Leentje Van Lommel

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

Insulin-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.

A polymorphism within the G6PC2 gene is associated with fasting plasma glucose levels

Several studies have shown that healthy individuals with fasting plasma glucose (FPG) levels at the high end of the normal range have an increased risk of mortality. To identify genetic determinants that contribute to interindividual variation in FPG, we tested 392,935 single-nucleotide polymorphisms (SNPs) in 654 normoglycemic participants for association with FPG, and we replicated the most strongly associated SNP (rs560887, P = 4 × 10–7) in 9353 participants. SNP rs560887 maps to intron 3 of the G6PC2 gene, which encodes glucose-6-phosphatase catalytic subunit–related protein (also known as IGRP), a protein selectively expressed in pancreatic islets. This SNP was associated with FPG (linear regression coefficient β = –0.06 millimoles per liter per A allele, combined P = 4 × 10–23) and with pancreatic β cell function (Homa-B model, combined P = 3 × 10–13) in three populations; however, it was not associated with type 2 diabetes risk. We speculate that G6PC2 regulates FPG by modulating the set point for glucose-stimulated insulin secretion in pancreatic β cells.

Interleukin-6 regulates pancreatic alpha-cell mass expansion

Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced β-cell function and mass, an increased proportion of α-cells relative to β-cells, and α-cell dysfunction. Here we show that the α cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the α-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates α-cell proliferation, prevents apoptosis due to metabolic stress, and regulates α-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased α-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of α-cell mass display decreased fasting glucagon levels. However, despite these α-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic α-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional β-cell compensation to increased metabolic demand.

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.

Loss of high-frequency glucose-induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5-/- mice

Glucose homeostasis is critically dependent on insulin release from pancreatic β-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (Vm) and the cytosolic calcium level ([Ca2+]cyt). We propose that TRPM5, a Ca2+-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca2+-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5−/− cells. Ca2+-imaging and electrophysiological analysis show that glucose-induced oscillations of Vm and [Ca2+]cyt have on average a reduced frequency in Trpm5−/− islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5−/− pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5−/− mice.

The Vitamin D Analog, TX527, Promotes a Human CD4+CD25highCD127low Regulatory T Cell Profile and Induces a Migratory Signature Specific for Homing to Sites of Inflammation

The use of hypocalcemic vitamin D analogs is an appealing strategy to exploit the immunomodulatory actions of active vitamin D in vivo while circumventing its calcemic side effects. The functional modulation of dendritic cells by these molecules is regarded as the key mechanism underlying their ability to regulate T cell reactivity. In this article, we demonstrate the capacity of the vitamin D analog, TX527, to target T cells directly. Microarray analysis of purified human CD3+ T cells, cultured in the presence of TX527, revealed differential expression of genes involved in T cell activation, proliferation, differentiation, and migratory capacity. Accordingly, functional analysis showed a TX527-mediated suppression of the T cell proliferative capacity and activation status, accompanied by decreased expression of effector cytokines (IFN-γ, IL-4, and IL-17). Furthermore, TX527 triggered the emergence of CD4+CD25highCD127low regulatory T cells featuring elevated levels of IL-10, CTLA-4, and OX40 and the functional capacity to suppress activation and proliferation of effector T cells. Moreover, the vitamin D analog profoundly altered the homing receptor profile of T cells and their migration toward chemokine ligands. Remarkably, TX527 not only modulated skin-homing receptors as illustrated for the parent compound, but also reduced the expression of lymphoid organ-homing receptors (CD62L, CCR7, and CXCR4) and uniquely promoted surface expression of inflammatory homing receptors (CCR5, CXCR3, and CXCR6) on T cells. We conclude that TX527 directly affects human T cell function, thereby inhibiting effector T cell reactivity while inducing regulatory T cell characteristics, and imprints them with a specific homing signature favoring migration to sites of inflammation.

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 Crohns 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.

Mucosal gene expression of cell adhesion molecules, chemokines, and chemokine receptors in patients with inflammatory bowel disease before and after infliximab treatment

OBJECTIVES:Inflammatory bowel disease (IBD) is characterized by a continuous influx of leukocytes into the gut wall. This migration is regulated by cell adhesion molecules (CAMs), and selective antimigration therapies have been developed. This study investigated the effect of infliximab therapy on the mucosal gene expression of CAMs in IBD.METHODS:Mucosal gene expression of 69 leukocyte/endothelial CAMs and E-cadherin was investigated in 61 IBD patients before and after first infliximab infusion and in 12 normal controls, using Affymetrix gene expression microarrays. Quantitative reverse transcriptase-PCR (qRT-PCR), immunohistochemistry, and western blotting were used to confirm the microarray data.RESULTS:When compared with control colons, the colonic mucosal gene expression of most leukocyte/endothelial adhesion molecules was upregulated and E-cadherin gene expression was downregulated in active colonic IBD (IBDc) before therapy, with no significant colonic gene expression differences between ulcerative colitis and colonic Crohns disease. Infliximab therapy restored the upregulations of leukocyte CAMs in IBDc responders to infliximab that paralleled the disappearance of the inflammatory cells from the colonic lamina propria. Also, the colonic gene expression of endothelial CAMs and of most chemokines/chemokine receptors returned to normal after therapy in IBDc responders, and only CCL20 and CXCL1-2 expression remained increased after therapy in IBDc responders vs. control colons. When compared with control ileums, the ileal gene expression of MADCAM1, THY1, PECAM1, CCL28, CXCL1, -2, -5, -6, and -11, and IL8 was increased and CD58 expression was decreased in active ileal Crohns disease (CDi) before therapy, and none of the genes remained dysregulated after therapy in CDi responders vs. control ileums. This microarray study identified a number of interesting targets for antiadhesion therapy including PECAM1, IL8, and CCL20, besides the currently studied α4β7 integrin–MADCAM1 axis.CONCLUSIONS:Our data demonstrate that many leukocyte/endothelial CAMs and chemokines/chemokine receptors are upregulated in inflamed IBD mucosa. Controlling the inflammation with infliximab restores most of these dysregulations in IBD. These results show that at least part of the mechanism of anti-tumor necrosis factor-α therapy goes through downregulation of certain adhesion molecules.

Predictive value of epithelial gene expression profiles for response to infliximab in Crohn's disease.

Background: Infliximab (IFX) has become the mainstay of therapy of refractory Crohns disease (CD). However, a subset of patients shows incomplete or no response to this agent. In this study we investigated whether we could identify a mucosal gene panel to predict (non)response to IFX in CD. Methods: Mucosal biopsies were obtained during endoscopy from 37 patients with active CD (19 Crohns colitis [CDc] and 18 Crohns ileitis [CDi]) before and after first IFX treatment. Response was defined based on endoscopic and histologic findings. Total RNA was analyzed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) was used to confirm microarray data. Results: At baseline, significant gene expression differences were found between CDc and CDi. For predicting response in CDc, comparative analysis of CDc pretreatment expression profiles identified 697 significant probe sets between CDc responders (n = 12) and CDc nonresponders (n = 7). Class prediction analysis of CDc top 20 and top 5 significant genes allowed complete separation between CDc responders and CDc nonresponders. The CDc top 5 genes were TNFAIP6, S100A8, IL11, G0S2, and S100A9. Only one patient with CDi completely healed the ileal mucosa. Even using less stringent response criteria, we could not identify a predictive gene panel for IFX responsiveness in CDi. Conclusions: This study identified a 100% accurate predictive gene signature for (non)response to IFX in CDc, whereas no such a predictive gene set could be identified for CDi. Inflamm Bowel Dis 2010

Plasticity in the adult rat pancreas: transdifferentiation of exocrine to hepatocyte-like cells in primary culture.

Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte‐like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse‐transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer‐binding protein‐ß protein, a liver‐enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and α‐fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX‐treated cells using oligonucleotide microarrays and found that multiple liver‐specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX‐treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes. (HEPATOLOGY 2004;39:1499–1507.)