Search

Abstract Background Approximately 1000 protein encoding genes common for vertebrates are still unannotated in avian genomes. Are these genes evolutionary lost or are they not yet found for technical reasons? Using genome landscapes as a tool to visualize large-scale regional effects of genome evolution, we reexamined this question. Results On basis of gene annotation in non-avian vertebrate genomes, we established a list of 15,135 common vertebrate genes. Of these, 1026 were not found in any of eight examined bird genomes. Visualizing regional genome effects by our sliding window approach showed that the majority of these "missing" genes can be clustered to 14 regions of the human reference genome. In these clusters, an additional 1517 genes (often gene fragments) were underrepresented in bird genomes. The clusters of “missing” genes coincided with regions of very high GC content, particularly in avian genomes, making them “hidden” because of incomplete sequencing. Moreover, proteins encoded by genes in these sequencing refractory regions showed signs of accelerated protein evolution. As a proof of principle for this idea we experimentally characterized the mRNA and protein products of four "hidden" bird genes that are crucial for energy homeostasis in skeletal muscle: ALDOA, ENO3, PYGM and SLC2A4. Conclusions A least part of the “missing” genes in bird genomes can be attributed to an artifact caused by the difficulty to sequence regions with extreme GC% (“hidden” genes). Biologically, these “hidden” genes are of interest as they encode proteins that evolve more rapidly than the genome wide average. Finally we show that four of these “hidden” genes encode key proteins for energy metabolism in flight muscle.

Abstract Background Rapid accumulation of vertebrate genome sequences render comparative genomics a powerful approach to study macro-evolutionary events. The assessment of phylogenic relationships between species routinely depends on the analysis of sequence homology at the nucleotide or protein level. Results We analyzed mRNA GC content, codon usage and divergence of orthologous proteins in 55 vertebrate genomes. Data were visualized in genome-wide landscapes using a sliding window approach. Landscapes of GC content reveal both evolutionary conservation of clustered genes, and lineage-specific changes, so that it was possible to construct a phylogenetic tree that closely matched the classic “tree of life”. Landscapes of GC content also strongly correlated to landscapes of amino acid usage: positive correlation with glycine, alanine, arginine and proline and negative correlation with phenylalanine, tyrosine, methionine, isoleucine, asparagine and lysine. Peaks of GC content correlated strongly with increased protein divergence. Conclusions Landscapes of base- and amino acid composition of the coding genome opens a new approach in comparative genomics, allowing identification of discrete regions in which protein evolution accelerated over deep evolutionary time. Insight in the evolution of genome structure may spur novel studies assessing the evolutionary benefit of genes in particular genomic regions.

The family of proglucagon peptides Includes glucagon and glucagon-like peptide 1 (GLP-1), two unique peptides derived from the same prohormone. Despite numerous similarities between the peptides, these have long been viewed as having opposing actions on metabolism. GLP-1 is described as a postprandial hormone that stimulates anabolic actions via insulin, while glucagon is viewed as a fasting hormone that drives catabolic actions to maintain euglycemia. Here, we revisit a classic article in Diabetes that first established that glucagon and GLP-1 have more in common than previously appreciated, including actions at the same receptor. Furthermore, we discuss how the impact of this observation has guided research decades later that has reshaped the view of how proglucagon hormones regulate metabolism., (© 2023 by the American Diabetes Association.)

During mouse pregnancy placental lactogens stimulate prolactin receptors on pancreatic islet beta cells to induce expression of the tryptophan hydroxylase Tph1 , resulting in the synthesis and secretion of serotonin. Presently, the functional relevance of this phenomenon is unclear. One hypothesis is that serotonin-induced activation of 5-HT 2B receptors on beta cells stimulates beta cell proliferation during pregnancy. We tested this hypothesis via three different mouse models: (i) total Tph1 KO mice, (ii) 129P2/OlaHsd mice, which are incompetent to upregulate islet Tph1 during pregnancy, whereas Tph1 is normally expressed in the intestine, mammary glands, and placenta, and (iii) Htr2b -deficient mice. We observed normal pregnancy-induced levels of beta cell proliferation in total Tph1 KO mice, 129P2/OlaHsd mice, and in Htr2b -/- mice. The three studied mouse models indicate that islet serotonin production and its signaling via 5-HT 2B receptors are not required for the wave of beta cell proliferation that occurs during normal mouse pregnancy.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKA iEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

Background: Bedside lung ultrasound (LUS) is an affordable diagnostic tool that could contribute to identifying COVID-19 pneumonia. Different LUS protocols are currently used at the emergency department (ED) and there is a need to know their diagnostic accuracy., Design: A multicentre, prospective, observational study, to compare the diagnostic accuracy of three commonly used LUS protocols in identifying COVID-19 pneumonia at the ED., Setting/patients: Adult patients with suspected COVID-19 at the ED, in whom we prospectively performed 12-zone LUS and SARS-CoV-2 reverse transcription PCR., Measurements: We assessed diagnostic accuracy for three different ultrasound protocols using both PCR and final diagnosis as a reference standard., Results: Between 19 March 2020 and 4 May 2020, 202 patients were included. Sensitivity, specificity and negative predictive value compared with PCR for 12-zone LUS were 91.4% (95% CI 84.4 to 96.0), 83.5% (95% CI 74.6 to 90.3) and 90.0% (95% CI 82.7 to 94.4). For 8-zone and 6-zone protocols, these results were 79.7 (95% CI 69.9 to 87.6), 69.0% (95% CI 59.6 to 77.4) and 81.3% (95% CI 73.8 to 87.0) versus 89.9% (95% CI 81.7 to 95.3), 57.5% (95% CI 47.9 to 66.8) and 87.8% (95% CI 79.2 to 93.2). Negative likelihood ratios for 12, 8 and 6 zones were 0.1, 0.3 and 0.2, respectively. Compared with the final diagnosis specificity increased to 83.5% (95% CI 74.6 to 90.3), 78.4% (95% CI 68.8 to 86.1) and 65.0% (95% CI 54.6 to 74.4), respectively, while the negative likelihood ratios were 0.1, 0.2 and 0.16., Conclusion: Identifying COVID-19 pneumonia at the ED can be aided by bedside LUS. The more efficient 6-zone protocol is an excellent screening tool, while the 12-zone protocol is more specific and gives a general impression on lung involvement., Trial Registration Number: NL8497., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Different types of proteins diverge at vastly different rates. Moreover, the same type of protein has been observed to evolve with different rates in different phylogenetic lineages. In the present study we measured the rates of protein evolution in Eutheria (placental mammals) and Metatheria (marsupials) on a genome-wide basis and we propose that the gene position in the genome landscape has an important influence on the rate of protein divergence., Results: We analyzed a protein-encoding gene set (n = 15,727) common to 16 mammals (12 Eutheria and 4 Metatheria). Using sliding windows that averaged regional effects of protein divergence we constructed landscapes in which strong and lineage-specific regional effects were seen on the molecular clock rate of protein divergence. Within each lineage, the relatively high rates were preferentially found in subtelomeric chromosomal regions. Such regions were observed to contain important and well-studied loci for fetal growth, uterine function and the generation of diversity in the adaptive repertoire of immunoglobulins., Conclusions: A genome landscape approach visualizes lineage-specific regional differences between Eutherian and Metatherian rates of protein evolution. This phenomenon of chromosomal position is a new element that explains at least part of the lineage-specific effects and differences between proteins on the molecular clock rates., (© 2021. The Author(s).)

FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, but its specific role in pancreatic β-cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, whereas PCs that potentially could provide redundancy are expressed at considerably lower levels. β-cell-specific Furin knockout (β Fur KO) mice are glucose intolerant as a result of smaller islets with lower insulin content and abnormal dense-core secretory granule morphology. mRNA expression analysis and differential proteomics on β Fur KO islets revealed activation of activating transcription factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). β Fur KO cells show impaired cleavage or shedding of vacuolar-type ATPase (V-ATPase) subunits Ac45 and prorenin receptor, respectively, and impaired lysosomal acidification. Blocking V-ATPase pharmacologically in β-cells increased mTORC1 activity, suggesting involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β-cells lacking Furin , causing β-cell dysfunction., (© 2020 by the American Diabetes Association.)

Background: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype., Methods: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status., Results: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFN γ -induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences., Conclusions: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology., (Copyright © 2020 by the American Society of Nephrology.)

Vitamin D and all its metabolites are bound to a specific vitamin D binding protein, DBP. This protein was originally first discovered by its worldwide polymorphism and called Group-specific Component (GC). We now know that DBP and GC are the same protein and appeared early in the evolution of vertebrates. DBP is genetically the oldest member of the albuminoid family (including albumin, α-fetoprotein and afamin, all involved in transport of fatty acids or hormones). DBP has a single binding site for all vitamin D metabolites and has a high affinity for 25OHD and 1,25(OH)2D, thereby creating a large pool of circulating 25OHD, which prevents rapid vitamin D deficiency. DBP of higher vertebrates (not amphibians or reptiles) binds with very high affinity actin, thereby preventing the formation of polymeric actin fibrils in the circulation after tissue damage. Megalin is a cargo receptor and is together with cubilin needed to reabsorb DBP or the DBP-25OHD complex, thereby preventing the urinary loss of these proteins and 25OHD. The total concentrations of 25OHD and 1,25(OH)2D in DBP null mice or humans are extremely low but calcium and bone homeostasis remain normal. This is the strongest argument for claiming that the "free hormone hypothesis" also applies to the vitamin D hormone, 1,25(OH)2D. DBP also transports fatty acids, and can play a role in the immune system. DBP is genetically very polymorphic with three frequent alleles (DBP/GC 1f, 1s, and 2) but in total more than 120 different variants but its health consequences, if any, are not understood. A standardization of DBP assays is essential to further explore the role of DBP in physiology and diseases., (Copyright © 2020 Bouillon, Schuit, Antonio and Rastinejad.)

Objective: Cytochrome c oxidase (COX) deficiency is a major mitochondrial respiratory chain defect that has vast genetic and phenotypic heterogeneity. This study aims to identify novel causative genes of COX deficiency with only striated muscle-specific symptoms., Methods: Whole exome sequencing was performed in 2 unrelated individuals who were diagnosed with congenital myopathy and presented COX deficiency in muscle pathology. We assessed the COX6A2 variants using measurements of enzymatic activities and assembly of mitochondrial respiratory chain complexes in the samples from the patients and knockout mice., Results: Both patients presented muscle weakness and hypotonia in 4 limbs along with facial muscle weakness. One patient had cardiomyopathy. Neither patient exhibited involvement from other organs. Whole exome sequencing identified biallelic missense variants in COX6A2, which is expressed only in the skeletal muscle and heart. The variants detected were homozygous c.117C > A (p.Ser39Arg) and compound heterozygous c.117C > A (p.Ser39Arg) and c.127T > C (p.Cys43Arg). We found specific reductions in complex IV activities in the skeletal muscle of both individuals. Assembly of complex IV and its supercomplex formation were impaired in the muscle., Interpretation: This study indicates that biallelic variants in COX6A2 cause a striated muscle-specific form of COX deficiency. ANN NEUROL 2019;86:193-202., (© 2019 American Neurological Association.)

Background: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available., Methods: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661., Findings: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection., Interpretation: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection., Funding: The Seventh Framework Programme (FP7) of the European Commission., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Early treatment of Crohn's disease [CD] is required in order to optimize patient outcomes. To this end, we need to gain a better understanding of the molecular changes at the onset of CD., Methods: As a model for the earliest mucosal CD lesions, we study post-operative recurrent CD [Rutgeerts score ≥ i2b]. We are the first to analyse gene and microRNA [miRNA] expression profiles in ileal biopsies from these patients, and compare them with those of newly diagnosed [≤18 months] and late-stage [>10 years after diagnosis] CD patients., Results: Except for one gene [WNT5A], there are no differential genes in CD patients without post-operative recurrence [i0], showing that previous disease did not influence gene expression in the neoterminal ileum, and that this model can be used to study early mucosal CD lesions. Gene expression and co-expression network dysregulation is more pronounced in newly diagnosed and late-stage CD than in post-operative recurrent CD, with most important modules associated with [a]granulocyte adhesion/diapedesis, and cholesterol biosynthesis. In contrast, we found a role for snoRNAs/miRNAs in recurrent CD, highlighting the potential importance of regulatory RNAs in early disease stages. Immunohistochemistry confirmed the expression of key dysregulated genes in damaged/regenerating epithelium and immune cells in recurrent CD., Conclusions: Aside from regulatory RNAs, there are no clear gene signatures separating post-operative recurrent, newly diagnosed, and late-stage CD. The relative contribution of dysregulated genes and networks differs, and suggests that surgery may reset the disease at the mucosal site, and therefore post-operative recurrent CD might be a good model a good model to study to study early mucosal CD lesions., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Peroxisomes play a crucial role in lipid and reactive oxygen species metabolism, but their importance for pancreatic β-cell functioning is presently unknown. To examine the contribution of peroxisomal metabolism to β-cell homeostasis in mice, we inactivated PEX5, the import receptor for peroxisomal matrix proteins, in an inducible and β-cell restricted manner (Rip-Pex5 -/- mice)., Methods: After tamoxifen-induced recombination of the Pex5 gene at the age of 6 weeks, mice were fed either normal chow or a high-fat diet for 12 weeks and were subsequently phenotyped., Results: Increased levels of very long chain fatty acids and reduced levels of plasmalogens in islets confirmed impairment of peroxisomal fatty acid oxidation and ether lipid synthesis, respectively. The Rip-Pex5 -/- mice fed on either diet exhibited glucose intolerance associated with impaired insulin secretion. Ultrastructural and biochemical analysis revealed a decrease in the density of mature insulin granules and total pancreatic insulin content, which was further accompanied by mitochondrial disruptions, reduced complex I activity and massive vacuole overload in β-cells. RNAseq analysis suggested that cell death pathways were affected in islets from HFD-fed Rip-Pex5 -/- mice. Consistent with this change we observed increased β-cell apoptosis in islets and a decrease in β-cell mass., Conclusions: Our data indicate that normal peroxisome metabolism in β-cells is crucial to preserve their structure and function., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Malaria reduces host fitness and survival by pathogen-mediated damage and inflammation. Disease tolerance mechanisms counter these negative effects without decreasing pathogen load. Here, we demonstrate that in four different mouse models of malaria, adrenal hormones confer disease tolerance and protect against early death, independently of parasitemia. Surprisingly, adrenalectomy differentially affects malaria-induced inflammation by increasing circulating cytokines and inflammation in the brain but not in the liver or lung. Furthermore, without affecting the transcription of hepatic gluconeogenic enzymes, adrenalectomy causes exhaustion of hepatic glycogen and insulin-independent lethal hypoglycemia upon infection. This hypoglycemia is not prevented by glucose administration or TNF-α neutralization. In contrast, treatment with a synthetic glucocorticoid (dexamethasone) prevents the hypoglycemia, lowers cerebral cytokine expression and increases survival rates. Overall, we conclude that in malaria, adrenal hormones do not protect against lung and liver inflammation. Instead, they prevent excessive systemic and brain inflammation and severe hypoglycemia, thereby contributing to tolerance.

The minigene encoding human growth hormone (hGH) has been incorporated into over 300 transgenic mouse lines to improve transgene expression. However, unexpected and functional hGH expression can drastically alter physiology. We list here the mouse lines in which ectopic hGH has been confirmed, and we provide a wiki for lines awaiting analysis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Lymphocyte recruitment to the inflamed gut is increased in UC. Inhibition of this cell trafficking by vedolizumab (VDZ) was successful in inducing and maintaining remission and in induction of endoscopic mucosal healing. There are no data on histological healing with VDZ. We studied histological changes following VDZ therapy and compared gene expression in patients with UC before and after therapy., Design: Forty-one patients with UC from GEMINI I and LTS were studied before and at three time points (weeks 6/12/52) following VDZ therapy. Colonic biopsies were scored using the Geboes index and correlated with Mayo endoscopic subscore. Gene expression was analysed using Affymetrix gene arrays., Results: Fifty-five per cent of patients achieving endoscopic healing (= Mayo endoscopic subscore 0-1) with VDZ at the studied time points also had histological healing (= Geboes grade 0-1). In most healers, some residual histological changes (eg, disturbed architecture and increased mononuclear cell infiltrate) were still observed, although this was less at week 52. VDZ restored expression of many inflammatory genes in patients with endoscopic healing only at week 52 and not before. In VDZ healers, the expression of many genes remained dysregulated at weeks 6/12/52 compared with controls., Conclusions: VDZ induces histological healing in >50% of patients with endoscopic healing, with maximal effect at week 52. VDZ also restored, although incompletely, the colonic expression of many immune-related genes in patients with UC achieving endoscopic healing at week 52. However, persistent histological and gene dysregulations did remain even in healers, suggesting that maintenance therapy will be necessary to control the intestinal inflammation., Trial Registration Numbers: NCT00783718 and NCT00790933; post-results., Competing Interests: Competing interests: GDH—consulting fees: Genentech, Centocor, Galapagos. MF—grant support: Takeda; lecture fees: Abbvie, Boehringer-Ingelheim, Chiesi, Falk, Ferring, Janssen, Mitsubishi Tanabe, MSD, Takeda, Tillotts, Zeria; consulting fees: Abbvie, Boehringer-Ingelheim, Ferring, Janssen, MSD. GVA—grant support: Abbvie, MSD; lecture fees: Abbvie, Ferring, MSD, Janssen, Takeda; consulting fees: Abbvie, MSD, Takeda. PR—grant support: Abbvie, Centocor, Merck, UCB; lecture fees: Centocor, Merck, Abbvie; consulting fees: Centocor, Merck, UCB, Abbvie, Millenium/Takeda, Genentech/Hoffman LaRoche, Merck/Serono, Bristol Myers Squibb, Robarts, Tillotts, Pfizer, Falk Pharma. SV—grant support from Abbvie, MSD; lecture fees: Abbvie, MSD, Takeda, Ferring, Falk Pharma, Hospira, Tillotts; grant support from Abbvie, MSD; consulting fees: Abbvie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, Janssen., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: Intestinal barrier defects are common in patients with inflammatory bowel disease (IBD). To identify which components could underlie these changes, we performed an in-depth analysis of epithelial barrier genes in IBD., Methods: A set of 128 intestinal barrier genes was selected. Polygenic risk scores were generated based on selected barrier gene variants that were associated with Crohn's disease (CD) or ulcerative colitis (UC) in our study. Gene expression was analyzed using microarray and quantitative reverse transcription polymerase chain reaction. Influence of barrier gene variants on expression was studied by cis-expression quantitative trait loci mapping and comparing patients with low- and high-risk scores., Results: Barrier risk scores were significantly higher in patients with IBD than controls. At single-gene level, the associated barrier single-nucleotide polymorphisms were most significantly enriched in PTGER4 for CD and HNF4A for UC. As a group, the regulating proteins were most enriched for CD and UC. Expression analysis showed that many epithelial barrier genes were significantly dysregulated in active CD and UC, with overrepresentation of mucus layer genes. In uninflamed CD ileum and IBD colon, most barrier gene levels restored to normal, except for MUC1 and MUC4 that remained persistently increased compared with controls. Expression levels did not depend on cis-regulatory variants nor combined genetic risk., Conclusions: We found genetic and transcriptomic dysregulations of key epithelial barrier genes and components in IBD. Of these, we believe that mucus genes, in particular MUC1 and MUC4, play an essential role in the pathogenesis of IBD and could represent interesting targets for treatment.

The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes ("disallowed genes") that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes-which are also deeply repressed in FACS-purified beta cells-remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of Dnmt3a in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.

Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca 2+ -activated cation channel expressed in type II taste receptor cells and pancreatic β-cells. Here we show that stevioside, rebaudioside A and their aglycon steviol potentiate the activity of TRPM5. We find that SGs potentiate perception of bitter, sweet and umami taste, and enhance glucose-induced insulin secretion in a Trpm5-dependent manner. Daily consumption of stevioside prevents development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5 -/- mice. These results elucidate a molecular mechanism of action of SGs and identify TRPM5 as a potential target to prevent and treat type 2 diabetes.

Aims/hypothesis: Type 1 diabetes is an endocrine disease where a long preclinical phase, characterised by immune cell infiltration in the islets of Langerhans, precedes elevated blood glucose levels and disease onset. Although several studies have investigated the role of the immune system in this process of insulitis, the importance of the beta cells themselves in the initiation of type 1 diabetes is less well understood. The aim of this study was to investigate intrinsic differences present in the islets from diabetes-prone NOD mice before the onset of insulitis., Methods: The islet transcriptome and proteome of 2-3-week-old mice was investigated by microarray and 2-dimensional difference gel electrophoresis (2D-DIGE), respectively. Subsequent analyses using sophisticated pathway analysis and ranking of differentially expressed genes and proteins based on their relevance in type 1 diabetes were performed., Results: In the preinsulitic period, alterations in general pathways related to metabolism and cell communication were already present. Additionally, our analyses pointed to an important role for post-translational modifications (PTMs), especially citrullination by PAD2 and protein misfolding due to low expression levels of protein disulphide isomerases (PDIA3, 4 and 6), as causative mechanisms that induce beta cell stress and potential auto-antigen generation., Conclusions/interpretation: We conclude that the pancreatic islets, irrespective of immune differences, may contribute to the initiation of the autoimmune process., Data Availability: All microarray data are available in the ArrayExpress database ( www.ebi.ac.uk/arrayexpress ) under accession number E-MTAB-5264.

Background and Aims: Increased levels of tissue inhibitor of metalloproteinase-1 [TIMP-1] have been detected in both inflammatory and fibrotic lesions in Crohn's disease. In a murine model of chronic inflammation, fibrosis was associated with an increase in TIMP-1 and inhibition of matrix metalloproteinase [MMP]-mediated degradation. We investigated the effect of TIMP-1 deficiency in acute and chronic murine models of colitis., Methods: Colitis was induced via oral administration of dextran sodium sulphate [DSS] to B6.129S4-Timp1 tm1Pds /J knock-out [KO] and C57BL/6J wild-type [WT] mice. Levels of inflammation and fibrosis were assessed and gelatin zymographies and gene expression microarrays were performed., Results: Compared with WT mice, TIMP-1 KO mice had higher inflammatory parameters after acute DSS administration and developed less fibrosis after chronic DSS administration. MMP-2 levels were increased in WT versus TIMP-1 KO mice with acute colitis, whereas a trend for higher proMMP-9 levels was observed in WT versus TIMP-1 KO mice with chronic colitis. In control conditions, several immune-related genes [e.g Ido1, Cldn8] were differentially expressed between young TIMP-1 KO and WT mice, but to a lesser extent between older TIMP-1 KO and WT mice. In response to DSS, the gene expression pattern was significantly different between young TIMP-1 KO and WT mice, whereas it was similar in older TIMP-1 KO and WT mice., Conclusions: TIMP-1 deficiency leads to differential expression of immune-related genes and to attenuated development of fibrosis. Unravelling the role of TIMP-1 in intestinal remodelling is necessary to develop more effective and more targeted therapeutic strategies for intestinal fibrosis., (Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Vitamin D deficiency is associated with beta-cell dysfunction and a higher risk of diabetes, but mice and humans with an absence of the vitamin D receptor (VDR) display normal glucose tolerance. Here, we investigated the direct effects of absence of VDR or absence of ligand activation of VDR on beta-cell function. For this purpose, we generated mice, with a mutation in the AF2 domain of Vdr (VDRΔAF2), preventing ligand-driven transcriptional activation of vitamin D responsive genes. VDRΔAF2 mice were compared to Vdr full knockout (VDR -/- ) and wild type (WT) mice. In order to avoid hypocalcemia, which has a direct effect on beta-cell function, mice were fed a high calcium, high lactose diet yielding comparable serum calcium in all mice. While VDR -/- mice developed extensive alopecia by the age of 24 weeks, the fur of VDRΔAF2 remained normal. All VDRΔAF2 mice weighed significantly less than WT, while male but not female VDR -/- mice had a lower body weight than WT mice. Dual-energy X-ray absorptiometry showed that both VDRΔAF2 (17.2% (females) and 16.6% (males)) and VDR -/- (15.7% and 14.8%) mice have a lower percentage of body fat (vs 19.3% and 22.2% in WT). Serum 25(OH)D 3 concentrations were lower for both VDRΔAF2 (-4.55 fold, P<0.001) and VDR -/- (-3.7 fold, P<0.001) as compared to 12 week old WT mice, while serum 1,25(OH) 2 D 3 was increased for both strains 94.5 fold (P<0.01) and 92.8 fold (P<0.001) for VDRΔAF2 and VDR -/- vs WT, respectively). In vivo glucose tolerance tests performed at 12 and 24 weeks of age, as well as ex vivo glucose stimulated insulin secretion on freshly isolated islets, revealed no major differences between the three strains. Microarray analysis on freshly isolated islets showed only 1 differentially expressed gene, phosphodiesterase 10a (Pde10a), which was 2.16 and 1.75 fold up-regulated in VDRΔAF2 and VDR -/- islets as compared to WT islets, respectively (P≤0.001). We conclude that in the presence of normocalcemia, absence of VDR or its ligand-activated transcription of genes has no direct regulatory effect on murine glucose homeostasis or gene expression in islets of Langerhans., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glucose homeostasis greatly depends on the match between fluctuating insulin demands and adjusted rates of insulin secretion, which is the function of pancreatic beta cells. Emerging evidence suggests that when neonatal beta cells mature, they acquire two faces of differentiated function: an expected "visible face" that depends on specific beta cell proteins needed for regulated insulin release, but also a "hidden face" that represses ubiquitous proteins to prevent inappropriate beta cell function such as elevated basal hormone secretion or insulin release triggered by exercise. This review highlights this novel concept, and we first propose that hidden faces may also be relevant for other specialized tissue functions, such as ketogenesis in the liver. Next, we discuss three scenarios in which aberrant gene expression causes abnormal glucose-induced insulin release and the epigenetic regulation of the hidden face in beta cells. We conclude with perspectives for new research, including beta cell replacement to cure diabetes.

Pregnancy is a key mammalian reproductive event in which growth and differentiation of the fetus imposes extra metabolic and hormonal demands on the mother. Its successful outcome depends on major changes in maternal blood circulation, metabolism and endocrine function. One example is the endocrine pancreas, where beta cells undergo a number of changes in pregnancy that result in enhanced functional beta cell mass in order to compensate for the rising metabolic needs for maternal insulin. During the last 5 years, a series of studies have increased our understanding of the molecular events involved in this functional adaptation. In the mouse, a prominent functional change during pregnancy is the capacity of some beta cells to produce serotonin. In this review we will discuss the mechanism and potential effects of pregnancy-related serotonin production in beta cells, considering functional consequences at the local intra-islet and systemic level.

Background: The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype., Methods: Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines., Results: Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R., Conclusions: This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

Aims/hypothesis: It is generally accepted that structural and functional quantitative imaging of individual islets would be beneficial to elucidate the pathogenesis of type 1 diabetes. We here introduce functional optical coherence imaging (FOCI) for fast, label-free monitoring of beta cell destruction and associated alterations of islet vascularisation., Methods: NOD mouse and human islets transplanted into the anterior chamber of the eye (ACE) were imaged with FOCI, in which the optical contrast of FOCI is based on intrinsic variations of the index of refraction resulting in a faster tomographic acquisition. In addition, the phase sensitivity allows simultaneous label-free acquisition of vascularisation., Results: We demonstrate that FOCI allows longitudinal quantification of progressive autoimmune insulitis, including the three-dimensional quantification of beta cell volume, inflammation and vascularisation. The substantially increased backscattering of islets is dominated by the insulin-zinc nanocrystals in the beta cell granules. This translates into a high specificity for the functional beta cell volume of islets. Applying FOCI to a spontaneous mouse model of type 1 diabetes, we quantify the modifications of the pancreatic microvasculature accompanying the progression of diabetes and reveal a strong correlation between increasing insulitis and density of the vascular network of the islet., Conclusions/interpretation: FOCI provides a novel imaging technique for investigating functional and structural diabetes-induced alterations of the islets. The label-free detection of beta cell volume and infiltration together with vascularisation offers a unique extension to study ACE-transplanted human islets. These results are contributing to a deeper understanding of human islet transplant rejection and label-free in vivo monitoring of drug efficacy.

Background: Inflammatory bowel disease (IBD) is characterized by a chronic inflammation of the gut, partly driven by defects in the innate immune system. Considering the central role of inflammasome signaling in innate immunity, we studied inflammasome components in IBD mucosa., Methods: Expression of genes encoding inflammasome sensor subunits was investigated in colonic mucosal biopsies from 2 cohorts of patients with IBD and controls., Results: A significant upregulation (>2-fold change in expression, false discovery rate <0.05) of the PYHIN inflammasomes AIM2 and IFI16 in active IBD versus controls was found. Also IFI16 was significantly increased in inactive IBD versus controls. Moreover, responders to anti-tumor necrosis factor therapy showed decreased expression of these inflammasomes although IFI16 remained significantly increased in responders showing endoscopic healing versus controls. AIM2 was mainly expressed in epithelial cells, whereas IFI16 was expressed in both lymphocytes and epithelial cells. Functional activation of predominant AIM2/IFI16-mediated inflammasomes in active IBD colon was shown by the presence of the downstream effectors CASP1 and HMGB-1 in inflamed mucosa., Conclusions: Our results highlight the importance of PYHIN inflammasome signaling in IBD and also link anti-tumor necrosis factor responsiveness to inflammasome signaling. Together, this points to the potential value of the inflammasome pathway as a new therapeutic target for IBD treatment.

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.

With the development of agriculture and food processing techniques, humanity has recently challenged the rules of a billion-year-old experiment called evolution. In this experiment the availability of food in a particular niche has been one of the major driving forces to shape particular species. Comparative genomics is a new research discipline that investigates two or more genomes from different species in order to find specific genetic adaptations that explain a 'workable match' between genetic make-up and environmental constraints such as nutrition. Three recent examples in the literature illustrate how selection of particular genes can contribute to species-specific adaptations that allow them to recognise, secure and digest particular types of food and metabolise its ingredients. There is growing consensus that the recent changes in human diet and physical activity play an active role in the rapid growth of the prevalence of obesity and diabetes. The working hypothesis of the present article is that in the future a more advanced level of comparative genomics of the many natural workable matches of natural species will lead to a much better understanding of the dynamics and regulation of integrated metabolism. It is anticipated that this deeper understanding will lead to novel insights into the mechanism of human diabetes and new strategies for diabetes prevention and treatment. This is one of a series of commentaries under the banner '50 years forward', giving personal opinions on future perspectives in diabetes, to celebrate the 50th anniversary of Diabetologia (1965-2015).

Pancreatic β cells support glucose homeostasis with great precision by matching insulin release to the metabolic needs of the moment. Previous gene-expression analysis indicates that adult β cells not only produce cell-specific proteins, but also repress a small set of housekeeping genes - such as those encoding lactate dehydrogenase A (LDHA), solute transporter MCT1, and hexokinase 1 (HK1) - that would otherwise interfere with normal β cell function. In this issue of the JCI, Dhawan et al. elucidate a molecular mechanism involved in β cell-specific repression of Ldha and Hk1 that is mediated by induction of the de novo DNA methyltransferase DNMT3A during the first weeks after birth. Failure to induce DNMT3A-dependent methylation disrupts normal glucose-induced insulin release in adult life. The results of this study reinforce the idea that the phenotype of adult β cells has two faces and that failure to achieve selective gene repression undermines β cell support of normal glucose homeostasis.

Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the 'islet pregnancy gene signature', which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH) 2 D 3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH) 2 D 3 , hypoxia did not change the tolerogenic function of 1,25(OH) 2 D 3 -treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH) 2 D 3 -conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH) 2 D 3 , because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In ulcerative colitis (UC) the butyrate metabolism is impaired, leading to energy-deficiency in the colonic cells. The effect of inflammation on the butyrate metabolism was investigated. HT-29 cells were incubated with pro-inflammatory cytokines (TNF-α and/or IFN-γ) for 1 and 24 h. Cells were additionally stimulated with butyrate to investigate its anti-inflammatory potential. Butyrate uptake and oxidation were measured using (14)C-labeled butyrate. Gene expression of the butyrate metabolism enzymes, interleukin 8 (IL-8; inflammatory marker) and villin-1 (VIL-1; epithelial cell damage marker) was measured via quantitative RT-PCR. Significantly increased IL-8 expression and decreased VIL-1 expression after 24 h incubation with TNF-α and/or IFN-γ confirmed the presence of inflammation. These conditions induced a decrease of both butyrate uptake and oxidation, whereas the gene expression was not reduced. Simultaneous incubation with butyrate counteracted the reduced butyrate oxidation. In contrast, 1 h incubation with TNF-α induced a significant increased IL-8 expression and decreased butyrate uptake. Incubation with TNF-α and/or IFN-γ for 1 h did not induce cell damage nor influence butyrate oxidation. The inflammation-induced downregulation of the butyrate metabolism was not caused by a reduced gene expression, but appeared consequential to a decreased butyrate uptake. Increasing the luminal butyrate levels might have therapeutic potential in UC., (© 2014 Wiley Periodicals, Inc.)

Background: Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA., Methodology: Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays., Results: When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression., Conclusion: Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on β cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic β cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the β cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We present a male patient with sporadic Aarskog syndrome, cleft palate, mild intellectual disability, and autism spectrum disorder (ASD). A submicroscopic discontiguous deletion was detected on chromosome Xp11.2 encompassing FGD1, FAM120C, and PHF8. That the deletion encompassed FGD1 (exons 2-8) explains the Aarskog features while the deletion of PHF8 most likely explains the cleft palate and mild intellectual disability. We identify FAM120C as a novel X-linked candidate gene for autism for two reasons: first, a larger deletion encompassing FAM120C segregates with autism in a previously reported family and second, there is recent evidence that FAM120C interacts with CYFIP1, part of the FMRP (Fragile X Mental Retardation Protein) network. In the current study, resequencing of FAM120C in 87 Belgian male patients with autism spectrum disorder identified no novel mutations. Expression of Fam120c in mouse tissues showed enriched expression in pituitary, cerebellum, cortex, and pancreatic islets of Langerhans. Additionally, we found a cortical expression pattern of Fam120c similar to that of Fmr1. In conclusion, FAM120C is a novel candidate gene for autism spectrum disorder based on genetic evidence and the brain expression pattern. Thereby we highlight a role for FMRP network genes in ASD., (© 2014 Wiley Periodicals, Inc.)

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

We recently proposed that the transient receptor potential melastatin 5 (TRPM5) cation channel contributes to glucose-induced electrical activity of the β cell and positively influences glucose-induced insulin release and glucose homeostasis. In this study, we investigated Trpm5 expression and function in pancreatic islets from mouse models of type II diabetes. Gene expression analysis revealed a strong reduction of Trpm5 mRNA levels in pancreatic islets of db/db and ob/ob mice. The glucose-induced Ca(2+) oscillation pattern in db/db and ob/ob islets mimicked those of Trpm5 (-/-) islets. Leptin treatment of ob/ob mice not only reversed the diabetic phenotype seen in these mice but also upregulated Trpm5 expression. Leptin treatment had no additional effect on Trpm5 expression levels when plasma insulin levels were comparable to those of the vehicle-injected control group. In murine β cell line, MIN6, insulin downregulated TRPM5 expression in a dose-dependent manner, unlike glucose or leptin. In conclusion, our data show that increased plasma insulin levels downregulate TRPM5 expression in pancreatic islets from leptin-deficient mouse models of type 2 diabetes.

Background: Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), a disintegrin and metalloprotease with thrombospondin motifs [ADAM(TS)s] and growth factors are involved in inflammation and tissue damage and repair, all occurring in inflammatory bowel disease (IBD). We studied the impact of anti-inflammatory therapy with infliximab on mucosal expression of these tissue remodeling genes in patients with IBD., Methods: Mucosal gene expression of 23 MMPs, 4 TIMPs, 50 ADAM(TS)s, and 158 growth factors was investigated in 61 patients with IBD before and after the first infliximab therapy and in 12 controls, with microarrays and quantitative RT-PCR. Protein localization, mucosal gelatinase levels, and net gelatinolytic activity were investigated by immunohistochemistry, zymography analysis, and gelatin degradation assay, respectively., Results: In patients with active IBD before infliximab versus controls, gene expression of many MMPs, TIMPs, ADAM(TS)s, and growth factors was upregulated, whereas colonic expression of MMP28 and TGFA and ileal expression of ADAMDEC1 and AGT were downregulated. After controlling inflammation with infliximab, most gene dysregulations observed at baseline were restored in responders. Increased ratio of MMP1/TIMP1 expression at baseline in active IBD was restored in responders with colonic mucosal healing. With immunohistochemistry, protein localization differences of MMP1, MMP3, REG1A, and TIMP1 were shown between active IBD and control mucosa. With zymography analysis and gelatin degradation assay, higher gelatinase levels and net gelatinolytic activity were measured before infliximab and levels normalized after infliximab., Conclusions: Our data suggest that suppression of inflammation results in the arrest of epithelial damage and subsequent mucosal healing. Therefore, the therapeutic potential of agents targeting MMPs or growth factors as primary therapy seems rather complex.

Background: Scarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells., Aim and Methods: To identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search., Results: Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp) 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment., Conclusion: Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.

Glucose-stimulated insulin secretion is ensured by multiple molecular, cellular and tissue events. In this issue of Diabetologia, Low et al (DOI: 10.1007/s00125-013-3019-5 ) have taken an important new step towards understanding the hierarchical organisation of these events, by monitoring in vitro the individual exocytosis of multiple beta cells within intact mouse islets. The authors show that glucose stimulation markedly increases the number of exocytotic events per cell and, to a lesser extent, the number of beta cells contributing to this event. In this commentary we discuss these novel observations and propose that metabolic and electrical coupling of islet beta cells is responsible for a more homogeneous glucose-induced secretory response of cells in an intact islet as compared with isolated beta cells.

Streptozotocin (STZ) is widely used as diabetogenic agent in animal models for diabetic nephropathy (DN). However, it is also directly cytotoxic to kidneys, making it difficult to distinguish between DN-related and STZ-induced nephropathy. Therefore, an improved protocol to generate mice for DN studies, with a quick and robust achievement of the diabetic state, without direct kidney toxicity is required. To investigate the mechanism leading to STZ-induced nephropathy, kidney damage was induced with a high dose of STZ. This resulted in delayed gastric emptying, at least partially caused by impaired desacyl ghrelin clearance. STZ uptake in the kidneys is to a large extent mediated by the sodium/glucose cotransporters (Sglts) because the Sglt inhibitor phlorizin could reduce STZ uptake in the kidneys. Consequently, the direct toxic effects in the kidney and the gastric dilatation were resolved without interfering with the β-cell toxicity. Furthermore, pancreatic STZ uptake was increased, hereby decreasing the threshold for β-cell toxicity, allowing for single low non-nephrotoxic STZ doses (70 mg/kg). In conclusion, this study provides novel insights into the mechanism of STZ toxicity in kidneys and suggests a more efficient regime to induce DN with little or no toxic side effects.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching., (Copyright © 2013 Elsevier Inc. All rights reserved.)

There is a need for plasma-based tests that can directly measure the extent of β-cell injury in vivo in patients receiving islet grafts and in animal models. In this study, we propose protein phosphatase 1, regulatory (inhibitor) subunit 1A (PPP1R1A) as a novel biomarker for acute β-cell destruction. Liquid chromatography-tandem mass spectrometry proteome analysis of fluorescence-activated cell sorter-purified β-cells, tissue-comparative Western blotting, and immunohistochemistry indicated relatively high molar abundance and selectivity of PPP1R1A in β-cells. PPP1R1A was discharged into the extracellular space of chemically injured rat and human islets in vitro, proportionate to the extent of β-cell death. Streptozotocin injection in rats led to a progressive PPP1R1A depletion from the cytoplasm of disintegrating β-cells and a marked surge in plasma levels detectable by an affinity-capture method. A similar massive PPP1R1A discharge in blood was also detected in three patients immediately after intraportal islet transplantation. Our findings provide first proof-of-principle for PPP1R1A as real-time biomarker of β-cell destruction in animal models and patients and warrant development of more sensitive methods for its further validation in clinical trials.

Introduction: Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease., Materials and Methods: C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T 2 relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis., Results: Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn's colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T2 relaxometry of the colon showed a clear shift towards higher T2 values in the acute stage and a gradual regression of T2 values with increasing cycles of DSS., Conclusions: Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn's disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T2 relaxometry is a promising non-invasive assessment of inflammation and fibrosis.

Controversy has arisen about the nature of circulating human CD20(+)CD27(+)CD43(+)CD70(-)CD69(-) B cells. Although originally described as being the human counterpart of murine B-1 B cells, some studies have raised the possibility that these might instead be plasmablasts. In this article, we have further characterized the putative B-1 cells and compared them directly with memory B cells and plasmablasts for several functional characteristics. Spontaneous antibody production of different isotypes as well as the induced production of antigen-specific antibodies after vaccination with a T-cell-dependent antigen did not reveal differences between the putative B-1 cells and genuine CD20(-) plasmablasts. Gene expression profiling of different B-cell subsets positioned the phenotype of putative B-1 cells closer to CD20(-) plasmablasts than to memory B cells. Moreover, putative B-1 cells could be differentiated into CD20(-) plasmablasts and plasma cells in vitro, supporting a pre-plasmablast phenotype. In conclusion, characterization of the putative B-1 cells revealed a functional phenotype and a gene expression profile that corresponds to cells that differentiate into CD20(-) plasmablasts. Our data offer perspectives for the investigation of differentiation of B cells into antibody secreting cells.