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.
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.
Aims/hypothesis Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition ...receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. Methods Male and female littermate Tlr2 ⁺/⁺ and Tlr2 ⁻/⁻ mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. Results While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2 ⁻/⁻ mice were protected from the adverse effects of HFD compared with Tlr2 ⁺/⁺ littermate controls. Female Tlr2 ⁻/⁻ mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2 ⁻/⁻ mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2 ⁻/⁻ mice did not increase IL-1β expression in response to a NEFA mixture, whereas Tlr2 ⁺/⁺ control tissues did. Conclusion/interpretation These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.
Aims/hypothesis
Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose ...improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious.
Methods
To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays.
Results
Culture in either 2–5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration.
Conclusions/interpretation
We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia.
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.
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.
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.
Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the ...latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated.
We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein Xbp1 mRNA splicing and XBP1/activating transcription factor ATF 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets.
Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose.
Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.
The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The ...anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion.
Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance.
We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase.
Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.
Abstract Premature progesterone rise during gonadotrophin-releasing hormone (GnRH) antagonist cycles for IVF is a frequent phenomenon and has been associated with lower pregnancy and implantation ...rates. This study evaluated endometrial gene expression on the day of oocyte retrieval according to the concentration of serum progesterone on the day of human chorionic gonadotrophin (HCG) administration in GnRH-antagonist/recombinant FSH IVF cycles with fresh embryo transfer. Endometrial biopsies ( n = 14) were analysed with Affymetrix HG U133 Plus 2.0 Arrays. Patients were divided into three groups according to their progesterone serum concentration on the day of HCG administration: ⩽0.9 ng/ml (group A), 1–1.5 ng/ml (group B) and >1.5 ng/ml (group C). Gene expression analysis showed a small number of significantly differentially expressed probe sets between groups A and B (five up/23 down in B) and a large difference between groups B and C (607 up/212 down; P ⩽ 0.05, fold change ⩾1.4). Validation was performed with quantitative real-time PCR on selected genes. As far as is known, this is the first study to demonstrate a distinct difference in endometrial gene expression profile between patients with a progesterone serum concentration above and below the threshold of 1.5 ng/ml on the day of HCG administration. During an IVF treatment in a stimulated cycle, some patients undergo an early elevation of progesterone during the follicular phase of their menstrual cycle. This is associated with lower pregnancy and implantation rates. In this study, we evaluated endometrial gene expression on the day of oocyte retrieval according to the concentration of serum progesterone on the day of human chorionic gonadotrophin (HCG) administration in stimulated IVF cycles with fresh embryo transfer. Fourteen endometrial biopsies were divided into three groups according to their progesterone serum concentration: ⩽0.9 ng/ml (group A), 1–1.5 ng/ml (group B) and >1.5 ng/ml (group C). Microarray gene expression analysis showed a small difference between groups A and B and a large difference between groups B and C. Technical validation with a more quantitative PCR technique was performed on selected genes. As far as is known, this is the first study to demonstrate a distinct difference in endometrial gene expression profile between patients with a progesterone serum concentration above and below the threshold of 1.5 ng/ml on the day of HCG administration, which might reflect an important issue for implantation failure in IVF patients with premature progesterone rise.
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. 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. 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.
▸ We investigate the effects of 1,25(OH)2D3 on global gene expression in pancreatic islets. ▸ Cytoskeletal and intracellular trafficking genes are induced by 1,25(OH)2D3 in islets. ▸ Genes involved ...in intercellular junctions in islets are upregulated by 1,25(OH)2D3. ▸ 1,25(OH)2D3 affects expression of genes associated with lipid metabolism in islets. ▸ Genes involved in ion transport and homeostasis in islets are induced by 1,25(OH)2D3.
Vitamin D deficiency has been linked to type 1 and 2 diabetes, whereas supplementation may prevent both diseases. However, the extent of the effects of vitamin D or its metabolites directly on pancreatic islets is still largely unknown. The aim of the present study was to investigate how active vitamin D, 1,25(OH)2D3, affects beta cells directly by establishing its effects on global gene expression in healthy murine islets.
Pancreatic islets were isolated from 2 to 3 week old C57BL/6 mice and cultured in vitro with 1,25(OH)2D3 or vehicle for 6 and 24h. Total RNA was extracted from the islets and the effects on global gene expression were analyzed using Affymetrix microarrays.
Exposure to 1,25(OH)2D3 compared to vehicle resulted in 306 and 151 differentially expressed genes after 6 and 24h, respectively (n=4, >1.3-fold, p<0.02). Of these 220 were up-regulated, whereas 86 displayed a decreased expression after 6h. Furthermore, expression levels were increased for 124 and decreased for 27 genes following 24h of exposure. Formation of intercellular junctions, cytoskeletal organization, and intracellular trafficking as well as lipid metabolism and ion transport were among the most affected gene classes. Effects on several genes already identified as being part of vitamin D signaling in other cell types were observed along with genes known to affect insulin release, although with our assay we were not able to detect any effects of 1,25(OH)2D3 on glucose-stimulated insulin release from healthy pancreatic islets.
The effects of 1,25(OH)2D3 on the expression of cytoskeletal and intracellular trafficking genes along with genes involved in ion transport may influence insulin exocytosis. However, an effect of 1,25(OH)2D3 on insulin release could not be detected for healthy islets in contrast to islets subjected to pathological conditions such as cytokine exposure and vitamin D deficiency as suggested by other studies. Thus, in addition to previously identified tolerogenic effects on the immune system, 1,25(OH)2D3 may affect basic functions of pancreatic beta cells, with the potential to render them more resistant to the detrimental conditions encountered during type 1 and 2 diabetes.
This article is part of a Special Issue entitled ‘Vitamin D Workshop’.
PDF
