C B Wollheim

Lund University, Lund, Skåne, Sweden

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Publications (391)2354.94 Total impact

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    ABSTRACT: Aims/hypothesis: The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Methods: Using partial correlation networks we identified a group of co-expressed genes ('module') including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a 'tethered ligand' to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. Results: We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single beta-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca(2+) release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced beta-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. Conclusions/interpretation: The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and beta-cell exhaustion in T2D.
    No preview · Article · Jan 2016 · Islets
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    S Xu · S M Nam · J-H Kim · R Das · S-K Choi · T T Nguyen · X Quan · S J Choi · C H Chung · E Y Lee · I-K Lee · A Wiederkehr · C B Wollheim · S-K Cha · K-S Park
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    ABSTRACT: Pathologic alterations in podocytes lead to failure of an essential component of the glomerular filtration barrier and proteinuria in chronic kidney diseases. Elevated levels of saturated free fatty acid (FFA) are harmful to various tissues, implemented in the progression of diabetes and its complications such as proteinuria in diabetic nephropathy. Here, we investigated the molecular mechanism of palmitate cytotoxicity in cultured mouse podocytes. Incubation with palmitate dose-dependently increased cytosolic and mitochondrial reactive oxygen species, depolarized the mitochondrial membrane potential, impaired ATP synthesis and elicited apoptotic cell death. Palmitate not only evoked mitochondrial fragmentation but also caused marked dilation of the endoplasmic reticulum (ER). Consistently, palmitate upregulated ER stress proteins, oligomerized stromal interaction molecule 1 (STIM1) in the subplasmalemmal ER membrane, abolished the cyclopiazonic acid-induced cytosolic Ca(2+) increase due to depletion of luminal ER Ca(2+). Palmitate-induced ER Ca(2+) depletion and cytotoxicity were blocked by a selective inhibitor of the fatty-acid transporter FAT/CD36. Loss of the ER Ca(2+) pool induced by palmitate was reverted by the phospholipase C (PLC) inhibitor edelfosine. Palmitate-dependent activation of PLC was further demonstrated by following cytosolic translocation of the pleckstrin homology domain of PLC in palmitate-treated podocytes. An inhibitor of diacylglycerol (DAG) kinase, which elevates cytosolic DAG, strongly promoted ER Ca(2+) depletion by low-dose palmitate. GF109203X, a PKC inhibitor, partially prevented palmitate-induced ER Ca(2+) loss. Remarkably, the mitochondrial antioxidant mitoTEMPO inhibited palmitate-induced PLC activation, ER Ca(2+) depletion and cytotoxicity. Palmitate elicited cytoskeletal changes in podocytes and increased albumin permeability, which was also blocked by mitoTEMPO. These data suggest that oxidative stress caused by saturated FFA leads to mitochondrial dysfunction and ER Ca(2+) depletion through FAT/CD36 and PLC signaling, possibly contributing to podocyte injury.
    Full-text · Article · Nov 2015 · Cell Death & Disease
  • Claes B Wollheim · Pierre Maechler
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    ABSTRACT: Type 2 diabetes occurs when the pancreatic beta cells fail to meet the increased insulin requirement in insulin-resistant individuals. A new therapeutic approach targeting glutamate receptors on beta cells improves insulin secretion and preserves beta cell mass.
    No preview · Article · Apr 2015 · Nature medicine
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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.
    No preview · Article · Apr 2015 · AJP Endocrinology and Metabolism
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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.
    No preview · Article · Mar 2015 · Biochemical Journal
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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.
    Full-text · Article · Dec 2014 · Journal of Biological Chemistry
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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.
    Full-text · Article · Dec 2014 · Human Molecular Genetics
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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.
    Full-text · Article · Sep 2014 · Proceedings of the National Academy of Sciences
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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.
    Full-text · Article · Jul 2014 · Human Molecular Genetics
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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.
    Full-text · Article · Mar 2014 · PLoS Genetics
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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.
    No preview · Article · Aug 2013 · Molecular and Cellular Endocrinology
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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.
    Full-text · Article · Jul 2013 · BMC Medical Genetics

  • No preview · Article · Feb 2013
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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.
    Preview · Article · Jan 2013 · Biochemical Journal
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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.
    No preview · Article · Dec 2012 · Molecular and Cellular Endocrinology
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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.
    Full-text · Article · Nov 2012 · Cell metabolism
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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.
    Full-text · Article · Sep 2012 · Diabetologia
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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.
    Full-text · Article · Jul 2012 · Biochimica et Biophysica Acta
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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.
    No preview · Article · Jul 2012 · Molecular and Cellular Endocrinology
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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.
    Full-text · Article · Jul 2012 · Cell metabolism

Publication Stats

21k Citations
2,354.94 Total Impact Points


  • 2012-2015
    • Lund University
      • Department of Clinical Sciences, Malmö
      Lund, Skåne, Sweden
  • 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
  • 2004-2012
    • Universitätsklinikum Freiburg
      Freiburg an der Elbe, Lower Saxony, Germany
    • RIKEN
      Вако, Saitama, Japan
  • 2003-2011
    • Centre Hospitalier Universitaire de Dijon
      Dijon, Bourgogne, France
    • University of Lausanne
      Lausanne, Vaud, Switzerland
  • 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
  • 1994-2001
    • University Hospital of Lausanne
      • Département de médecine
      Lausanne, Vaud, Switzerland
    • Universität des Saarlandes
      Saarbrücken, Saarland, Germany
  • 1983-2001
    • French National Centre for Scientific Research
      • Institut de Génomique Fonctionnelle (IGF)
      Lutetia Parisorum, Île-de-France, France
  • 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
  • 1985-1989
    • University of Liverpool
      • Department of Cellular and Molecular Physiology
      Liverpool, England, United Kingdom
  • 1984
    • Università di Pisa
      • Department of Clinical and Experimental Medicine
      Pisa, Tuscany, Italy