Sebastiaan M Bol

Perry D Moerland

Sophie Limou

Yvonne Van Remmerden

Cédric Coulonges

Daniëlle Van Manen

Joshua T Herbeck

Jacques Fellay

Margit Sieberer

Jantine G Sietzema

Ce ´dric Coulonges

Danie ¨lle Van Manen

Ruben Van 't Slot

Jeremy Martinson

Jean-Franc ¸ois Zagury

Hanneke Schuitemaker

Ange ´lique

B Van 't Wout

Genome-Wide Association Study Identifies Single Nucleotide Polymorphism in DYRK1A Associated with Replication of HIV-1 in Monocyte-Derived Macrophages

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The development of highly active antiretroviral therapy (HAART) has been the biggest achievement in HIV/AIDS medicine. The current drug regimens can suppress plasma viral load to (near) undetectable levels. However, often residual levels of ongoing viremia can be detected [START_REF] Dornadula | Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy[END_REF][START_REF] Fischer | Residual HIV-RNA levels persist for up to 2.5 years in peripheral blood mononuclear cells of patients on potent antiretroviral therapy[END_REF], even after treatment intensification [START_REF] Dinoso | Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy[END_REF], and complete eradication of HIV-1 from an infected patient has not been achieved with HAART [START_REF] Hutter | Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation[END_REF]. HAART does not affect cells that are latently infected because in these cells the virus is not actively replicating. Activation of this viral reservoir of latently infected resting CD4 + T cells [START_REF] Chun | Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines[END_REF] and residual replication in monocytes/macrophages (reviewed in [START_REF] Crowe | Macrophages and residual HIV infection[END_REF]) is believed to be responsible for the increase in plasma viral RNA levels that is observed after discontinuing HAART. Furthermore, cells residing in the gut associated lymphoid tissue (GALT) [START_REF] Guadalupe | Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection[END_REF][START_REF] Chun | Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy[END_REF], the testis [START_REF] Zhang | Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy[END_REF][START_REF] Mayer | Persistence of human immunodeficiency virus in semen after adding indinavir to combination antiretroviral therapy[END_REF] and the central nervous system (CNS) [START_REF] Letendre | Validation of the CNS Penetration-Effectiveness rank for quantifying antiretroviral penetration into the central nervous system[END_REF][START_REF] Lambotte | Persistence of replication-competent HIV in the central nervous system despite long-term effective highly active antiretroviral therapy[END_REF] may keep producing virus despite the use of antiretrovirals, because drug penetration of these tissues is suboptimal. As a result of this viral rebound and the existence of these sanctuary sites, HIV-1 infected individuals need to be on anti-HIV-1 medication for life. The emerging drug resistance [START_REF] Degruttola | The relation between baseline HIV drug resistance and response to antiretroviral therapy: re-analysis of retrospective and prospective studies using a standardized data analysis plan[END_REF][START_REF] Richman | The prevalence of antiretroviral drug resistance in the United States[END_REF] and the severe side effects (reviewed by [START_REF] Hawkins | Understanding and managing the adverse effects of antiretroviral therapy[END_REF]), high costs and suboptimal tissue penetration of HAART necessitate transition from treatment to cure as soon as possible.

The viral reservoir is established early after primary HIV-1 infection [START_REF] Chun | Early establishment of a pool of latently infected, resting CD4(+) T cells during primary HIV-1 infection[END_REF][START_REF] Schacker | Rapid accumulation of human immunodeficiency virus (HIV) in lymphatic tissue reservoirs during acute and early HIV infection: implications for timing of antiretroviral therapy[END_REF]. Although the precise mechanism is unknown, it has been shown that infected macrophages play a critical role in rendering resting CD4 + (but CCR5 2 ) T cells permissive for HIV-1 infection [START_REF] Swingler | HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection[END_REF]. Targeting these HIV-1 infected macrophages will not only help to eradicate the viral reservoir, but will also limit the spread of HIV-1 to CD4 + T cells [START_REF] Mann | HIV-1 transmission and function of virus-infected monocytes/macrophages[END_REF][START_REF] Carr | Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes[END_REF], prevent recruitment and activation of these cells [START_REF] Swingler | HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages[END_REF], and reduce macrophage mediated apoptosis of T cells [START_REF] Finkel | Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV-and SIV-infected lymph nodes[END_REF][START_REF] Badley | Macrophage-dependent apoptosis of CD4+ T lymphocytes from HIV-infected individuals is mediated by FasL and tumor necrosis factor[END_REF][START_REF] Herbein | Distinct mechanisms trigger apoptosis in human immunodeficiency virus type 1-infected and in uninfected bystander T lymphocytes[END_REF][START_REF] Herbein | Apoptosis of CD8+ T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4[END_REF]. In addition, inhibiting or reducing HIV-1 replication in macrophages/microglia will be of importance in preventing the onset of AIDS dementia and other neurocognitive disorders associated with HIV-1 infection in the brain, because macrophages/microglia play a crucial role in the pathogenesis of these [START_REF] Koenig | Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy[END_REF][START_REF] Glass | Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia[END_REF][START_REF] Anthony | Influence of HAART on HIV-related CNS disease and neuroinflammation[END_REF].

In the battle to cure HIV-1 infected individuals, identification of novel targets for the development of drugs that can be used to inhibit HIV-1 replication in macrophages will be required, especially since macrophages are relatively resistant to the cytopathic effect of HIV-1 replication [START_REF] Gendelman | Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes[END_REF]. Furthermore, tissue macrophages residing in the sanctuary sites described above are exposed to suboptimal levels of antiretrovirals, and the efficacy of protease inhibitors (PIs) and other late stage inhibitors in macrophages is substantially reduced [START_REF] Aquaro | Macrophages and HIV infection: therapeutical approaches toward this strategic virus reservoir[END_REF][START_REF] Perno | In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages[END_REF]. This lower efficacy of PIs in macrophages forms an obstacle in ending the ongoing residual replication, since only intervention of the virus' replication cycle at stages post reverse transcription and integration will prevent the production of new virions. Once the provirus is integrated in the host genome, integrase and reverse transcriptase inhibitors will no longer be effective in the cell.

Since the HIV-1 genome encodes only 15 proteins [START_REF] Frankel | HIV-1: fifteen proteins and an RNA[END_REF], it is dependent on numerous host proteins for its replication [START_REF] Goff | Host factors exploited by retroviruses[END_REF]. Intervention of the interaction between HIV-1 and these HIV-1 dependency factors (HDFs) can potentially be used to inhibit replication of the virus. Currently, the majority of drugs target viral enzymes: HIV-1 protease, reverse transcriptase and integrase (reviewed in [START_REF] Broder | The development of antiretroviral therapy and its impact on the HIV-1/AIDS pandemic[END_REF]). The abundance of the HDFs offers great promise for finding drugs that may be less prone to emerging drug resistance of the virus, may better penetrate tissues and reach sanctuary sites, and may effectively prevent the formation of new virions in both monocytes/macrophages and CD4 + T cells. However, it will be crucial to find a balance between limiting the availability of HDFs and inhibiting viral replication, since most host proteins essential for HIV-1 replication may also play critical roles in the host cell.

Recently, a large number of host proteins that might be good targets for novel drugs to inhibit HIV-1 replication was identified [START_REF] Brass | Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen[END_REF][START_REF] Ko ¨nig | Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication[END_REF][START_REF] Yeung | A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication[END_REF][START_REF] Zhou | Genome-scale RNAi screen for host factors required for HIV replication[END_REF]. These candidate proteins were found by measuring a significant change in HIV-1 replication after a genomescale knock-down of host proteins, in different cell-lines. Since macrophages are important as reservoir and in sanctuary sites, it is also important to identify HDFs or host antiviral factors in these cells. Because primary macrophages are notoriously difficult to transfect and since HIV-1 replication in monocyte-derived macrophages (MDM) can vary enormously between donors [START_REF] Bol | Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages[END_REF][START_REF] Chang | Twin studies demonstrate a host cell genetic effect on productive human immunodeficiency virus infection of human monocytes and macrophages in vitro[END_REF][START_REF] Eisert | Analysis of cellular factors influencing the replication of human immunodeficiency virus type I in human macrophages derived from blood of different healthy donors[END_REF][START_REF] Fouchier | HIV-1 macrophage tropism is determined at multiple levels of the viral replication cycle[END_REF][START_REF] Naif | Definition of the stage of host cell genetic restriction of replication of human immunodeficiency virus type 1 in monocytes and monocyte-derived macrophages by using twins[END_REF] we decided to use a different approach and to actually exploit this genetic variation between individuals. When monocyte isolation, cell culture methods, medium and virus are all strictly controlled, remaining variation in HIV-1 replication observed in MDM from different donors can be assumed to be primarily of genetic origin. Finding an association between host genetic traits like a single nucleotide polymorphism (SNP) and HIV-1 replication in MDM would suggest that the locus tagged by this SNP is of importance for replication of HIV-1 in these cells. We therefore searched genome-wide for associations between SNPs and in vitro HIV-1 replication specifically in monocyte-derived macrophages.

Results

Association between single nucleotide polymorphism in the kinase DYRK1A and HIV-1 replication in monocytederived macrophages A total of 494,656 SNPs passing quality control were tested for association with levels of HIV-1 replication in monocyte-derived macrophages (MDM) using linear regression. With data from 191 healthy blood donors whose MDM ranked in the bottom quartile with lowest (n = 95 donors) or top quartile with highest (n = 96 donors) Gag p24 production 14 days post infection with HIV-1 [START_REF] Bol | Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages[END_REF] (Table 1), we found strongest associations for SNPs in the genes PDE8A (rs2304418, p = 2.4610 26 and rs12909130, p = 8.3610 26 ), UBR7 (rs2905, p = 7.0610 26 ), MOAP1 (rs1046099, p = 9.9610 26 and rs1270629, p = 1.09610 25 ), DYRK1A (rs12483205, p = 2.2610 25 ) and SPOCK3 (rs17519417, p = 2.5610 25 ) (Figure 1, Table S1). The two SNPs in PDE8A were found to be in high linkage disequilibrium (LD; r 2 = 0.97), whereas only a moderate degree of LD was found between rs1046099 and rs1270629 in MOAP1 (r 2 = 0.54) (Table 2). Table 2 shows all other SNPs associated with HIV-1 replication in MDM (cutoff p value = 5610 25 ; n = 16), and includes information about LD, location, number of donors homozygous for the minor allele (MIN) and empirical p values after permutation testing. Genotyping results for none of these 16 SNPs violated Hardy-Weinberg equilibrium.

The empirical p values for linear regression using 10 7 permutations of the genotype for each SNP in Table 2 were in close agreement with the asymptotic p values (Table 2). However, none of these SNPs remained statistically significant after a conservative correction for multiple testing (Bonferroni threshold of p,1610 27 ; Figure S1). We next investigated the association between the SNPs in the five most promising genes (rs2304418 in PDE8A, rs2905 in UBR7, rs1046099 and rs1270629 in MOAP1, rs12483205 in DYRK1A and rs17519417 in SPOCK3) in a second group of blood donors (replication cohort). MDM from an independent group of 32 healthy blood donors were infected with HIV-1 and Gag p24 levels were measured 14 days after infection. One donor was found to be homozygous for the 32 base pair deletion in CCR5, rendering the cells completely resistant to infection with CCR5-using HIV-1, and results obtained with macrophages from this donor were excluded from further analysis. The associations for the SNPs in PDE8A, UBR7, MOAP1 and SPOCK3 with Gag p24 production by MDM could not be replicated in this small group of 31 donors (data not shown). However, in this replication cohort we again found a strong association between SNP rs12483205 in DYRK1A and in vitro HIV- p values only for comparison between donors with MDM that produced low levels of Gag p24 and donors with MDM that had high p24 production after infection with HIV-1. b Information was not available.

1 replication in macrophages (p = 0.0034) (Figure 2, Table S2), also after correction for cell number and normalization (p = 0.0081; n = 28, information on cell number was missing for 3 donors) (data not shown, Table S2). This association remained significant after correction for multiple testing (Bonferroni corrected p = 0.020 (0.003466 SNPs for which we tried to replicate the association)) and when calculating the empirical p value for linear regression using 10 5 permutations of the genotypes (p = 0.004) (Table S1).

The DYRK1A SNP rs12483205 genotypes were then also determined for the 202 donors with MDM that had intermediate Gag p24 levels in the supernatant [START_REF] Bol | Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages[END_REF]. Adding results from this group of donors did not change the strength of the association between the rs12483205 genotype and HIV-1 replication in MDM (p = 2.09610 25 ; n = 393) (Figure S2A, Table S3). As expected, combined analysis of both the initial group of donors (n = 393) and the replication cohort (n = 28) revealed an increase in the strength of the association for rs12483205 in DYRK1A (p = 4.84610 26 ) (Figure S2B, Table S4).

Effect of rs12483205 in DYRK1A is independent of the CCR5 D32 genotype

The 32 base pair deletion in CCR5 strongly affects replication of HIV-1, also in macrophages in vitro [START_REF] Bol | Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages[END_REF]. MDM from donors that were heterozygous for this 32 base pair deletion (n = 33) had significantly lower HIV-1 replication than donors without the deletion (n = 158) (p = 0.0036, two-sample t-test) (Table S5). There were significantly more CCR5 wt/D32 heterozygous donors present in the rs12483205 heterozygous (HZ) and rs12483205 MIN groups (p = 0.02, Fisher Exact test). After adjusting for the CCR5 wt/D32 heterozygous genotype by using it as a covariate in multivariate analysis, the strength of the association between the The effect of the SNP in DYRK1A was replicated with monocyte-derived macrophages from an independent group of 31 donors. Major genotype is the heterozygous genotype (,50%); both classes of homozygous genotypes equally present (,25% each). doi:10.1371/journal.pone.0017190.t002

Figure 2. Significant association between rs12483205 and in vitro replication of HIV-1 in macrophages derived from an independent group of 31 healthy blood donors. The negative association between the rs12483205 minor allele and Gag p24 levels in MDM culture supernatant 14 days after inoculation with HIV-1, was found to match with the results from the genome-wide association study. Open circles represent results from donors with the CCR5 D32 wild-type genotype, filled circles from donors with the CCR5 wt/D32 heterozygous genotype. MAJ, homozygous for the major allele; HZ, heterozygote; MIN, homozygous for the minor allele. doi:10.1371/journal.pone.0017190.g002

N N

DYRK1A rs12483205 genotype and HIV-1 replication in MDM decreased (from p = 2.2610 25 to p = 1.2610 24 ), but persisted (Table S5). Moreover, the association of rs12483205 in DYRK1A with HIV-1 replication in MDM in the replication cohort of 31 donors, was independent of the CCR5 D32 genotype (p = 0.0022, and p = 0.007 when corrected for cell number and normalized for date of isolation; Table S2), suggesting only a spurious relationship between the CCR5 locus on chromosome 3 and SNP rs12483205 in DYRK1A located on chromosome 21. Indeed, this was supported by results from the multivariate analysis on the total group of 393 donors (p = 3.8610 25 , Table S3) and on the combined group of 421 (393+28 donors from the replication cohort) individuals (p = 9.7610 26 , Table S4).

Minor variant of SNP rs12483205 in DYRK1A is associated with slower disease progression

Next, we investigated the association between HIV-1 load or disease course in vivo and the six most promising SNPs identified in our GWAS (rs12483205 in DYRK1A, rs2304418 in PDE8A, rs2905 in UBR7, rs1046099 and rs1270629 in MOAP1 and rs17519417 in SPOCK3). Results from the Amsterdam Cohort Study (ACS) GWAS on HIV/AIDS disease progression (Van Manen et al., manuscript in preparation) and three previously published HIV-1 cohort studies with GWAS data were used: the CHAVI cohort [START_REF] Fellay | Common genetic variation and the control of HIV-1 in humans[END_REF], the GRIV [START_REF] Limou | Genomewide association study of an AIDS-nonprogression cohort emphasizes the role played by HLA genes (ANRS Genomewide Association Study 02)[END_REF][START_REF] Clerc | Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03)[END_REF] and the MACS [START_REF] Herbeck | Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS[END_REF]. To avoid possible overlap between the replication cohorts, MACS samples were removed from the CHAVI cohort and the analysis was performed only using data from the European subset (EURO-CHAVI).

Illumina Human Hap BeadChips were used for genotyping DNA samples in the EURO-CHAVI cohort (550 K/1 M), the GRIV (300 K) and the ACS (300 K), whereas in the MACS they used Affymetrix Human 500 K GeneChips for the first stage discovery analysis [START_REF] Herbeck | Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS[END_REF]. Using different genotyping platforms results in genotype data for a different set of SNPs, which occasionally makes it necessary to estimate the unobserved genotypes, a process called SNP imputation [START_REF] Marchini | A new multipoint method for genome-wide association studies by imputation of genotypes[END_REF]. When the imputation score (a measure for the reliability of the imputation) was below 0.8, imputation was considered unreliable and data was therefore not used. Results for SNPs rs2905 (UBR7), rs1046099 (MOAP1), rs1270629 (MOAP1) and rs12483205 (DYRK1A) in the MACS, and rs1046099 (MOAP1) and rs17519417 (SPOCK3) in both the GRIV and the ACS, were based on SNP imputation. The imputation score for rs12483205 in DYRK1A was 0.94, and ,0.8 for rs2905, rs1046099 and rs1270629. For rs1046099 and rs17519417 the imputation scores were 0.88 and 1 respectively, in the GRIV and in the ACS.

Table 3 shows the results for association testing between disease progression or viral load and the identified SNPs in DYRK1, PDE8A, UBR7, MOAP1 and SPOCK3. No significant associations or trends were found for the SNPs in PDE8A, UBR7 or MOAP1. However, SNPs rs12483205 in DYRK1A and rs17519417 in SPOCK3 were significantly associated with in vivo endpoints in at least one of the four independent cohorts analyzed. While no association was observed in the GRIV and the ACS, we found an association between rs12483205 in DYRK1A and disease progression in the MACS (p = 0.0048, Table 3) and in the EURO-CHAVI cohort (p = 0.035, Table 3). In both cohort studies the minor allele of rs12483205 was associated with slower disease progression, which was consistent with the decreased in vitro replication of HIV-1 in MDM for the minor allele of rs12483205.

Furthermore, there was an association between SNP genotype and progression to AIDS for rs17519417 in SPOCK3 in the MACS [START_REF] Herbeck | Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS[END_REF] (p = 0.012, Table 3), with the C allele associated with slower progression. This was also in agreement with the association with lower in vitro HIV-1 replication in MDM in our study.

SNP rs12483205 is localized near the 59 untranslated region of DYRK1A transcript variant 3

The DYRK1A mRNA has multiple splice forms. According to the NCBI Entrez Gene database [START_REF] Tatusova | Genomic databases and resources at the National Center for Biotechnology Information[END_REF] the alternative splicing can yield four different DYRK1A transcript variants, referred to as transcripts 1, 2, 3 and 5 (Figure 3). In addition to differences in the number or size of exons, there is also variation in the length of both the 59 and 39 untranslated region (UTR). Transcript variant 1 encodes the longest isoform (763 amino acids), which is a fraction larger than isoform 2 (754 amino acids). Isoforms 3 and 5 lack a C-terminal part of the full length protein (179 and 234 amino acids respectively), and consequently miss among others a poly-His domain. SNP rs12483205 in DYRK1A lies ,900 base pairs downstream of the most distant 59 UTR fragment of transcript variant 3 (Figure 3). PCR analysis with primers able to discriminate between all four known transcript variants (Figure S3) confirmed the presence of transcript variants 1, 2 and 3 in MDM, and in the glioma cell line U87 that was used as a positive control for the PCR (Figure 4). Transcript variant 5 was present in U87 cells, but was not detected in MDM (Figure 4). The DYRK1A gene region is devoid of known SNPs that are in high LD with rs12483205; currently available data on LD in this region does not reveal any SNP with an r 2 .0.5 in the Caucasian population [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF][START_REF] Johnson | SNAP: a web-based tool for identification and annotation of proxy SNPs using HapMap[END_REF][START_REF] Xu | SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies[END_REF].

Discussion

Genome-wide studies provide a hypothesis free and unbiased approach to identify novel associations between genetic factors and the studied phenotype. Selecting donors with a more extreme in vitro phenotype allowed us to identify a number of promising SNPs in genes not previously linked to HIV-1, that are associated with replication levels of HIV-1 in monocyte-derived macrophages (MDM). Importantly, the association between rs12483205 in DYRK1A and in vitro HIV-1 replication could be replicated using MDM from an independent small group of donors, and was independent of the CCR5 D32 genotype. Moreover, this SNP in DYRK1A was significantly associated with in vivo endpoints in two independent cohorts. SNPs in four other genes were not replicated in the small in vitro replication cohort, possibly reflecting insufficient power. However, the identified SNP in SPOCK3 was replicated in one of the in vivo GWAS.

The use of, or even the dependence on, certain cellular factors by HIV-1 can differ between the various target cells such as T cells and macrophages. For example, the cellular factors GATA-3, ETS-1 and NF-ATc are T cell specific [START_REF] Yang | Human T cell transcription factor GATA-3 stimulates HIV-1 expression[END_REF][START_REF] Kinoshita | The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells[END_REF], whereas C/EBPb is required for HIV-1 replication in macrophages, but not in T cells [START_REF] Henderson | CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4(+) T cells[END_REF][START_REF] Lee | CCAAT/enhancer binding proteins are not required for HIV-1 entry but regulate proviral transcription by recruiting coactivators to the long-terminal repeat in monocytic cells[END_REF]. To test whether our top SNPs were macrophagespecific, we investigated if these SNPs were also associated with HIV-1 replication in CD4 + T cells. Genotyping DNA from 128 blood donors for whom the level of HIV-1 replication in PHAstimulated CD4 + T cells was previously determined [START_REF] Ciuffi | Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection[END_REF], revealed no association between the level of HIV-1 replication in T cells and the genotype of the five SNPs tested. This finding could indeed be an indication that the effects of some of the SNPs that we have identified are macrophage-specific, but also could reflect lack of power (n = 128, and not only extreme phenotypes). The finding that the SNP rs12483205 in DYRK1A was associated with in vivo endpoints could indicate a role for HIV-1 infection of macrophages in disease progression.

DYRK1A is a kinase that phosphorylates serine and threonine residues. The gene encoding for this kinase is located on the Down syndrome critical region on chromosome 21 which is thought to be responsible for the features of Down syndrome. For this reason, the emphasis of DYRK1A research has been on its role in trisomy 21 and neurological dysfunction [START_REF] Dierssen | DYRK1A (dual-specificity tyrosinephosphorylated and -regulated kinase 1A): a gene with dosage effect during development and neurogenesis[END_REF][START_REF] Galceran | The MNB/DYRK1A protein kinase: genetic and biochemical properties[END_REF][START_REF] Gardiner | Transcriptional dysregulation in Down syndrome: predictions for altered protein complex stoichiometries and post-translational modifications, and consequences for learning/behavior genes ELK, CREB, and the estrogen and glucocorticoid receptors[END_REF][START_REF] Hammerle | The MNB/ DYRK1A protein kinase: neurobiological functions and Down syndrome implications[END_REF][START_REF] Park | Function and regulation of Dyrk1A: towards understanding Down syndrome[END_REF][START_REF] Rachidi | Mental retardation and associated neurological dysfunctions in Down syndrome: a consequence of dysregulation in critical chromosome 21 genes and associated molecular pathways[END_REF]. Substrates for DYRK1A comprise among many others the transcription factors CREB [START_REF] Yang | Protein kinase Dyrk1 activates cAMP response element-binding protein during neuronal differentiation in hippocampal progenitor cells[END_REF], FKHR [START_REF] Woods | The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site[END_REF], GLI1 [START_REF] Mao | Regulation of Gli1 transcriptional activity in the nucleus by Dyrk1[END_REF], NFAT [START_REF] Gwack | A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT[END_REF][START_REF] Arron | NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21[END_REF] and STAT3 [START_REF] Becker | Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity[END_REF]. This large number of protein interactions and substrates described for DYRK1A strongly suggests it plays an important role in the regulation of gene expression and cell function. While the molecular mechanisms underlying the effect of DYRK1A on HIV-1 replication in MDM remain to be unraveled, the critical role of kinases in general for the replication of HIV-1 in macrophages has already been established [START_REF] Luciano-Montalvo | Cystatin B associates with signal transducer and activator of transcription 1 in monocyte-derived and placental macrophages[END_REF][START_REF] Zhang | Primitive hematopoietic cells resist HIV-1 infection via p21[END_REF][START_REF] Wu | Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation[END_REF][START_REF] Bergamaschi | The CDK inhibitor p21Cip1/WAF1 is induced by FcgammaR activation and restricts the replication of human immunodeficiency virus type 1 and related primate lentiviruses in human macrophages[END_REF][START_REF] Sung | miR-198 inhibits HIV-1 gene expression and replication in monocytes and its mechanism of action appears to involve repression of cyclin T1[END_REF][START_REF] Dong | Transcriptional restriction of human immunodeficiency virus type 1 gene expression in undifferentiated primary monocytes[END_REF].

Both SNPs in MOAP1 that we found to be associated with HIV-1 replication in MDM are in close proximity to the UBR7 gene region (44 and 47 kb for rs1046099 and rs1270629 respectively), yet the degree of LD is fairly low (r 2 = 0.28 and 0.35 for rs1046099 and rs1270629 respectively) which could indicate that the observed associations for the SNPs in UBR7 and MOAP1 with HIV-1 replication are independent of each other. MOAP1 is a homodimeric protein that binds both proapoptotic (BAX) and prosurvival (BCL2) molecules [START_REF] Tan | MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains[END_REF]. MOAP1 has no known direct effect on HIV-1 replication. However, it is well recognized that HIV-1 infection affects the regulation of programmed cell death (reviewed in [START_REF] Gougeon | Apoptosis as an HIV strategy to escape immune attack[END_REF]) and that host factors induce apoptosis during the infection process to prevent viral dissemination [START_REF] Lum | Induction of cell death in human immunodeficiency virus-infected macrophages and resting memory CD4 T cells by TRAIL/Apo2l[END_REF][START_REF] Huang | TRAILmediated apoptosis in HIV-1-infected macrophages is dependent on the inhibition of Akt-1 phosphorylation[END_REF]. This host factor-induced apoptosis was found to be associated with reduced HIV-1 replication in MDM [START_REF] Lum | Induction of cell death in human immunodeficiency virus-infected macrophages and resting memory CD4 T cells by TRAIL/Apo2l[END_REF][START_REF] Huang | TRAILmediated apoptosis in HIV-1-infected macrophages is dependent on the inhibition of Akt-1 phosphorylation[END_REF]. Only very little is known about UBR7. However, studies on other members of the UBR family support the hypothesis that UBR7 is be associated with HIV-1 replication. Proteins of the UBR family are E3 ligases that recognize degradation signals at the N-terminus of proteins (Ndegrons) and conjugate ubiquitin to the target protein [START_REF] Tasaki | A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons[END_REF]. The HIV-1 protein integrase contains an N-terminal phenylalanine that is recognized as a degradation signal by UBR1, UBR2 and UBR4 [START_REF] Tasaki | A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons[END_REF][START_REF] Mulder | Degradation of HIV-1 integrase by the N-end rule pathway[END_REF] and indeed, these proteins control the level of HIV-1 integrase [START_REF] Tasaki | A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons[END_REF]. Knocking down UBR1, UBR2 and UBR4 resulted in higher levels of HIV-1 integrase [START_REF] Tasaki | A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons[END_REF], yet inhibition of the proteasome resulted in a further increase of HIV-1 integrase [START_REF] Tasaki | A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons[END_REF], suggesting the presence of other E3 ligases that target Ndegrons of HIV-1 integrase. The results of our study could indicate that UBR7 affects HIV-1 replication in MDM through regulation of HIV-1 integrase.

In addition to the SNP in DYRK1A we also found associations for rs17519417 in SPOCK3 with time to progression to AIDS. SPOCK3, also referred to as Testican 3, is a proteoglycan and one of the components of the extracellular matrix. It inhibits processing of metalloproteinase 2 (MMP-2) [START_REF] Nakada | Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes[END_REF]. There are indications that MMP-2 might play a role in the pathogenesis of AIDS-related Kaposi's sarcoma [START_REF] Albini | Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2[END_REF][START_REF] Toschi | Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor[END_REF] or HIV-1 associated dementia [START_REF] Lopez-Herrera | HIV-1 interaction with human mannose receptor (hMR) induces production of matrix metalloproteinase 2 (MMP-2) through hMR-mediated intracellular signaling in astrocytes[END_REF][START_REF] Conant | Cerebrospinal fluid levels of MMP-2, 7, and 9 are elevated in association with human immunodeficiency virus dementia[END_REF][START_REF] Chong | Increased activity of matrix metalloproteinase-2 in human glial and neuronal cell lines treated with HIV-1 gp41 peptides[END_REF].

While we could not replicate the GWAS result for the SNP in PDE8A, results from other in vitro studies strongly suggest that PDE8A plays an important role in the replication cycle of HIV-1. Phosphodiesterase 8A (PDE8A) specifically hydrolyses cAMP to AMP. Previous studies that showed strong inhibition of HIV-1 replication after efficient knock-down [START_REF] Zhou | Genome-scale RNAi screen for host factors required for HIV replication[END_REF], interaction between HIV-1 Tat protein and PDE8A [START_REF] Fu | Human immunodeficiency virus type 1, human protein interaction database at NCBI[END_REF][START_REF] Zauli | HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway[END_REF] and the deleterious effect of high levels of cAMP on HIV-1 replication [START_REF] Hayes | Prostaglandin E(2) inhibits replication of HIV-1 in macrophages through activation of protein kinase A[END_REF], all profoundly suggest that this type of phosphodiesterase is indeed important for in vitro HIV-1 replication.

To further validate our findings it will be important to also investigate the role of the identified SNPs and genes in HIV-1 related pathologies that are more specifically affected by macrophages, such as HIV-1 associated dementia and AIDS related lymphomas [START_REF] Huysentruyt | The role of macrophages in the development and progression of AIDS-related non-Hodgkin lymphoma[END_REF]. Here, the contribution of macrophages could be more eminent than in cohorts that study time to progression to AIDS, which might be more T cell dependent, especially since sometimes AIDS is defined by CD4 + T cell counts.

Follow-up experiments that will identify the causal SNP and study its effect on the primary protein structure, protein folding, alternative splicing or expression (level, localization and timing) will be essential to better understand the precise mechanism by which this SNP affects HIV-1 replication in MDM. This will be a valuable step that might help determine if, and to what extent the host protein can be efficiently targeted by novel antiretroviral drugs. Finding a SNP in a gene encoding the kinase DYRK1A that affects HIV-1 replication, holds great promise for finding molecules to be safely used as potentially novel anti-HIV-1 medication [START_REF] Netterwald | Who likes kinases? Drug Discovery & Development[END_REF].

Materials and Methods

Ethics statement

This study has been conducted in accordance with the ethical principles set out in the declaration of Helsinki, written informed consent was obtained from all participants and was approved by the Medical Ethics Committee of the Academic Medical Center in Amsterdam and the Ethics Advisory Body of the Sanquin Blood Supply Foundation, The Netherlands.

Study population

We previously determined the ability of HIV-1 to replicate in monocyte-derived macrophages (MDM) from 429 different healthy seronegative blood donors [START_REF] Bol | Donor variation in in vitro HIV-1 susceptibility of monocyte-derived macrophages[END_REF]. In brief, Gag p24 levels were measured in MDM culture supernatant 14 days post infection by an in-house enzyme-linked immunosorbent assay. To correct for differences in the number of viable MDM present at day 14 post infection, p24 levels were expressed per 10,000 cells. Since monocyte isolations were performed in four time frames and by two operators, p24 levels were normalized by dividing through the median per period and operator. These normalized p24 levels were subsequently used as a measure for in vitro HIV-1 replication in MDM.

After excluding donors that were homozygous for the 32 base pair deletion in CCR5 (n = 5) or not from European decent (n = 30), we selected 192 individuals whose MDM gave the highest (n = 96) or the lowest (n = 96) p24 production in vitro, for SNP genotyping; thus representing two groups of donors with MDM that had a more extreme phenotype (Table 1). Inclusion of donors with extreme phenotypes is known to increase power in genetic association studies [START_REF] Van Gestel | Power of selective genotyping in genetic association analyses of quantitative traits[END_REF]. These two groups with a more extreme phenotype were separated by a group with intermediate HIV-1 replication in MDM which consisted of 202 donors. There was no difference in age or distribution of males and females between the group with MDM that had low HIV-1 replication and the group with MDM that had high Gag p24 production (p = 0.95, Pearson chi-square test and p = 0.37, two sample t-test, respectively; Table 1). One donor from the group with low in vitro HIV replication in MDM was excluded for further analysis because the corresponding DNA samples did not pass the quality control (SNP call rate ,0.98; see paragraph ''Quality control of SNP genotyping data'' below).

Cells (monocytes and lymphocytes) used to replicate the genome-wide association study (GWAS) findings were derived from blood samples collected from an independent group of 32 healthy blood donors (replication cohort).

Genotyping

Peripheral blood lymphocytes (PBL) from each donor were used for DNA isolation using the QIAamp ''DNA blood mini kit'' (Qiagen, Valencia, CA, USA). For the two groups of donors whose MDM had highest (n = 96 donors) or lowest (n = 96 donors) in vitro HIV Gag p24 production, we used the Illumina Infinium Human Hap 610-Quad BeadChip (Illumina, San Diego, CA, USA) [START_REF] Steemers | Whole genome genotyping technologies on the BeadArray platform[END_REF] for SNP genotyping. ABI TaqManH SNP genotyping assays were used to genotype DNA from the replication cohort for rs12483205 (C_31609775_10), rs12909130 (C_1342209_10) as a proxy for rs2304418, rs2905 (C_3236245_20), rs1046099 (C_7585751_10) and rs17519417 (C_33238869_10) (Applied Biosystems, Carlsbad, CA, USA). All TaqManH assays were run on a LightCyclerH 480 system (Roche, Basel, Switzerland) using the following amplification cycles: 10 min 95uC; 50 cycles of 15 sec 95uC, 1 min 60uC.

Quality control of SNP genotyping data

One donor DNA sample did not pass the minimal genotyping call rate threshold of 98% and this sample was excluded for further SNP association analysis. The Illumina 610Q SNP bead chip contained 620,901 markers to detect common genetic variation, including 21,890 markers for copy number variation (CNV). After excluding a total of 126,245 SNPs (minor allele frequency ,0.05, or SNP call frequency ,0.98, or SNPs for which there were no donors homozygous for the minor allele) and copy number variation markers, 494,656 SNPs were left to be tested for association between genotype and in vitro HIV-1 replication MDM. Violation of Hardy-Weinberg equilibrium was assessed post-analysis for all SNPs with p,5610 25 . Gender calls in BeadStudio (version 3.1.3.0) were compared with self-reported gender and were found to match for all donors (n = 191).

Identification of population stratification

Donors who reported that either one or more parents or grand parents were born outside of Europe had been excluded from further analysis (as described in the paragraph ''Study population'' above). The genetic homogeneity of the genotyped donors whose MDM gave lowest (n = 96) or highest (n = 96) Gag p24 production in vitro was confirmed by both Structure [START_REF] Price | Principal components analysis corrects for stratification in genome-wide association studies[END_REF] and Eigenstrat [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] analysis. As expected, the Q-Q plot displaying the normality of the p value distribution did not show deviations from what is expected under the null hypothesis (l = 1.0024; l = 1.0 is expected with a null hypothesis; Figure S4), indicating little effect of population stratification.

Statistical analysis

We set a lower detection limit (background signal) for the normalized p24 values based on the highest results that we found for a donor that was homozygous for the 32 base pair deletion in CCR5. Normalized p24 values were log10 transformed to allow for parametric testing.

Association was tested assuming an additive relationship between the genetic variants (wild-type, heterozygous and homozygous for the minor allele) and phenotype, using linear regression. Permutation testing was done to empirically determine the corresponding p value based on the observed linear regression F-statistic relative to the distribution of the F-statistic estimated from 10 7 permutations of the genotypes. All R code and data used for the calculations can be found as supplementary online material (Tables S1, S2, S3, S4 and S5 and text file ''R Scripts S1'').

Linkage disequilibrium (LD) between SNPs was calculated using data obtained from genotyping DNA from our studied population (n = 191) with the Illumina SNP chip, using Haploview software (version 4.2) [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. SNP genotype imputations for our study population were performed using Impute software [START_REF] Marchini | A new multipoint method for genome-wide association studies by imputation of genotypes[END_REF] and the Caucasian HapMap population (release 21) as the reference panel.

DYRK1A transcript detection in monocyte-derived macrophages

Seven day old MDM and U87 cells were used for RNA isolation and subsequent cDNA synthesis using the Roche ''high pure RNA isolation'' and ''transcriptor first strand cDNA synthesis'' kit (Roche, Basel, Switzerland). To uniquely detect the presence of transcript variants 1, 2, 3 and 5, we used primer pairs 59-TGATATTGTCATGTTACAGAGGCGG-39 (forward) and 59-CTGACGCACCTGGGGACTG-39 (reverse), 59-TGTCTCTG-AGGTTCTTTTCCAGTG-39 (forward) and 59-AGCACCC-TCTCAATTCCCAATGCC-39 (reverse), 59-GCTCGCACGTG-GTTCATTTGCT-39 (forward) and 59-TCCTTAGACAGGA-ACGTCATGAACCT-39 (reverse), and 59-CAGGAGGACCT-GGTGGGCGA-39 (forward) and 59-TGCTGACGCACCT-GAGCTTG-39 (reverse) respectively (Figure S3). For the detection of transcript variants 1, 2, 3 and 5 the following amplification cycles were used: 2 min 95uC; 35 cycles of 30 sec 95uC, 30 sec 62uC (64uC for transcript 1), 30 sec 72uC (90 sec for transcript 1); 10 min 72uC.

Figure 1 .

 1 Figure 1. Association between HIV-1 replication in monocyte-derived macrophages (MDM) and the genotypes for the SNPs rs12483205 in DYRK1A, rs2304418 in PDE8A, rs2905 in UBR7, rs1046099 and rs1270629 in MOAP1, and rs17519417 in SPOCK3. Only donors with MDM with low (n = 95) or high (n = 96) HIV-1 replication in vitro were included in the genome-wide SNP analysis. This selection of donors with a more extreme phenotype explains the absence of circles in the middle section of the graphs. MAJ, homozygous for the major allele; HZ, heterozygote; MIN, homozygous for the minor allele. doi:10.1371/journal.pone.0017190.g001

* 2 Empirical

 2 p value calculated for linear regression using 10 7 permutations of the genotypes. *3 Measure for the magnitude of linkage disequilibrium (r 2 ) between SNPs with identical symbols (m, b, c, ., &, N or X).

  All p values ,0.1 are shown. *Effect of the minor allele consistent with our findings. # Based on SNP imputation; imputation score .0.8. N.A., not available (imputation score ,0.8); EURO-CHAVI, European subset of the CHAVI cohort (n = 1,280 for viral load and n = 634 for disease progression) [44]; GRIV, Genomics of Resistance to Immunodeficiency Virus cohort (275 non-progressors vs. 1,352 controls [45], and 85 rapid progressors vs. 1,352 controls [46]); MACS, MultiCenter AIDS Cohort Study cohort (n = 156) [47]; ACS, Amsterdam Cohort Study (n = 404; Van Manen et al., manuscript in preparation). doi:10.1371/journal.pone.0017190.t003

Figure 3 .

 3 Figure 3. Schematic representation of the DYRK1A gene region, depicting all four transcript variants (1, 2, 3 and 5) and the localization of SNP rs12483205. Untranslated regions (UTR) are shown as open blocks, whereas exons are shown as filled blocks. SNP rs12483205 lies in close proximity to a part of the 59 UTR unique for splice variant 3. doi:10.1371/journal.pone.0017190.g003

Figure 4 .

 4 Figure 4. Detection of different DYRK1A transcript variants in macrophages. Amplicons were generated by PCR on cDNA from U87 cells, used as a positive control for the PCR, and from monocyte-derived macrophages (MDM) obtained from three individuals, using DYRK1A transcript variant specific primers. DYRK1A transcript variants 1, 2 and 3 were detected in MDM and U87 cells (panel A, B and C respectively). Transcript variant 5, however, was only convincingly detected in U87 cells, and not in MDM. Numbers on the left side of each picture indicate the size (in base pairs, bp) for the corresponding DNA fragment of the DNA ladder or PCR amplicons. 1 kb, 1 kb DNA ladder; 100 bp, 100 bp DNA ladder. doi:10.1371/journal.pone.0017190.g004

Table 1 .

 1 Characteristics of healthy donors whose macrophages showed either low, intermediate, or high HIV-1 replication in vitro.

	In vitro HIV-1 replication in monocyte-derived macrophages
	Low	Intermediate	High	p value a

n/a, not applicable. SD, standard deviation. a

Table 2 .

 2 SNPs (p,561025 ) associated with in vitro HIV-1 replication in monocyte-derived macrophages.

			Closest						# MIN
		SNP	gene	Location	Chr. Position	p value	Empirical p *2	donors	Linkage disequilibrium (r 2 ) *3
	1	rs2304418	PDE8A	Intronic	15	85640983	2.41610 26	2.30610 26	15	0.97m
	2	rs2905	UBR7	39 UTR	14	93693422	6.96610 26	8.00610 26	16		0.35c	0.28&
	3	rs12909130	PDE8A	Intronic	15	85590501	8.31610 26	8.70610 26	15	0.97m
	4	rs1046099	MOAP1	39 UTR	14	93649501	9.94610 26	1.12610 25	17			0.28& 0.54N
	5	rs1270629	MOAP1	Flanking 39 UTR	14	93646409	1.09610 25	1.15610 25	6		0.35c	0.54N
	6	rs2828074		Intergenic	21	24710202	1.41610 25	1.45610 25	-*4
	7	rs12483205 *1	DYRK1A	Intronic	21	38740824	2.16610 25	2.26610 25	8
	8	rs17519417	SPOCK3	Intronic	4	167659185 2.52610 25	2.41610 25	-*4
	9	rs16884060		Intergenic	5	10060297	3.20610 25	3.12610 25	4
	10 rs7856177	SPTLC1	Intronic	9	94839342	3.42610 25	3.64610 25	1		0.26.
	11 rs8070997	ACCN1	Intronic	17	31428130	3.66610 25	3.38610 25	2
	12 rs1792745	LOC642484	Intronic	18	53804993	3.86610 25	4.45610 25	8
	13 rs7042102	SPTLC1	Flanking 39 UTR	9	94763790	4.01610 25	4.25610 25	28	1.00b	0.26.
	14 rs10739923	SPTLC1	Flanking 39 UTR	9	94746291	4.01610 25	4.23610 25	28	1.00b
	15 rs12361072		Intergenic	11	23086211	4.93610 25	5.46610 25	34			1.00X
	16 rs2468574		Intergenic	11	23096397	4.93610 25	5.46610 25	34			1.00X
	Chr., chromosome.

*1

Table 3 .

 3 Results (p values) for association testing using the additive analysis model, between disease progression or viral load and the SNPs in DYRK1A, PDE8A, UBR7, MOAP1 and SPOCK3.

		EURO-CHAVI		GRIV		MACS		ACS
				non-	rapid	rapid -			AIDS
		viral load	progression	progressors -	progressors -	moderate -	AIDS	AIDS	related
	SNP (gene)	at set point	(CD4 T cells)	controls	controls	non-progressors	(def. '87)	(def. '93)	death
	rs12483205 (DYRK1A)	.0.1	0.035*	.0.1	.0.1	0.0048* #	0.059* #	0.059	.0.1
	rs2304418 (PDE8A)	.0.1	.0.1	.0.1	.0.1	.0.1	.0.1	.0.1	.0.1
	rs2905 (UBR7)	.0.1	.0.1	.0.1	.0.1	N.A.	N.A.	.0.1	.0.1
	rs1046099 (MOAP1)	.0.1	.0.1	.0.1	.0.1	N.A.	N.A.	0.075 #

PLoS ONE | www.plosone.org

February 2011 | Volume 6 | Issue 2 | e17190

Acknowledgments

We are indebted to all blood donors for their participation in this study, and thank Susan Cuvalay, Rachid Essinouh, Reno Frissen, Colinda Kroes, Cheryl Land, and all other employees at Sanquin Blood Bank for logistics. We would like to thank Evelien Bunnik for critical reading of the manuscript, Amalio Telenti and Marga Rotger for kindly sharing DNA from the donors used for their in vitro HIV-1 replication study in CD4 + T cells, Sigrid Le Clerc for sharing her part of the GRIV GWAS data, and Olivier Delaneau for his helpful advice on SNP imputation. Finally, we acknowledge Thomas Lumley for help with the design of the statistical analysis. MACS data in this manuscript were collected by the Multicenter AIDS Cohort Study (MACS) with centers (Principal Investigators) at The Johns Hopkins Bloomberg School of Public Health (Joseph B. Margolick, Lisa P. Jacobson), Howard Brown Health Center, Feinberg School of Medicine, Northwestern University, and Cook County Bureau of Health Services (John P. Phair, Steven M. Wolinsky), University of California, Los Angeles (Roger Detels), and University of Pittsburgh (Charles R. Rinaldo).

The authors acknowledge funding from the Landsteiner Foundation Blood Research (registration number 0526) and the European Union (Marie Curie International Reintegration Grant 029167). The MACS is funded by the National Institute of Allergy and Infectious Diseases, with additional supplemental funding from the National Cancer Institute. UO1-AI-35042, UL1-RR025005 (GCRC), UO1-AI-35043, UO1-AI-35039, UO1-AI-35040, UO1-AI-35041. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supporting Information

in the gene DYRK1A, for the total group of 393 blood donors (A), and this group of donors joined with donors from the replication cohort for which we had normalized data (n = 28; total n = 421) (B). DNA from donors with MDM that had low (n = 95) or high (n = 96) HIV-1 replication in vitro was used for the genome-wide association screen. Inclusion of genotype and normalized p24 data from donors with MDM that had intermediate Gag p24 production did not change the strength of the association (p = 2.09610 25 ), whereas combining the initial group of donors (n = 393) and the replication cohort (n = 28) increased the strength of the association (p = 4.84610 26 ). MAJ, homozygous for the major allele; HZ, heterozygote; MIN, homozygous for the minor allele. (TIF)

Figure S3 Schematic representation of the alignment of all four known DYRK1A mRNAs. Primers are depicted as arrows and were used to amplify a unique region for each of the transcripts. The start and stop codons are shown as green and red vertical lines respectively. (TIF) Figure S4 Q-Q plot showing the expected and observed distribution of the p values. The line and the corresponding Lambda (l) suggest there are no systematic differences in allele frequencies between subpopulations in our study population due to differences in genetic background of donors. The plot was created using the WGA viewer software version 1.26G [START_REF] Ge | WGAViewer: software for genomic annotation of whole genome association studies[END_REF]. (TIF) Table S1 (TXT) Table S2 (TXT) Table S3 (TXT) Table S4 (TXT)


Janne Prawitt

Sandrine Caron

Bart Staels

Bile acid metabolism and the pathogenesis of type 2 diabetes

Keywords: Animals, Bile Acids and Salts, metabolism, Diabetes Mellitus, Type 2, metabolism, physiopathology, Dyslipidemias, metabolism, physiopathology, Humans, Obesity, metabolism, physiopathology Bile acids, T2D, FXR, TGR5, bile acid sequestrants, obesity, dyslipidemia, NAFL

T2D is a growing health problem world-wide, but the currently available strategies for therapy and prevention are insufficient.

Recent observations indicate that

bile acid homeostasis is altered in T2D. Bile acids are metabolic regulators that act as signaling molecules through receptor-dependent and -independent pathways. The most prominent signaling molecules mediating bile acid signaling are the nuclear receptor FXR and the membrane receptor TGR5. Both are implicated in the regulation of lipid, glucose and energy metabolism. Dysregulation of these pathways might contribute to the development of T2D and associated metabolic complications. Interestingly, data from studies with bile acids or bile acid sequestrants indicate that the manipulation of bile acid homeostasis might be an attractive approach for T2D therapy. In this review, we summarize the mechanisms of bile-acid-mediated metabolic control that might be of relevance in the pathogenesis of T2D.

Introduction

Type 2 diabetes (T2D) is characterized by relative insulin deficiency due to the resistance of target tissues towards insulin action and the concomitant decline of insulin secretion from the pancreas. T2D pathogenesis includes genetic as well as environmental factors, but despite extensive research in this field the detailed mechanisms are yet incompletely known. The development of T2D is commonly associated with obesity and often with hypertension and dyslipidemia. These latter complications promote the development of cardiovascular disease which presents the most common mortality linked to T2D .

[1 ]

In the past 15 years, a growing body of evidence has shown that bile acid metabolism is altered in T2D patients and, conversely, that manipulation of the bile acid pool can improve glycemic control in such patients. Bile acids are increasingly recognized for their function as metabolic regulators. Via the activation of different signaling pathways, they participate in the control of bile acid, lipid and glucose metabolism. In this review, we summarize the mechanisms through which bile acids exert metabolic control and discuss their possible implication in the pathogenesis of T2D.

Bile acid metabolism

The synthesis and metabolism of bile acids is complex and highly regulated. Bile acids are synthesized from cholesterol in the liver.

Specific transporters located in the basolateral membrane of the hepatocyte actively secrete bile acids as well as cholesterol and phospholipids into the bile. The ingestion of a meal triggers the release of bile from the gallbladder into the intestinal lumen, where bile acids form micelles with dietary lipids and lipid-soluble vitamins, thereby facilitating their absorption. Specific transporters expressed in the distal ileum assure the re-absorption of ~95 of bile acids conveying them back to the liver, while the remaining 5 are excreted in % % the feces. In humans, the bile acid pool undergoes this enterohepatic cycle around 12x/day. Even though the bile acid pool size is maintained constant, the flux of bile acids varies during the day. Promoted by the ingestion of a meal, bile acid flux and plasma bile acid concentrations are highest postprandially .

[2 ]

Bile acids consist of a 24-carbon steroid core and a side chain carrying a carboxyl group. The nature of the bile acid species is determined by the number and position of hydroxyl groups on the steroid core, forming a hydrophobic and a hydrophilic side of the molecule. This amphiphatic property is essential for the bile acid s biological function, i.e. facilitating the excretion of hydrophobic ' molecules into the bile and mediating the absorption of dietary lipids from the intestinal lumen. However, it also turns bile acids into powerful detergents which at high concentrations may be cytotoxic (toxicity increases with hydrophobicity). For this reason, the size and composition of the bile acid pool is strictly controlled. Since the biological activity of a bile acid depends on its chemical structure, the pool composition (and thus hydrophobicity) may further determine to what extent bile acid signaling pathways are activated .

[3 ]

Bile acids are synthesized in a multiple step process . The liver produces the primary bile acids cholic acid (CA) and [START_REF] Russell | The enzymes, regulation, and genetics of bile acid synthesis[END_REF] chenodeoxycholic acid (CDCA) in humans, and CA and muricholic acid (MCA) in mice. The majority of bile acids is formed via the so-called classical pathway and only a small quantity (in humans estimated at ~6 ) via the alternative pathway. Cholesterol-7

% α

-hydroxylase (CYP7A1) catalyzes the first step of the classical pathway and is submitted to a negative feedback regulation mediated by bile acids themselves. CYP7A1 is considered to be the key enzyme of bile acid synthesis, CYP7A1-deficient mice die from liver failure within three weeks after birth . The key enzymes of the alternative pathway are sterol 27-hydroxylase (CYP27A1) and [START_REF] Russell | The enzymes, regulation, and genetics of bile acid synthesis[END_REF] 25-hydroxycholesterol-7 -hydroxylase (CYP7B1). Even though mice deficient for CYP7B1 are able to maintain a normal bile acid pool, a α case of a human newborn has been reported where a CYP7B1 mutation led to severe liver dysfunction, demonstrating the importance of the alternative pathway in humans . While CYP7A1 defines the size of the bile acid pool, its composition strongly depends on the [START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] activity of the sterol-12 -hydroxylase (CYP8B1). This enzyme catalyzes the formation of CA by diverting intermediates from CDCA α synthesis (or MCA synthesis in mice), thus defining the CA/CDCA ratio in the pool. As CYP7A1, CYP8B1 is negatively regulated by bile acids . The synthesized primary bile acids are conjugated to glycine or taurine before secretion into the bile. Once they have reached the [START_REF] Russell | The enzymes, regulation, and genetics of bile acid synthesis[END_REF] intestine, they may undergo deconjugation and dehydroxylation by bacteria of the gut microflora. The resulting secondary bile acids in humans are mainly deoxycholic acid (DCA) and lithocholic acid (LCA). During the second passage through the liver, a small part of the secondary bile acids is again modified by sulfonation or glucuronidation giving rise to tertiary bile acids.

Bile acids exert negative feedback on their own synthesis as mentioned above, but also on the expression of transport molecules assuring the enterohepatic cycle. Thus, bile acids stimulate their basolateral efflux into the bile (via the bile salt export pump BSEP), inhibit their ileal absorption (via the apical sodium-dependent bile acid transporter ASBT), stimulate their efflux from the enterocyte into the blood (via the organic solute transporter OST) and inhibit their apical uptake into the liver (via the sodium taurocholate-cotransporting polypeptide NTCP). The details of bile acid synthesis and transport regulation have been reviewed in detail elsewhere , .

[

Bile acid homeostasis in T2D

Due to their long-known role in the digestion of dietary fat, bile acids have classically been associated with lipid metabolism. Over the past decades, a limited number of studies has described alterations of the bile acid pool in T2D patients and animal models. Even though the available data are not fully consistent, they present a link of bile acid and glucose homeostasis.

An early study in patients with uncontrolled T2D reported an increase in the bile acid pool size and fecal bile acid excretion which decreased upon insulin treatment . Consistently, insulin was found to inhibit the expression of CYP7A1 and CYP27A1 in rat [START_REF] Bennion | Effects of diabetes mellitus on cholesterol metabolism in man[END_REF] hepatocytes, the key enzymes of both bile acid synthesis pathways . However, a second study observed no differences in bile acid [START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] synthesis between uncontrolled and insulin-treated diabetic patients . A recent investigation evaluated bile acid kinetics more [START_REF] Abrams | Metabolism of cholesterol and plasma triglycerides in nonketotic diabetes mellitus[END_REF] systematically in T2D patients . Even though no difference was found in the size of the total bile acid pool, the contribution of specific [START_REF] Brufau | Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism[END_REF] bile acid species to the pool was altered in T2D. Thus, the pool of the secondary bile acid DCA was elevated as was the DCA input rate, whereas the CDCA pool decreased . The CA pool was unchanged, even though the CA synthesis rate was elevated in diabetes. Another [START_REF] Brufau | Plasma bile acids are not associated with energy metabolism in humans[END_REF] study used a metabolomic approach to show lower CA and elevated DCA levels in plasma of self-reported, controlled diabetic patients compared to controls . Hence, CA might be increasingly converted into DCA in the gut of patients with T2D. Interestingly, changes in

[9 ]
the gut microflora have lately been associated with the development of metabolic diseases. This area of research might provide further insights into associated changes of bile acid metabolism. Despite the increasing interest in the link between bile acid metabolism and diabetes, the evidence from animal models is still very scarce. In insulin resistant ob/ob mice, leptin administration diminished the size and further decreased the hydrophobicity of the bile acid pool, resulting in an enhanced efflux of cholesterol into the bile . Unfortunately,

[10 ]
possible consequences of these alterations on glucose homeostasis were not analyzed. Herrema et al. investigated bile acid kinetics in more detail in diabetic db/db mice and observed an elevation of total bile acid pool size and synthesis, due to an increase in the fractional pool size and synthesis rate of CA .

[11 ]

Even though not devoid of discrepancies, the above presented data clearly suggest that changes in bile acid homeostasis are either a cause or a consequence of the metabolic disturbances observed during T2D. This raises the question whether the manipulation of bile acid homeostasis may improve metabolic complications linked to T2D. In this regard, an interesting study looked at bile acid metabolism in high-fat diet-fed rats that were submitted to ileal interposition surgery. This operation moves upwards a distal part of the ileum which is repositioned in the proximal jejunum. The authors found that plasma bile acid levels increased in operated rats, with a higher ratio of primary to secondary bile acids, and that bile acid excretion decreased, indicating an early reabsorption which short-circuits the enterohepatic cycle. Most interestingly, obesity, glucose tolerance and high cholesterol levels improved subsequent to the operation even though high-fat feeding continued . Similar observations were made in gastric bypass patients who, compared to weight-matched

[12 ]
controls, had increased plasma bile acid levels which inversely correlated with fasting plasma lipids and postprandial blood glucose .

[13 ]

Another recent report confirmed that the increase of the bile acid pool size, in this case by overexpression of CYP7A1 in mice, protected from Western diet-induced obesity, insulin resistance and hepatic steatosis .

[14 ]

More evidence for a beneficial effect of the modulation of bile acid metabolism comes from trials in which bile acid sequestrants were administered to T2D patients. These non-absorbable polymers complex bile acids in the intestinal lumen and thus divert them from the enterohepatic cycle. Consequently, the bile acid pool composition is modulated . Successfully applied to lower elevated [START_REF] Brufau | Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism[END_REF] LDL-cholesterol and prevent cardiovascular disease since a few decades, bile acid sequestrants were found to improve glycemic control in T2D patients. In patients not adequately controlled by common anti-diabetic therapeutics such as insulin, sulfonylurea or metformin, the administration of a bile acid sequestrant decreased plasma glucose and HbA1c concentrations . A pilot study in T2D patients and [START_REF] Prawitt | Bile acid sequestrants: glucose-lowering mechanisms[END_REF] [16 ]

a report from diabetic mice suggest that an increase in whole-body insulin sensitivity might underlie the improvement in glucose [START_REF] Kobayashi | Prevention and treatment of obesity, insulin resistance, and diabetes by bile acid-binding resin[END_REF] homeostasis upon treatment with bile acid sequestrants. Two other recent investigations found an increase in GLP1 (glucagon-like peptide 1) secretion from the intestine of sequestrant-treated diabetic rats, which might mediate the increased responsiveness towards insulin ,

Bile acids as signaling molecules

Over the past decade, several pathways have been identified, which mediate the regulatory effects of bile acids. The membrane receptor FPR (formyl peptide receptor) was reported to be activated by CDCA and DCA with an immunosuppressive outcome. The nuclear receptors PXR (pregnane X receptor) and VDR (vitamin D receptor) were shown to bind the toxic bile acid LCA and mediate its elimination from the liver and the intestine, providing protection from cholestasis and colon cancer. The most prominent candidates, however, are the nuclear receptor FXR (farnesoid X receptor) and the G-protein-coupled membrane receptor TGR5 .

[2 ]

FXR

The nuclear receptor FXR is highly expressed in liver and intestine, the main sites of bile acid metabolism, but also in adipose tissue, pancreas and adrenals . CDCA is most effective in activating FXR, while LCA, DCA and CA bind with lower affinity and specificity.

[2 ]

Upon ligand-binding, FXR acts either as a monomer or forms a heterodimer with RXR (retinoid X receptor) to subsequently transactivate or repress the expression of genes that contain a specific FXR response element in their promoter . In addition to the direct regulation [START_REF] Prawitt | How to modulate FXR activity to treat the Metabolic Syndrome[END_REF] of target gene expression, numerous FXR functions are mediated by the induction of SHP (short heterodimer protein) which in turn represses target genes in bile acid, lipid and glucose metabolism . In the intestine, FXR further induces the expression and subsequent [START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] secretion of FGF (fibroblast growth factor) 19 (corresponding to FGF15 in mice), which as a hormone can transmit the BA-induced signal to specific target tissues. By binding to the FGF receptor 4 (FGFR4) in the liver, FGF15 inhibits bile acid synthesis via a c-Jun N terminal kinase (JNK) mediated pathway .

-

TGR5

The identification of the G protein coupled receptor TGR5 (also Gpbar1, M-Bar, BG37) as a bile acid receptor , has opened [ 22 23 ] new insights in the mechanisms of bile acid signaling. Mainly expressed in the gall bladder, ileum, colon, brown and white adipose tissue, and to a lesser extent in skeletal muscle, liver and immune cells, TGR5 is activated by nanomolar concentrations of LCA and TLCA and micromolar concentrations of CA, DCA and CDCA . After ligand-binding to plasma membrane TGR5, the receptor is internalized, the

G subunit released and adenylate cyclase activated. The consecutively formed cAMP can activate PKA (protein kinase A) or the α S transcription factor CREB (cAMP response element binding protein), mediating bile acid functions in immunosuppression, energy and glucose homeostasis .

[3 ]

Receptor-independent pathways

Cell signaling networks, e.g. the MAPK pathway, can also be activated by bile acids independent of the mentioned receptors (reviewed in ), but their implication in the control of metabolic homeostasis still needs to be determined.

[24 ]

Metabolic regulation by bile acids

Based on the variety of bile acid-targeted receptors and signaling pathways, it is obvious that bile acids exert multiple regulatory functions. Here, we will focus on the role of bile acids in the regulation of pathways that are affected in T2D and associated metabolic complications ( ). Figure 1 Glucose metabolism and insulin sensitivity T2D is commonly preceded by a state of insulin resistance. In peripheral tissues, insulin resistance leads to a decreased uptake of glucose from the blood, whereas hepatic insulin resistance results in enhanced de novo glucose production. Both phenomena contribute to elevated plasma glucose levels, first in the postprandial and later also in the fasted state. Bile acids are involved in the regulation of hepatic glucose metabolism by FXR-mediated pathways. The expression of FXR itself is decreased in rat models of type 1 and T2D, an effect that could be reversed by the administration of insulin . The evaluation of the fasting-refeeding transition in FXR-deficient mice revealed [START_REF] Duran-Sandoval | Glucose regulates the expression of the farnesoid X receptor in liver[END_REF] the importance of functional FXR for the maintenance of stable blood glucose concentrations. In the absence of FXR, the induction of glycolytic (liver pyruvate kinase) and lipogenic genes (fatty acid synthase, acetyl CoA carboxylase) was accelerated, associated with a transient hypoglycaemia . In contrast, the induction of glucose-responsive genes was lower upon FXR activation. Via FXR, bile acids [START_REF] Duran-Sandoval | The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition[END_REF] further regulate hepatic glucose production by gluconeogenesis. In one set of studies, FXR activation by CA decreased the expression of the gluconeogenic genes PEPCK (phosphoenol pyruvate kinase), G6Pase (glucose-6-phosphatase) and FBP1 (fructose-1,6-bisphosphatase) , . In contrast, FXR activation by the synthetic agonist GW4064 was found to induce the expression of PEPCK in another [ 27 28 ] [29 ] investigation. In line with this finding, PEPCK and G6Pase expression were repressed in FXR-deficient mice , . Despite the [26 28 ] controversy of the available data, bile acids clearly impact on the regulation of hepatic glucose metabolism.

Intact insulin sensitivity is a prerequisite for the maintenance of glucose homeostasis. Several groups have reported that FXR-deficiency leads to peripheral insulin resistance in mice with a reduction of glucose disposal and decreased adipose tissue and skeletal muscle insulin signaling, however they presented conflicting data on the status of the liver , , . By contrast, the activation [28 30 31 ] of FXR by GW4064 in insulin-resistant ob/ob mice reduced hyperinsulinemia and improved glucose tolerance . Another study [START_REF] Cariou | The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice[END_REF] applying the semi-synthetic FXR agonist 6-ethyl-CDCA to diabetic fa/fa rats confirmed the beneficial effect of FXR activation on insulin resistance in liver and skeletal muscle . In addition to this key role of FXR in the control of insulin sensitivity, FXR expression has [START_REF] Cipriani | FXR activation reverses insulin resistance and lipid abnormalities and protects against liver steatosis in Zucker (fa/fa) obese rats[END_REF] recently been reported to play a role in the pancreas, where it regulates glucose-induced insulin secretion , and protects against [33 34 ] lipotoxicity , a crucial feature in the development of T2D.

[33 ]

Modulation of the bile acid pool by intestinal sequestration has been shown to improve insulin resistance in rat models of diabetes by promoting the secretion of GLP1 from intestinal L-cells , . The incretin GLP1 is released in response to the ingestion of a meal and, [18 19 ] in addition to controlling appetite and gastrointestinal function, promotes insulin secretion from the pancreas. This is why GLP1 based strategies are currently being used for the therapy of T2D. Both studies , exclude that the observed effect is FXR-dependent. Most [18 19 ] interestingly, recent reports provide evidence that bile acids induce GLP1 secretion by TGR5-mediated pathways. LCA and DCA TGR5-dependently induced intracellular cAMP concentrations and GLP1 secretion from intestinal L-cells . In a second study, in vitro [START_REF] Katsuma | Bile acids promote glucagon-like peptide-1 secretion through tgr5 in a murine enteroendocrine cell line stc-1[END_REF] the TGR5 agonist oleanolic acid attenuated obesity and improved insulin resistance in mice upon high-fat diet-feeding . The most [START_REF] Sato | Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea[END_REF] detailed investigation has been provided by Thomas et al.

. The authors show that glucose tolerance reduced by TGR5-deficiency and [START_REF] Thomas | This elaborate study is the first to propose a mechanism by which bile acids might improve glucose homeostasis[END_REF] improved by TGR5 overexpression in high-fat diet-fed mice via increased GLP1 and insulin secretion. Pharmacological activation of TGR5 by the semi-synthetic agonist INT777 also ameliorated insulin sensitivity and raised GLP1 secretion in obese mice. The authors proposed that TGR5 activation raises the intracellular ATP/ADP ratio and enhances calcium influx which promotes GLP1 secretion. Thus, the TGR5-mediated triggering of GLP1 secretion presents a powerful mean for bile acids to control glucose homeostasis.

Very few data have demonstrated that receptor-independent pathways account for the beneficial action of bile acids on glucose homeostasis. The administration of taurine-conjugated ursodeoxycholic acid (TUDCA) to genetically obese mice improved insulin resistance by attenuating endoplasmatic reticulum (ER) stress . A very recent study in human obese subjects found an increase of [START_REF] Ozcan | Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes[END_REF] insulin sensitivity in liver and muscle, but not adipose tissue upon TUDCA administration . However, no effect on markers of ER [START_REF] Kars | 1905 One of the rare investigations of receptor-independent effects of bile acids on insulin sensitivity in human patients 40[END_REF] stress was observed.

Energy metabolism

As mentioned above, visceral obesity is often associated with T2D, which is characterized by a dysregulation of energy metabolism. It is still under discussion to which extent and by which mechanisms obesity is causal for the development of insulin resistance and T2D, but the epidemiological link is clearly established .

[1 ]

Even though adipose tissue is not a tissue classically involved in bile acid metabolism, it expresses FXR , . In FXR-deficient . Further, FXR activation was shown to increase the insulin-mediated [START_REF] Abdelkarim | The Farnesoid X Receptor Regulates Adipocyte Differentiation and Function by Promoting Peroxisome Proliferator-activated Receptor-gamma and Interfering with the Wnt/ beta -Catenin Pathways[END_REF] uptake of glucose .

[40 ]

Activation of FXR induces the expression and secretion of FGF19 in the intestine. Fu et al. have shown that the administration of FGF19 to high-fat diet-fed mice increased energy expenditure, reversed weight gain and as a consequence improves insulin resistance [42 ] . Changes of the amount or nature of bile acids passing through the intestine can thus have strong systemic effects on metabolic regulation by modulating FXR-mediated FGF19 secretion.

The discovery of TGR5 as a bile acid receptor identified another pathway for the bile acid-mediated regulation of energy metabolism.

The supplementation of a high-fat diet with CA increased energy expenditure in brown adipose tissue of mice with a beneficial outcome for obesity and insulin resistance . The authors suggest a TGR5-cAMP-triggered increase in triiodothyronine due to the activation of [43 ] deiodinase in brown adipose tissue, resulting in increased uncoupling. Even though an attractive concept, several facts put the proposed mechanism into question. Neither the overexpression nor the absence of TGR5 in mice leads to a difference in body weight and the susceptibility to diet-induced obesity is gender-specific . Further, thyroid hormones have recently been shown to exert a central rather [44 ] than peripheral effect on energy metabolism of brown adipose tissue .

[45 ]

Lipid metabolism and NAFL/NASH

Even though the dysregulation of lipid and lipoprotein metabolism is not considered as a direct cause for T2D, it is strongly associated with obesity, insulin resistance and T2D itself. Dyslipidemia increases the risk for cardiovascular incidences in T2D. The implication of bile acids in the regulation of triglyceride and cholesterol metabolism via hepatic FXR became manifest through the observation of dyslipidemia in FXR-deficient mice .

[46 ]

CDCA administration was shown to decrease plasma lipids in hypertriglyceridemic patients, long before FXR was identified .

[47 ]

Watanabe et al. delineated a FXR-SHP-mediated mechanism which repressed the induction of SREBP1c by LXR and thus reduced lipogenesis and VLDL secretion . Recently, the administration of bile acid sequestrants was reported to induce lipogenesis dependent [48 ] of FXR and LXR . The reduction of plasma triglycerides by FXR activation was further attributed to an increased clearance from the bloodstream by lipoprotein lipase (LPL). LPL activity is enhanced by the induction of its activator apolipoprotein CII and the repression of its inhibitor apolipoprotein CIII in the liver upon FXR activation .

[2 ]

Given that bile acids are synthesized from cholesterol, the direct link of these two metabolic pathways is obvious. Bile acids exert additional control in cholesterol metabolism by regulating numerous FXR target genes . , FXR activation induced the [START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] In vitro expression of the LDL receptor and repressed its inhibitor PCSK9. However, this mechanism did not seem to apply since CDCA in vivo administration to patients increased LDL cholesterol levels . The key structural component of HDL, apolipoprotein AI, was reported to [START_REF] Prawitt | How to modulate FXR activity to treat the Metabolic Syndrome[END_REF] be a negative FXR target gene . Data from mice further proposed that FXR stimulates the uptake of HDL cholesterol esters via hepatic [START_REF] Lefebvre | Role of bile acids and bile acid receptors in metabolic regulation[END_REF] SRBI induction . Consequently, FXR activation in patients lowered HDL cholesterol levels .

[46 ]

[47 ]

The occurrence of non-alcoholic liver (NAFL) and its progression towards non-alcoholic steatohepatitis (NASH) in association with insulin resistance and T2D development has been re-evaluated in the past years. Several studies suggest that NAFLD might be one of the causal factors for the manifestation of T2D in obese subjects . TUDCA administration was shown to decrease NAFLD in genetically [49 ] obese mice and TGR5 activation in diet-induced obesity , but no directly-underlying mechanisms were proposed. CDCA and CA [38 ] [37 ] further prevented fructose-induced hepatic steatosis by protecting against the entry of bacterial endotoxins from the intestine . Several

[50 ]

studies have further linked bile acids to NASH via FXR. FXR-deficient mice displayed marked inflammatory cell infiltration and increased hepatic collagen content, whereas FXR activation decreased inflammatory cell infiltration and fibrosis in mice by repression of the fibrotic genes TGF1 , -SMA and TIMP-1 .

β α [START_REF] Prawitt | How to modulate FXR activity to treat the Metabolic Syndrome[END_REF]

Conclusions

A growing body of evidence acknowledges bile acids as metabolic regulators. By binding to the nuclear receptor FXR or the membrane receptor TGR5, they participate in the control of glucose metabolism, insulin sensitivity, energy homeostasis as well as lipid metabolism. During the pathogenesis of T2D, the composition of the circulating bile acid pool is altered, probably contributing to the dysregulation of metabolic homeostasis in T2D. The manipulation of the bile acid pool itself by bile acid substitution or intestinal sequestration, or bile acid receptors such as FXR and TGR5 present promising strategies to develop therapeutics for T2D. However, given the diversity of the metabolic pathways controlled by bile acid signaling, more mechanistic and clinical studies are needed to precipitate the optimal approach.

Figure 1

Schematic overview of the functions of bile acids (BA) in the regulation of BA, energy, glucose and lipid metabolism via FXR-and TGR5-mediated signaling pathways. For details please see text. GLP1, glucagon-like peptide 1; FGF, fibroblast growth factor; BAT, brown adipose tissue.

Biol Chem . 2002 ; 277 : 34117 -34124

Acknowledgements:

The authors are supported by the EU Grant HEPADIP (N 018734) and received funding from Daiichi Sankyo.

°Footnotes: