C B Wollheim

University of Geneva, Genève, Geneva, Switzerland

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Publications (385)2332.84 Total impact

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    ABSTRACT: Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters prevented ΔΨm hyperpolarization, superoxide generation and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants, mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, insulin content and secretion in INS-1E cells as well as dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content, defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states. Copyright © 2015, American Journal of Physiology - Endocrinology and Metabolism.
    AJP Endocrinology and Metabolism 04/2015; 308(11):ajpendo.00009.2015. DOI:10.1152/ajpendo.00009.2015 · 3.79 Impact Factor
  • Claes B Wollheim · Pierre Maechler ·

    Nature medicine 04/2015; 21(4):310-311. DOI:10.1038/nm.3835 · 27.36 Impact Factor
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    ABSTRACT: Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 Diabetes, but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 cells and compared it to insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells, respectively, secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. While glycolytic metabolism was similar in the two cell-lines, Krebs-cycle metabolism, respiration and ATP-levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate, abolished glucose-provoked ATP production and hormone secretion from αTC1-6, but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol-3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerolphosphate shuttle compared to malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerolphosphate shuttle augments the malate-aspartate shuttle in the INS-1 832/13 but not in αTC1-6 cells. These results were confirmed in mouse islets, where phenyl succinate abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerolphosphate shuttle, was higher in sorted primary ß- than α-cells. Thus, suppressed glycerolphosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signaling is independent on mitochondrial shuttles.
    Biochemical Journal 03/2015; 468(1). DOI:10.1042/BJ20140697 · 4.40 Impact Factor
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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; 290(7). DOI:10.1074/jbc.M114.632547 · 4.57 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; 24(7). DOI:10.1093/hmg/ddu610 · 6.39 Impact Factor
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    ABSTRACT: Genetic variation can modulate gene expression, and thereby phenotypic 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 hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 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 understanding of how genetic variation can influence glucose metabolism.
    Proceedings of the National Academy of Sciences 09/2014; 14(38). DOI:10.1073/pnas.1402665111 · 9.67 Impact Factor
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    ABSTRACT: Genome-wide association studies have revealed >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; 23(24). DOI:10.1093/hmg/ddu359 · 6.39 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. DOI:10.1371/journal.pgen.1004160 · 7.53 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; 381(1-2). DOI:10.1016/j.mce.2013.08.003 · 4.41 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. DOI:10.1186/1471-2350-14-76 · 2.08 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; 450(3). DOI:10.1042/BJ20121349 · 4.40 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; 367(1-2). DOI:10.1016/j.mce.2012.12.001 · 4.41 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. DOI:10.1016/j.cmet.2012.10.009 · 17.57 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; 55(12). DOI:10.1007/s00125-012-2716-9 · 6.67 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. DOI:10.1016/j.bbamcr.2012.07.005 · 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. DOI:10.1016/j.mce.2012.07.003 · 4.41 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. DOI:10.1016/j.cmet.2012.06.006 · 17.57 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. DOI:10.1210/me.2012-1004 · 4.02 Impact Factor
  • N Perakakis · D Danassi · E Tsaroucha · K Laubner · N Rimmer · M Alt · H Wang · CB Wollheim · J Seufert · G Päth ·

    Diabetologie und Stoffwechsel 05/2012; 7(S 01). DOI:10.1055/s-0032-1314537 · 0.33 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. DOI:10.1016/j.jprot.2012.04.023 · 3.89 Impact Factor

Publication Stats

19k Citations
2,332.84 Total Impact Points


  • 1973-2015
    • University of Geneva
      • • Department of Cellular Physiology and Metabolism
      • • Department of Internal Medicine
      • • Department of Biochemistry
      • • Department of Rehabilitation and Geriatrics
      • • Department of Surgery
      • • Division of Infectious Diseases
      Genève, Geneva, Switzerland
  • 2012-2014
    • Lund University
      • Department of Clinical Sciences, Malmö
      Lund, Skåne, Sweden
  • 2004-2012
    • Universitätsklinikum Freiburg
      Freiburg an der Elbe, Lower Saxony, Germany
    • RIKEN
      Вако, Saitama, Japan
  • 2011
    • The Rockefeller University
      New York City, New York, United States
  • 2003-2011
    • Centre Hospitalier Universitaire de Dijon
      Dijon, Bourgogne, France
    • University of Lausanne
      Lausanne, Vaud, Switzerland
  • 2010
    • Royal College of Surgeons in Ireland
      • Department of Physiology and Medical Physics
      Dublin, L, Ireland
  • 2008
    • China-Japan Friendship Hospital
      Peping, Beijing, China
    • Peking Union Medical College Hospital
      Peping, Beijing, China
  • 2007-2008
    • Tohoku University
      • Department of Developmental Biology and Neuroscience
    • Novartis Institutes for BioMedical Research
      Cambridge, Massachusetts, United States
  • 2002
    • University of Texas at Dallas
      Richardson, Texas, United States
  • 2001
    • University of Barcelona
      Barcino, Catalonia, Spain
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1994-2001
    • University Hospital of Lausanne
      • Département de médecine
      Lausanne, Vaud, Switzerland
    • Universität des Saarlandes
      Saarbrücken, Saarland, Germany
  • 1999
    • Hackensack University Medical Center
      Hackensack, New Jersey, United States
  • 1997-1998
    • Centre universitaire romand de médecine légale Lausanne - Genève (CURML)
      Genève, Geneva, Switzerland
  • 1986-1997
    • University of Padova
      • Department of Biomedical Sciences - DSB
      Padua, Veneto, Italy
  • 1993
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1992
    • University of Iowa
      • Department of Internal Medicine
      Iowa City, Iowa, United States
  • 1985-1989
    • University of Liverpool
      • Department of Cellular and Molecular Physiology
      Liverpool, England, United Kingdom