C B Wollheim

Lund University, Lund, Skåne, Sweden

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Publications (372)2285.25 Total impact

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    ABSTRACT: In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilised clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarisation of the electrical gradient was not altered. In permeabilised cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 12/2014; · 4.60 Impact Factor
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    ABSTRACT: Normal glucose homeostasis is characterized by appropriate insulin secretion and low HbA1c. Gene expression signatures associated with these two phenotypes could be essential for islet function and patho-physiology of type 2 diabetes (T2D). Herein, we employed a novel approach to identify candidate genes involved in T2D by correlating islet microarray gene expression data (78 donors) with insulin secretion and HbA1c level. Expression of 649 genes (p<0.05) was correlated with insulin secretion and HbA1c. Of them, 5 genes (GLR1A, PPP1R1A, PLCDXD3, FAM105A and ENO2) correlated positively with insulin secretion/negatively with HbA1c and one gene (GNG5) correlated negatively with insulin secretion/positively with HbA1c were followed up. The 5 positively correlated genes have lower expression levels in diabetic islets, whereas, GNG5 expression is higher. Exposure of human islets to high glucose for 24 hrs resulted in up-regulation of GNG5 and PPP1R1A expression, while expression of ENO2 and GLRA1 was down-regulated. No effect was seen on the expression of FAM105A and PLCXD3. siRNA silencing in INS-1 832/13 cells showed reduction in insulin secretion for PPP1R1A, PLXCD3, ENO2, FAM105A and GNG5 but not GLRA1. Although, no SNP in these gene loci passed the genome-wide significance for association with T2D in DIAGRAM+ database, four SNPs influenced gene expression in cis in human islets. In conclusion, we identified and confirmed PPP1R1A, FAM105A, ENO2, PLCDX3 and GNG5 as potential regulators of islet function. We provide a list of candidate genes as a resource for exploring their role in the pathogenesis of T2D. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Human Molecular Genetics 12/2014; · 6.68 Impact Factor
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    ABSTRACT: Genetic variation can modulate gene expression, and thereby pheno-typic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglyce-mic individuals, siRNA of tetraspanin 33 (TSPAN33), 5′-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain contain-ing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understand-ing of how genetic variation can influence glucose metabolism. T ype 2 diabetes (T2D) is an increasing global health problem (1). Although genome-wide association studies (GWAS) have yielded more than 70 loci associated with T2D or related traits (2, 3), they have not provided the expected breakthrough in our understanding of the pathogenesis of the disease. They have nonetheless pointed at a central role of the pancreatic islets and β-cell dysfunction in the development of the disease (4, 5). It therefore seems pertinent to focus on human pancreatic islets to obtain insights into the molecular mechanisms causing the disease (6, 7). Given that most SNPs associated with T2D lie in noncoding regions, the majority of causal variants are likely to regulate gene expression rather than protein function per se. Therefore, combi-nation of DNA and RNA sequencing in the same individuals may help to disentangle the role these SNPs play in the pathogenesis of the disease (8). Although the human pancreatic islet transcriptome has been previously described (6, 9–18), using microarrays or RNA sequencing of a limited number of nondiabetic individuals, this has not allowed a more global analysis of the complexity of the islet transcriptome in T2D. Here we combined genotypic imputation, expression microarrays, and exome and RNA sequencing (Exome-Seq and RNA-Seq) in a large number of human pancreatic islets from deceased donors with and without T2D. This study identified a number of novel genes, including long intergenic noncoding RNAs (lincRNAs), whose expression and/or splicing influences insulin secretion and is associated with glycemia. In addition, we provide a catalog of RNA editing and allele-specific expression events in human pancreatic islets (SI Appendix, Fig. S1). Results
    Proceedings of the National Academy of Sciences 09/2014; 14. · 9.81 Impact Factor
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    ABSTRACT: Genome-wide association studies have revealed more than 60 loci associated with type 2 diabetes (T2D), but the underlying causal variants and functional mechanisms remain largely elusive. Although variants in TCF7L2 confer the strongest risk of T2D among common variants by presumed effects on islet function, the molecular mechanisms are not yet well understood. Using RNA-Sequencing, we have identified a TCF7L2-regulated transcriptional network responsible for its effect on insulin secretion in rodent and human pancreatic islets. ISL1 is a primary target of TCF7L2 and regulates proinsulin production and processing via MAFA, PDX1, NKX6.1, PCSK1, PCSK2 and SLC30A8, thereby providing evidence for a coordinated regulation of insulin production and processing. The risk T-allele of rs7903146 was associated with increased TCF7L2 expression, and decreased insulin content and secretion. Using gene expression profiles of 66 human pancreatic islets donors', we also show that the identified TCF7L2-ISL1 transcriptional network is regulated in a genotype-dependent manner. Taken together, these results demonstrate that not only synthesis of proinsulin is regulated by TCF7L2, but also processing and possibly clearance of proinsulin and insulin. These multiple targets in key pathways may explain why TCF7L2 has emerged as the gene showing one of the strongest associations with T2D.
    Human Molecular Genetics 07/2014; · 6.68 Impact Factor
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    ABSTRACT: Impaired insulin secretion is a hallmark of type 2 diabetes (T2D). Epigenetics may affect disease susceptibility. To describe the human methylome in pancreatic islets and determine the epigenetic basis of T2D, we analyzed DNA methylation of 479,927 CpG sites and the transcriptome in pancreatic islets from T2D and non-diabetic donors. We provide a detailed map of the global DNA methylation pattern in human islets, β- and α-cells. Genomic regions close to the transcription start site showed low degrees of methylation and regions further away from the transcription start site such as the gene body, 3'UTR and intergenic regions showed a higher degree of methylation. While CpG islands were hypomethylated, the surrounding 2 kb shores showed an intermediate degree of methylation, whereas regions further away (shelves and open sea) were hypermethylated in human islets, β- and α-cells. We identified 1,649 CpG sites and 853 genes, including TCF7L2, FTO and KCNQ1, with differential DNA methylation in T2D islets after correction for multiple testing. The majority of the differentially methylated CpG sites had an intermediate degree of methylation and were underrepresented in CpG islands (∼7%) and overrepresented in the open sea (∼60%). 102 of the differentially methylated genes, including CDKN1A, PDE7B, SEPT9 and EXOC3L2, were differentially expressed in T2D islets. Methylation of CDKN1A and PDE7B promoters in vitro suppressed their transcriptional activity. Functional analyses demonstrated that identified candidate genes affect pancreatic β- and α-cells as Exoc3l silencing reduced exocytosis and overexpression of Cdkn1a, Pde7b and Sept9 perturbed insulin and glucagon secretion in clonal β- and α-cells, respectively. Together, our data can serve as a reference methylome in human islets. We provide new target genes with altered DNA methylation and expression in human T2D islets that contribute to perturbed insulin and glucagon secretion. These results highlight the importance of epigenetics in the pathogenesis of T2D.
    PLoS Genetics 03/2014; 10(3):e1004160. · 8.17 Impact Factor
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    ABSTRACT: Here, we have investigated the role of inorganic phosphate (Pi) transport in mitochondria of rat clonal β-cells. In -toxin-permeabilized INS-1E cells, succinate and glycerol-3-phosphate increased mitochondrial ATP release which depends on exogenous ADP and Pi. In the presence of substrates, addition of Pi caused mitochondrial matrix acidification and hyperpolarisation which promoted ATP export. Dissipation of the mitochondrial pH gradient or pharmacological inhibition of Pi transport blocked the effects of Pi on electrochemical gradient and ATP export. Knock-down of the phosphate transporter PiC, however, neither prevented Pi-induced mitochondrial activation nor glucose-induced insulin secretion. Using (31)P-NMR we observed reduction of Pi pools during nutrient stimulation of INS-1E cells. Interestingly, Pi loss was less pronounced in mitochondria than in the cytosol. We conclude that matrix alkalinisation is necessary to maintain a mitochondrial Pi pool, at levels sufficient to stimulate energy metabolism in insulin-secreting cells beyond its role as a substrate for ATP synthesis.
    Molecular and Cellular Endocrinology 08/2013; · 4.24 Impact Factor
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    ABSTRACT: Insulin secretion is enhanced upon the binding of Glucagon-like peptide-1 (GLP-1) to its receptor (GLP1R) in pancreatic beta cells. Although a reduced expression of GLP1R in pancreatic islets from type 2 diabetic patients and hyperglycaemic rats has been established, it is still unknown if this is caused by differential DNA methylation of GLP1R in pancreatic islets of type 2 diabetic patients. In this study, DNA methylation levels of 12 CpG sites close to the transcription start site of GLP1R were analysed in pancreatic islets from 55 non-diabetic and 10 type 2 diabetic human donors as well as in beta and alpha cells isolated from human pancreatic islets. DNA methylation of GLP1R was related to GLP1R expression, HbA1c levels, and BMI. Moreover, mRNA expression of MECP2, DNMT1, DNMT3A and DNMT3B was analysed in pancreatic islets of the non-diabetic and type 2 diabetic donors. One CpG unit, at position +199 and +205 bp from the transcription start site, showed a small increase in DNA methylation in islets from donors with type 2 diabetes compared to non-diabetic donors (0.53%, p=0.02). Furthermore, DNA methylation levels of one CpG site located 376 bp upstream of the transcription start site of GLP1R correlated negatively with GLP1R expression (rho=-0.34, p=0.008) but positively with BMI and HbA1c (rho=0.30, p=0.02 and rho=0.30, p=0.03, respectively). This specific CpG site is located in an area with known SP1 and SP3 transcription factor binding sites. Moreover, when we compared the DNA methylation of the GLP1R promoter in isolated human beta and alpha cells, we found that it was higher in alpha-compared with beta-cells (p=0.009). Finally, there was a trend towards decreased DNMT3A expression (p=0.056) in type 2 diabetic compared with non-diabetic islets. Together, our study shows that while BMI and HbA1c are positively associated with DNA methylation levels of GLP1R, its expression is negatively associated with DNA methylation of GLP1R in human pancreatic islets.
    BMC Medical Genetics 07/2013; 14(1):76. · 2.45 Impact Factor
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    ABSTRACT: Insulin secretion is coupled to changes in ß-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH, and insulin secretion were measured within 15 minutes after stimulation of clonal INS-1 832/13 ß-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the ß-cell metabolic response. This was fulfilled by the NADPH/NADP+-ratio, which was elevated (8-fold, p<0.01) at 6 min after glucose stimulation. The NADPH/NADP+-ratio paralleled an increase in ribose-5-phosphate (+2.5-fold; p<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone suppressed ribose-5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 ß-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose-5-phosphate, which was prevented by trans-dehydroepiandrosterone. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in ß-cell stimulus-secretion coupling.
    Biochemical Journal 01/2013; · 4.78 Impact Factor
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    ABSTRACT: To shed light on islet cell molecular phenotype in human type 2 diabetes (T2D), we studied the trascriptome of non-diabetic (ND) and T2D islets to then focus on the ubiquitin-proteasome system (UPS), the major protein degradation pathway. We assessed gene expression, amount of ubiquitinated proteins, proteasome activity, and the effects of proteasome inhibition and prolonged exposure to palmitate. Microarray analysis identified more than one thousand genes differently expressed in T2D islets, involved in many structures and functions, with consistent alterations of the UPS. Quantitative RT-PCR demonstrated downregulation of selected UPS genes in T2D islets and beta cell fractions, with greater ubiquitin accumulation and reduced proteasome activity. Chemically induced reduction of proteasome activity was associated with lower glucose-stimulated insulin secretion, which was partly reproduced by palmitate exposure. These results show the presence of many changes in islet transcriptome in T2D islets and underline the importance of the association between UPS alterations and beta cell dysfunction in human T2D.
    Molecular and Cellular Endocrinology 12/2012; · 4.24 Impact Factor
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    ABSTRACT: A plethora of candidate genes have been identified for complex polygenic disorders, but the underlying disease mechanisms remain largely unknown. We explored the pathophysiology of type 2 diabetes (T2D) by analyzing global gene expression in human pancreatic islets. A group of coexpressed genes (module), enriched for interleukin-1-related genes, was associated with T2D and reduced insulin secretion. One of the module genes that was highly overexpressed in islets from T2D patients is SFRP4, which encodes secreted frizzled-related protein 4. SFRP4 expression correlated with inflammatory markers, and its release from islets was stimulated by interleukin-1β. Elevated systemic SFRP4 caused reduced glucose tolerance through decreased islet expression of Ca(2+) channels and suppressed insulin exocytosis. SFRP4 thus provides a link between islet inflammation and impaired insulin secretion. Moreover, the protein was increased in serum from T2D patients several years before the diagnosis, suggesting that SFRP4 could be a potential biomarker for islet dysfunction in T2D.
    Cell metabolism 11/2012; 16(5):625-33. · 17.35 Impact Factor
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    ABSTRACT: AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.
    Diabetologia 09/2012; · 6.88 Impact Factor
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    ABSTRACT: Glucose-induced insulin secretion from pancreatic β-cells depends on mitochondrial activation. In the organelle, glucose-derived pyruvate is metabolised along the oxidative and anaplerotic pathway to generate downstream signals leading to insulin granule exocytosis. Entry into the oxidative pathway is catalysed by pyruvate dehydrogenase (PDH) and controlled in part by phosphorylation of the PDH E1α subunit blocking enzyme activity. We find that glucose but not other nutrient secretagogues induce PDH E1α phosphorylation in INS-1E cells and rat islets. INS-1E cells and primary β-cells express pyruvate dehydrogenase kinase (PDK) 1, 2 and 3, which mediate the observed phosphorylation. In INS-1E cells, suppression of the two main isoforms, PDK1 and PDK3, almost completely prevented PDH E1α phosphorylation. Under basal glucose conditions, phosphorylation was barely detectable and therefore the enzyme almost fully active (90% of maximal). During glucose stimulation, PDH is only partially inhibited (to 78% of maximal). Preventing PDH phosphorylation in situ after suppression of PDK1, 2 and 3 neither enhanced pyruvate oxidation nor insulin secretion. In conclusion, although glucose stimulates E1α phosphorylation and therefore inhibits PDH activity, this control mechanism by itself does not alter metabolism-secretion coupling in INS-1E clonal β-cells.
    Biochimica et Biophysica Acta 07/2012; 1823(10):1815-24. · 4.66 Impact Factor
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    ABSTRACT: Human Krüppel-like factor 11 (hKLF11) has been characterised to both activate and inhibit human insulin promoter (hInsP) activity. Since KLF11 is capable to differentially regulate genes dependent on recruited cofactors, we investigated the effects of hKLF11 on cotransfected hInsP in both β-cells and non-β-cells. hKLF11 protein interacts with hp300 but not with hPDX1. Overexpressed hKLF11 stimulates PDX1-transactivation of hInsP in HEK293 non-β-cells, but confers inhibition in INS-1E β-cells. Both hKLF11 functions can be neutralised by the p300 inhibitor E1A, increased hp300 levels (INS-1E), dominant negative (DN)-PDX1 and by mutation of the PDX1 binding site A3 or the CACCC box. In summary, hKLF11 differentially regulates hInsP activity depending on the molecular context via modulation of p300:PDX1 interactions with the A3 element and CACCC box. We postulate that KLF11 has a role in fine-tuning insulin transcription in certain cellular situations rather than representing a major transcriptional activator or repressor of the insulin gene.
    Molecular and Cellular Endocrinology 07/2012; 363(1-2):20-6. · 4.24 Impact Factor
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    ABSTRACT: Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.
    Cell metabolism 07/2012; 16(1):122-34. · 17.35 Impact Factor
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    ABSTRACT: Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.
    Molecular Endocrinology 05/2012; 26(7):1203-12. · 4.20 Impact Factor
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    ABSTRACT: Insulin secretory granules (ISGs) are pivotal organelles of pancreatic ß-cells and represent a key participant to glucose homeostasis. Indeed, insulin is packed and processed within these vesicles before its release by exocytosis. It is therefore crucial to acquire qualitative and quantitative data on the ISG proteome, in order to increase our knowledge on ISG biogenesis, maturation and exocytosis. Despites efforts made in the past years, the coverage of the ISG proteome is still incomplete and comprises many potential protein contaminants most likely coming from suboptimal sample preparations. We developed here a 3-step gradient purification procedure combined to Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to further characterize the ISG protein content. Our results allowed to build three complementary proteomes containing 1/ proteins which are enriched in mature ISGs, 2/ proteins sharing multiple localizations including ISGs, and finally 3/ proteins sorted out from immature ISGs and/or co-purifying contaminants. As a proof of concept, the ProSAAS, a neuronal protein found in ISGs was further characterized and its granular localization proved. ProSAAS might represent a novel potential target allowing to better understand the defaults in insulin processing and secretion observed during type 2 diabetes progression. This article is part of a special issue entitled: Translational Proteomics.
    Journal of proteomics 04/2012; 75(15):4620-31. · 5.07 Impact Factor
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    ABSTRACT: Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.
    Molecular &amp Cellular Proteomics 03/2012; 11(8):244-54. · 7.25 Impact Factor
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    Kyu-Sang Park, Andreas Wiederkehr, Claes B Wollheim
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    ABSTRACT: Mitochondrial dynamics and distribution is critical for their role in bioenergetics and cell survival. We investigated the consequence of altered fission/fusion on mitochondrial function and motility in INS-1E rat clonal β-cells. Adenoviruses were used to induce doxycycline-dependent expression of wild type (WT-Mfn1) or a dominant negative mitofusin 1 mutant (DN-Mfn1). Mitochondrial morphology and motility were analyzed by monitoring mitochondrially-targeted red fluorescent protein. Adenovirus-driven overexpression of WT-Mfn1 elicited severe aggregation of mitochondria, preventing them from reaching peripheral near plasma membrane areas of the cell. Overexpression of DN-Mfn1 resulted in fragmented mitochondria with widespread cytosolic distribution. WT-Mfn1 overexpression impaired mitochondrial function as glucose- and oligomycin-induced mitochondrial hyperpolarization were markedly reduced. Viability of the INS-1E cells, however, was not affected. Mitochondrial motility was significantly reduced in WT-Mfn1 overexpressing cells. Conversely, fragmented mitochondria in DN-Mfn1 overexpressing cells showed more vigorous movement than mitochondria in control cells. Movement of these mitochondria was also less microtubule-dependent. These results suggest that Mfn1-induced hyperfusion leads to mitochondrial dysfunction and hypomotility, which may explain impaired metabolism-secretion coupling in insulin-releasing cells overexpressing Mfn1.
    Korean Journal of Physiology and Pharmacology 02/2012; 16(1):71-7. · 1.26 Impact Factor
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    ABSTRACT: Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells.
    The Journal of Lipid Research 12/2011; 53(3):412-20. · 4.73 Impact Factor
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    ABSTRACT: To evaluate whether healthy or diabetic adult mice can tolerate an extreme loss of pancreatic α-cells and how this sudden massive depletion affects β-cell function and blood glucose homeostasis. We generated a new transgenic model allowing near-total α-cell removal specifically in adult mice. Massive α-cell ablation was triggered in normally grown and healthy adult animals upon diphtheria toxin (DT) administration. The metabolic status of these mice was assessed in 1) physiologic conditions, 2) a situation requiring glucagon action, and 3) after β-cell loss. Adult transgenic mice enduring extreme (98%) α-cell removal remained healthy and did not display major defects in insulin counter-regulatory response. We observed that 2% of the normal α-cell mass produced enough glucagon to ensure near-normal glucagonemia. β-Cell function and blood glucose homeostasis remained unaltered after α-cell loss, indicating that direct local intraislet signaling between α- and β-cells is dispensable. Escaping α-cells increased their glucagon content during subsequent months, but there was no significant α-cell regeneration. Near-total α-cell ablation did not prevent hyperglycemia in mice having also undergone massive β-cell loss, indicating that a minimal amount of α-cells can still guarantee normal glucagon signaling in diabetic conditions. An extremely low amount of α-cells is sufficient to prevent a major counter-regulatory deregulation, both under physiologic and diabetic conditions. We previously reported that α-cells reprogram to insulin production after extreme β-cell loss and now conjecture that the low α-cell requirement could be exploited in future diabetic therapies aimed at regenerating β-cells by reprogramming adult α-cells.
    Diabetes 09/2011; 60(11):2872-82. · 7.90 Impact Factor

Publication Stats

14k Citations
2,285.25 Total Impact Points


  • 2011–2014
    • Lund University
      • • Department of Clinical Sciences, Malmö
      • • Department of Clinical Sciences
      Lund, Skåne, Sweden
    • Nestlé Institute of Health Sciences S.A.
      Lausanne, Vaud, Switzerland
    • The Rockefeller University
      New York City, New York, United States
  • 2004–2012
    • Universitätsklinikum Freiburg
      Freiburg an der Elbe, Lower Saxony, Germany
  • 1973–2012
    • University of Geneva
      • • Department of Cellular Physiology and Metabolism
      • • Department of Internal Medicine
      • • Department of Biochemistry
      • • Department of Surgery
      • • Division of Infectious Diseases
      Genève, Geneva, Switzerland
  • 2010–2011
    • Royal College of Surgeons in Ireland
      • Department of Physiology and Medical Physics
      Dublin, L, Ireland
  • 2008
    • Tohoku University
    • China-Japan Friendship Hospital
      Peping, Beijing, China
    • Peking Union Medical College Hospital
      Peping, Beijing, China
  • 2007
    • Novartis Institutes for BioMedical Research
      Cambridge, Massachusetts, United States
    • Roche
      Bâle, Basel-City, Switzerland
  • 2002
    • University of Münster
      Muenster, North Rhine-Westphalia, Germany
  • 1989–2001
    • University Hospital of Lausanne
      • Département de médecine
      Lausanne, Vaud, Switzerland
  • 1999
    • Hackensack University Medical Center
      Hackensack, New Jersey, United States
  • 1997
    • University of Padova
      • Department of Biomedical Sciences - DSB
      Padua, Veneto, Italy
  • 1988–1994
    • Centre universitaire romand de médecine légale Lausanne - Genève (CURML)
      Genève, Geneva, Switzerland
  • 1993
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1992
    • University of Iowa
      • Department of Internal Medicine
      Iowa City, Iowa, United States
  • 1990
    • Philipps University of Marburg
      Marburg, Hesse, Germany
  • 1985
    • University of Liverpool
      Liverpool, England, United Kingdom