Anica Schraenen

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8−/−) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8−/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8+/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8−/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8−/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

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.

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

Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression

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.

β-Cell–Specific Gene Repression: A Mechanism to Protect Against Inappropriate or Maladjusted Insulin Secretion?

What makes β-cells unique is their continuous responsibility to produce, store and release the required amount of insulin to keep blood glucose normal. This is no trivial task because daily insulin demands fluctuate acutely (as happens during meals) or chronically (e.g., the adaptation to obesity or pregnancy). Moreover, normal blood glucose has two borders that need continuous protection. Insufficient insulin secretion should be avoided as this leads to hyperglycemia. Moreover, insulin excess causes hypoglycemia, a situation that jeopardizes brain function. Therefore, the optimal amount of β-cell activity is needed for normal health and this depends on an adequate plasticity of the functional β-cell mass (1). This Perspective examines the idea that protection of the two borders of blood glucose requires a genetically programmed β-cell phenotype with two unique faces (Fig. 1). The first depends on transcription factors that activate expression of specific proteins that mediate β-cell function. The second face depends on β-cell–specific repression of a small set of genes. We start to understand how expression of the latter genes may impair normal β-cell function. The best examples of expression of such “disallowed” genes in β-cells lead to inappropriate insulin release (2). FIG. 1. A balance between two faces of the mature β-cell. The general working hypothesis in this Perspective is that the mature β-cell needs to defend both the upper and lower normal limits of circulating glucose levels, thereby preventing the occurrence of episodes of hyperglycemia and hypoglycemia. A genetically determined β-cell program is responsible for the appropriate expression level of proteins in β-cells that allow a physiological response in terms of sufficient release of insulin when the organism needs the hormone. In parallel, some genes are specifically repressed in β-cells in order to avoid situations where β-cells respond inappropriately (induction by the wrong stimuli) or in a maladjusted manner …

Mice deficient in the respiratory chain gene Cox6a2 are protected against high-fat diet-induced obesity and insulin resistance

Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle-specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance. This phenotype results from elevated energy expenditure and a skeletal muscle fiber type switch towards more oxidative fibers. At the molecular level we observe increased formation of reactive oxygen species, constitutive activation of AMP-activated protein kinase, and enhanced expression of uncoupling proteins. Our data indicate that COX6A2 is a regulator of respiratory uncoupling in muscle and we demonstrate that a novel and direct link exists between muscle respiratory chain activity and diet-induced obesity/insulin resistance.

Strong Upregulation of AIM2 and IFI16 Inflammasomes in the Mucosa of Patients with Active Inflammatory Bowel Disease

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.

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone

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.

Prolactin receptors and placental lactogen drive male mouse pancreatic islets to pregnancy-related mRNA changes.

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.