Genetic and functional analysis of chymotrypsin-like protease (CTRL) in chronic pancreatitis.

BACKGROUND: Genetic predisposition is crucial in the pathogenesis of early-onset chronic pancreatitis (CP). So far, several genetic alterations have been identified as risk factors, predominantly in genes encoding digestive enzymes. However, many early-onset CP cases have no identified underlying cause. Chymotrypsins are a family of serine proteases that can cleave trypsinogen and lead to its degradation. Because genetic alterations in the chymotrypsins CTRC, CTRB1, and CTRB2 are associated with CP, we genetically and functionally investigated chymotrypsin-like protease (CTRL) as a potential risk factor. METHODS: We screened 1005 non-alcoholic CP patients and 1594 controls for CTRL variants by exome sequencing. We performed Western blots and activity assays to analyse secretion and proteolytic activity. We measured BiP mRNA expression to investigate the potential impact of identified alterations on endoplasmic reticulum (ER) stress. RESULTS: We identified 13 heterozygous non-synonymous CTRL variants: five exclusively in patients and three only in controls. Functionality was unchanged in 6/13 variants. Four alterations showed normal secretion but reduced (p.G20S, p.G56S, p.G61S) or abolished (p.S208F) activity. Another three variants (p.C201Y, p.G215R and p.C220G) were not secreted and already showed reduced or no activity intracellularly. However, intracellular retention did not lead to ER stress. CONCLUSION: We identified several CTRL variants, some showing potent effects on protease function and secretion. We observed these effects in variants found in patients and controls, and CTRL loss-of-function variants were not significantly more common in patients than controls. Therefore, CTRL is unlikely to play a relevant role in the development of CP.

Gender-specific changes of the gut microbiome correlate with tumor development in murine models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal outcome. To improve understanding of sequential microbiome changes during PDAC development we analyzed mouse models of pancreatic carcinogenesis (KC mice recapitulating pre-invasive PanIN formation, as well as KPC mice recapitulating invasive PDAC) during early tumor development and subsequent tumor progression. Diversity and community composition were analyzed depending on genotype, age, and gender. Both mouse models demonstrated concordant abundance changes of several genera influenced by one or more of the investigated factors. Abundance was significantly impacted by gender, highlighting the need to further elucidate the impact of gender differences. The findings underline the importance of the microbiome in PDAC development and indicate that microbiological screening of patients at risk and targeting the microbiome in PDAC development may be feasible in future.

Loss-of-function variant in chymotrypsin like elastase 3B (CELA3B) is associated with non-alcoholic chronic pancreatitis.

BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.

Variants in the pancreatic CUB and zona pellucida-like domains 1 (CUZD1) gene in early-onset chronic pancreatitis - A possible new susceptibility gene.

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.

Colocalization analysis of pancreas eQTLs with risk loci from alcoholic and novel non-alcoholic chronic pancreatitis GWAS suggests potential disease causing mechanisms.

BACKGROUND: Previous genome-wide association studies (GWAS) identified genome-wide significant risk loci in chronic pancreatitis and investigated underlying disease causing mechanisms by simple overlaps with expression quantitative trait loci (eQTLs), a procedure which may often result in false positive conclusions. METHODS: We conducted a GWAS in 584 non-alcoholic chronic pancreatitis (NACP) patients and 6040 healthy controls. Next, we applied Bayesian colocalization analysis of identified genome-wide significant risk loci from both, our recently published alcoholic chronic pancreatitis (ACP) and the novel NACP dataset, with pancreas eQTLs from the GTEx V8 European cohort to prioritize candidate causal genes and extracted credible sets of shared causal variants. RESULTS: Variants at the CTRC (p = 1.22 × 10-21) and SPINK1 (p = 6.59 × 10-47) risk loci reached genome-wide significance in NACP. CTRC risk variants colocalized with CTRC eQTLs in ACP (PP4 = 0.99, PP4/PP3 = 95.51) and NACP (PP4 = 0.99, PP4/PP3 = 95.46). For both diseases, the 95% credible set of shared causal variants consisted of rs497078 and rs545634. CLDN2-MORC4 risk variants colocalized with CLDN2 eQTLs in ACP (PP4 = 0.98, PP4/PP3 = 42.20) and NACP (PP4 = 0.67, PP4/PP3 = 7.18), probably driven by the shared causal variant rs12688220. CONCLUSIONS: A shared causal CTRC risk variant might unfold its pathogenic effect in ACP and NACP by reducing CTRC expression, while the CLDN2-MORC4 shared causal variant rs12688220 may modify ACP and NACP risk by increasing CLDN2 expression.

Loss of function TRPV6 variants are associated with chronic pancreatitis in nonalcoholic early-onset Polish and German patients.

PURPOSE: Loss of function variants of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) have been recently associated with chronic pancreatitis (CP) in Japanese, German and French patients. Here, we investigated the association of TRPV6 variants with CP in independent European cohorts of early-onset CP patients from Poland and Germany. PATIENTS AND METHODS: We enrolled 152 pediatric CP patients (median age 8.6 yrs) with no history of alcohol/smoking abuse and 472 controls from Poland as well as 157 nonalcoholic young CP patients (median age 20 yrs) and 750 controls from Germany. Coding regions of TRPV6 were screened by Sanger and next generation sequencing. Selected, potentially pathogenic TRPV6 variants were expressed in HEK293T cells and TRPV6 activity was analyzed using ratiometric Ca2+ measurements. RESULTS: Overall, we identified 10 novel (3 nonsense and 7 missenses) TRPV6 variants in CP patients. TRPV6 p.V239SfsX53 nonsense variant and the variants showing significant decrease in intracellular Ca2+ concentration in HEK293T cells (p.R174X, p.L576R, p.R342Q), were significantly overrepresented in Polish patients as compared to controls (6/152, 3.9% vs. 0/358, 0%; P = 0,0007). Nonsense TRPV6 variants predicted as loss of function (p.V239SfsX53 and p.R624X) were also significantly overrepresented in German patients (3/157; 2.0% vs 0/750; 0%, P = 0.005). CONCLUSIONS: We showed that TRPV6 loss of function variants are associated with elevated CP risk in early-onset Polish and German patients confirming that TRPV6 is a novel CP susceptibility gene.

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

Sequencing of the complex CTRB1-CTRB2 locus in chronic pancreatitis.

BACKGROUND: /Objectives: A recent Genome-wide Association Study (GWAS) in alcoholic chronic pancreatitis (ACP) identified a novel association with the CTRB1-CTRB2 (chymotrypsinogen B1, B2) locus, linked to a 16.6 kb inversion that was confirmed in non-alcoholic chronic pancreatitis (NACP). Moreover, recent findings on the function of CTRB1 and CTRB2 suggest a protective role in pancreatitis development. The aim of the present study was to investigate the CTRB1-CTRB2 locus for rare genetic variants associated with chronic pancreatitis (CP). METHODS: We analyzed 134 patients with ACP and 203 patients with NACP and compared them to up to 258 healthy controls. Genotyping was performed with polymerase chain reaction, followed by Sanger sequencing of all exons and the exon-intron-boundaries of CTRB1 and CTRB2. Finally, in silico analyses of the identified variants were conducted. RESULTS: None of the seven rare missense variants or the single 5'-UTR variant in CTRB1 and CTRB2 was associated with ACP or NACP. In silico analysis predicted that variant p. Trp5Leu in CTRB1 and variant c.-4C > T in CTRB2 might alter protein expression and variants p. Asp222His in CTRB1 and p. Ala247Thr in CTRB2 might affect protein function. However, all of these variants were also described in public databases. CONCLUSIONS: The present study did not reveal an association of rare variants in CTRB1 and CTRB2 with ACP or NACP. Although rare missense variants were almost exclusively found in patients, only four variants were predicted to affect protein expression or function. Thus, a major influence of rare variants in the CTRB1-CTRB2 locus on CP development is unlikely.

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.

Dynamic modelling of an ACADS genotype in fatty acid oxidation - Application of cellular models for the analysis of common genetic variants.

BACKGROUND: Genome-wide association studies of common diseases or metabolite quantitative traits often identify common variants of small effect size, which may contribute to phenotypes by modulation of gene expression. Thus, there is growing demand for cellular models enabling to assess the impact of gene regulatory variants with moderate effects on gene expression. Mitochondrial fatty acid oxidation is an important energy metabolism pathway. Common noncoding acyl-CoA dehydrogenase short chain (ACADS) gene variants are associated with plasma C4-acylcarnitine levels and allele-specific modulation of ACADS expression may contribute to the observed phenotype. METHODS AND FINDINGS: We assessed ACADS expression and intracellular acylcarnitine levels in human lymphoblastoid cell lines (LCL) genotyped for a common ACADS variant associated with plasma C4-acylcarnitine and found a significant genotype-dependent decrease of ACADS mRNA and protein. Next, we modelled gradual decrease of ACADS expression using a tetracycline-regulated shRNA-knockdown of ACADS in Huh7 hepatocytes, a cell line with high fatty acid oxidation-(FAO)-capacity. Assessing acylcarnitine flux in both models, we found increased C4-acylcarnitine levels with decreased ACADS expression levels. Moreover, assessing time-dependent changes of acylcarnitine levels in shRNA-hepatocytes with altered ACADS expression levels revealed an unexpected effect on long- and medium-chain fatty acid intermediates. CONCLUSIONS: Both, genotyped LCL and regulated shRNA-knockdown are valuable tools to model moderate, gradual gene-regulatory effects of common variants on cellular phenotypes. Decreasing ACADS expression levels modulate short and surprisingly also long/medium chain acylcarnitines, and may contribute to increased plasma acylcarnitine levels.

Protease-Sensitive Pancreatic Lipase Variants Are Associated With Early Onset Chronic Pancreatitis.

OBJECTIVES: Premature activation of the digestive protease trypsin within the pancreatic parenchyma is a critical factor in the pathogenesis of pancreatitis. Alterations in genes that affect intrapancreatic trypsin activity are associated with chronic pancreatitis (CP). Recently, carboxyl ester lipase emerged as a trypsin-independent risk gene. Here, we evaluated pancreatic lipase (PNLIP) as a potential novel susceptibility gene for CP. METHODS: We analyzed all 13 PNLIP exons in 429 nonalcoholic patients with CP and 600 control subjects from Germany, in 632 patients and 957 controls from France, and in 223 patients and 1,070 controls from Japan by DNA sequencing. Additionally, we analyzed selected exons in further 545 patients with CP and 1,849 controls originating from Germany, United States, and India. We assessed the cellular secretion, lipase activity, and proteolytic stability of recombinant PNLIP variants. RESULTS: In the German discovery cohort, 8/429 (1.9%) patients and 2/600 (0.3%) controls carried a PNLIP missense variant (P = 0.02, odds ratio [OR] = 5.7, 95% confidence interval [CI] = 1.1-38.9). Variants detected in patients were prone to proteolytic degradation by trypsin and chymotrypsin. In the French replication cohort, protease-sensitive variants were also enriched in patients with early-onset CP (5/632 [0.8%]) vs controls (1/957 [0.1%]) (P = 0.04, OR = 7.6, 95% CI = 0.9-172.9). In contrast, we detected no protease-sensitive variants in the non-European populations. In the combined European data, protease-sensitive variants were found in 13/1,163 cases (1.1%) and in 3/3,000 controls (0.1%) (OR = 11.3, 95% CI = 3.0-49.9, P < 0.0001). CONCLUSIONS: Our data indicate that protease-sensitive PNLIP variants are novel genetic risk factors for the development of CP.

Genome-wide association study identifies inversion in the CTRB1-CTRB2 locus to modify risk for alcoholic and non-alcoholic chronic pancreatitis.

OBJECTIVE: Alcohol-related pancreatitis is associated with a disproportionately large number of hospitalisations among GI disorders. Despite its clinical importance, genetic susceptibility to alcoholic chronic pancreatitis (CP) is poorly characterised. To identify risk genes for alcoholic CP and to evaluate their relevance in non-alcoholic CP, we performed a genome-wide association study and functional characterisation of a new pancreatitis locus. DESIGN: 1959 European alcoholic CP patients and population-based controls from the KORA, LIFE and INCIPE studies (n=4708) as well as chronic alcoholics from the GESGA consortium (n=1332) were screened with Illumina technology. For replication, three European cohorts comprising 1650 patients with non-alcoholic CP and 6695 controls originating from the same countries were used. RESULTS: We replicated previously reported risk loci CLDN2-MORC4, CTRC, PRSS1-PRSS2 and SPINK1 in alcoholic CP patients. We identified CTRB1-CTRB2 (chymotrypsin B1 and B2) as a new risk locus with lead single-nucleotide polymorphism (SNP) rs8055167 (OR 1.35, 95% CI 1.23 to 1.6). We found that a 16.6 kb inversion in the CTRB1-CTRB2 locus was in linkage disequilibrium with the CP-associated SNPs and was best tagged by rs8048956. The association was replicated in three independent European non-alcoholic CP cohorts of 1650 patients and 6695 controls (OR 1.62, 95% CI 1.42 to 1.86). The inversion changes the expression ratio of the CTRB1 and CTRB2 isoforms and thereby affects protective trypsinogen degradation and ultimately pancreatitis risk. CONCLUSION: An inversion in the CTRB1-CTRB2 locus modifies risk for alcoholic and non-alcoholic CP indicating that common pathomechanisms are involved in these inflammatory disorders.

Allele-specific quantitative proteomics unravels molecular mechanisms modulated by cis-regulatory PPARG locus variation.

Genome-wide association studies identified numerous disease risk loci. Delineating molecular mechanisms influenced by cis-regulatory variants is essential to understand gene regulation and ultimately disease pathophysiology. Combining bioinformatics and public domain chromatin information with quantitative proteomics supports prediction of cis-regulatory variants and enabled identification of allele-dependent binding of both, transcription factors and coregulators at the type 2 diabetes associated PPARG locus. We found rs7647481A nonrisk allele binding of Yin Yang 1 (YY1), confirmed by allele-specific chromatin immunoprecipitation in primary adipocytes. Quantitative proteomics also found the coregulator RING1 and YY1 binding protein (RYBP) whose mRNA levels correlate with improved insulin sensitivity in primary adipose cells carrying the rs7647481A nonrisk allele. Our findings support a concept with diverse cis-regulatory variants contributing to disease pathophysiology at one locus. Proteome-wide identification of both, transcription factors and coregulators, can profoundly improve understanding of mechanisms underlying genetic associations.

5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells.

5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment.

Effects of Genetic Loci Associated with Central Obesity on Adipocyte Lipolysis.

OBJECTIVES: Numerous genetic loci have been associated with measures of central fat accumulation, such as waist-to-hip ratio adjusted for body mass index (WHRadjBMI). However the mechanisms by which genetic variations influence obesity remain largely elusive. Lipolysis is a key process for regulation of lipid storage in adipocytes, thus is implicated in obesity and its metabolic complications. Here, genetic variants at 36 WHRadjBMI-associated loci were examined for their influence on abdominal subcutaneous adipocyte lipolysis. SUBJECTS AND METHODS: Fasting subcutaneous adipose tissue biopsies were collected from 789 volunteers (587 women and 202 men, body mass index (BMI) range 17.7-62.3 kg/m2). We quantified subcutaneous adipocyte lipolysis, both spontaneous and stimulated by the catecholamine isoprenaline or a cyclic AMP analogue. DNA was extracted from peripheral blood mononuclear cells and genotyping of SNPs associated with WHRadjBMI conducted. The effects on adipocyte lipolysis measures were assessed for SNPs individually and combined in a SNP score. RESULTS: The WHRadjBMI-associated loci CMIP, PLXND1, VEGFA and ZNRF3-KREMEN1 demonstrated nominal associations with spontaneous and/or stimulated lipolysis. Candidate genes in these loci have been reported to influence NFκB-signaling, fat cell size and Wnt signalling, all of which may influence lipolysis. SIGNIFICANCE: This report provides evidence for specific WHRadjBMI-associated loci as candidates to modulate adipocyte lipolysis. Additionally, our data suggests that genetically increased central fat accumulation is unlikely to be a major cause of altered lipolysis in abdominal adipocytes.

Metabolic switch during adipogenesis: From branched chain amino acid catabolism to lipid synthesis.

Fat cell metabolism has an impact on body homeostasis and its proper function. Nevertheless, the knowledge about simultaneous metabolic processes, which occur during adipogenesis and in mature adipocytes, is limited. Identification of key metabolic events associated with fat cell metabolism could be beneficial in the field of novel drug development, drug repurposing, as well as for the discovery of patterns predicting obesity risk. The main objective of our work was to provide comprehensive characterization of metabolic processes occurring during adipogenesis and in mature adipocytes. In order to globally determine crucial metabolic pathways involved in fat cell metabolism, metabolomics and transcriptomics approaches were applied. We observed significantly regulated metabolites correlating with significantly regulated genes at different stages of adipogenesis. We identified the synthesis of phosphatidylcholines, the metabolism of even and odd chain fatty acids, as well as the catabolism of branched chain amino acids (BCAA; leucine, isoleucine and valine) as key regulated pathways. Our further analysis led to identification of an enzymatic switch comprising the enzymes Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthase) and Auh (AU RNA binding protein/enoyl-CoA hydratase) which connects leucine degradation with cholesterol synthesis. In addition, propionyl-CoA, a product of isoleucine degradation, was identified as a putative substrate for odd chain fatty acid synthesis. The uncovered crosstalks between BCAA and lipid metabolism during adipogenesis might contribute to the understanding of molecular mechanisms of obesity and have potential implications in obesity prediction.

Medium-chain dicarboxylic acylcarnitines as markers of n-3 PUFA-induced peroxisomal oxidation of fatty acids.

SCOPE: Omega-3 polyunsaturated fatty acids (n-3 PUFA) found in fish oil activate PPAR-α, stimulate peroxisomal fatty acid (FA) ß-oxidation and prevent impairments on glucose homeostasis. METHODS AND RESULTS: Glucose metabolism and FA oxidation were studied in C57/Bl6 mice fed with diets containing either 3.6 and 31.5% fish oil or lard. To assess the effects of peroxisomal proliferation on FA oxidation independent of n-3 PUFA intake, mice were treated with the PPAR-α agonist WY-14643. n-3 PUFA-fed mice were protected from glucose intolerance and dyslipidemia compared to animals fed a lard-based high-fat diet. Most importantly, mice fed on the hyperlipidic diet based on fish oil as well as the WY-14643 treated mice showed twofold increase of odd, medium-chain, dicarboxylic acylcarnitines in the liver suggesting that not only ß-oxidation, but also α- and ω-oxidation of FA were increased. Finally, an oxidation assay using liver homogenates and palmitic acid as substrate revealed an over tenfold increased production of similar acylcarnitines, indicating that FA are their precursors. CONCLUSION: This study shows at the metabolite level that peroxisome proliferation induced either by fish oil or WY-14643 is associated with increased α- and ω-oxidation of FA producing specific acylcarnitines that can be utilized as biomarkers of peroxisomal FA oxidation.

Restless legs syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon.

Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.

Postprandial activation of metabolic and inflammatory signalling pathways in human peripheral mononuclear cells.

High-fat, high-carbohydrate (HFHC) meals induce an inflammatory response in mononuclear cells (MNC). Here, we studied the interaction between metabolic and inflammatory signalling pathways by the measurement of postprandial effects of three different test meals on intracellular Akt, S6 kinase (S6K)/mammalian target of rapamycin and NF-κB signalling in human MNC. We recruited six healthy, lean individuals. Each individual ingested three different meals in the morning separated by at least 3 d: a HFHC meal; an oral lipid-tolerance test meal; a healthy breakfast. Blood samples were obtained before and 1, 2, 4, 6 and 8 h after ingestion. Plasma insulin and IL-6 levels were measured. Intracellular metabolic and inflammatory signalling pathways were assessed by measuring the phosphorylation of Akt kinase and S6K, the degradation of inhibitory κB-α (IκB-α) protein and the DNA binding activity of NF-κB in MNC. mRNA expression levels of the Akt and NF-κB target genes Mn superoxide dismutase (MnSOD), CC-chemokine-receptor 5 (CCR5), intercellular adhesion molecule 1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) were measured by quantitative RT-PCR. We found a positive correlation of Akt phosphorylation with NF-κB activation (NF-κB binding activity: r 0·4500, P= 0·0003; IκB-α protein levels: r -0·5435, P< 0·0001), a negative correlation of plasma insulin levels with NF-κB binding activity (r -0·3993, P= 0·0016) and a positive correlation of plasma insulin levels with S6K activation (r 0·4786, P< 0·0001). The activation of Akt and pro-inflammatory NF-κB signalling was supported by the up-regulation of the respective target genes MnSOD and CCR5. In conclusion, the present data suggest a postprandial interaction between the metabolic and inflammatory signalling pathways Akt and NF-κB in MNC.

Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.