IMF and MA, and the LEPR polymorphism on BF and IMF. But when the candidate gene effect was included in a QTL regression analysis these associations were not observed, suggesting that they must not be the causal mutations responsible for the effects found. Differences in the results of both analyses showed the inadequacy of the animal model approach for the evaluation of positional candidate genes in populations with linkage disequilibrium, when the probabilities of the parental origin of the QTL alleles are not included in the model.     

H-FABP、MC4R、ADD1基因多态性在3个猪群中分布及其与肌内脂肪和背膘的相关研究

具有不同遗传特性的猪种具有不同的肉质性状。尤其是地方品种和引进品种间在肉质性状存在极大的差异。在已有的研究中H-FABP,MC4R,ADD1基因同肌内脂肪或背膘相关。利用梅山猪、苏太猪和杜×长×大猪为试验动物,研究上述3个基因的多态性分布和多态性同肌内脂肪和背膘的相关性。结果表明：3个基因的多态性分布在不同猪种间存在极显著的差异,这种差异可能是肌内脂肪（IMF）或背膘（BF）不同的主要原因之一。连锁分析表明：H-FABP和ADD1基因多态性同IMF有显著的相关,但是同BF没有显著的相关;MC4R基因的多态性同IMF和BF都有显著相关性。说明：H-FABP和ADD1基因多态性有可能应用到提高IMF,同时不影响BF的育种实践中。      

A study of associations of the H-FABP genotypes with fat and meat production of pigs

Although the content of intramuscular fat (IMF) influences significantly meat quality, it can be estimated only after the slaughter of animals. Variants of the H-FABP gene were suggested as candidate genes influencing the variability of IMF. The effect of H-FABP - HinfI polymorphism on the content of IMF, backfat thickness, the weight and percentages of major meat parts and of the leg in carcass weight was studied in a group of 97 pigs (46 gilts and 51 barrows) of Large White and Landrace breeds using the test of fattening capacity and carcass value. In the set of experimental animals, the frequencies of genes were H = 0.75 +/- 0.03 and h = 0.25 +/- 0.03. Biometric analyses did not corroborate differences among different H-FABP - HinfI genotypes and all the traits under study. Only in genotypes HH and Hh the differences between least-square means of phenotypic IMF values under study were close to the limit of significance (P = 0.06).     

绵羊肌肉H-FABP和PPARγ基因表达的发育性变化及其对肌内脂肪含量的影响

选取不同日龄的雄性哈萨克羊和新疆细毛羊,屠宰后取背最长肌,用索氏抽提法检测肌内脂肪（Intamuscular fat,IMF）含量,用荧光实时定量PCR法检测心脏脂肪酸结合蛋白（heart fatty acid-binding protein,H-FABP）和过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptorγ,PPARγ）基因表达的发育性变化极其对IMF含量的影响.结果表明：（1）随着日龄的增加,IMF含量在雄性哈萨克羊中持续上升,且在各生长期间的差异显著（P＜0.05）,而在新疆细毛羊的各生长期间无显著差异（P＞0.05）.雄性哈萨克羊的IMF含量在30～90日龄间极显著高于新疆细毛羊（P＜0.01）;（2）雄性哈萨克羊肌肉H-FABP基因的表达量在2日龄时最高,30日龄时降到最低,然后持续上升,各日龄间差异显著（P＜0.05）.新疆细毛羊中的表达量在2日龄时也最高,然后持续下降,到90日龄时降到了最低点,然后上升,各日龄间差异也显著（P＜0.05）;（3）雄性哈萨克羊肌肉PPARγ基因的表达量随着日龄的增加而呈下降趋势,各日龄间差异显著（P＜0.05）;新疆细毛羊中的表达量在2～60日龄期间持续上升且各日龄间差异显著（P＜0.05）,但在90日龄时降到最低点,然后上升;（4）在哈萨克羊中,H-FABP基因在30～90日龄间的表达量与IMF含量的相关系数为0.737（P＜0.01）,而PPARγ基因在2-90日龄间的表达量与之的相关系数为-0.835（P＜0.01）.     

H-FABP基因的多态性和营养因素对猪肉质的影响

遗传和营养因素都能影响猪肉的品质。但是, 到目前为止同时研究遗传和营养因素对肉质影响的报道很少。在本研究中, 136头PIC5系杂交猪, 体重65 kg, 被随机分成4组, 各组分别给予不同日粮。在饲养35 d、体重大约90 kg时统一屠宰并且进行肉质测定、H-FABP基因分型及其与肉质性状的关联分析。结果表明: (1)所采用的3种日粮对肉色、屠宰后24 h的pH、肌内脂肪和肌肉蛋白含量有极显著的影响; (2)H-FABP基因型对肌内脂肪和肌肉蛋白含量存在极显著的影响; (3)H-FABP基因多态性和营养因素的交互作用对pH 和肌内脂肪含量均有显著的影响, 对照组的AA基因型具有最高pH值, 高维生素E组的AA基因型具有最高肌内脂肪值。实验结果提示在关于猪肉质的育种和生产过程中应该同时考虑营养因素和遗传因素。     

FABP5 is a critical regulator of methionine- and estrogen-induced SREBP-1c gene expression in bovine mammary epithelial cells

The intracellular fatty acid-binding proteins (FABPs) are a well-conserved family that function as lipid chaperones. Ongoing studies are focused on identification of the mechanistic complexity and vast biological diversity of different isoforms of FABPs. However, the molecular mechanism of FABP5 in the regulation of milk fat synthesis in the mammary gland of dairy cows is still largely unknown. Here, we report that FABP5 acts as a critical regulator of terol response element-binding protein-1c (SREBP-1c) gene expression induced by methionine (Met) and estrogen (E2) in bovine mammary epithelial cells (BMECs). We observed that the expression of FABP5 was markedly higher in dairy cow mammary tissue during the lactating period than the puberty period and the dry period. FABP5 is located in the cytoplasm, and Met and E2 significantly increase the protein levels of FABP5 in BMECs. Using gene function study approaches, we revealed that FABP5 positively regulates SREBP-1c gene expression and promotes milk fat synthesis. We confirmed that FABP5 is required for Met- and E2-induced SREBP-1c gene expression and milk fat synthesis. We further uncovered that fatty acids are needed for FABP5-mediated SREBP-1c gene expression. Thus, our study demonstrates that FABP5 is a critical regulator of Met- and E2-induced SREBP-1c gene expression leading to milk fat synthesis.     

猪H-FABP基因多态性与肌内脂肪含量的关联分析

以山西白猪为实验材料,采用索氏脂肪抽提法对其背最长肌的肌内脂肪含量进行了测定。根据心脏脂肪酸结合蛋白(heart fatty acid-binding protein,H-FABP)基因的PCR-RFLP多态性,应用SPSS软件,用最小二乘法拟合一般线性模型(GLM)分析了H-FABP基因的不同位点的基因型对肌内脂肪含量的影响。结果表明,基因型hh、dd、BB对肌内脂肪含量影响的效应值最大。证明H-FABP基因可以作为肌内脂肪含量的侯选基因。     

Comparative genome analysis with the human genome reveals chicken genes associated with fatness and body weight

The selection of meat-type chickens (broilers) for rapid growth has been accompanied by excessive fat deposition. In this study, we analysed 53 candidate genes that are associated with obesity and obesity-related traits in humans, for which we found chicken orthologues by BLAST searches. We have identified single nucleotide polymorphisms (SNPs) with significant differences in allele frequencies between broilers and layers in each of the following six candidate genes: adrenergic, beta-2-, receptor, surface (ADRB2); melanocortin 5 receptor (MC5R); leptin receptor (LEPR), McKusick-Kaufman syndrome (MKKS), milk fat globule-EGF factor 8 protein (MFGE8) and adenylate kinase 1 (AK1). To examine associations with fatness and/or body weight, we used birds of extreme phenotypes in F(2) and backcross populations with varying levels of abdominal fat weight per cent (%AFW) and body weight. We then assessed the level of gene expression by real-time PCR. In two genes, ADRB2 and MFGE8, we found significant association with %AFW. The ADRB2 gene was found to have a significantly higher expression in the liver of lean chickens compared with those of the fat individuals. We believe that this approach can be applied for the identification of other quantitative genes.     

Association of melanocortin 4 receptor (MC4R) and high mobility group AT-hook 1 (HMGA1) polymorphisms with pig growth and fat deposition traits

The aim of this study was to analyse the combined effect of melanocortin 4 receptor (MC4R) and high mobility group AT-hook 1 (HMGA1) polymorphisms on growth and fatness traits in Duroc pigs. No significant interaction was observed between MC4R and HMGA1 for back-fat traits. An additive mode of inheritance of both gene effects was found for average daily gain and lean meat content. Maximum mean differences from combined genotypic effects were over 2 mm for back fat, 70 g/day for average daily gain and 2% for lean meat content. Therefore, utilization of polymorphisms in both MC4R and HMGA1 for marker-assisted selection could result in an economic benefit to the pig industry.     

齐口裂腹鱼和鲤鱼H-FABP基因的克隆及其表达谱

心型脂肪酸结合蛋白（heart fatty acid binding protein, H-FABP）的水平与影响肉质性状的肌内脂肪含量有关,鱼类H-FABP的表达水平对其肌内脂肪含量是否相关仍未见报道.本研究获得齐口裂腹鱼和鲤鱼心脏型脂肪酸结合蛋白基因序列,利用半定量RT-PCR分析其表达特性并测定肌内脂肪含量,比较H-FABP基因在不同生活环境的2种鲤科鱼肌内脂肪沉积中的作用.结果显示,齐口裂腹鱼和鲤鱼H-FABP基因的ORF为402 bp,编码133个氨基酸,它们的氨基酸序列相同,与人、猪、小鼠、斑马鱼、大西洋鲑、虹鳟等的同源性为71.3%~ 90%;H-FABP基因在2种鲤科鱼的心、肌肉、脂肪、肝、脑、脾、肾和鳃等组织中均有表达,肝中的表达量显著高于其它组织（P<0.05）,H-FABP基因的肌肉表达谱在齐口裂腹鱼和鲤鱼中存在明显差异：齐口裂腹鱼中的表达随生长发育呈上升趋势,在大体重鱼（500 g）中的表达显著高于小体重鱼（P<0.05),其表达与肌内脂肪含量呈显著正相关（R=0.370,P<0.05）;H-FABP基因在鲤鱼生长发育中呈下降趋势,而小体重鱼（50~60 g）中的表达显著高于其它大体重鱼(P<0.05),其表达与肌内脂肪含量呈显著负相关（R=-7.083,P<0.01）.据此推测,齐口裂腹鱼和鲤鱼肌肉组织H-FABP基因表达与肌内脂肪关联性的差异可能与2种鱼的生活环境不同有关.     

SIRT1 Deacetylates and Inhibits SREBP-1C Activity in Regulation of Hepatic Lipid Metabolism

The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in diet-induced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.     

西部地区主要猪种H-FABP基因多态性,IMF含量及不同基因型脂肪细胞脂滴量的关系

H-FABP是FABP家族成员之一,在长链脂肪酸的吸收和代谢平衡中发挥关键作用,但它对猪IMF含量的影响还知之甚少,对中国西部地区的猪种更是如此.文章利用PCR-RFLP（HinfⅠ、HaeⅢ和Msp Ⅰ 3种限制性内切酶）分子标记技术,检测了杜洛克猪、长白猪、大白猪、内江猪、荣昌猪、汉江黑猪、汉中白猪、八眉猪和野猪共计265头猪H-FABP基因5＇-上游区和第二内含子区的遗传变异,利用最小二乘模型分析了H-FABP基因对猪肌内脂肪含量的遗传效应,并运用猪脂肪细胞培养,油红O染色和TG测定等技术检测了H-FABP基因不同基因型脂肪细胞内脂滴的形态和沉积的量.结果表明：（1）在HinfⅠ-RFLP位点上,上述品种和野猪均存在多态性,其中大白猪、八眉猪、汉江黑猪、汉中白猪和野猪表现为低度多态,杜洛克、长白猪、内江猪和荣昌猪为中度多态;除汉江黑猪（P＜0.05）和野猪（P＜0.01）外,其他猪种基因频率和基因型频率都处于Hardy-Weinderg平衡状态（P＞0.05）;而在HaeⅢ-RFLP和Msp Ⅰ-RFLP位点上,仅内江猪、荣昌猪、汉江黑猪和八眉猪为单态;（2）9种基因型对肌内脂肪（IMF）含量的影响,HH＞Hh＞hh,DD＜Dd＜dd,AA＜Aa＜aa,遗传效应值分别为：3.89,3.42,3.17,2.27,2.49,2.91,2.28,2.70,2.95,H-FABP基因可显著地提高IMF含量（P＜0.05）;（3）aaddHH型的脂肪细胞脂滴的形态,密度和含量与其他基因型细胞差异显著（P＜0.05）.结果提示：可通过提高＂aaddHH＂基因型的频率来增加IMF含量,达到改善猪肉质的目的.     

Functional and association studies on the pig HMGCR gene, a cholesterol-synthesis limiting enzyme

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the rate-limiting enzyme in the biosynthesis of cholesterol. We have studied the role of the HMGCR gene in pig lipid metabolism by means of expression and structural analysis. We describe here the complete coding region of this gene in pigs and report two synonymous single nucleotide polymorphisms in the coding region. We have, additionally, studied the association of one of these polymorphisms (HMGCR:c.807A>C) with several lipid deposition- and cholesterol-related traits in a half-sib population generated from a commercial Duroc line, showing in some families a positive relationship of HMGCR:c.807A allele with serum low-density lipoprotein (LDL)-bound cholesterol and triglyceride levels, and also with intramuscular fat (IMF) content of gluteus medius muscle. We have also assessed the expression levels in muscle and in liver from 68 Duroc individuals corresponding to the most extreme animals for the analysed traits. Liver HMGCR expression correlated negatively with the serum high-density lipoprotein (HDL) levels, carcass lean percentage and stearic acid content, while muscle expression correlated also negatively with the carcass lean percentage, stearic and linoleic acids content, but showed a positive correlation with the serum lipid cholesterol (HDL, LDL and total cholesterol), IMF and muscle oleic and palmitic fatty acid content. With this information, we have performed an association analysis of expression data with lipid metabolism phenotypic levels and the HMGCR genotype. The results indicate that HMGCR expression levels in muscle are different in the two groups of pigs with extreme values for fat deposition and total cholesterol levels, and also between animals with the different HMGCR genotypes.