A comprehensive analysis of QTL for abdominal fat and breast muscle weights on chicken chromosome 5 using a multivariate approach.

Quantitative trait loci (QTL) influencing the weight of abdominal fat (AF) and of breast muscle (BM) were detected on chicken chromosome 5 (GGA5) using two successive F(2) crosses between two divergently selected 'Fat' and 'Lean' INRA broiler lines. Based on these results, the aim of the present study was to identify the number, location and effects of these putative QTL by performing multitrait and multi-QTL analyses of the whole available data set. Data concerned 1186 F(2) offspring produced by 10 F(1) sires and 85 F(1) dams. AF and BM traits were measured on F(2) animals at slaughter, at 8 (first cross) or 9 (second cross) weeks of age. The F(0), F(1) and F(2) birds were genotyped for 11 microsatellite markers evenly spaced along GGA5. Before QTL detection, phenotypes were adjusted for the fixed effects of sex, F(2) design, hatching group within the design, and for body weight as a covariable. Univariate analyses confirmed the QTL segregation for AF and BM on GGA5 in male offspring, but not in female offspring. Analyses of male offspring data using multitrait and linked-QTL models led us to conclude the presence of two QTL on the distal part of GGA5, each controlling one trait. Linked QTL models were applied after correction of phenotypic values for the effects of these distal QTL. Several QTL for AF and BM were then discovered in the central region of GGA5, splitting one large QTL region for AF into several distinct QTL. Neither the 'Fat' nor the 'Lean' line appeared to be fixed for any QTL genotype. These results have important implications for prospective fine mapping studies and for the identification of underlying genes and causal mutations.

Triglyceride synthesis and secretion and lipogenesis implicated gene expression in the chicken hepatocarcinoma cell line LMH.

Avian lipogenesis was studied in the chicken hepatocarcinoma LMH cell line. The differentiated and lipogenic status of these cells was evidenced by the presence of the albumin mRNA as well as of some mRNA coding for enzymes involved in lipogenesis (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase) and for apoproteins (apoprotein B and A1). These results were further confirmed by the analysis of triglyceride synthesis and secretion rates in growing cells. A time course analysis showed that triglyceride metabolism was affected by cell density. Hormone responsiveness of triglyceride production was also analyzed. Insulin, triiodothyronine and glucagon to a lesser extent were shown to regulate lipogenesis of LMH cells. The results were compared with those obtained in primary cultures of chicken hepatocytes.

Characterization of a cDNA sequence encoding the peroxisome proliferator activated receptor alpha in the chicken.

Peroxisome proliferator-activated receptors (PPAR) belong to the superfamily of the nuclear hormone receptors that play an important role in lipid homeostasis. A partial complementary deoxyribonucleic acid clone encoding a PPAR alpha from chicken liver was isolated and sequenced. Comparison with human, mouse, rat, and Xenopus PPAR alpha cDNA indicates a high degree of homology, especially at the level of the inferred peptide sequence (greater than 90%). The tissue pattern of expression indicates that PPAR alpha expression in the chicken is similar to that reported in other species, i.e., high expression levels in the liver, heart, and kidney, but also occurs in the lipogenic uropygial gland specific to birds.

DNA polymorphisms of lipogenesis genes and analysis of linkage with fatness in turkeys.

DNA variability of lipogenesis genes was evaluated by Restriction Fragment Length Polymorphism (RFLP) on four selected lines of turkeys. Thirteen polymorphisms were obtained, four on the acetyl-coenzyme A carboxylase gene, one on the apoprotein AI gene, two on the malic enzyme gene, four on the fatty acid synthase gene, and two on the stearoyl-coenzyme A mesaturase gene. Six of these RFLP were studied in more detail on 64 female turkeys sampled in two lines. Large differences in terms of number of DNA profiles and haplotype frequencies were observed between the two genes, in accordance with the variability of recorded fatness performances. An additive model including the effects of RFLP profiles was used to assess linkage between polymorphisms on lipogenesis genes and fatness measured either in vivo by an ultrasonic instrument or, after slaughter, by the ratio of skin plus subcutaneous fat weight to the whole leg weight. In each line, two polymorphisms suggested a statistical association with fat deposit, one on the malic enzyme gene detected by DNA digestion with HindIII and one on the fatty acid synthase gene detected by MspI. A multiple comparison of means associated with each RFLP profile enabled the identification of two lean profiles in one strain, and a fat profile in the other.

Association of fatty acid synthase gene and malic enzyme gene polymorphisms with fatness in turkeys.

A candidate gene approach was carried out on a commercial line of turkeys to assess the association between fatness variability and polymorphisms of genes involved in lipid metabolism. Four restriction fragment length polymorphisms (RFLP) were typed on the fatty acid synthase gene (MspI/pF5), on the malic enzyme gene (HindIII/em), as well as on the acetyl coenzyme A carboxylase and delta9 desaturase genes. Fatness level was estimated in vivo by an ultrasonic instrument. Fat yield was assessed after slaughter by calculating the ratio of the leg skin plus subcutaneous fat weight to the whole leg weight. Finally, the lipid content was determined by extraction from the boneless leg. The 84 female turkeys sampled were full and half-sibs born from eight sires, seven of which were heterozygous for MspI/pF5 or HindIII/em RFLP and one of which was double homozygous at these loci. The analyses of variance used to compare the genotypes at each RFLP suggested a major role associated with the fatty acid synthase gene polymorphism in the explanation of fatness variability. One homozygous genotype for MspI/pF5 was about 1.5 standard deviations leaner than the other two homozygous genotypes. An analysis of the average effects of gene substitution confirmed the association between leanness and one allele of the fatty acid synthase polymorphism. It also identified a significant association between leanness and one malic enzyme RFLP allele, congruent with a strictly additive determinism for the effect associated with this polymorphism. This experiment provided new evidence of the association between both fatty acid synthase and malic enzyme gene polymorphisms and fatness variability in turkeys.

Identifying genes involved in the variability of genetic fatness in the growing chicken.

A precise knowledge of the genome involved in the expression of a quantitative trait could provide a useful tool in breeding programs; molecular genetic methods are capable of yielding this kind of information. An experimental procedure is presented here for identifying genes whose expression is related to weight variability of abdominal adipose tissue in the growing chicken. Quantitative traits are the result of metabolic pathways exhibiting some major regulation stages that are controlled genetically. These steps involve genes that may act as "major genes". With regard to chicken fat metabolism, most fatty acids are synthesized in the liver and incorporated into very low density lipoprotein (VLDL) particles before their secretion into the plasma. Accordingly, the present study focused on the expression of liver genes. The mRNA of lipogenic enzymes (acetyl-coenzyme-A carboxylase, fatty acid synthase, malic enzyme, and delta 9-desaturase) were analyzed. Also studied were apoprotein (apo)A1, apoVLDL-II, and apoB mRNA from 9-wk-old male chickens from two lines selected for high and low abdominal fat pads. Significant differences for apoA1 mRNA levels occurred between fat and lean birds. Moreover, the total quantity of mRNA provided an accurate estimation of the abdominal fat pad (r = .74 with P < .05).

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Fat crystallisation at oil-water interfaces.

This review focuses on recent advances in the understanding of lipid crystallisation at or in the vicinity of an interface in emulsified systems and the consequences regarding stability, structure and thermal behaviour. Amphiphilic molecules such as emulsifiers are preferably adsorbed at the interface. Such molecules are known for their ability to interact with triglycerides under certain conditions. In the same manner that inorganic crystals grown on an organic matrix see their nucleation, morphology and structure controlled by the underlying matrix, recent studies report a templating effect linked to the presence of emulsifiers at the oil/water interface. Emulsifiers affect fat crystallisation and fat crystal behaviour in numerous ways, acting as impurities seeding nucleation and, in some cases, retarding or enhancing polymorphic transitions towards more stable forms. This understanding is of crucial importance for the design of stable structures within emulsions, regardless of whether the system is oil or water continuous. In this paper, crystallisation mechanisms are briefly described, as well as recent technical advances that allow the study of crystallisation and crystal forms. Indeed, the study of the interface and of its effect on lipid crystallisation in emulsions has been limited for a long time by the lack of in-situ investigative techniques. This review also highlights reported interfacial effects in food and pharmaceutical emulsion systems. These effects are strongly linked to the presence of emulsifiers at the interface and their effects on crystallisation kinetics, and crystal morphology and stability.

Messenger RNA levels and transcription rates of hepatic lipogenesis genes in genetically lean and fat chickens.

Levels of body fat content in commercial meat chickens have prompted research in order to control the development of this trait. Based on experimentally selected divergent lean and fat lines, many studies have shown that liver metabolism has a major role in the fatness variability. In order to identify which genes are involved in this variability, we investigated the expression of several genes implicated in the hepatic lipid metabolism. The studied genes code for enzymes of fatty acid synthesis [ATP citrate-lyase (ACL), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), stearoyl-CoA desaturase (SCD1)], for an apolipoprotein [apolipoprotein A1 (APOA1)], and for the CCAAT/enhancer binding protein alpha (C/EBPalpha), which is a transcription factor implied in the regulation of several genes of lipid metabolism. The results show that the fat-line chickens display significantly higher hepatic transcription rates and mRNA levels than the lean-line chickens for the ACL, ME and APOA1 genes. This suggests that these genes could be responsible for the phenotypic fatness variability.

Hepatic lipogenesis gene expression in two experimental egg-laying lines divergently selected on residual food consumption.

Two Rhode Island Red egg-laying lines have been divergently selected on residual food intake (low intake R- line, high intake R+ line) for 19 generations. In addition to direct response, correlated responses have altered several other traits such as carcass adiposity and lipid contents of several tissues, the R+ animals being leaner than the R- ones. In a search for the biological origin of the differences observed in fat deposit, the hepatic mRNA amounts of genes involved in lipid metabolism were investigated. No difference was found between lines for mRNA levels of ATP citrate-lyase, acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and CCAAT/enhancer binding protein alpha, a transcription factor acting on several lipogenesis genes. The genes coding for stearoyl-CoA desaturase and apolipoprotein A1 displayed significantly lower mRNA levels in the R+ cockerels compared to the R-. All together these mRNA levels explained 40% of the overall variability of abdominal adipose tissue weight, suggesting an important role of both genes in the fatness variability.

Hormonal regulation of stearoyl coenzyme-A desaturase 1 activity and gene expression in primary cultures of chicken hepatocytes.

Previous studies have provided evidence for the important role of liver stearoyl-CoA desaturase (SCD) in excessive adiposity in the chicken and suggest that the difference in SCD activity between fat and lean chickens could be explained by a difference in SCD1 gene expression. In the present study, the regulation of SCD1 gene expression was analyzed as the result of insulin and glucagon action, using primary cultures of 6-week-old chicken hepatocytes. Insulin increased SCD1 activity and mRNA levels, whereas glucagon decreased dramatically both the enzyme activity and the mRNA levels. Nuclear run-on transcription assays and mRNA stability investigations demonstrated that insulin and glucagon effects on SCD1 gene expression was primarily transcriptional. Furthermore, the results indicated that the glucagon-mediated inhibition of SCD1 gene transcription was more potent than just counteracting the insulin-mediated effect. These data represent the first demonstration that the glucagon effect on the SCD1 gene expression is primarily transcriptional. Moreover, among hepatic genes involved in lipid metabolism in chicken, SCD1 is the first gene shown to be regulated at the transcriptional level by insulin, in the absence of triiodothyronine. These data point out the potency of the growing chicken hepatocyte culture model in contrast with the embryonic cell culture model as regards the investigations of the insulin effect on gene expression.

ApoVLDLII gene transcription in immature cockerels without estradiol stimulation.

1. Previous studies have led to the conclusion that the gene encoding the apo very low density lipoprotein II (ApoVLDLII) is under full estrogen control. However, by using a sensitive hybridization technique we found a weak expression of this gene in some males in the absence of hormone. 2. As the vitellogenin II gene, which is also estrogen dependent, remains inactive in these animals it is likely that they carry a deregulated allele of the ApoVLDLII gene. 3. The highest level of ApoVLDLII mRNA is found in genetically fat males suggesting a possible involvement of the ApoVLDLII gene in fatness determinism.

Hepatic lipogenesis in genetically lean and fat chickens. In vitro studies.

1. Acetyl-coenzyme A carboxylase, malic enzyme, glucose 6-phosphate dehydrogenase and delta 9-desaturase activity was measured in liver extracts from 5- to 11-week-old genetically lean or fat chickens. 2. A significant difference between the two lines of chickens was shown as concerns desaturating activity only, which was 45% higher in the fat animals than in the lean ones. 3. This result is consistent with the hypothesis of a higher rate of lipoprotein processing and secretion in the liver of the fat line chickens.

Lipid biosynthesis and deposition in genetically lean and fat chickens. Comparative in vivo studies with 14C acetate.

Following intravenous administration of 14C-acetate to genetically lean or fat chickens, the radioactivity in their liver, plasma, striated muscle and abdominal adipose tissue lipids and that in the breathed out CO2 were studied in function of time. As compared to the lean ones, the fat chickens showed a higher labelling of their triglyceride fatty acids in the plasma and adipose tissue, but not in the liver. Thus a higher rate of triglyceride secretion from the liver would be responsible for the higher abdominal fat weight in the fat line chickens.

Isolation and long-term maintenance of differentiated adult chicken hepatocytes in primary culture.

Adult chicken hepatocytes were obtained by an adaptation of the two step in situ collagenase perfusion. Usually 0.5 to 1 x 10(9) cells were obtained, with 75 to 95% viability. Hepatocytes attached within 2 h when plated on plastic cell culture dishes and spread in 4 h, surviving for several months in a specific serum-free medium. These cells retained a typical parenchymal cell morphology and the ability to produce a specific protein (albumin) throughout the culture period. We hereby provide a suitable model for studying hepatic metabolism in birds.

Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes.

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. beta-actin and albumin mRNA, as well as albumin secretion, were also assessed. Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.

Identification of 16 chicken microchromosomes by molecular markers using two-colour fluorescence in situ hybridization (FISH).

A feature of avian karyotypes is the presence of microchromosomes. As a typical avian genome, the chicken karyotype (2n = 78) consists of nine pairs of macrochromosomes, including the W and Z sexual chromosomes, and 30 pairs of indistinguishable microchromosomes usually ordered arbitrarily by decreasing size. Despite their reduced size, microchromosomes represent one-third of the genome and have a high gene density. So as to provide a tool to identify them, we developed a set of large insert-containing clones to be used as tags in two-colour fluorescence in situ hybridization experiments. Seventeen clones, six of which contain a microsatellite sequence and two others the fatty acid synthase gene or genes from the major histocompatibility complex, all presenting a strong hybridization signal, were selected for this purpose and enabled us to identify 16 different microchromosomes. The ability to recognize individual microchromosomes will be of great value for cytogenetic gene mapping, assignation of linkage groups from genetic maps and other studies on avian genome structure.

Effects of polyunsaturated fatty acids and clofibrate on chicken stearoyl-coA desaturase 1 gene expression.

In chicken, adiposity is influenced by hepatic stearoyl-CoA desaturase (SCD) 1. This gene is up-regulated by low-fat high-carbohydrate diet and down-regulated by addition of polyunsaturated fatty acids (PUFA). In this study, we present evidence for an inhibition of chicken SCD1 expression by PUFA using reporter gene constructs in transient transfection assays. This inhibition does not involve the peroxisome proliferator-activated receptor pathway, in contrast with what has been observed in rodents. We were able to localise a PUFA as well as an insulin response element within the -372/+125 bp region of the promoter. Sequence analyses of this region allowed identification of several cis-regulatory elements: A sterol regulatory element (SRE) and a juxtaposed NF-Y element which have been shown to be involved in the regulation of mouse SCD genes by PUFA. In addition, we identified an overlapping Sp1/USF motif, which was described to play a role in insulin/glucose and PUFA regulation of fatty synthase, ATP-citrate-lyase, and leptin genes. These data provide the first characterisation of the chicken SCD1 promoter and putative cis-sequences involved in the regulation of this gene by PUFA and insulin.

Cloning and characterization of the 5' end and promoter region of the chicken acetyl-CoA carboxylase gene.

Acetyl-CoA carboxylase is a rate-limiting enzyme in the biogenesis of long-chain fatty acids. In the present study, the 5' end and flanking region of the acetyl-CoA carboxylase (ACC) gene was analysed in the chicken. A genomic clone was isolated containing the first three exons, the third one containing the ATG codon. Using nuclease-mapping experiments and primer-extension analyses, the transcription-initiation site was located 153 nucleotides upstream of the ATG codon. In contrast with rat ACC gene expression, reverse transcriptase PCR analysis performed on chicken liver mRNA did not reveal alternative splicing in the 5'-untranslated region of these messengers. The promoter region is very G+C rich, and contains no TATA or CAAT boxes. Analysis by transient transfection in a human hepatoma cell line (HepG2) demonstrates that the promoter activity requires the presence of symmetrical sequences located upstream of the GC boxes. Transcription of this gene is found to be controlled by tri-iodothyronine in HepG2 cells, but the sequence responsible for the tri-iodothyronine response is not the consensus tri-iodothyronine-responsive element localized in the promoter. These results bring new insights to the regulation of the chicken ACC gene which differs from that of the rat.

Characterization of the chicken fatty acid synthase gene 5' part and promoter region.

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5' part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3-kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5' end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5' flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5' flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125-bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty-acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.