In vivo expression and functional characterization of the zinc transporter ZnT8 in glucose-induced insulin secretion.

International audienceInsulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells

Mucosal Gene Expression of Antimicrobial Peptides in Inflammatory Bowel Disease Before and After First Infliximab Treatment

Background: Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD. Methodology/Principal Findings: Mucosal gene expression of 81 AMPs was assessed in 61 IBD patients before and 4-6 weeks after their first infliximab infusion and in 12 control patients, using Affymetrix arrays. Quantitative real-time reverse-transcription PCR and immunohistochemistry were used to confirm microarray data. The dysregulation of many AMPs in colonic IBD in comparison with control colons was widely restored by infliximab therapy, and only DEFB1 expression remained significantly decreased after therapy in the colonic mucosa of IBD responders to infliximab. In ileal Crohn's disease (CD), expression of two neuropeptides with antimicrobial activity, PYY and CHGB, was significantly decreased before therapy compared to control ileums, and ileal PYY expression remained significantly decreased after therapy in CD responders. Expression of the downregulated AMPs before and after treatment (DEFB1 and PYY) correlated with villin 1 expression, a gut epithelial cell marker, indicating that the decrease is a consequence of epithelial damage. Conclusions/Significance: Our study shows that the dysregulation of AMPs in IBD mucosa is the consequence of inflammation, but may be responsible for perpetuation of inflammation due to ineffective clearance of microorganisms

Using Ribosomal Protein Genes as Reference: A Tale of Caution

Background: Housekeeping genes are needed in every tissue as their expression is required for survival, integrity or duplication of every cell. Housekeeping genes commonly have been used as reference genes to normalize gene expression data, the underlying assumption being that they are expressed in every cell type at approximately the same level. Often, the terms "reference genes'' and "housekeeping genes'' are used interchangeably. In this paper, we would like to distinguish between these terms. Consensus is growing that housekeeping genes which have traditionally been used to normalize gene expression data are not good reference genes. Recently, ribosomal protein genes have been suggested as reference genes based on a meta-analysis of publicly available microarray data. Methodology/Principal Findings: We have applied several statistical tools on a dataset of 70 microarrays representing 22 different tissues, to assess and visualize expression stability of ribosomal protein genes. We confirmed the housekeeping status of these genes, but further estimated expression stability across tissues in order to assess their potential as reference genes. One- and two-way ANOVA revealed that all ribosomal protein genes have significant expression variation across tissues and exhibit tissue-dependent expression behavior as a group. Via multidimensional unfolding analysis, we visualized this tissue-dependency. In addition, we explored mechanisms that may cause tissue dependent effects of individual ribosomal protein genes. Conclusions/Significance: Here we provide statistical and biological evidence that ribosomal protein genes exhibit important tissue-dependent variation in mRNA expression. Though these genes are most stably expressed of all investigated genes in a meta-analysis they cannot be considered true reference genes

Characterization of proposed human B-1 cells reveals pre-plasmablast phenotype

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 rather be plasmablasts. We here further characterized the putative B-1 cells and compared them directly to 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.status: publishe

A complex Xp11.22 deletion in a patient with syndromic autism: Exploration of FAM120C as a positional candidate gene for autism

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.status: publishe

Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity

The proprotein convertase 1/3 (PC1/3), encoded by 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.status: publishe

Effects of increasing glucose concentrations on gene mRNA expression in cultured rat pancreatic islets

Background and Aims, Materials and Methods: Survival and function of rat β-cells are best preserved by culture at 10 mM glucose (G10) and rapidly deteriorate at lower or higher glucose concentrations. To evaluate how glucose exerts these effects on the β-cell phenotype, we measured gene mRNA levels in rat islets precultured for 1wk in G10 and further cultured 18h in G2, G5, G10 or G30 (Affymetrix Rat Genome 230 2.0 oligonucleotide arrays, n=4). Match/mismatch probe signal intensities and differences in mRNA abundance, expressed as absolute signal log2 ratios (SLR), were calculated with GCOS software. Results: From the 31099 probe sets on the arrays, 18081 were reliably detected in at least one glucose concentration. The SLR for at least one of the comparisons between the different glucose concentrations was ≥ 1 and ≥ 0.5 in 1039 and 3530 probe sets respectively. Using Self-Organizing Map cluster analysis, 3120 of the 3530 affected probe sets were classified in 6 clusters with monophasic concentration-dependencies that differed in overall direction (increase/decrease) and glucose threshold. The remaining 410 probe sets were grouped into 5 small clusters with complex mRNA expression profiles (V-shaped or inverted V-shaped with minimum or maximum in G5 or G10). Analysis of gene function in each cluster suggested the presence of glucose co-regulated metabolic and signalling pathways (glycolysis, cholesterol synthesis, unfolded protein response…). Conclusion: These results will help identifying key molecules that maintain the differentiated β-cell phenotype as well as those responsible for β-cell deterioration after exposure to chronic hypo- or hyperglycemi

Gene Expression Profiling of Early Hepatic Stellate Cell Activation Reveals a Role for Igfbp3 in Cell Migration

<div><p>Background</p><p>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. </p> <p>Aim and methods</p><p>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. </p> <p>Results</p><p>Gene expression profiling confirmed early changes for known genes related to HSC activation such as <i>alpha</i><i>smooth</i><i>muscle</i><i>actin</i> (<i>Acta2</i>)<i>, lysyl</i><i>oxidase</i> (<i>Lox</i>) and <i>collagen</i>, type <i>I</i>, alpha <i>1</i> (<i>Col1a1</i>). 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. <i>Insulin-like</i><i>growth</i><i>factor</i><i>binding</i> protein <i>3</i> (<i>Igfbp3</i>) was identified as a gene strongly affected by VPA treatment. During normal HSC activation <i>Igfbp3</i> is up regulated and this can thus be prevented by VPA treatment <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>. siRNA-mediated silencing of <i>Igfbp3</i> in primary mouse HSCs induced matrix metalloproteinase (Mmp) <i>9</i> mRNA expression and strongly reduced cell migration. The reduced cell migration after <i>Igfbp3</i> knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. </p> <p>Conclusion</p><p>Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents <i>Igfbp3</i> transcription during activation of HSCs <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>.</p> </div