Frans Schuit

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.

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta‐cells differ in their individual rates of glucose‐induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta‐cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta‐cells were 2‐ to 4‐fold higher than in low responsive beta‐cells, whereas their leucine and glutamine oxidation was only 10–50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13–19 pmol/min/10(3) beta‐cells) were at least 50‐fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3–0.7 pmol/min/10(3) beta‐cells) ranged within those of total glucose utilization (0.2–0.4 pmol/min/10(3) beta‐cells). High responsive beta‐cells exhibited a 60% higher glucokinase activity than low responsive beta‐cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta‐cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta‐cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

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.

Physical Exercise–Induced Hypoglycemia Caused by Failed Silencing of Monocarboxylate Transporter 1 in Pancreatic β Cells

Exercise-induced hyperinsulinism (EIHI) is a dominantly inherited hypoglycemic disorder characterized by inappropriate insulin secretion during anaerobic exercise or on pyruvate load. We aimed to identify the molecular basis of this novel disorder of beta -cell regulation. EIHI mapped to chromosome 1 (LOD score 3.6) in a genome scan performed for two families with 10 EIHI-affected patients. Mutational analysis of the promoter of the SLC16A1 gene, which encodes monocarboxylate transporter 1 (MCT1), located under the linkage peak, revealed changes in all 13 identified patients with EIHI. Patient fibroblasts displayed abnormally high SLC16A1 transcript levels, although monocarboxylate transport activities were not changed in these cells, reflecting additional posttranscriptional control of MCT1 levels in extrapancreatic tissues. By contrast, when examined in beta cells, either of two SLC16A1 mutations identified in separate pedigrees resulted in increased protein binding to the corresponding promoter elements and marked (3- or 10-fold) transcriptional stimulation of SLC16A1 promoter-reporter constructs. These studies show that promoter-activating mutations in EIHI induce SLC16A1 expression in beta cells, where this gene is not usually transcribed, permitting pyruvate uptake and pyruvate-stimulated insulin release despite ensuing hypoglycemia. These findings describe a novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues.

Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets.

Aims/hypothesisInadequate 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.Materials and methodsWe compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca2+ 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.ResultsThapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30xa0mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10xa0mmol/l; (2) moderate increase parallel to the UPR between 10 and 30xa0mmol/l. These glucose effects occurred within 2xa0h, were mimicked by other metabolic substrates, but were independent of changes in Ca2+ 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.Conclusions/interpretationNutrient 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.

Physiologic relevance of heterogeneity in the pancreatic beta-cell population

SummaryIn vitro studies on purified rat beta cells have indicated a functional diversity among insulincontaining cells. Intercellular differences were found in the rates of glucose-induced insulin synthesis and release. They are attributed to differences in cellular thresholds for glucose utilization and oxidation, as can be caused by varying activities in rate limiting steps such as glucokinase-dependent phosphorylation. The percent of functionally active beta cells increases dose-dependently with the glucose concentration, making cellular heterogeneity and its regulation by glucose major determinants for the dose-response curves of the total beta-cell population. Beta cells which are already responsive to low glucose concentrations are characterized by a higher content in pale immature granules; their activated biosynthetic and secretory activity accounts for preferential release of newly-formed hormone by the total beta-cell population. At any glucose level, the amplitude of insulin release depends on the percent glucose-activated cells and their cyclic AMP content, an integrator of (neuro)hormonal influences. The in vitro described heterogeneity in beta-cell functions may bear physiological relevance as several of its characteristics are also detectable in intact pancreatic tissue; furthermore, in vitro signs of heterogeneity can be altered by prior in vivo treatment indicating that they express properties of the cells in their in situ configuration. Elevated basal levels of (pro)insulin may reflect the existence of an increased number of beta cells that are activated at low physiologic glucose concentrations. Reductions in stimulated insulin levels can be caused by decreased numbers of beta cells that are activated at the prevailing glucose concentration or by insufficient cyclic AMP levels in beta cells, possibly as a result of inadequate signalling from hormones of local or distal origin. Only few markers are currently available with which to explore these mechanisms in vivo. Additional markers and tests should help assess the possible role of variations in beta-cell heterogeneity in the pathogenesis of diabetes mellitus.

Tissue-specific disallowance of housekeeping genes: The other face of cell differentiation

We report on a hitherto poorly characterized class of genes that are expressed in all tissues, except in one. Often, these genes have been classified as housekeeping genes, based on their nearly ubiquitous expression. However, the specific repression in one tissue defines a special class of disallowed genes. In this paper, we used the intersection-union test to screen for such genes in a multi-tissue panel of genome-wide mRNA expression data. We propose that disallowed genes need to be repressed in the specific target tissue to ensure correct tissue function. We provide mechanistic data of repression with two metabolic examples, exercise-induced inappropriate insulin release and interference with ketogenesis in liver. Developmentally, this repression is established during tissue maturation in the early postnatal period involving epigenetic changes in histone methylation. In addition, tissue-specific expression of microRNAs can further diminish these repressed mRNAs. Together, we provide a systematic analysis of tissue-specific repression of housekeeping genes, a phenomenon that has not been studied so far on a genome-wide basis and, when perturbed, can lead to human disease.

Effects of Chronically Elevated Glucose Levels on the Functional Properties of Rat Pancreatic β-Cells

This study examines the effects of chronically elevated glucose levels on the survival and function of purified rat β-cells. Prolonged exposure (9 days) of β-cell aggregates to 20 mmol/l glucose did not lead to cell losses, but reduced the amount of insulin secreted in response to glucose. This decrease was not caused by cellular desensitization but resulted from the lower cellular insulin content after a prolonged imbalance between stimulated rates of insulin synthesis and release. Virtually all β-cells exhibited a state of metabolic and biosynthetic activation, which was maintained for at least 2 h in glucose-depleted media. Their rates of protein and insulin synthesis were amplified by glucose, reaching (half-) maximal stimulation at lower glucose concentrations (2 and 5 mmol/l, respectively) than control cells cultured at 10 mmol/l glucose (5 and 10 mmol/l, respectively). As for insulin release, the net glucose effect on insulin synthesis was markedly reduced as compared with that in control cells. This was also the case after culture at 6 mmol/l glucose. In the latter condition, the lower glucose-inducible activities were caused by cellular desensitization, with 50% of the β-cells unresponsive to glucose and the other 50% responding with a lower sensitivity (half-maximal stimulation at 7 mmol/l glucose). Comparison of β-cells cultured at the three glucose concentrations indicated that prolonged exposure to elevated glucose levels increases the number of degranulated cells, of cells with a high proportion of immature insulin granules, and of cells with glycogen deposition-morphologic features previously described in conditions of hyperglycemia. It is concluded that chronic exposure (9 days) of rat β-cells to elevated glucose levels induces a prolonged state of β-cell activation and glucose hypersensitivity rather than a glucotoxicity or glucose desensitization. This shift in the functional state of the β-cell population is responsible for a reduced insulin release in response to glucose, as observed in other conditions of prolonged exposure to high glucose levels.

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy.

Aims/hypothesisUpregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes.MethodsRNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells.ResultsmRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24xa0h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h).Conclusions/interpretationA very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified.

Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations

Aims/hypothesisSurvival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2xa0mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10xa0mmol/l are deleterious.MethodsTo identify the mechanisms of such beta cell plasticity, we tested the effects of 18xa0h culture at 2, 5, 10 and 30xa0mmol/l glucose on the transcriptome of rat islets pre-cultured for 1xa0week at 10xa0mmol/l glucose using Affymetrix Rat 230 2.0 arrays.ResultsCulture in either 2–5 or 30xa0mmol/l instead of 10xa0mmol/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 30xa0mmol/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 10xa0mmol/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/interpretationWe 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.