Cytogenetic mapping of eight genes encoding fatty acid binding proteins (FABPs) in the pig genome

In the present study cytogenetic localization of eight fatty acid binding protein genes in the pig genome was shown. BAC clones, containing sequences of selected genes (FABP1, FABP2, FABP3, FABP4, FABP5, FABP6, FABP7 and FABP8) were derived from porcine BAC libraries and mapped by FISH to porcine chromosomes (SSC) 3q12, 8q25, 6q26, 4q12, 4q12, 16q22, 1p22 and 4q12, respectively. Detailed analyses of regions containing gene clusters (FABP4, FABP5, FABP8) in chromosome 4 were performed and their order was established. It was shown that these three genes are located beyond the FAT1 region. Assignment of the FABP genes to chromosome regions harboring quantitative trait loci (QTL) for fat deposition is discussed.     

Nucleotide variability and linkage disequilibrium patterns at the porcine FABP5 gene

Fatty acid binding protein 5 (FABP5) is a major positional and physiological candidate gene for the porcine FAT1 QTL on SSC4. Here we characterize the nucleotide polymorphism and haplotype variability of FABP5 and we compare it with that of FABP4, given their close physical location and similar metabolic roles. DNA resequencing of the FABP5 gene region in 29 pigs from 14 breeds and in European and Japanese wild boars revealed 36 polymorphisms in 5.2 kb, and a nucleotide diversity of 0.19%, comparable to values reported in other domestic species but sixfold lower than that previously found for FABP4. Remarkably, both the nucleotide variability and the haplotype structure of FABP5 and FABP4 were dramatically different, and the Hudson-Kreitman-Aguadé test was highly significant. Nevertheless, both genes also had similarities. The neighbour-joining trees of their haplotypes did not show a geographical arrangement for any of the genes. Besides, both genes presented a similar extent and pattern of linkage disequilibrium. Haplotype blocks did not extend for large stretches ( approximately 1 kb in both genes), and the number of tag SNPs required to capture all variability was higher than previously expected. Our findings indicate that FABP4 and FABP5 have undergone different selective or evolutive processes. The fact that haplotype blocks were so small may require us to increase the number of SNPs in prospective whole-genome association studies in the pig.     

鸡脂肪酸结合蛋白基因的克隆和测序分析

根据哺乳动物脂肪酸结合蛋白基因序列设计一对引物对鸡基因组进行PCR扩增,将163bp扩增片段进行克隆和测序,并与猪的脂肪酸结合蛋白（fatty acid binding protein,FABP）基因序列进行同源性比较。该基因因片段与猪的心脏脂肪酸结合蛋白（heart fatty acid binding protein,H-FABP）基因有68%的同源性,与猪的脂肪型脂肪酸结合蛋白（adipocyte fatty acid binding protein,A-FABP）基因有75%的同源性,演绎成氨基酸之后与猪的脂肪型脂肪酸结合蛋白相应的氨基酸有75%的同源性。Northern结果表明该基因只在脂肪组织中表达。     

The identification of QTL that affect the fatty acid composition of milk on sheep chromosome 11

In this work, we analysed 11 genetic markers localized on OAR11 in a commercial population of Spanish Churra sheep to detect QTL that underlie milk fatty acid (FA) composition traits. Following a daughter design, we analysed 799 ewes distributed in 15 half‐sib families. Eight microsatellite markers and three novel SNPs identified in two genes related to fatty acid metabolism, acetyl‐CoA carboxylase α (ACACA) and fatty acid synthase (FASN), were genotyped in the whole population under study. The phenotypic traits considered in the study included 22 measurements related to the FA composition of the milk and three other milk production traits (milk protein percentage, milk fat percentage and milk yield). Across‐family regression analysis revealed four significant QTL at the 5% chromosome‐wise level influencing contents of capric acid (C10:0), lauric acid (C12:0), linoleic conjugated acid (CLA) and polyunsaturated fatty acids (PUFA) respectively. The peaks of the QTL affecting C10:0 and PUFA contents in milk map close to the FASN gene, which has been evaluated as a putative positional candidate for these QTL. The QTL influencing C12:0 content reaches its maximum significance at 58 cM, close to the gene coding for the glucose‐dependent insulinotropic polypeptide. We were not able to find any candidate genes related to fat metabolism at the QTL influencing CLA content, which is located at the proximal end of the chromosome. Further research efforts will be needed to confirm and refine the QTL locations reported here.     

Unexpected high polymorphism at the FABP4 gene unveils a complex history for pig populations

Fatty acid bing protein 4 (FABP4) plays a key role in fat regulation in mammals and is a strong positional candidate gene for the FAT1 quantitative trait locus located on porcine chromosome 4. DNA resequencing of the FABP4 gene region in 23 pigs from 10 breeds and wild boar revealed 134 variants in 6.4 kb, representing a silent nucleotide diversity of piS=0.01, much higher than reported so far in animal domestic species. Moreover, this diversity was highly structured. Also strikingly, the FABP4 phylogenetic tree did not show any geographical or breed origin clustering, with distant breeds sharing similar haplotypes and some of the most heterozygous samples pertaining to highly inbred animals like Iberian Guadyerbas (inbreeding coefficient approximately 0.3) or British Tamworth. In contrast, the cytochrome b (mtDNA) phylogenetic tree was coherent with geographical origin. The estimated age of the most recent common ancestor for the most divergent Iberian or Tamworth haplotypes was much older than domestication. An additional panel of 100 pigs from 8 different breeds and wild boar from Spain, Tunisia, Sardinia, and Japan was genotyped for seven selected single nucleotide polymorphisms and shows that high variability at the porcine FABP4 is the rule rather than the exception. Pig populations, even highly inbred, can maintain high levels of variability for surprisingly long periods of time.     

Time-Series Clustering of lncRNA-mRNA Expression during the Adipogenic Transdifferentiation of Porcine Skeletal Muscle Satellite Cells

Skeletal muscle satellite cells (SMSCs), which are multifunctional muscle-derived stem cells, can differentiate into adipocytes. Long-chain non-coding RNA (lncRNA) has diverse biological functions, including the regulation of gene expression, chromosome silencing, and nuclear transport. However, the regulatory roles and mechanism of lncRNA during adipogenic transdifferentiation in muscle cells have not been thoroughly investigated. Here, porcine SMSCs were isolated, cultured, and induced for adipogenic differentiation. The expressions of lncRNA and mRNA at different time points during transdifferentiation were analysed using RNA-seq analysis. In total, 1005 lncRNAs and 7671 mRNAs showed significant changes in expression at differential differentiation stages. Time-series expression analysis showed that the differentially expressed (DE) lncRNAs and mRNAs were clustered into 5 and 11 different profiles with different changes, respectively. GO, KEGG, and REACTOME enrichment analyses revealed that DE mRNAs with increased expressions during the trans-differentiation were mainly enriched in the pathways for lipid metabolism and fat cell differentiation. The genes with decreased expressions were mainly enriched in the regulation of cell cycle and genetic information processing. In addition, 1883 DE mRNAs were regulated by 193 DE lncRNAs, and these genes were related to the controlling in cell cycle mainly. Notably, three genes in the fatty acid binding protein (FABP) family significantly and continuously increased during trans-differentiation, and 15, 13, and 11 lncRNAs may target FABP3, FABP4, and FABP5 genes by cis- or trans-regulation, respectively. In conclusion, these studies identify a set of new potential regulator for adipogenesis and cell fate and help us in better understanding the molecular mechanisms of trans-differentiation.     

Disentangling Two QTL on Porcine Chromosome 12 for Backfat Fatty Acid Composition

A previous study allowed the identification of two QTL regions at positions 11–34 cM (QTL1) and 68–76 cM (QTL2) on porcine chromosome SSC12 affecting several backfat fatty acids in an Iberian x Landrace F2 intercross. In the current study, different approaches were performed in order to better delimit the quoted QTL regions and analyze candidate genes. A new chromosome scan, using 81 SNPs selected from the Porcine 60KBeadChip and six previously genotyped microsatellites have refined the QTL positions. Three new functional candidate genes (ACOX1, ACLY, and SREBF1) have been characterized. Moreover, two putative promoters of porcine ACACA gene have also been investigated. New isoforms and 24 SNPs were detected in the four candidate genes, 19 of which were genotyped in the population. ACOX1 and ACLY SNPs failed to explain the effects of QTL1 on palmitic and gadoleic fatty acids. QTL2, affecting palmitoleic, stearic, and vaccenic fatty acids, maps close to the ACACA gene location. The most significant associations have been detected between one intronic (g.53840T > C) and one synonymous (c.5634T > C) ACACA SNPs and these fatty acids. Complementary analyses including ACACA gene expression quantification and association studies in other porcine genetic types do not support the expected causal effect of ACACA SNPs.     

Evolutionary diversification of the avian fatty acid-binding proteins

Phylogenetic analysis of avian and other vertebrate fatty acid binding proteins (FABPs) supported the hypothesis that several gene duplications within this family occurred prior to the most recent common ancestor (MRCA) of tetrapods and bony fishes. The chicken genome encodes two liver-expressed FABPs: (1) L-FABP or FABP1; and (2) Lb-FABP. We propose that the latter be designated FABP10, because in our phylogenetic analysis it clustered with zebrafish FABP10. Bioinformatic analysis of across-tissue gene expression patterns in the chicken showed some congruence with phylogenetic relationships. On the basis of expression, chicken FABP genes seemed to form two major groups: (1) a cluster of genes many of which showed predominant expression in the digestive system (FABP1, FABP2, FABP6, FABP10, RBP1, and CRABP1); and (2) a cluster of genes most of which had predominant expression in tissues other than those of the digestive system, including muscle and the central nervous system (FABP3, FABP4, FABP5, FABP7, and PMP2). Since these clusters corresponded to major clusters in the phylogenetic tree as well, it seems a plausible hypothesis that the earliest duplication in the vertebrate FABP family led to the divergence of a gut-specialized gene from a gene expressed mainly in nervous and muscular systems. Data on gene expression in livers of two lines of chickens selected for high growth and low growth showed differences between FABP1 and FABP10 expressions in the liver, supporting the hypothesis of functional divergence between the two chicken liver-expressed FABPs related to food intake.     

Hierarchical subfunctionalization of fabp1a, fabp1b and fabp10 tissue-specific expression may account for retention of these duplicated genes in the zebrafish (Danio rerio) genome

Fatty acid-binding protein type 1 (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is encoded by a single gene in mammals. We cloned and sequenced cDNAs for two distinct FABP1s in zebrafish coded by genes designated fabp1a and fabp1b. The zebrafish proteins, FABP1a and FABP1b, show highest sequence identity and similarity to the human protein FABP1. Zebrafish fabp1a and fabp1b genes were assigned to linkage groups 5 and 8, respectively. Both linkage groups show conserved syntenies to a segment of mouse chromosome 6, rat chromosome 4 and human chromosome 2 harboring the FABP1 locus. Phylogenetic analysis further suggests that zebrafish fabp1a and fabp1b genes are orthologs of mammalian FABP1 and most likely arose by a whole-genome duplication event in the ray-finned fish lineage, estimated to have occurred 200-450 million years ago. The paralogous fabp10 gene encoding basic L-FABP, found to date in only nonmammalian vertebrates, was assigned to zebrafish linkage group 16. RT-PCR amplification of mRNA in adults, and in situ hybridization to whole-mount embryos to fabp1a, fabp1b and fapb10 mRNAs, revealed a distinct and differential pattern of expression for the fabp1a, fabp1b and fabp10 genes in zebrafish, suggesting a division of function for these orthogolous and paralogous gene products following their duplication in the vertebrate genome. The differential and complementary expression patterns of the zebrafish fabp1a, fapb1b and fabp10 genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated fabp1a and fabp1b genes, and the fabp10 gene in the zebrafish genome.     

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.     

Depot specific differences during adipogenesis of porcine stromal-vascular cells

Recently a role of adipose tissue as an endocrine organ secreting factors involved in the regulation of whole-body energy homeostasis has emerged. Preadipocytes in different fat depots have distinct adipogenic potential and the metabolic activity differs between mature adipocytes of different depot origins. Here we describe the proliferation and differentiation of stromal-vascular cells derived from subcutaneous and visceral fat depots of adult pigs. We demonstrate that subcutaneous porcine preadipocytes proliferate more actively and that individual subcutaneous adipocytes have a more rapid accumulation of triacylglycerols than visceral cells. During differentiation, subcutaneous and visceral preadipocytes showed similar gene expression patterns with increased expression of adiponectin (APM1), adipocyte-specific fatty acid binding protein (FABP4), catalase (CAT), and peroxisome proliferator-activated receptor gamma 2 (PPARG2). Furthermore, initial data showing depot-originated effects on the expression of CAT, carnitine palmitoyl transferase 1B (CPT1B) and FABP4 suggest possible depot specific differences in the function and metabolism of mature porcine adipocytes.     

Increased lipolysis in transgenic animals overexpressing the epithelial fatty acid binding protein in adipose cells

Fatty acid binding proteins (FABPs) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967-972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered. These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type.     

Phloretin promotes adipocyte differentiation in vitro and improves glucose homeostasis in vivo

Adipocyte dysfunction is associated with many metabolic diseases such as obesity, insulin resistance and diabetes. Previous studies found that phloretin promotes 3T3-L1 cells differentiation, but the underlying mechanisms for phloretin's effects on adipogenesis remain unclear. In this study, we demonstrated that phloretin enhanced the lipid accumulation in porcine primary adipocytes in a time-dependent manner. Furthermore, phloretin increased the utilization of glucose and nonesterified fatty acid, while it decreased the lactate output. Microarray analysis revealed that genes associated with peroxisome proliferator-activated receptor-γ (PPARγ), mitogen-activated protein kinase and insulin signaling pathways were altered in response to phloretin. We further confirmed that phloretin enhanced expression of PPARγ, CAAT enhancer binding protein-α (C/EBPα) and adipose-related genes, such as fatty acids translocase and fatty acid synthase. In addition, phloretin activated the Akt (Thr308) and extracellular signal-regulated kinase, and therefore, inactivated Akt targets protein. Wortmannin effectively blocked the effect of phloretin on Akt activity and the protein levels of PPARγ, C/EBPα and fatty acid binding protein-4 (FABP4/aP2). Oral administration of 5 or 10 mg/kg phloretin to C57BL BKS-DB mice significantly decreased the serum glucose level and improved glucose tolerance. In conclusion, phloretin promotes the adipogenesis of porcine primary preadipocytes through Akt-associated signaling pathway. These findings suggested that phloretin might be able to increase insulin sensitivity and alleviate the metabolic diseases.     

Structure-guided design,synthesis and in vitro evaluation of a series of pyrazole-based fatty acid binding protein (FABP) 3 ligands

We designed a series of pyrazole-based carboxylic acids as candidate ligands of heart fatty acid binding protein (H-FABP, or FABP3), based on a comparison of the X-ray crystallographic structures of adipocyte fatty acid binding protein (FABP4)–selective inhibitor (BMS309403) complex and FABP3–elaidic acid complex. Some of the synthesized compounds exhibited dual FABP3/4 ligand activity, and some exhibited selectivity for FABP3.     

A quantitative trait locus for oleic fatty acid content on Sus scrofa chromosome 7

A partial genome scan using microsatellite markers was conducted to detect quantitative trait loci (QTLs) for 10 fatty acid contents of backfat on 15 chromosomes in a porcine resource population. Two QTLs were discovered on Sus scrofa chromosome 4 (SSC4) and SSC7. The QTL on SSC4 was located between marker loci sw1336 and sw512, and this QTL was detected (P < 0.05) only for linoleic acid. Its position was in proximity of those mapped for linoleic acid content in previous studies. The QTL on SSC7 was mapped between markers swr1343 and sw2155, and it was significant (P < 0.05) only for oleic acid. A novelty of the QTL for oleic acid was suggested because the QTL was located far from any other QTLs previously mapped for fatness traits. The QTL on SSC7 explained 19% of phenotypic variation for oleic acid content. Further studies on fine mapping and positional comparative candidate gene analysis would be the next step toward better understanding of the genetic architecture of fatty acid contents.     

A non-synonymous mutation in a conserved site of the MTTP gene is strongly associated with protein activity and fatty acid profile in pigs

Despite the economic interest of the fatty acid profile in pigs, no gene has been convincingly associated with this trait so far. Here, the porcine microsomal triglyceride transfer protein ( MTTP ) gene, which plays a crucial role in the assembly of nascent lipoproteins, has been analysed as a positional candidate gene for a QTL affecting the fatty acid composition that was previously identified on chromosome 8 in an Iberian by Landrace F₂ cross. By resequencing a panel of different breeds, a non-synonymous polymorphism in a conserved residue of the lipid transfer domain of MTTP was identified. Association analyses with this polymorphism showed a strong association with the fatty acid composition of porcine fat, much stronger than the QTL effect, in the F₂ cross and in a synthetic Sino-European line. In addition, in vitro activity assays in liver protein extracts have shown that this mutation is also associated with the lipid transfer activity of the MTTP protein ( P  <   0.1). These results suggest that the detected polymorphism is a potential causal factor of the fatty acid composition QTL. There appears to be an interaction between the porcine MTTP genotype and the type of fat source in the pig diet, which would agree with the previous results on the biology of MTTP biology.     

Comparative mapping of a region on chromosome 10 containing QTL for reproduction in swine

Several quantitative trait loci (QTL) for important reproductive traits (age of puberty, ovulation rate, nipple number and plasma FSH) have been identified on the long arm of porcine chromosome 10. Bi-directional chromosome painting has shown that this region is homologous to human chromosome 10p. Because few microsatellite or type I markers have been placed on SSC10, we wanted to increase the density of known ESTs mapped in this region of the porcine genome. Genes were chosen for their position on human chromosome 10, sequence availability from the TIGR pig gene indices, and their potential as a candidate gene. The PCR primers were designed to amplify across introns or 3'-UTR to maximize single nucleotide polymorphism (SNP) discovery. Parents of the mapping population (one sire and seven dams) were amplified and sequenced to find informative markers. The SNPs were genotyped using primer extension and mass spectrometry. These amplification products were also used to probe a BAC library (RPCI-44, Roswell Park Cancer Institute) for positive clones and screened for microsatellites. Six genes from human chromosome 10p (AKR1C2, PRKCQ, ITIH2, ATP5C1, PIP5K2A and GAD2) were mapped in the MARC swine mapping population. Gene order was conserved within these markers from centromere to telomere of porcine chromosome 10q, as compared with human chromosome 10p. Four of these genes (PIP5K2A, ITIH2, GAD2 and AKR1C2), which map under QTL, are potential candidate genes. Identification of porcine homologues near important QTL and development of a comparative map for this chromosome will allow further fine- mapping and positional cloning of candidate genes affecting reproductive traits.