Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms . These candidate genes are known to regulate TGFβ signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFβ-mediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease. Gene expression data submitted to the NCBI Gene Expression Omnibus Accession No: GSE49533http://www.ncbi.nlm.nih.gov/projects/SNP/snp_viewTable.cgi?handle=MUSPNEUMONIA.SNP data submitted to NCBI dbSNP Short Genetic Variation  Streptococcus pneumoniae is an important pathogen of humans. It is estimated that pneumococcal diseases cause 5–35% mortality worldwide while less severe pneumococcal infections give rise to high costs of healthcare Attempts to characterize the genetic factors in pneumococcal pneumonia susceptibility in humans have predominantly taken a candidate gene approach and been limited to a small number of pneumonia cases An alternative approach is to use mouse models of pneumococcal diseases. Presently, most of the data on host susceptibility in mice come from comparative studies of pairs of mouse strains representing opposite disease phenotypes . Such studies have focused on immune cell profiles and inflammatory responses (predominantly in lungs) at different stages of the disease S. pneumoniae infection resistance QTL 1 (Spir1) was identified on proximal chromosome 7 Previously we undertook mapping of the disease susceptibility loci using BALB/c and CBA/Ca crosses and a A huge drawback in linkage analysis is its low mapping resolution that rarely leads to gene discovery. Additionally, a genetic effect of the QTL may be attributed to the combined effect of several physically linked genetic elements and can be disrupted during an attempt to narrow the QTL Currently the Jackson Laboratory (JAX) holds the largest repository of mouse strains (JAX-mouse strains). It offers a wide range of inbred, mutant and genetically engineered mouse strains for which the dense genotyping data and other relevant information are freely available. Recently 17 of the inbred mouse strains from the Jackson Laboratory have been fully sequenced by the Sanger Institute Spir1 locus In this study a panel of inbred JAX-mouse strains was evaluated for their susceptibility to infection with pneumococci. Collected phenotype data were then analysed by Efficient Mixed Model Association (EMMA) and Haplotype Association Mapping (HAM) to identify susceptibility loci. The complete sequences within the identified disease QTLs were then examined to narrow the number of candidate genes to those carrying the phenotype-specific polymorphism. Comparative analysis of the pulmonary gene expression in resistant and susceptible mouse strains was also used to support hypotheses of candidate genes and pathways. This approach identified three putative novel susceptibility QTLs, significantly narrowed the previously identified Our results indicate an important role of lung vasculature remodelling and immune cell regulation in the resistance to pneumococcal infection. We demonstrated that a prominent feature associated with the resistance was lung perivascular area free from cellular infiltrates. Subsequently we discussed the possible role of lung fibroblasts and/or smooth muscle cells in the resistance to pneumococcal infections. We also hypothesize a role of candidate genes in regulation of the TGFβ pathway and the vascular and immune cell functions in relation to the resistance to pneumococcal infection.S. pneumoniae susceptibility S. pneumoniae strain D39 and observed for 168 h (7 days), after which time each mouse that was alive was considered to have survived the infection. The shortest survival time was 21 h (LP/J) and the longest (for non-survivors) was 154 h (A/J) and the average survival time (excluding survivors) was 49 h. 32% of all tested animals survived infection but among the tested mouse strains the survival rate ranged from 100% to 0%. Survival differed significantly (p<0.001) between the mouse strains as measured by the time (hours) animal survived post-infection  with Pearson Rperiment . Furtherperiment , Table 1−8 were considered significant. For the HAM model the 132K SNP panel and the average survival time were used and the P value cut-off for an inferred haplotype was 10−5. The QTL interval was defined as a region containing SNPs or haplotypes that passed the significance threshold plus 1 Mb upstream or downstream of this region.The survival time of the 26 inbred JAX-mouse strains were used to map candidate pneumonia susceptibility loci using two statistical approaches: the Efficient Mixed Model Associations (EMMA) and the Haplotype Association Mapping (HAM). The EMMA mapping was done using the EMMA server and the HAM mapping was done using PLINK software. Two SNP panels (132K and 4Mil) and data for individual mouse were used in the EMMA mapping. The P value was calculated for each SNP in the panels and the SNPs with a P value below 5×10S. pneumoniae infection resistance 1 QTL (Spir1) identified during linkage analysis of progeny of crosses between CBA/CaOlaHsd and BALB/cOlaHsd Despite conceptual differences of the two methods, the results obtained with each were very similar . The QTL−8) in the EMMA mapping, using the 4Mil SNP panel, were then extracted and the nucleotide variant at the selected SNP position was examined in the tested panel of 26 inbred JAX-mouse strains and in the two Harlan strains (CBA/CaOlaHsd and BALB/cOlaHsd). There were 17 significant SNPs within the QTL on chromosome 7, seven of them within coding sequences for Ryr1, RasGRP4 and Spint2 and two SNPs within regions annotated as regulatory by the Mouse Phenome Database (Ntrk2 gene) and five significant SNPs within the QTL on chromosome 18   . The QTLd Kif20b .Comparison of the significant SNP variants between the tested mouse strains showed a clear separation of the resistant strains from the others , althougRyr1, RasGRP4, Spint2, Ntrk2, Stambpl1, Acta2, Fas, Slc16a12 and Kif20b) previously identified by EMMA and described above, but phenotype-associated SNPs also were found in a further 13 genes , five susceptible strains  and four strains with intermediate phenotypes . In this approach a SNP was classified as phenotype-associated if it was shared exclusively by all the mouse strains representing a given phenotype . Only SNPs differentiating resistant mouse strains from both susceptible and intermediate strains were identified in this analysis. No SNPs were found associated only with susceptible or only with an intermediate phenotype. The resistance phenotype-associated SNPs were located within the intergenic regions and the nine genes (13 genes . However13 genes .Ryr1 gene (over 400), including SNPs causing amino acid change or substitutions within a splicing site . Interestingly, Smad2 located near to the QTL on chromosome 18 was differentially regulated in BALB/cOlaHsd, but not in CBA/CaOlaHsd, during the infection. Two additional members of the SMAD family: Smad1 and Smad6 were also differentially expressed during the disease in BALB/cOlaHsd and CBA/CaOlaHsd, respectively (Differences in pulmonary gene expression between highly resistant (BALB/cOlaHsd) and highly susceptible (CBA/CaOlaHsd) mouse strains, during the early stage of pneumococcal infection (6 h post-infection), were then investigated. Gene expression was measured using an Illumina MouseWG-6_V2_0_R3 microarray and result submitted to the NCBI Gene Expression Omnibus, Accession No: GSE49533. The results were then validated by the real time RT-PCR of 15 selected genes. On average, an 81% correlation was observed between the two methods . Genes wparisons . Five geectively .The magnitude of DEGs up-regulation was much higher (about 40-fold change) than that of down-regulation (about 3-fold change) . Approxiversus CBA/CaOlaHsd PBS-treated) were compared  and axon guidance signalling were uniquely associated with the pulmonary transcriptome of the resistant BALB/cOlaHsd mice during the infection . Interescompared . The patcompared . Interescompared . There wExamination of lung histopathology showed that about 30% of perivascular areas of resistant BALB/cOlaHsd and almost 80% of susceptible CBA/CaOlaHsd mice were affected by cellular infiltration at 24 h post-infection and this difference was statistically significant. However the difference between both mouse strains was not observed at 6 h post-infection when about 30%–40% of perivascular areas of both CBA/CaOlaHsd and BALB/cOlaHsd were affected by cellular infiltrates . InteresPneumococcal infections can lead to a high mortality and morbidity in humans. While substantial effort has been made to understand the pneumococcal attributes that explain this situation, almost nothing has been done to explain the fact that individuals vary in their innate susceptibility to these infections. Identification of the host factors explaining innate resistance or susceptibility would help development of alternative, host-directed therapies and screening tests to identify individuals at risk. In the present study we aimed to identify host factors determining resistance to pneumococcal infection, using mouse models of pneumonia and bacteraemia. We phenotyped a panel of genetically defined inbred mouse strains for their resistance to pneumococcal infection and looked for association between the host genetic background and disease phenotype. Furthermore we analysed differences in gene expression and lung pathology between two mouse strains representing opposite disease phenotypes (susceptible CBA/CaOlaHsd versus resistant BALB/cOlaHsd).S. pneumoniae resistance QTL 1 (Spir1), identified by our previous linkage analysis of BALB/cOlaHsd (refer thereafter as BALB/c) and CBA/CaOlaHsd crosses (refer thereafter as CBA/Ca) Spir1 locus from 13 Mb to 2.6 Mb. The most statistically significant SNPs identified by EMMA were located within nine genes, or in intergenic regions, and were able to differentiate the resistant from the susceptible mouse strains, therefore they were regarded as phenotype-specific (or phenotype-associated).The tested panel of inbred mouse strains displayed variable disease phenotypes, ranging from death to complete resistance. The time that animal survived the infection  was used to map pneumococcal pneumonia susceptibility/resistance loci and four candidate QTLs on chromosomes 7, 13, 18 and 19 were identified. It is noteworthy that the candidate QTL on chromosome 7 is located within the Slc16a12. A large number of phenotype-associated SNPs were identified within this gene, some of them leading to a change within the amino acid sequence and the splicing site. This is worth mentioning because one of the best-known genetic factors influencing susceptibility to bacterial infection is a solute carrier transporter, Slc11a1  Slc11a1 gene was not associated with pneumococcal disease in our GWAS but finding another member of the same family associated with resistance to pneumococcal infection may suggest a broader role of the solute carrier transporters during bacterial infection. The transporter associated with pneumococcal disease, Slc16a12, belongs to the monocarboxylate transporters We took advantage of the availability of the full nucleotide sequences of thirteen of the tested inbred stains However, the susceptibility to complex diseases such as infectious diseases, most likely depends on networks of genes, with additive genetic effects, or on groups of proteins forming interacting networks Smad4, Smad2 and Smad7 on chromosome 18 and Smad5 on chromosome 13) focused our attention on the TGFβ pathway, in which SMAD proteins are key factors Stambpl1 and Acta2 (both within the QTL on chromosome 19). The former gene encodes the STAM binding protein-like 1 that interacts with SMAD2 and SMAD7 The presence of a cluster of genes encoding members of the SMAD family within or nearby two of the disease QTLs , up-regulated only by BALB/c during pneumococcal infection. Egr1 is known to inhibit TGFβ-mediated Acta2 expression In addition to influencing certain immune cells, the TGFβ-pathway also regulates wound healing, angiogenesis and extracellular matrix production via activation of fibroblast differentiation Acta2 expression. The parenchymal fibroblasts, but not the bronchial ones can rapidly activate endogenous TGFβ via tension-mediated mechanism Fibroblasts are ubiquitous cells, which in normal lungs residue in vascular adventitia and airway wall tissue. Beside their vital role in maintaining tissue structure and integrity, fibroblasts also regulate immune cells trafficking, site-specific accumulation and survival Ryr1, Kcnk6, Ntrk2, Ppp1r14a and Kif20b). Ryr1 is a calcium channel Kcnk6 is a potassium channel Ppp1r14p is a phosphatase inhibitor Ntrk2 is a neutrophin receptor Kif20b encodes a kinesin Ntrk2 knock-out mice show significantly reduced blood vessel formation Kcnk6 knock-out mice show vascular dysfunction The large accumulation of immune cells within perivascular areas of CBA/Ca but not BALB/c mice suggests that the vasculature is involved in the resistance. We showed that perivascular areas of resistant BALB/c mice were either unaffected or mildly inflamed, even when massive inflammation within lung parenchyma was observed. The accumulation of leukocytes within perivascular areas could cause damage to the vessel structure and facilitate bacterial dissemination into the blood, which is a feature of infection of CBA/Ca but not of BALB/c mice. The hypothesis that alteration of the vasculature is a key aspect of susceptibility is further supported by analysis of our pulmonary gene expression data that showed one of the most enriched functional gene clusters during the pneumococcal infection is for the remodelling of lung vasculature. Furthermore, we have found a group of candidate genes with identified phenotype-associated polymorphism to be involved in smooth muscle functioning (Retnlg), which is secreted by adipocytes, was almost 3-fold higher in the susceptible CBA/Ca mice during pneumococcal infection.Interestingly, we found that the enrichment of genes belonging to the adipocytokine signalling pathway was unique for the CBA/Ca pulmonary transcriptome. This is worth noting because the adipocytokines are secreted predominantly by adipose tissue, which in the lung is located within perivascular areas. This perivascular adipose tissue (PVAT) plays an important role in the regulation of vascular structure and homeostasis Dcc (netrin receptor Dcc) was encoded within the QTL on chromosome 18, in direct proximity of the cluster of highly significant intergenic SNPs. In addition to the axon guidance pathway being involved in the formation of the neuronal network that can control blood vessels and bronchial contractility, it can also regulate leukocyte transmigration from blood to lung tissue PVAT is also an important element of a brain-blood vessel axis because nerve endings are present in the PVAT, enabling neuronal control of vessel function Ntrk2 (in the QTL on chromosome 13), for a neutrophin receptor, is also involved in cross-talk between nervous and immune systems Ntrk2 functions places this gene in a central position linking airway smooth muscle, nervous and immune system, three areas where differences were found between resistant and susceptible strains during pneumococcal infection. In this context it is also notable that Ntrk2 was recently described as a protecting factor in pneumococcal meningitis It is noteworthy though, that a candidate gene Fas, Map4k1, RasGRP4 and Spint2  were involved in regulation of immune cells. For example, RasGRP4 encodes guanine nucleotides exchange factor, which was recently reported to influence ROS production in neutrophils Spint2 encodes a Kunitz-type serine protease inhibitor. Interestingly, a Kunitz-type protease inhibitor was reported to suppress pro-inflammatory cytokine production in macrophages and neutrophils Fas and Map4k1 both of which showed phenotype-associated amino acid substitutions. Their expression also differed between CBA/Ca and BALB/c in disease or between control animals. Although Fas regulates apoptosis in a wide range of cells, Map4k1 is expressed exclusively in hematopoietic compartment. Map4k1 plays a pivotal role in regulation of activation induced T-cell apoptosis There is clear evidence that regulation of immune cell populations differs between resistant and susceptible strains during pneumococcal infection. This can be seen in timing and quantity of cellular infiltrations The regulation of immune cell populations is a key factor in resistance to pneumococci that can lead to either resolution (associated with resistance) or uncontrolled inflammation (associated with susceptibility). The trafficking of immune cells from the bloodstream to tissues parenchyma is regulated by fibroblasts and may be critical for the host early defences against pneumococci All animal experiments were performed in accordance to the UK Home Office guidelines. The University of Leicester Ethics Committee and the UK Home Office approved the study protocols. Animals were housed and experiments carried out at designated facility at the University of Leicester.The female mice of 26 inbred Jax-mouse strains, 10 mice per strain were purchased from The Jackson Laboratory: 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BALB/cJ, BTBR T+ tf/J, C3H/HeJ, C57BL/J, C57BL/6J, C57BL/10J, C57BLKS/J, C57BR/cdJ, C58/J, CBA/J, DBA/1J, DBA/2J, FVB/NJ, I/LnJ, LP/J, MA/MyJ, NOD/ShiLtJ, NZW/LacJ, PL/J, SEA/GnJ, SJL/J and SM/J while two strains BALB/cOlaHsd and CBA/CaOlaHsd were purchased from Harlan, UK. Mice were housed in the Division of Biomedical Services, University of Leicester. All mice were fed on fundamental diet. Mice used for the experiments were 9 to 12 week old.Streptococcus pneumonia serotype 2 strain D39 (NCTC7466) from the National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom was used. To prepare a challenge stock bacteria were grown overnight on blood agar base (BAB) plate with 5% horse blood in CO2 jar at 37°C. Next day bacterial colonies were transferred from the BAB plate to THY media (Gibco) until OD600 reached 0.2. The primary culture was diluted 100 times in THY and grown at 37°C in anaerobic condition until the culture reached OD600 corresponding to 1–3×108 CFU/ml . At this point culture was mixed with glycerol , aliquot and frozen at −70°C. Culture was stored up to 3 months.8 CFU/ml. Mice were lightly anaesthetized with 2.5 to 5.0% (v/v) fluothane  over oxygen (1.5 to 2 l/min) and intranasally infected with 20 µl (5×106) of the challenge dose. 24 h post-infection about 50 µl of blood were taken from the tail vain and bacteria load in blood was measured by viable count . Mice were closely monitored and the pain score chart was filled every 6 h until animal was lethargic or until the end of the experiment (7 days) when animal was classified as a survivor. This time point was then used as a surrogate for the survival end point. Animals were humanly euthanized by cervical dislocation when they reached the end point, either when lethargic or at the end of the experiment (168 h). At the time of death blood and lung were collected and bacterial load was measured by viable count.To prepare the challenge dose, aliquot of bacteria stock was centrifuged (13 K rpm) and pellet resuspended in PBS to final concentration 2.5×10The time the animal survived the infection , was used for genome-wide associations mapping to identify disease QTLs. Collected data for each mouse were first analysed using statistical software SPSS. Bartlett's test for equal variances was calculated for each mouse strain and phenotype. One-way ANOVA and Tukey's Multiple Comparison post-test were used to check for statistically significant differences between strains. Strains were classified as resistant, sensitive or intermediate based on the ANOVA results of the survival time for each strain. The rate of survival was represented as the percentage of animals that survived the infection (168 h).Pulmonary gene expression in CBA/CaOlaHsd and BALB/cOlaHsd mice  was assessed at 6 h post infection. Mice inoculated with 20 µl of PBS and sacrificed 6 h post-inoculation were treated as control. Six control animals and five infected animals per strain were used in the gene expression experiment. Control and infected mice were sacrificed at 6 h time point and whole lung was removed. Lungs were cut into small squares and immediately immersed in RNAlater (Qiagen). Lung RNA was isolated using RNAeasy mini kit (Qiagen) according to manufacturer instructions. cRNA was synthesized using Ambion MessageAmp kit for Illumina arrays according to manufacturer instruction. The whole lung gene expression was done using chip MouseWG-6_V2_0_R3 on Illumina platform. Gene expression data deposited within the NCBI Gene Expression Omnibus (GEO), Accession No: GSE49533.Microarray data were analysed using GenomeStudio (Illumina) and ArrayTrack For quantitative RT-PCR, 500 ng of total RNA was reverse transcribed using the RevertAid H Minus First Strand cDNASyntheis Kit according to manufacturer protocol (Fermentas). Real time quantitative PCR was performed in triplicate on a LightCycler 480 Real-Time PCR System (Roche) in a final volume of 5 µl using 1× Maxima SYBR Green qPCR master mix (Fermentas) and 3.3 µM of forward and reverse primers for each mRNA tested. Cycling conditions were 95°C for 15 minutes, and 38 cycles of amplification at 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds. Primers were designed to amplify the same transcripts as those tested on the Illumina MouseWG-6_V2_0_R3 whole genome gene expression beadchips . Gapdh aGenotype files  for phenotyped inbred Jax-mouse strains were downloaded from Mouse Phenome Database (MPD). To search for haplotype associations  the mean survival time (hours) was used. Data were analysed using gPLINK version 2.050 Measurements for survival in hours for 10 animals per mouse strain were used to search for association using Efficient Mixed Model Association, EMMA −5 for 130K SNP panel and p-value equal or below 5×10−8 when 4Mil SNP panel was used. The interval of the QTL was calculated as the region containing significant SNPs with 1 Mb downstream and upstream of this region The QTLs were consider a candidate disease QTL if at least 3 SNPs within 0.5 Mb were found displaying significance equal or below p-value 10For sequencing of BALB/cOlaHsd and CBA/CaOlaHsd mice, DNA was extracted from mouse-tail tissue using the Nucleon kit (Gen-probe). Sequencing was performed at the Wellcome Trust Centre for Human Genetic's High Throughput Genomics Facility (UK), generating 8- to 10-fold coverage of a single lane of PE100 nt per sample on the HiSeq2000. Paired-end Illumina reads from CBA/CaOlaHsd were aligned to the reference genome  using BWA, the same was done for BALB/cOlaHsd. Detection of SNPs in each alignment was made using the Genome Analysis Toolkit (GATK) with default parameters. We adopted a filtering strategy to identify high-confidence SNPs and reduce false positives. SNP sites that failed GATK's internal status check, quality <300 or had an allele ratio <0.8 were removed from any further analysis. Differing SNPs between CBA/CaOlaHsd and BALB/cOlaHsd were then compared to the precompiled SNPs found in 11 strains from the Mouse Genome Project . We generated a list of SNPs between CBA/CaOlaHsd and BALB/cOlaHsd that were copied in CBA/J and BALB/cJ; this was used as the final list of differentiating SNPs. List of SNPs was deposited in NCBI dbSNP Gene list were generated for each selected QTL using MGI Genes and Markers Query Form. Genes within selected QTLs were then compared with the lists of differentially expressed genes during pneumococcal infection of BALB/c and CBA/Ca strains (ArrayTrack). The list of differentially expressed genes between non-disease condition in BALB/cOlaHsd and CBA/CaOlaHsd was also analysed. The sequence of the genes within QTL region was examined using Mouse Phenome Database (MPD) SNP variation query form (NCBIM37). The gene was classified as most likely causal candidate if found within susceptibility QTL and either showed genetic polymorphism within or nearby its coding region or showed change in expression during pneumococcal infection. The identified nucleotide substitutions within candidate genes that led to amino acid change were also analysed using the PROVEAN tool At necropsy, tissue samples were immersed in 10% v/v neutral buffered formaldehyde solution prior to conventional processing and embedding in paraffin wax. Histopathological assessment was performed on tissue section stained with haematoxylin and eosin (BDH). Lungs samples were then examined microscopically and vessels with visible perivascular areas were counted as affected (accumulation of cellular influx) or unaffected (clear perivascular areas). The number of affected vessels per lung was then calculated as a percentage of total number of counted vessels (affected and unaffected) in the lung section. Lungs of five animals per time point (6 h and 24 h) and per mouse strain were examined.Table S1The genome-wide significant SNPs  identified during the EMMA mapping and their alteration within tested inbred mouse strains. The SNP ID, Ensemble annotation for the SNP  and log10 p-value scores for the EMMA mapping [GWAS log(p)] are presented. The red colour indicates resistant mouse strain or SNP associated with the resistance to pneumococcal infection.(XLSX)Click here for additional data file.Table S2Correlation between expression scores obtained by microarray and RT-qPCR technique.(XLSX)Click here for additional data file.Table S3Primer pairs used for RT-qPCR of selected mouse genes.(XLSX)Click here for additional data file.


GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations.Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls ; a pattern that is reversed to XL hypomethylation found in PHPIb. Interestingly, XL hypermethylation was associated with reduced XLalphaS protein levels in the patients' platelets. Methylation for NESP and ExonA/B was significantly different for some but not all patients, though most patients have site-specific CpG methylation abnormalities in these amplicons. Since some AHO features are present in other imprinting disorders, the methylation of IGF2, H19, SNURF and GRB10 was quantified. Surprisingly, significant IGF2 hypermethylation  and SNURF hypomethylation  was found in patients vs. controls, while H19 and GRB10 methylation was normal.We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated In conclusion, this is the first report of methylation defects including GNAS in patients with an AHO-like phenotype without endocrinological abnormalities. Additional studies are still needed to correlate the methylation defect with the clinical phenotype. GNAS gene have been reported to cause Albright's Hereditary Osteodystrophy , a complex and broad phenotype mostly characterized by short stature, obesity, round face, subcutaneous calcifications, brachydactyly and cognitive impairment GNAS loss-of-function mutations on maternally inherited alleles have pseudohypoparathyroidism type Ia  that is characterized by AHO and resistance to multiple stimulatory G protein-coupled hormones (e.g. Parathormone (PTH) and others) GNAS mutations are reported as having only AHO features or pseudopseudohypoparathyroidism (PPHP)  patients GNAS defects in PHP-Ia patients without mutations in the GNAS coding region (GNAS (epi)genetic defects. The latter is further supported by the Gs functional overlapping between PHP-Ia and PHP-Ib recently reported, where Gsalpha hypofunction, determined either in isolated erythrocyte membranes or in platelets, has been detected also in patients with GNAS imprinting mutations, AHO features and hormone resistance GNAS mutations. The severity of the AHO phenotype varies greatly between patients, and some patients have only few features of the syndrome.Heterozygous inactivating mutations affecting the m (PPHP) Table 1 GNAS also the IGF2/H19 cluster and the SNURF/SNRPN regions have been described to play a causative role in embryonic growth defects. DNA methylation defects involving imprinting control region 1 (ICR1) of the IGF2/H19 locus for which methylation abnormalities result in two growth disorders with opposite phenotypes: the overgrowth disorder Beckwith-Wiedemann syndrome H19-ICR1 hypermethylation and the growth retardation disorder Silver–Russell syndrome H19-ICR1 loss of methylation. Prader-Willi and Angelman syndromes SNURF/SNRPN region is hypermethylated in some Prader-Willi syndrome patients Some clinical characteristics of AHO are also reported in imprinting syndromes Silver-Russell, Beckwith-Wiedemann, Prader-Willi and Angelman that are mainly characterized by defects in growth, behaviour and/or development. To further support the common soil of imprinting disorders, an ‘imprinting gene network’ that regulates embryonic growth and differentiation dependent on Zac-1 ) regulation has been identified GNAS , IGF2/H19 and SNURF in 17 patients with some typical AHO features that mainly include in common growth retardation and brachydactyly. Methylation studies of GRB10 are also performed, as the imprinting of this gene is not actually linked to growth regulation but rather to behaviour GNAS coding mutations.We here study the methylation of the growth regulatory imprinted genes Verbal informed consent to collect blood samples for advanced non-routine diagnostic procedures was obtained from the participants and/or their legal representatives. This strategy is in agreement with the Belgian Law and local regulations and was specifically approved for this study by the Ethics Committee of the Katholieke Universiteit Leuven- University of Leuven. The Ethics Committee of the Katholieke Universiteit Leuven- University of Leuven, also waived the need for formal approval by the ethical review board.Patients enrolled in this study were followed at or referred to the pediatric endocrinology department of the University Hospital in Leuven (Belgium).Patients were selected based on having AHO features, mostly with severe short stature, mental retardation or behavioural problems, clinodactyly or short metacarpals. Few patients also showed obesity and none of them presented with subcutaneous calcifications. One patient (patient 5) showed heterotopic ossifications, and was diagnosed with Progressive Osseous Heteroplasia 1  or the stable prostacyclin analogue Iloprost  were added one minute prior to induction of aggregation with collagen (2 µg/ml). The 50% inhibitory concentration (IC50) was evaluated for each Gs agonist from the patient's response curve and compared to the mean IC50 measured on platelets of a group of controls (n = 24) for the same agonist The platelet aggregation-inhibition test was performed as described GNAS were amplified and sequenced using conditions previously described GNAS region, we performed genotyping of different SNPs by PCR and direct sequencing within the NESP55, XL and Exon A/B regions  was used for bisulfite treatment with the MethylDetector™ bisulfite modification kit  as described SNURF methylation was studied via Sequenom EpiTYPER technology using primers and conditions already reported IGF2, H19 (ICR1 region) and GRB10 regions were designed using the Sequenom EpiDesigner software. Primers and amplicons characteristics are reported in andNESP, XL, Exon A/B and The sequence and chromosomal location of all amplicons are shown in IGF2 and SNURF amplicons, we have screened for the presence of SNPs in the same region that was used for the Sequenom analysis and its surrounding region. A list of all the IGF2 and SNURF SNPs are reported in  and S3 that could exclude also deletions in the loci as most patients are heterozygous for the intronic SNPs rs734351 and rs2855523.To rule out the presence of SNPs that could interfere with the methylation detection sensitivity in the 3VO4, 1 mmol/liter EDTA, 1 mmol/liter phenylmethylsulfonyl fluoride, 2 mmol/liter dithioerythreitol, 1% aprotinin, and 2 mmol/liter NaF, and incubated on ice for 60 min. Platelet extracts (50 μg) were mixed with Laemmli sample buffer and resolved by SDS/PAGE. Blots were revealed with a monoclonal anti-Gsα antibody Platelet immunoblot analysis for XLalphas, Gsalphas and CAP-1 was performed as described Average of CpGs methylation for each amplicon was calculated for both controls and patients samples. Statistical analysis was performed using PRISM 5.0a software. Two-tailed unpaired T-test (p<0.05) was used to study group methylation differences between PPHP patients and healthy controls for all the imprinting control regions studied and to evaluate protein expression differences.GRB10, n = 47 for Exon A/B, n = 45 for IGF2, n = 33 for H19, n = 35 for SNURF) . Values with a Z-score ≤−2 and ≥+2 were considered significantly hypo- or hypermethylated, respectively. Normality test was assessed with SPSS 12.5 software to study the control population values distribution.A more individual statistical approach was then performed comparing each patient's Sequenom CpG value or amplicon average with the distribution of values of the same variable measured in a group of healthy controls , as compared to the healthy controls. This platelet aggregation-inhibition Gs test was performed in 24 healthy controls and we compared their mean IC50 values for patients.We studied platelet Gs activity in 13 PPHP patients with variable AHO features as reported in GNAS screening for inactivation mutations was performed using leukocyte gDNA for sequencing the PCR amplified 13 exons, including exon/intron boundaries. No GNAS coding mutations were found in any of the patients. All patients were heterozygous for at least one of the studied GNAS region SNPs, excluding small chromosomal deletions within the GNAS cluster  and had platelet Gs hypofunction,  cluster TableS1GNAS methylation was screened for the three amplicons NESP, XL and ExonA/B using the Sequenom EpiTYPER as we previously optimized for PHP-Ib and PHP-Ia cases vs. controls (36±3 vs. 29±3% (mean±SD); T-test, p<0.001; To evaluate whether the XL hypermethylation would be associated with decreased XLalphaS expression, immunoblot analysis was performed using platelet extracts as we previously also did for a PHP-Ib patient with XL hypomethylation and increased XLalphaS levels in platelets IGF2 and 25 CpGs in the ICR1 of the H19 locus , though a significant overall hypermethylation was observed for patients 2 and 4 . For the H19 region also some specific CpGs show significant differences in methylation but only for a few patients and clustering within patients seemed not be present. Spearman correlation between IGF2 methylation and height of patients was not significant.We next studied 30 CpGs in the DMR1 of SNURF included 18 CpGs and a significant hypomethylation in the SNURF amplicon was found for patients vs. controls  . The analysis of single CpGs showed some significant differences for some patients with both hyper- and hypomethylated sites  could present with GNAS methylation abnormalities if coding GNAS mutations are also excluded. We studied GNAS methylation in 16 patients with clinical diagnosis of PPHP and 1 POH patient without GNAS mutations but having platelet Gs hypofunction and an AHO phenotype that mainly involves short stature and brachydactyly and/or other types of bone abnormalities. GNAS methylation was quantified for the three differentially methylated regions using the Sequenom EpiTYPER as we previously did for PHP-Ib and PHP-Ia cases The human The main defect in our patients is the significant XL hypermethylation that could be linked to their Short for Gestational Age (SGA) and shortness phenotype. Interestingly, it is known that the main phenotype for XLalphaS deficient mice is the regulation of postnatal growth with neonatal feeding problems, leanness, inertiae and a high mortality rate IGF2, H19 and GRB10 together with GNAS have been described to be part of an imprinted gene network that regulate embryonic growth and differentiation dependent on Zac-1 regulation in mice IGF2, H19, SNURF and GRB10. Surprisingly, we could observe significant hypermethylation for IGF2  and hypomethylation  for SNURF while H19 and GRB10 showed no overall differences between patients and controls. The physiological relevance of these findings in relation to the clinical phenotypes remains to be studied. However, some other groups already reported so-called multilocus methylation abnormalities . In conclusion we studied GNAS, IGF2, H19, SNURF and GRB10 methylation in patients with and AHO-like phenotype and Gs hypofunction but no GNAS coding mutations. We could broaden the spectrum of (epi)genetic defects associated with an AHO phenotype by identifying an epigenetic defect in XL, IGF2 and SNURF in 16 PPHP patients and 1 POH case. More studies on multiple imprinting control regions in more PPHP patients are warranted to further investigate the combination of epigenetic defects in relation to phenotypes.Mutations in a trans-acting factor involved in establishing or maintaining methylation at multiple chromosomal loci however could also explain the presence of such overall methylation abnormalities. The latter hypothesis has been demonstrated in the Beckwith-Wiedemann syndrome Figure S1GNAS schematic representation of genomic regions studied via Sequenom EpiTYPER. GNAS schematic representation of genomic regions studied via Sequenom EpiTYPER. Features of the paternal and the maternal allele are shown above and below the line, respectively. The arrows show initiation and direction of transcription. Paternal and maternal transcripts are highlighted in blue and pink, respectively. The first exons of the protein coding transcripts are shown as black boxes and the first exons of the noncoding transcripts (Nespas and exon A/B) are shown as gray boxes. Differentially methylated regions (DMRs) are shown by + symbols (indication of methylation). For each amplicon reported in the black frames CpG sites are underlined, CpGs studied via Sequenom are additionally depicted in italic and bold. Red dinucleotides refer to SNPs analysed in the same regions. The figure is not to scale. Adapted from Izzi et al. Curr Mol Med 2012.(TIF)Click here for additional data file.Figure S2IGF2/H19 schematic representation of genomic regions studied via Sequenom EpiTYPER. Features of the paternal and the maternal allele are shown above and below the line, respectively. The arrows show initiation and direction of transcription. Paternal IGF2 transcript is highlighted in blue. The first exons of the protein coding transcripts are shown as black boxes. Differentially methylated regions (DMRs) are shown by + symbols (indication of methylation). For each amplicon reported in the black frames CpG sites are underlined, CpGs studied via Sequenom are additionally depicted in italic and bold. Red dinucleotides refer to SNPs analysed in the same regions. The figure is not to scale. Adapted from Jeong et al. Nature Genetics (2004) 36, 1036–1037.(TIF)Click here for additional data file.Figure S3SNURF (A) and GRB10 (B) schematic representation of genomic regions studied via Sequenom EpiTYPER. Features of the paternal and the maternal allele are shown above and below the line, respectively. The arrows show initiation and direction of transcription. Paternal SNURF transcript is highlighted in blue. The first exons of the protein coding transcripts are shown as black boxes. Differentially methylated regions (DMRs) are shown by + symbols (indication of methylation). For each amplicon reported in the black frames CpG sites are underlined, CpGs studied via Sequenom are additionally depicted in italic and bold. Red dinucleotides refer to SNPs analysed in the same regions. The figure is not to scale. B adapted from Hikichi et al. Nucleic Acids Research (2003) 31 (5): 1398–1406.(TIF)Click here for additional data file.Table S1(XLSX)Click here for additional data file.Table S2(XLSX)Click here for additional data file.Table S3(XLSX)Click here for additional data file.


Male obese Zucker rats (11-weeks old) had increased body weight , liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis  and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1- Branched chain amino acids  are important nutrient signals increasing insulin secretion in islet beta cells and mammalian Target of Rapamycin (mTOR) signaling in most tissues. In addition, they regulate satiety and affect glucose metabolism through peripheral and central mechanisms 13C]-Leu and gas-chromatography mass spectrometry (GC-MS), elevated protein turnover and proteolysis in obese humans has been reported To better understand how BCAAs associate with the pathogenesis of obesity and diabetes, it is necessary to determine the mechanisms underlying their elevation. Plasma BCAA concentrations result from the difference between their rates of appearance and disappearance. These are due on the one hand to dietary intake and tissue proteolysis, and counterbalanced by protein synthesis and BCAA catabolism. It is possible that elevated plasma BCAA concentrations in obesity and diabetes are secondary to decreased protein synthesis and/or elevated protein degradation resulting from insulin resistance as originally speculated by Felig and coworkers Given the highly regulated nature of BCAA catabolism, diminished or incomplete BCAA catabolism could also contribute to elevations in BCAAs. Recent metabolomic, proteomic and genomic studies have suggested altered BCAA metabolism as a potential etiology with some studies focusing on adipose tissue 14C]-α-ketoisocaproate (KIC) using methods outlined by Wolfe and colleagues 3H]-Phe and the flooding dose method. To elucidate the role of changes in the rate-controlling step in BCAA oxidation, total and actual BCKDC activities were also determined. Amino acid and acylcarnitine profiling were also performed to elucidate the status of later steps in BCAA metabolism, incomplete fatty acid oxidation and protein turnover. To the best of our knowledge, acylcarnitines have not been previously characterized in this model. In addition to providing insight into BCAA derived metabolites, other acylcarnitines have been linked to obesity comorbidities such as insulin resistance, alterations in intestinal bacterial flora or have been found to polarize macrophages to the M1 pro-inflammatory phenotype associated with metabolic syndrome Thus, beyond elevated food intake, it is not clear what mechanisms additionally contribute to the branched chain aminoacidemia of obesity. Therefore, in this study the potential role of BCAA appearance from food intake and proteolysis, along with their rates of disappearance due to protein synthesis and oxidation were determined in obese Zucker fa/fa rats, a genetic model of obesity and insulin resistance. We additionally sought to understand how nutrient signaling to translation initiation in obesity, e.g., All procedures were conducted after review and approval by the Penn State Hershey Institutional Animal Care and Use Committee (IACUC). The Animal Resource Program, operated by the Department of Comparative Medicine, is accredited by AAALAC International. All animal living conditions are consistent with standards laid forth in the Guide for the Care and Use of Laboratory Animals, 8th edition, published by the National Research Council.14C-Leu), (B) protein synthesis using the [3H]-Phe flooding-dose method, and (C) metabolomics/enzyme activity assays. To facilitate comparison of the results from these studies, each of the cohorts experienced similar handling, single housing, infusion , nutritional state and surgical interventions  with the exception that animals in the latter cohort were housed for three days prior to catheterization in Nalgene metabolic cages to obtain 24 h urine samples. Although urine was collected daily, only the urine on the third collection day was aliquoted on ice and frozen at −84°C for biochemical analyses. Venous (jugular) and arterial (carotid) catheters were surgically implanted using sterile procedures in all rats under isoflurane anesthesia as previously described 1H-NMR was performed on awake animals for ∼1 minute after body weight determination, as previously described Male obese (fa/fa) Zucker and lean control (Fa/?) rats at the age of ∼9 weeks were obtained from Charles River Laboratories . After quarantine they were acclimated to single housing for ∼1 wk while being maintained on a 12∶12 h light dark cycle at ∼21°C and provided free access to water and a standard rodent chow . The concentration of protein (18%) and leucine (1.8%) in this diet were supplied by the manufacturer and more than one lot of the diet was used in these studies. Three cohorts of narrow body weight range Zucker rats (n = 10/lean and obese genotype) were used in three separate experiments: (A) Leu metabolism  it decreases the variability of measurement of Leu turnover and protein synthesis in randomly fed rats where the timing of food intake is not controlled, (b) the animals are not normally in the overnight fasted state and (c) an overnight fast can affect both plasma BCAA concentrations and the concentration of enzymes involved in BCAA metabolism At the conclusion of each study animals were anesthetized using isoflurane at which time additional blood was collected for hormone or metabolomic studies. The heparinized blood was maintained on ice until plasma was isolated following centrifugation at 4°C. The plasma was then frozen in liquid nitrogen. At this time tissues were also collected and freeze clamped at the temperature of liquid nitrogen. The tissues were quickly wrapped in labeled foil and kept in liquid nitrogen. Later tissues were powdered under liquid nitrogen. The frozen tissue powders and plasma were transferred to and maintained at −84°C until subsequent biochemistry or analytical studies.Amino acid concentrations were analyzed by two methods that provided different subsets of amino acids and had different sensitivities. The first method measured 10% sulfosalicylic acid (SSA)-extracted amino acids using a BioChrom 30 Amino Acid Analyzer  with a cation exchange column with post-column ninhydrin detection at 440 and 570 nm housed in the Hormone Assay and Analytical core of the Vanderbilt Diabetes and Research Training Center. The second method has been previously described This UPLC-MS/MS method was employed in order to measure 3-MeHis and resolve it from 1-methyl-histidine (1-MeHis). The distinction is important because the degradation of anserine, from which 1-MeHis arises, and muscle proteins, from which 3-MeHis arises, is thought to occur through different mechanisms. There are two caveats with the assay of these isomers. First, the nomenclature for these two molecules has been reversed in recent years and this change has been adopted by chemical vendors, but not in the biological literature. This has caused confusion For tissue amino acid analysis, powders of freeze-clamped gastrocnemius and liver were aliquoted (200–300 mg) and then extracted with ice-cold 10% 5-sulfosalicylic acid  following Polytron homogenization. The homogenates were centrifuged at 3,000 g for 15 min. Plasma was deproteinized with 10% SSA  and centrifuged at 8,000 g for 10 min. Aliquots of supernatants were used for amino acid analysis using the above BioChrom 30 Amino Acid Analyzer method. Tissue amino acid concentrations were normalized to the amount of precipitated protein. Protein in the precipitates was measured using the BCA™ protein assay kit  after pellets were solubilized with 2.5 ml of 1 N NaOH facilitated by vortexing and heating at 80°C for 30 min.14C specific radioactivity (SA) was determined as previously described 4OH. One ml of the amino acid eluate was used to determine radioactivity by liquid scintillation counting. Recoveries of [1-14C]-Leu added to cold plasma and tissue samples carried through this procedure were ∼98%. Leu 14C SA was calculated by dividing 14C radioactivity  by the amount of Leu present as determined by HPLC.Leu-Plasma glucose and insulin was measured as previously described 14C-radioactivity in the eluted fractions corresponding to KIC was determined using a Beckman 6600IC scintillation counter. Non-radioactive rat plasma was studied after addition of [U-14C]-KIC standard and was carried through the above procedure, and a 68% recovery of [U-14C]-KIC standard in KIC fractions was used to correct actual 14C-KIC radioactivity in plasma samples. Plasma and tissue 14C-KIC SA was calculated by dividing radioactivity by KIC concentration in each sample.For specific activity determination, the eluates were collected using a BioRad Model 2110 Fraction Collector . The DPMs of Urine creatinine concentration was measured spectrophotometrically using the Jaffe reaction. Urine total and free carnitine, butyrobetaine (the precursor of carnitine) and seventy acylcarnitine species were quantified in the CLIA compliant laboratory of Dr. C.L. Hoppel  as previously described 14C]-Leu by monitoring the 14CO2 in the expired air while the rats rested in a sealed chamber designed for calorimetry studies  and finely regulated to 1600 ml/min with a Bel-Art Scienceware (#404070125) Aluminum Riteflow Mounted Flowmeter. The air flowed through one rigid inlet tube ; air exited the chamber via two outlet tubes of the same diameter to prevent backpressure and flowed into a CO2 trap containing 10 ml of Hyamine 10X hydroxide -ethanol .At 0900 h, after 2h of food deprivation but free access to water, previously catheterized rats were placed into a closed chamber  without food. The chamber as supplied was fitted with a convection fan along with sealed connectors for catheters, air flow-in and -out as shown in 14C]-NaHCO3 facilitates quicker equilibrium of the 14CO2 pool so that the time for [1-14C]-Leu infusion for measuring Leu oxidation can be shorter than infusing [1-14C]-Leu alone 14C]-NaHCO3  and [1-14C]-Leu . Bicarbonate pools were first brought into isotopic equilibrium with [14C]-NaHCO3  at a priming dose of 3.6 µCi (30 µl/min for 3 min) and then continuously infused  for 117 min. [1-14C]-Leu  was primed with a dose of 18 µCi  for 117 min. 14CO2 samples were collected every 10 min throughout the infusions. The recovery of 14CO2 of infused [14C]-NaHCO3 as expired 14CO2 was determined for CO2 fixation efficiency and retention calculation 14C]-Leu infusion.The protocol for the Leu infusion studies was developed from the monograph by Wolfe 14C]-NaHCO3 samples trapped from expired gas and [1-14C]-Leu infusates were diluted 300x with 1 N NaOH and H2O, respectively. The diluted infusates and the 14CO2 Hyamine samples were mixed with 10 ml of Hionic-Fluor  and DPMs- in each sample was determined by scintillation counting.At the conclusion of the infusion protocol, rats were anesthetized with sodium pentobarbital (70 mg/kg) injected into the artery catheter. Indicated tissue samples were quickly collected, freeze-clamped using liquid nitrogen, these samples were quickly foil wrapped and stored at −80°C for biochemical analysis. [14C]-Leu SA 14C]-KIC is thought to better represent the intracellular [14C]-Leu specific activity, plasma 14C-KIC SA was used to calculate Leu turnover and oxidation rates, unless otherwise indicated. The formulas used to were as follows:All calculations were carried out at near isotope and substrate steady-state using the formulas below derived by Wolfe and colleagues KIC (dpm/µmol)*wt (kg)], where F is Leu turnover rate (i.e. Leu flux or appearance rate from proteolysis if there was no food absorption); I is [1-14C]-Leu infusion rate, SAKIC is plasma [14C]-KIC SA; and wt is either body weight or FFM.F (µmol/min/kg) = I (dpm/min)/[SA14CO2 (dpm/min)/[SAKIC(dpm/µmol)*wt (kg)], where 14CO2 is the rate of 14CO2 production corrected by a CO2 fixation efficiency and retention factor. This factor was individually determined for each animal. SAKIC is plasma KIC 14C SA, and wt is either body weight or FFM as indicated.Ox (µmol/min/kg)  = PD (g protein/kg/h) = [F (µmol/min/kg) * 60]/590 (µmol/g protein)], where F is Leu turnover, and 590 represents an average mol of Leu per g of whole-body protein. PS (g protein/kg/h) = [(PD-Ox) * 60]/590.Protein synthesis rates were also determined using the flooding dose approach as previously described 13CO2 generated from α-keto[1-13C]-isocaproate via BCKDC was measured using an isotope ratio mass spectrometry system Branched chain keto acid dehydrogenase complex (BCKDC) extracts from rat tissues were prepared as reported previously t-tests were used to compare the difference between the lean and obese groups. To reduce the risk of false positives in HPLC data, we corrected for multiple comparisons using the method of Benjamini and Hochberg as supplied with Graphpad Prism 6.0 software, Data are expressed as mean ± SEM, and P ≤ 0.05 was considered as statistically significant. Statistical analysis was performed using Graphpad Prism 6.0 software; two-tailed unpaired Each of the three cohorts of rats had similar body weights , 2, 3. ABody composition changes were also apparent in wet weights of tissues, exemplified by those from cohort B . Body weAs anticipated, the obese rats consumed more food, protein and Leu per day along with more water . HoweverTo decrease the variability of plasma measures and other metabolic assessments, overnight food deprivation is frequently used. However as mentioned in the Methods, we have found that this duration of food deprivation alters plasma BCAAs and enzymes in lean and obese animals Twenty-four hour urine collections were made after the animals had a 48 h acclimation period in the metabolic cages. Consistent with their increased body weight , obese rFor reference the metabolic pathway for the branched chain amino acids and their ketoacids is shown in Plasma concentrations of Leu, Val and Ile  were increased 46–68% in obese rats compared to lean rats . Plasma Urinary concentrations of BCAAs were increased in obese rats by 130–170% . During 14C]-Leu 14C]-Leu the plasma 14C-KIC SA within each group did not differ at three time points during the last hr of the infusion indicating a steady-state metabolism of infused 14C-Leu , in agreement with a previous report The elevations in the plasma and urine BCAAs and their metabolites in the obese rats were ∼2–8 fold higher than the ∼20% increased Leu intake corrected for body weight suggesting that changes in BCAA metabolism also occur. Leu turnover studies using -Leu is derived from BCKDC-catalyzed oxidative decarboxylation, our Leu oxidation data reflect an elevated whole-body BCKDC-catalyzed activity in obese rats. Increased BCAA oxidation in obesity seems inconsistent with our previous findings of decreased content, activity and phosphorylation-mediated inactivation of key regulatory enzymes of BCAA catabolism in liver and adipose tissue of obese and T2D animal models The rate of whole body Leu oxidation was increased in obese rats regardless of whether data were normalized to body weight (+22%) or FFM +60%, . Since 1Compared to other tissues, liver had the highest BCKDC activity in lean rats. Total and actual BCKDC activity in liver was decreased by 54% and 68%, respectively, in obese Zucker rats compared to lean controls. For comparison purposes, hepatic citrate synthase activity, measured as a mitochondrial marker, decreased only 19%. Thus, a portion of the decreased BCKDC activity is attributable to decreased hepatic mitochondrial content in obese rats. As the body composition of the obese rats was different, we also calculated enzyme activities per organ. When hepatic BCKDC activity is considered in this context, that is with liver mass as the denominator, the actual BCKDC activity did not differ between the lean and obese animals. Citrate synthase activity per liver increased 65% supporting the idea that the enlarged liver also has more mitochondria, but this change was not proportional to the increase in the liver mass, which was increased 107% in obesity . Renal B13C methodology was used and this necessitated that the BCKDC activity be expressed in different units. Unfortunately, in this experiment the lamda phosphatase activation of the enzyme did not work so only actual BCKDC activities are available. In addition, the adipose tissue activities were too close to the level of detection and contamination of room air CO2, so they are not provided. Actual activity measurements were therefore only available for muscle , actual and total tissue BCKDC activity (lower) and protein degradation per FFM (higher) changed in a direction that would positively contribute to elevating circulating BCAAs and BCKAs in obesity. Opposing these rises in BCAAs, whole body protein synthesis per FFM was also increased, however, tissue specific changes were restricted to a few tissues. The effect on protein synthesis may be due in part to elevated nutrient signaling arising from the branched chain aminoacidemia. BCKDC activity decreased in all tissues and previous studies indicate it should decline even further in adipose tissue, for review see An obvious factor contributing to elevated BCAAs and BCKAs in the obese rats appears to be increased BCAA intake, because animals were sampled in the fed to early-postabsorptive state. The mitochondrial isoform of branched chain amino acid transaminase (BCATm) catalyzes the first step in BCAA metabolism in most peripheral tissues. However this enzyme is absent in liver thereby allowing BCAAs in particular to rise postprandially so they may act as a circulating signal of a protein-containing meal. We found that BCAA intake per g body weight was 23% greater in obese animals. While the percent rise in food intake is not as great as changes in BCKAs or metabolites in various compartments, food intake is clearly a contributing factor. It is noteworthy that virtually all other studies examining blood BCAA in obese humans or rodents were conducted in the overnight-fasted state, suggesting that obesity-associated increases in blood BCAA in those cases could not be due solely to differences in dietary input.14C-Leu radiotracer was also increased, with the caveat that evolved 14CO2 only marks the first step in Leu oxidation and does not imply complete oxidation. A decrease in the concentration/activity of enzymes downstream in the BCAA metabolic pathway relative to the incoming substrate load might contribute to accumulation of upstream metabolites reflective of incomplete BCAA oxidation. An example of this is our finding of elevated urinary 3-OH-isovalerycarnitine in obese rats. Elevation of this urine metabolite is used clinically to signal a block or deficiency in 3-methylcrotonyl-CoA carboxylase. Consistent with this possibility, genomic studies indicate down-regulation of gene expression along the whole BCAA metabolic pathway in liver and/or adipose tissue in the insulin-resistant or obese state, for reviews see in situ, and the tissue origins of systemic and urinary BCAA derivatives are areas that warrant further study.Another potential factor that can contribute to elevated BCAAs in obesity is their rate of disappearance via complete BCAA oxidation, which could potentially be reduced in the obese state. Our observation of increased urinary BCAA-derived metabolites in obese Zucker rats is consistent with increased overall BCAA catabolism, at least through the first steps of BCAA oxidation. Consistent with this view, whole-body Leu oxidation as measured by 1-14CO2 evolution in all the tissues studied. When calculations were performed using organ mass as the denominator, a different picture emerged. That is actual BCKDC activity is still decreased in kidney, heart and muscle, but the whole liver BCKDC actual activity is not changed due to obesity.Our oxidation measurements indicated that Leu oxidation was increased in obese rats consistent with some previous clinical studies, e.g. We could not measure BCKDC activity in adipose tissue due to very low activity and in other previous studies in vitro activities from adipose tissue have also been low compared to other tissues Thus, we speculate that increased whole-body BCAA oxidation in obesity may be the result of a substrate effect in liver (a key tissue for BCKA catabolism) based on two observations: (1) the amount of actual hepatic activity per animal (i.e. per liver) in obese animals does not change compared to lean animals and (2) the substrate for hepatic BCKDC, BCKAs, were elevated to a greater extent in the liver of obese animals (∼3-fold) than in either muscle or plasma. While we were surprised that the concentrations of BCAAs per gm of liver were not different, this may also be due to changes in body composition, that is a dilution of hepatic BCAAs due to the more than doubling of the liver size. Acute elevations in BCKAs normally activate BCKDC We also found decreased BCKDC activity in the heart. Previous studies have shown that a regulatory defect in BCKDC due to loss of the BCKDC phosphatase (PP2Cm) leads to a partial reduction in BCKDC activity and elevations of plasma BCKAs and BCAAs Two other factors potentially underlying the elevations in BCAAs in obesity are protein synthesis and protein degradation. Hyperaminoacidemia and elevated plasma Leu have been linked to increased mTOR activity The increase in the size of liver and fat in this model is consistent with elevated rates of protein synthesis. This increased muscle protein synthesis in obese rats is contrary to our data showing that muscle mass is decreased or unchanged in obesity and therefore suggests a possible compensatory role for protein degradation. Indeed, whole-body proteolysis was elevated when normalized to lean body mass. Those findings are consistent with previous studies in Zucker rats in the food deprived, postabsorbtive state finding increase protein degradation relative to protein synthesis It is noteworthy that hyperaminoacidemia in lean animals is usually associated with increased protein synthesis and decreased protein degradation, e.g. A limitation of our current study is that we did not measure protein turnover in individual tissues as would be possible by measuring arterial-venous differences. Hence using our approaches we did not assess the relative role of each tissue to proteolysis, which may be differ owing to the decreased demand for physical activity and smaller muscle sizes in the obese animals along with the larger liver.The finding, that obese Zucker rats simultaneously increased the rate of two opposing and energy-consuming processes, protein synthesis (based on lean body mass) and protein degradation, suggests a futile cycle for amino acids similar to that in obese rats for glucose, pyruvate and triglyceride In our study plasma alanine increased however paradoxically plasma glycine decreased. Increased plasma alanine and decreased glycine was also reported by Felig et al. A number of urinary acylcarnitines and 3-OH acylcarnitines were elevated by obesity including acylcarnitines of intermediate chain length (exemplified by decanoylcarnitine and the S- and R-isomers of 3-hydroxy-decanoylcarnitine) that may be indicative of increased and incomplete β-oxidation Our data show that isobutyrylcarnitine (metabolite of Ile) is elevated 874% in obesity, more than any other metabolite including the BCAAs. Despite the magnitude of this elevation, other unique downstream metabolites of Ile were not detected in lean or obese animals, in contrast to a number of metabolites found for Leu and Val. Increased isobutyrylcarnitine along with propionylcarnitine were previously identified in insulin-resistant obese human plasma We observed an ∼800% increase in an unidentified C4-OH acylcarnitine. One possibility is that this C4-OH-acylcarnitine is derived from 2-hydroxy-butyrate a metabolite whose blood concentration correlates with insulin resistance and glucose intolerance A urine amino acid arising from collagen, 4-OH-proline, was greatly elevated in obese Zucker rats and has not been previously linked to obesity. However, it is recognized to be elevated in obese individuals during food restriction In conclusion, we report that BCKAs were elevated more than BCAAs in plasma and tissues of obese rats. Thus, BCKAs may represent a more sensitive metabolic signature compared to BCAAs for obesity or insulin resistance. In future studies, following BCKAs concentrations and metabolic capacity may be important for several reasons. As mentioned already, circulating BCKAs are thought to be better representatives of the intracellular pool of BCAAs Our results also show that the elevated plasma BCAAs in obese Zucker rats arise in part from increased BCAA intake, a decrease in the actual activity of BCKDC in peripheral tissues and increased protein degradation. Excess BCAAs appeared to increase protein synthesis and BCAA oxidation; these processes probably contribute to dampening the rise in systemic BCAA concentrations. Elevated leucine and protein turnover also appear to contribute. Alterations in protein turnover could underlie the disproportionately smaller muscles in these rats as previously suggested by Chan et al


Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral  and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of  Echinococcus granulosus affects a wide range of livestock mammals and humans, causing cystic echinococcosis (hydatid disease), a zoonosis with significant economic and public health impact. The disease is characterised by the growth of a fluid-filled cyst in the host's viscera (mainly liver and lung). The most relevant antigen for hydatid disease diagnosis is antigen B (EgAgB), a highly abundant lipoprotein present in the cyst fluid. There is overwhelming literature regarding EgAgB antigenicity and molecular characterisation at the protein and gene levels, but the knowledge of the lipids physiologically bound to EgAgB protein subunits is very scarce. Indeed, there is only one report showing that delipidated EgAgB binds fatty acids in vitro. This work describes the physiological lipids of EgAgB, an important piece of information to complete our knowledge on EgAgB molecular composition. In contrast to what was thought, EgAgB consists of a variety of neutral and polar lipid classes, associated to protein subunits, forming a plasma lipoprotein-like particle of 229 kDa in size. Taken into account that E. granulosus cannot synthesise fatty acids and sterols, these data suggest that EgAgB plays a role in the uptake and transportation of these essential lipids across parasite structures.The larva of the cestode parasite  Echinococcus granulosus is the causative agent of cystic echinococcosis (hydatid disease) in a range of mammalian species (mainly domestic ungulates) as well as in humans. It is a unilocular fluid-filled cyst, which steadily grows inside host visceras (mostly liver and lung). One of the major molecules produced in large amounts by the cyst is a highly immunogenic lipoprotein named antigen B (EgAgB) The larval stage of the cestode parasite EgAgB1 to EgAgB5E. granulosus isolates from different geographic origins, a recent study has proposed that there are at least 10 EgAgB distinct genes, including four and three different genes corresponding to the EgAgB3 and EgAgB4 clades E. granulosus genome revealed the presence of seven EgAgB loci clustered on a discrete region of the genome, with one copy each of EgAgB1, EgAgB2, EgAgB4 and EgAgB5, as well as three slightly differing copies of EgAgB3A lot of efforts have been made to understand the molecular composition/organization of EgAgB , and then purified the EgAgB lipid moiety by extraction with organic solvents. Characterisation of immunopurified EgAgB of bovine origin showed that lipoprotein particles are constituted mostly by EgAgB8/1 apolipoprotein and that the native lipid moiety of this antigen comprises neutral and polar lipids that have not been previously described as ligands of this HLBP family.The present work was aimed at identifying the native lipid moiety of EgAgB in order to complete our knowledge on the EgAgB molecular composition; this information could simultaneously shed light into EgAgB structure and function. Of particular relevance was to determine whether EgAgB binds tert-butyl-4-hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA) and authentic lipid standards including cholesterol (CH), fatty acids (FA), triacylglycerols (TAGs), phosphatidylethanolamine (PE), cardiolipin (CLP), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine (PC) were acquired from Sigma Chemicals (USA). Solvents (HPLC grade or better) and α-naphthol were purchased from Merck (Germany) or Fisher Scientific (USA).Inorganic salts, 3,5-di-E. granulosus HCFs from cysts containing protoscoleces of bovine origin were obtained by aspiration of the content of cysts present in lungs and livers of naturally infected cattle. Cysts were collected during the routine work of local abattoirs in Montevideo (Uruguay). E. granulosus HCFs of human origin, collected from surgically-removed hepatic hydatid cysts, were generously donated by Dr A. Leites and Dr E. Torterolo . All HCF samples were preserved by addition of 5 mM EDTA and 20 µM BHT, and maintained at −20°C. For EgAgB purification, three batches of bovine HCF  and two samples of individual human HCF were used.g for 30 min at 4°C and the resulting supernatant filtered consecutively through 5, 2, 0.8 and 0.45 µm filter membranes (Millipore). The clarified HCF was firstly fractioned by anion exchange chromatography on a Q-Sepharose column  previously equilibrated in 20 mM phosphate buffer, pH 7.2 containing 200 mM NaCl, 5 mM EDTA and 20 µM BHT. After washing in equilibration buffer, the retained material was eluted by changing ionic strength to 400 mM NaCl in a single step. The eluted fraction  was used to purify the antigen to homogeneity by immunoaffinity chromatography based on the utilization of a monoclonal antibody (MoAb) -named EB7- that specifically recognises the native lipoprotein Native EgAgB was purified from HCF following a previously described protocol First dimension was performed with commercially available IPG-strips . Immunopurified EgAgB was prepared and concentrated by using the 2-D Clean-Up kit  and dissolved in rehydration solution , 0.002% bromophenol blue, DTT 17 mM). Samples in rehydration solution were loaded onto IPG-strips by passive rehydration during 12 h at room temperature. The isoelectric focusing was done in an IPGphor Unit (Pharmacia Biotech) employing the following voltage profile: constant phase of 300 V for 30 min; linear increase to 1000 V in 30 min; linear increase to 5000 V in 80 min and a final constant phase of 5000 V to reach total of 6.5 kVh. Prior running the second dimension, IPG-strips were reduced for 15 min in equilibration buffer  supplemented with DTT (10 mg/ml) and subsequently alkylated for 15 min in same equilibration buffer but supplemented with iodoacetamide (25 mg/ml). The second-dimensional separation (SDS-PAGE) was performed in 15% polyacrilamyde gels using a SE 260 mini-vertical gel electrophoresis unit . The molecular size markers used were Amersham Low Molecular Weight Calibration Kit for SDS Electrophoresis . The gels were silver stained according to http://www.matrixscience.com/search_form_select.html) and using the following search parameters: monoisotopic mass tolerance, 0.05 Da; fragment mass tolerance, 0.2 Da; carbamidomethyl cysteine and methionine oxidation as variable modifications and up to one missed tryptic cleavage allowed.Identification of protein spots was performed by mass spectrometry (MS) using a 4800 MALDI TOF/TOF™ (AB Sciex). Briefly, peptide mass fingerprinting plus MS/MS ion search of selected spots were carried out by in-gel trypsin treatment  at 37°C, overnight. Peptides were extracted from gels using 60% acetonitrile in 0.1% TFA, concentrated by vacuum drying. Peptides were further concentrated and desalted using C18 reverse phase micro-columns . Peptide elution from micro-column was performed directly into the mass spectrometer sample plate with 2 µl of matrix solution (α-cyano-4-hydroxycinnamic acid in 60% aqueous acetonitrile containing 0.1% TFA). Mass spectra of digestion mixtures were acquired in the MALDI-TOF/TOF mass spectrometer using the reflector mode. Spectra were externally calibrated using a mixture of peptide standards . For increased confidence of identification, selected peptides were further fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Proteins were identified by NCBI nr database searching using the MASCOT program (Matrix Science 3∶CH3OH mixture (2∶1) and vigorously shaken for 2 minutes. Next, the homogenate was filtrated and the extract washed with NaCl solution to reach a final concentration of 0.73% and a CHCl3∶CH3OH∶H2O ratio of 2∶1∶0.2 in volume. After vigorous agitation for 1 minute, the separation of phases was achieved by centrifugation at 2400 rpm for 20 min and the upper phase was removed by aspiration and discarded. The lower phase containing lipids was recovered and taken to dryness by rotary evaporation at 40°C under vacuum; last traces of solvent were removed by a stream of N2 (g). Finally, the lipid fraction was dissolved in CHCl3 to a concentration of 10 mg/mL. As a control of lipid contaminants in buffers and/or solvents, an equal volume of PBS-EDTA-BHT was used in parallel for extraction. Purified lipid extracts were stored at −20°C under N2(g) until analysis.Total lipids were extracted according to the methodology described by 2(g).The protein content of immunopurified EgAgB preparations was determined using bicinchoninic acid in a microtitre plate assay (BCA Protein Assay kit) with BSA as standard . Total lipids in EgAgB were determined gravimetrically by weighting purified lipids immediately after extraction and solvent removal under a stream of N3/CH3OH (2∶1 v/v) and then activated at 100°C for 30 min. Lipid samples (fractions obtained from HCF and EgAgB and standards) were spotted manually using a micro-syringe (Hamilton). Double development was initially carried out as follows: plates were first half-developed using a mobile phase for resolving polar lipids (PL), and after drying under a N2 (g) stream, were developed to completion in a mobile phase for neutral lipids (NL). Single development for resolving PL or NL was also performed. The solvent systems used as mobile phase were: for PL resolution- methyl acetate/isopropanol/chloroform/methanol/0.25%KCl  and for NL resolution- hexane/diethyl-ether/acetic acid . Lipid bands were visualised by spraying the plates with 8% (m/v) CuSO4 in a 10% (v/v) H3PO4 aqueous solution, and heating at 140°C; the identification of lipid classes was performed by comparison with primary and secondary standards run on the same HPTLC plate. The relative abundance of each lipid class respect to the total lipid content could not be estimated because not all lipids were resolved adequately in a single HPTLC using double development. We estimated the percentage of individual lipid classes in the total of NL or PL instead. 1-nonadecanol was used as internal standard for normalization. HPTLC plates were scanned and the intensity of the bands was determined using the ImageJ software (http://rsb.info.nih.gov/ij/). In addition, specific staining for sterol esters/sterols and glycolipids were carried out using FeCl3.6H2O and α-naphthol, respectively g, 4°C for 30 min) to obtain a cell membrane enriched fraction. Lipids were then extracted following the Folch method as described above.Separation of lipid class components was performed by high-performance thin layer chromatography (HPTLC) on Kieselgel 60 plates (Merck). HPTLC plates (10×10 cm) were pre-washed by migration in CHCl2SO4 in methanol at 50°C for 16 h. The purified FAMEs were dissolved in hexane and then subjected to GLC analysis on a Hewlett-Packard 5890 equipped with a Carbowax 20 capillary column and flame ionisation detector (FID). The oven temperature was initially set at 180°C for 10 min, and then increased at 2.5°C/min to 212°C, level at which was held for 10 min. The individual FAMEs peaks were identified by comparison of their retention times with those of authentic FAMEs standards.The fatty acid composition of EgAgB and HCF lipid fractions was analysed by gas-liquid chromatography of their methyl esters derivatives (FAMEs); for these studies material of both human and bovine origin were used. Lipid fractions were subjected to acid methanolysis with 1% Hdn/dc ratio set to 0.186 mL/g Light scattering analysis was carried out using a Superset 200 HR 10/30 SEC column , connected to an HPLC system (Schimatzu) at room temperature. Immunopurified EgAgB (200 µL of a 0.7 mg/mL solution in PBS) was applied onto the column previously equilibrated in PBS, and elution was monitored with on-line detection using the following detectors: multiangle laser light scattering , ultraviolet UV  and differential refractive index . In addition, plasma-derived high density lipoprotein (HDL) was analysed in the same conditions for comparison. For HDL preparation, human plasma was obtained from a healthy donor and HDL purification was carried out following conventional ultracentrifugation methods m/z of 1275.68 and 1399.69 that matched with the ELEEVFQLLR and YFFERDPLGQK sequences, respectively. These peptides lie in conserved regions of the amino acid sequence of EgAgB8/1. Therefore, we cannot discriminate which ones of the already described EgAgB8/1 isoforms is present in the samples. These isoforms include molecules having different theoretical isoelectric point .Most studies were performed with EgAgB of bovine origin since the availability of parasite material of human origin was very limited. EgAgB was purified to homogeneity from bovine HCF using a previously described procedure based on selective adsorption of this antigen on Q-Sepharose followed by immunoaffinity chromatography using immobilised MoAb EB7 ic point . In the ic point , arrow, Previous work suggested that EgAgB8 apolipoproteins are capable of binding fatty acids As an initial approach for examining the complexity of lipid classes, HCF and EgAgB lipids were analysed by HPTLC using double development. Under these conditions the majority of neutral and polar lipids are clearly separated, although the resolution of some lipid classes may not be optimal. By analysing in parallel a mixture of authentic lipid standards, we observed that the natively-bound lipid component of immunopurified EgAgB is highly heterogeneous, comprising several lipid classes, all of which are also found in the HCF. Indeed, the lipid fractions of both EgAgB and HCF showed to contain a wide variety of lipid classes from very hydrophobic ones (compatible with sterol esters and TAGs) to charged ones (phospholipids) ; this pa3 at acid pH  for normalization. This analysis was carried out only for HCF and EgAgB of bovine origin. As shown in  acid pH . The mosWith respect to polar lipids, the analysis of EgAgB and HCF in parallel with phospholipid standards showed that phosphatidylcholine was the main phospholipid present in both samples. Phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine were also detected in smaller amounts as well as traces of cardiolipin. Furthermore, minor components with higher mobility than phospholipids but lower than neutral lipids compatible with glycolipids were observed. The presence of glycolipids was confirmed by staining with α-naphthol, a dye that specifically reacts with sugar groups .The relative abundance of the major lipid classes found in immunopurified EgAgB and HCF within total neutral or polar lipids was estimated as shown in We next analysed the fatty acid composition of total lipids extracted from human and bovine EgAgB and HCF . Globallinset).Bovine EgAgB was analysed by SEC to study its physical state in aqueous solution. EgAgB eluted as a main peak centered at 12.5 mL, from which an apparent MW of 160 kDa was estimated by comparison with protein standards. This value is in agreement with previous results This work aimed at characterising the native lipid moiety of EgAgB. As we have already mentioned, studies were mainly performed with EgAgB of bovine origin, yielding data that improve our knowledge on EgAgB lipoprotein composition. In addition, the characterisation of EgAgB protein∶lipid ratio and EgAgB lipid composition  was carried out with limited amounts of human EgAgB, obtaining similar results that confirm our findings.in vitroet al.et al.E. granulosus metacestode EgAgB8/1 is highly expressed in the E. granulosus metacestode EgAgB was purified from HCF using a previously described method based on anion exchange followed by immunoaffinity on MoAb EB7-Sepharose in vitro, in which fatty acids were described as the main lipid ligands of EgAgB8/1 and EgAgB8/2 3inset). However, the content of TAG in EgAgB is much higher than in HDL3, which likely reflects differences in the lipid transport function of these lipoproteins. In addition, in the context of this structural organisation share by all plasma lipoproteins, and taken into account its size and chemical composition, each EgAgB particle would contain between 11 and 15 EgAgB8 apolipoprotein molecules inserted into the outer phospholipid monolayer. This new scenario is relevant when considering the biological effects of EgAgB through parasite's as well as host's receptors. The exposure of more than a dozen of apolipoproteins on the surface of the lipoprotein particle would facilitate the establishment of multiple interactions with receptors, increasing the avidity of the interaction and the signals derived from it. The formation of multiple EgAgB-B cell interactions likely contributes to the immunogenicity of this lipoprotein. On the other hand, it cannot be ruled out that lipids could participate, at least partially, in some of the EgAgB biological activities (for example those anti-inflammatory described in vitro). Surface charge and/or hydrophobic distribution resulting from a lipoprotein ensemble  may alter the type of receptors involved, affecting its physiological effects.Analysis of the lipid composition of immunopurified EgAgB of both bovine and human origin, revealed a high diversity in terms of lipid classes ranging from highly hydrophobic lipids  to a variety of phospholipids (mainly phosphatidylcholine). These findings indicate that EgAgB is a more complex lipoprotein than previously suggested from lipid-binding studies Taenia soliumE. granulosus metabolic demand of lipids. In this context is important to highlight that no enzymes for either fatty acid anabolism or squalene synthesis  have been found in the E. granulosus transcriptome (data base http://www.compsysbio.org/partigene/) or the Echinococcus multilocularis genome . In addition, metabolic studies have demonstrated that sterol synthesis in E. granulosus seems to stop at the level of farnesyl or nerolidol pyrophosphate and that the content of cholesterol in HCF derives from the cholesterol pool of the host E. granulosus needs to take these lipids from its host. Whether EgAgB apolipoproteins are directly involved in the uptake of fatty acids and sterols from host tissues remains to be elucidated, but most likely they contribute to transport these lipids within metacestode tissues. Delivery of sterols to metacestode target cells may be crucial for biosynthetic purposes , but also for triggering signaling pathways associated with parasite development and growth. In fact, signaling pathways involving sterol-responsive nuclear receptors are conserved from simple invertebrates to mammals and regulate metabolism and development E. granulosus trasncriptome (our unpublished observations). Interestingly, about twenty nuclear receptors have been recently identified in E. multilocularis and E. granulosus and one of them displayed structural similarities to the DAF-12 subfamily, which binds cholesterol modified compounds and regulates cholesterol homeostasis and longevity in metazoans de novo by E. granulosus, but may be synthesised from building blocks obtained from the host as it occurs in other cestodes Echinococcus, but evidence of TAG synthesis via α-glycerophosphate–phosphatidic acid-diglyceride pathway exists for the cestode Hymenolepis diminutaEchinococcus (our unpublished observations from the E. granulosus transcriptome). This scenario suggests that TAG mainly provide a source of fatty acids and glycerol for synthetic purposes via an enzymatic hydrolysis. Consistent with this view is the identification of two ESTs clusters (EGC01304 and EGC 03444) that encode putative lipases in the E. granulosus transcriptome data base. With respect to the high content of phospholipids in EgAgB (mainly phosphatidylcholine), these molecules likely play a structural role in the lipoprotein by exposing a polar outer surface required for lipoprotein solubilization in the aqueous milieu. Phospholipids are not synthesised de novo by flatworms, but they can be synthesised from building blocks obtained from the host (fatty acids and the head group) The identification of the native lipid ligands of EgAgB provides relevant information for studying the function of this HLBP family member. Evidence for HLBP-mediated fatty acid binding and transportation across parasite membrane has been obtained in E. multilocularis and E. granulosus genomes, antigen B gene cluster is flanked by EmLDLR or EgLDLR genes, which encode proteins that display significant sequence similarities to low density lipoprotein (LDL) receptors from other species, and contain one single class A LDL receptor domain The uptake and delivery of lipids by lipoproteins require the existence of lipoprotein receptors in target cells. The existence of parasite EgAgB receptors as well as the use of host receptors by EgAgB would be needed for EgAgB-mediated lipid traffic. In this sense it is worth to mention that in Overall, a new picture for EgAgB structure and function has emerged from this work. EgAgB is a complex 229 kDa lipoprotein capable of transporting a variety of lipid classes including essential lipids that are not synthesised by the parasite, such as fatty acids and sterols. EgAgB uptake and delivery of these lipids may not only contribute to biosynthetic purposes, but also to signalling events associated with parasite metabolism and development. Whether the hydrophobic ligand binding properties of EgAgB, reflected by its lipid class composition, are an intrinsic feature of cestode HLBP family remains to be determined.