Katleen Lemaire

A variant near MTNR1B is associated with increased fasting plasma glucose levels and type 2 diabetes risk

In genome-wide association (GWA) data from 2,151 nondiabetic French subjects, we identified rs1387153, near MTNR1B (which encodes the melatonin receptor 2 (MT2)), as a modulator of fasting plasma glucose (FPG; P = 1.3 × 10−7). In European populations, the rs1387153 T allele is associated with increased FPG (β = 0.06 mmol/l, P = 7.6 × 10−29, N = 16,094), type 2 diabetes (T2D) risk (odds ratio (OR) = 1.15, 95% CI = 1.08–1.22, P = 6.3 × 10−5, cases N = 6,332) and risk of developing hyperglycemia or diabetes over a 9-year period (hazard ratio (HR) = 1.20, 95% CI = 1.06–1.36, P = 0.005, incident cases N = 515). RT-PCR analyses confirm the presence of MT2 transcripts in neural tissues and show MT2 expression in human pancreatic islets and beta cells. Our data suggest a possible link between circadian rhythm regulation and glucose homeostasis through the melatonin signaling pathway.

A Saccharomyces cerevisiae G-protein coupled receptor, Gpr1, is specifically required for glucose activation of the cAMP pathway during the transition to growth on glucose

In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras‐mediated long‐lasting cAMP increase. Addition of glucose to cells grown on a non‐fermentable carbon source or to stationary‐phase cells triggers a transient burst in the intracellular cAMP level. This glucose‐induced cAMP signal is dependent on the G alpha‐protein Gpa2. We show that the G‐protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val‐132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose‐induced loss of heat resistance, which is consistent with its lack of glucose‐induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose‐sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK‐controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE‐controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose‐sensing system must signal glucose availability for the Sch9‐dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.

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.

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.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis

Background and aims: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. Methods: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. Results: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. Conclusion: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process.

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G‐protein‐coupled receptor (GPCR) system, Gpr1‐Gpa2, and uptake and phosphorylation of the sugar. In a hxt‐null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose‐induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR‐dependent glucose‐sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt‐null cells and subsequent addition of glucose restored the glucose‐induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular α and βd‐glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild‐type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild‐type cells result only from the low sensitivity of the Gpr1‐Gpa2 system and not from the intracellular sugar kinase‐dependent process. In conclusion, we have shown that the two essential requirements for glucose‐induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar‐sensing process requiring the hexose kinases.

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.

Sex and sugar in yeast: two distinct GPCR systems

Although eukaryotic G‐protein coupled receptor (GPCR) systems are well known for their ability to detect and mediate rapid responses to extracellular signals, the full range of stimuli to which they respond may not yet have been identified. Activation of GPCRs by hormones, pheromones, odorants, neurotransmitters, light and different taste compounds is well established. However, the recent discovery of a glucose‐sensing GPCR system in Saccharomyces cerevisiae has unexpectedly added common nutrients to this list of stimuli. This GPCR system mediates glucose activation of adenylate cyclase during the switch from respirative/gluconeogenic metabolism to fermentation. The GPCR system involved in pheromone signalling in S. cerevisiae has already served as an important model and tool for the study of GPCR systems in higher eukaryotic cell types. Here, we highlight the similarities and differences between these two signalling systems. We also indicate how the new glucose‐sensing system can serve as a model for GPCR function and as a tool with which to screen for heterologous components of signalling pathways as well as for novel ligands in high‐throughput assays.

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.