Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

Objective: Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor (TF) PDX1 leads to pancreatic agenesis, whereas heterozygous mutations can cause Maturity-Onset Diabetes of the Young 4 (MODY4). Although the function of Pdx1 is well studied in pre-clinical models during insulin-producing beta-cell development and homeostasis, it remains elusive how this TF controls human pancreas development by regulating a downstream transcriptional program. Also, comparative studies of PDX1 binding patterns in pancreatic progenitors and adult beta-cells have not been conducted so far. Furthermore, many studies reported the association between single nucleotide polymorphisms (SNPs) and T2DM, and it has been shown that islet enhancers are enriched in T2DM-associated SNPs. Whether regions, harboring T2DM-associated SNPs are PDX1 bound and active at the pancreatic progenitor stage has not been reported so far. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions. Results: ChIP-seq for PDX1 revealed a total of 8088 PDX1-bound regions that map to 5664 genes in iPSC-derived PPs. The PDX1 target regions include important pancreatic TFs, such as PDX1 itself, RFX6, HNF1B, and ME1S1, which were activated during the differentiation process as revealed by the active chromatin mark H3K27ac and mRNA expression profiling, suggesting that auto-regulatory feedback regulation maintains PDX1 expression and initiates a pancreatic TF program. Remarkably, we identified several PDX1 target genes that have not been reported in the literature in human so far, including RFX3, required for ciliogenesis and endocrine differentiation in mouse, and the ligand of the Notch receptor DLL1, which is important for endocrine induction and tip-trunk patterning. The comparison of PDX1 profiles from PPs and adult human islets identified sets of stage-specific target genes, associated with early pancreas development and adult beta-cell function, respectively. Furthermore, we found an enrichment of T2DM-associated SNPs in active chromatin regions from iPSC-derived PPs. Two of these SNPs fall into PDX1 occupied sites that are located in the intronic regions of TCF7L2 and HNF1B. Both of these genes are key transcriptional regulators of endocrine induction and mutations in cis-regulatory regions predispose to diabetes. Conclusions: Our data provide stage-specific target genes of PDX1 during in vitro differentiation of stem cells into pancreatic progenitors that could be useful to identify pathways and molecular targets that predispose for diabetes. In addition, we show that T2DM-associated SNPs are enriched in active chromatin regions at the pancreatic progenitor stage, suggesting that the susceptibility to T2DM might originate from imperfect execution of a beta-cell developmental program

The importance of visceral adipose tissue mass reduction for improving endothelial function in type 2 diabetes prone individuals

Obesity is an accepted risk factor for cardiovascular disease because of its many associated metabolic diseases (insulin resistance, hyperglycaemia, hypertriglyceridemia, dyslipoproteinemia, arterial hypertension, hyperuricemia etc.). Within the last years, research results showed that body weight alone is of only limited value for cardiovascular morbidity, even if the Body Mass Index (BMI) is determined that also takes account of the height. Against this background, the importance of body fat distribution and particularly the importance of visceral adipose tissue mass become potentially significant. Recent research data postulate an increased cardiovascular risk for visceral obesity compared with increased subcutaneous adipose tissue mass caused by increased expression and secretion of pro-inflammatory cytokines in visceral adipose tissue. On that assumption we performed a prospective intervention study to prove that reducing visceral adipose tissue mass is essential for improving endothelial function - as an early marker of atherogenesis – and for reducing systemic inflammation. Analyses were done in 189 individuals (age: 45.5 ± 0,8 years) at increased risk of type 2 diabetes (obesity, impaired glucose tolerance or positive family history). Endothelial function expressed as flow mediated dilation (FMD) of the brachial artery was measured by high resolution ultrasound (13 MHz). Further the vascular adhesion molecules sE-selectin, sV-CAM and sI-CAM were determined based on ELISA-technique as well as the body fat distribution measured by magnetic resonance tomography. There was a mean decrease in body weight (- 3%), total adipose tissue mass (TAT, -7.6%) und visceral adipose tissue mass (VAT, - 4.3%). The change in FMD was not associated with change in body weight (p = 0.35) or TAT (p = 0.21) but with a decrease in VAT (r = −0.189, p = 0.009). In a post hoc analysis, the subjects were divided by the median change in VAT into responders and non-responders. FMD increased only in the responders (from 6.2 ± 0.4% to 8.0 ± 0.5%, p = 0.0005) but not in the non-responders (p = 0.15). Also sE-selectin significantly decreased only in the responders (from 54 ± 4 ng/ml to 47 ± 3 ng/ml; p = 0.03). Our prospective study data prove that a lifestyle intervention in obese or in type 2 diabetes prone individuals that leads to reducing visceral adipose tissue mass is essential for improving endothelial function and not the body weight reduction or the reduction of total adipose tissue mass alone. It can be concluded that the reduction of visceral adipose tissue mass is an essential part of further research focused on improvement in primary prevention of cardiovascular disease.Adipositas stellt aufgrund seiner zahlreichen assoziierten Stoffwechselstörungen (Insulinresistenz, Hyperglykämie, Hypertriglyceridämie, Dyslipoproteinämie, arterielle Hypertonie, Hyperurikämie u.a.) einen anerkannten Risikofaktor für kardiovaskuläre Erkrankungen dar. Allerdings hat sich in den letzten Jahren zunehmend gezeigt, dass das Körpergewicht allein, selbst bereinigt um die Körpergröße (über den sog. Body-Mass-Index – BMI), keine zuverlässige prädiktive Aussagekraft für die vaskuläre Herz-Kreislauf-Morbidität aufweist. Vor diesem Hintergrund haben sich Körperfettverteilung und insbesondere die Bedeutung des viszeralen Fettkompartiments als potentiell bedeutsam herausgestellt. Bisherige Untersuchungen postulieren ein erhöhtes kardiovaskuläres Risiko bei bauchbetonter Adipositas gegenüber einer Vermehrung des subkutanen Fettgewebes evtl. über eine vermehrte Expression und Sekretion proinflammatorischer Zytokine im viszeralen Fettgewebe. Ausgehend von dieser Hypothese führten wir eine prospektive Interventionsstudie durch, um zu belegen, dass sich im Rahmen einer Reduktion der viszeralen Fettmenge eine Verbesserung der Endothelfunktion - als früher Atherosklerosemarker - sowie eine Verbesserung des systemischen Inflammationstatus ergibt. Untersucht wurden 189 Personen (45,5 ± 0,8 Jahre) Personen mit Übergewicht, pathologischer Glukosetoleranz oder einer positiven Familienanamnese auf Typ-2 Diabetes. Die periphere Gefäßfunktion wurde mit hochauflösendem Ultraschall (13 MHz) als flussmediierte Dilatation (FMD) der Arteria brachialis bestimmt. Als weitere Endothelmarker wurden die Adhäsionsmoleküle sE-Selectin, sV-CAM und sI-CAM mittels ELISA-Technik bestimmt. Die Körperfettverteilung wurde mittels Ganzkörper-Magnetresonanztomographie erfasst. Wir erreichten im Rahmen der Studienintervention eine mittlere Abnahme des Körpergewichts um 3%, eine Abnahme der Ganzkörperfettmenge (TAT) um 7.6%, sowie eine Reduktion des VAT um 4.3%. Erhärtend für die Ausgangshypothese war keine Korrelation zwischen der verbesserten peripheren Gefäßfunktion und der Abnahme des Körpergewichtes per se (p=0.35) oder der Gesamtkörperfettmasse (p=0.21) festzustellen, jedoch zwischen der FMD und der viszeralen Fettgewebsmasse (r=-0.189, p=0.009). Davon ausgehend wurde eine Responder- und eine Nicht-Responder-Gruppe anhand des Medians der erzielten Abnahme des viszeralen Fettgewebes gebildet. Dabei zeigte sich, dass sich die FMD nur bei den Respondern verbesserte (von 6.2 ± 0.4% auf 8.0 ± 0.5%; p=0.0005) nicht jedoch bei den Nicht-Respondern (p=0.15). Auch sE-Selectin sank signifikant ausschließlich bei den Respondern (von 54 ± 4 ng/ml bis 47 ± 3 ng/ml; p=0.03). Unsere prospektiven Studiendaten belegen, dass durch eine Lebensstilintervention bei Personen mit Übergewicht bzw. erhöhtem Risiko eines Diabetes mellitus Typ 2 nicht der Körpergewichtsverlust an sich bzw. die Abnahme des Gesamtkörperfettes, sondern primär die Reduktion der viszeralen („Bauch“)-Fettmenge mit einer verbesserten Endothel- / Gefäßfunktion korreliert. Daraus lässt sich schlussfolgern, dass Maßnahmen zur Reduktion des viszeralen Fettgewebes mit im Fokus weiterer Forschungsaktivitäten zur verbesserten Primärprävention kardiovaskulärer Erkrankungen stehen sollten

Peroxisome proliferator-activated receptor gamma (PPARG) modulates free fatty acid receptor 1 (FFAR1) dependent insulin secretion in humans

Genetic variation in FFAR1 modulates insulin secretion dependent on non-esterified fatty acid (NEFA) concentrations. We previously demonstrated lower insulin secretion in minor allele carriers of PPARG Pro12Ala in high-NEFA environment, but the mode of action could not been revealed. We tested if this effect is mediated by FFAR1 in humans. Subjects with increased risk of diabetes who underwent oral glucose tolerance tests were genotyped for 7 tagging SNPs in FFAR1 and PPARG Pro12Ala. The FFAR1 SNPs rs12462800 and rs10422744 demonstrated interactions with PPARG on insulin secretion. FFAR1 rs12462800 (p = 0.0006) and rs10422744 (p = 0.001) were associated with reduced insulin secretion in participants concomitantly carrying the PPARG minor allele and having high fasting FFA. These results suggest that the minor allele of the PPARG SNP exposes its carriers to modulatory effects of FFAR1 on insulin secretion. This subphenotype may define altered responsiveness to FFAR1-agonists, and should be investigated in further studies. (C) 2014 The Authors. Published by Elsevier GmbH

Generation of a human induced pluripotent stem cell (iPSC) line from a patient with family history of diabetes carrying a C18R mutation in the PDX1 gene

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a C18R heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 C18R iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms

Generation of a human induced pluripotent stem cell (iPSC) line from a patient carrying a P33T mutation in the PDX1 gene

Homozygous loss-of-function mutations in the gene coding for the homeobox transcription factor PDX1 leads to pancreatic agenesis, whereas certain heterozygous point mutations are associated with Maturity-Onset Diabetes of the Young 4 (MODY4) and Type 2 Diabetes Mellitus (T2DM). To understand the pathomechanism of MODY4 and T2DM, we have generated iPSCs from a woman with a P33T heterozygous mutation in the transactivation domain of PDX1. The resulting PDX1 P33T iPSCs generated by episomal reprogramming are integration-free, have a normal karyotype and are pluripotent in vitro and in vivo. Taken together, this iPSC line will be useful to study diabetes pathomechanisms

Associations of <i>PIK3CG</i> SNPs rs4288294 (A) and rs116697954 (B) with HDL2- and HDL3-cholesterol concentrations.

<p>The HDL-cholesterol subfractions HDL2 and HDL3 were obtained by ultracentrifugation. Adjustment of HDL2- and HDL3-cholesterol concentrations (N = 34) was achieved by multiple linear regression modelling with gender, age, and BMI as confounding variables. The SNPs were tested in the dominant inheritance model. HDL–high-density lipoprotein; SNP–single nucleotide polymorphism.</p