Publications by authors named "Marina Casimir"

13 Publications

  • Page 1 of 1

Development and Validation of a Targeted Next-Generation Sequencing Gene Panel for Children With Neuroinflammation.

JAMA Netw Open 2019 10 2;2(10):e1914274. Epub 2019 Oct 2.

Infection, Inflammation and Rheumatology Section, University College London Great Ormond Street Institute of Child Health, London, United Kingdom.

Importance: Neuroinflammatory disorders are a range of severe neurological disorders causing brain and spinal inflammation and are now increasingly recognized in the pediatric population. They are often characterized by marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis.

Objective: To develop and evaluate a next-generation sequencing panel targeting genes causing neuroinflammation or mimicking neuroinflammation.

Design, Setting, And Participants: Cohort study in which a total of 257 genes associated with monogenic neuroinflammation and/or cerebral vasculopathy, including monogenic noninflammatory diseases mimicking these entities, were selected. A customized enrichment capture array, the neuroinflammation gene panel (NIP), was created. Targeted high-coverage sequencing was applied to DNA samples taken from eligible patients referred to Great Ormond Street Hospital in London, United Kingdom, between January 1, 2017, and January 30, 2019, because of onset of disease early in life, family history, and/or complex neuroinflammatory phenotypes.

Main Outcomes And Measures: The main outcome was the percentage of individuals with definitive molecular diagnoses, variant classification, and clinical phenotyping of patients with pathogenic variants identified using the NIP panel. The NIP panel was initially validated in 16 patients with known genetic diagnoses.

Results: The NIP was both sensitive (95%) and specific (100%) for detection of known mutations, including gene deletions, copy number variants, small insertions and deletions, and somatic mosaicism with allele fraction as low as 3%. Prospective testing of 60 patients (30 [50%] male; median [range] age, 9.8 [0.8-20] years) presenting with heterogeneous neuroinflammatory phenotypes revealed at least 1 class 5 (clearly pathogenic) variant in 9 of 60 patients (15%); 18 of 60 patients (30%) had at least 1 class 4 (likely pathogenic) variant. Overall, a definitive molecular diagnosis was established in 12 of 60 patients (20%).

Conclusions And Relevance: The NIP was associated with molecular diagnosis in this cohort and complemented routine laboratory and radiological workup of patients with neuroinflammation. Unexpected genotype-phenotype associations in patients with pathogenic variants deviating from the classic phenotype were identified. Obtaining an accurate molecular diagnosis in a timely fashion informed patient management, including successful targeted treatment in some instances and early institution of hematopoietic stem cell transplantation in others.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.14274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824223PMC
October 2019

Resveratrol long-term treatment differentiates INS-1E beta-cell towards improved glucose response and insulin secretion.

Pflugers Arch 2019 02 11;471(2):337-345. Epub 2018 Oct 11.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva Medical Centre, 1 rue Michel-Servet, 1211, Geneva 4, Switzerland.

The clonal INS-1E beta-cell line has proven to be instrumental for numerous studies investigating the mechanisms of glucose-stimulated insulin secretion. The composition of its culture medium has not changed over the years, although some compounds have been recently highlighted for their effects on tissue differentiation. The present study investigated the effects of long-term treatment of INS-1E cells with 1 μM resveratrol on glucose-stimulated insulin secretion, testing an extended glucose dose response. The data demonstrate that chronic exposure to low-dose resveratrol expands the range of the glucose dose response of INS-1E cells beyond 15 mM glucose. We also assessed whether such beneficial effects could be retained after resveratrol withdrawal from the culture medium. This was not the case as INS-1E cells deprived of resveratrol returned to the phenotype of naïve cells, i.e., exhibiting a plateau phase at 15 mM glucose. Of note, although resveratrol has antioxidant properties, it cannot substitute for β-mercaptoethanol normally present in the medium of INS-1E cells as a reducing agent. In conclusion, the addition of resveratrol as a standard component of the culture medium of INS-1E cells improves glucose-stimulated insulin secretion.
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http://dx.doi.org/10.1007/s00424-018-2215-zDOI Listing
February 2019

Cytokines induce endoplasmic reticulum stress in human, rat and mouse beta cells via different mechanisms.

Diabetologia 2015 Oct 23;58(10):2307-16. Epub 2015 Jun 23.

ULB-Center for Diabetes Research, Universitè Libre de Bruxelles (ULB), Route de Lennik, 808-CP618, 1070, Brussels, Belgium.

Aims/hypothesis: Proinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca(2+) transporting ATPase sarco/endoplasmic reticulum Ca(2+) pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field.

Methods: Rat and mouse insulin-producing cells, human pancreatic islets and human EndoC-βH1 cells were exposed to the cytokines IL-1β, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells.

Results: NO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis.

Conclusions/interpretation: These observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.
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http://dx.doi.org/10.1007/s00125-015-3669-6DOI Listing
October 2015

The role of the transcription factor ETV5 in insulin exocytosis.

Diabetologia 2014 Feb 5;57(2):383-91. Epub 2013 Nov 5.

Department of Internal Medicine, University of Cincinnati, 2170 East Galbraith Road, Cincinnati, OH, 45237, USA.

Aims/hypothesis: Genome-wide association studies have revealed an association of the transcription factor ETS variant gene 5 (ETV5) with human obesity. However, its role in glucose homeostasis and energy balance is unknown.

Methods: Etv5 knockout (KO) mice were monitored weekly for body weight (BW) and food intake. Body composition was measured at 8 and 16 weeks of age. Glucose metabolism was studied, and glucose-stimulated insulin secretion was measured in vivo and in vitro.

Results: Etv5 KO mice are smaller and leaner, and have a reduced BW and lower fat mass than their wild-type controls on a chow diet. When exposed to a high-fat diet, KO mice are resistant to diet-induced BW gain. Despite a greater insulin sensitivity, KO mice have profoundly impaired glucose tolerance associated with impaired insulin secretion. Morphometric analysis revealed smaller islets and a reduced beta cell size in the pancreatic islets of Etv5 KO mice. Knockdown of ETV5 in an insulin-secreting cell line or beta cells from human donors revealed intact mitochondrial and Ca(2+) channel activity, but reduced insulin exocytosis.

Conclusion/interpretation: This work reveals a critical role for ETV5 in specifically regulating insulin secretion both in vitro and in vivo.
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http://dx.doi.org/10.1007/s00125-013-3096-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947344PMC
February 2014

In vivo role of focal adhesion kinase in regulating pancreatic β-cell mass and function through insulin signaling, actin dynamics, and granule trafficking.

Diabetes 2012 Jul 12;61(7):1708-18. Epub 2012 Apr 12.

Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic β-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)-driven Cre-loxP recombination system to specifically delete FAK in pancreatic β-cells. These RIPcre(+)fak(fl/fl) mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced β-cell viability and proliferation resulting in decreased β-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal-related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient β-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca(2+) influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking.
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http://dx.doi.org/10.2337/db11-1344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379666PMC
July 2012

SUMOylation regulates insulin exocytosis downstream of secretory granule docking in rodents and humans.

Diabetes 2011 Mar 24;60(3):838-47. Epub 2011 Jan 24.

Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.

Objective: The reversible attachment of small ubiquitin-like modifier (SUMO) proteins controls target localization and function. We examined an acute role for the SUMOylation pathway in downstream events mediating insulin secretion.

Research Design And Methods: We studied islets and β-cells from mice and human donors, as well as INS-1 832/13 cells. Insulin secretion, intracellular Ca(2+), and β-cell exocytosis were monitored after manipulation of the SUMOylation machinery. Granule localization was imaged by total internal reflection fluorescence and electron microscopy; immunoprecipitation and Western blotting were used to examine the soluble NSF attachment receptor (SNARE) complex formation and SUMO1 interaction with synaptotagmin VII.

Results: SUMO1 impairs glucose-stimulated insulin secretion by blunting the β-cell exocytotic response to Ca(2+). The effect of SUMO1 to impair insulin secretion and β-cell exocytosis is rapid and does not require altered gene expression or insulin content, is downstream of granule docking at the plasma membrane, and is dependent on SUMO-conjugation because the deSUMOylating enzyme, sentrin/SUMO-specific protease (SENP)-1, rescues exocytosis. SUMO1 coimmunoprecipitates with the Ca(2+) sensor synaptotagmin VII, and this is transiently lost upon glucose stimulation. SENP1 overexpression also disrupts the association of SUMO1 with synaptotagmin VII and mimics the effect of glucose to enhance exocytosis. Conversely, SENP1 knockdown impairs exocytosis at stimulatory glucose levels and blunts glucose-dependent insulin secretion from mouse and human islets.

Conclusions: SUMOylation acutely regulates insulin secretion by the direct and reversible inhibition of β-cell exocytosis in response to intracellular Ca(2+) elevation. The SUMO protease, SENP1, is required for glucose-dependent insulin secretion.
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http://dx.doi.org/10.2337/db10-0440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046844PMC
March 2011

A role for pancreatic beta-cell secretory hyperresponsiveness in catch-up growth hyperinsulinemia: Relevance to thrifty catch-up fat phenotype and risks for type 2 diabetes.

Nutr Metab (Lond) 2011 Jan 18;8(1). Epub 2011 Jan 18.

Department of Medicine / Physiology, University of Fribourg, Switzerland.

Current notions about mechanisms by which catch-up growth predisposes to later type 2 diabetes center upon those that link hyperinsulinemia with an accelerated rate of fat deposition (catch-up fat). Using a rat model of semistarvation-refeeding in which catch-up fat is driven solely by elevated metabolic efficiency associated with hyperinsulinemia, we previously reported that insulin-stimulated glucose utilization is diminished in skeletal muscle but increased in white adipose tissue. Here, we investigated the possibility that hyperinsulinemia during catch-up fat can be contributed by changes in the secretory response of pancreatic beta-cells to glucose. Using the rat model of semistarvation-refeeding showing catch-up fat and hyperinsulinemia, we compared isocalorically refed and control groups for potential differences in pancreatic morphology and in glucose-stimulated insulin secretion during in situ pancreas perfusions as well as ex vivo isolated islet perifusions. Between refed and control animals, no differences were found in islet morphology, insulin content, and the secretory responses of perifused isolated islets upon glucose stimulation. By contrast, the rates of insulin secretion from in situ perfused pancreas showed that raising glucose from 2.8 to 16.7 mmol/l produced a much more pronounced increase in insulin release in refed than in control groups (p < 0.01). These results indicate a role for islet secretory hyperresponsiveness to glucose in the thrifty mechanisms that drive catch-up fat through glucose redistribution between skeletal muscle and adipose tissue. Such beta-cell hyperresponsiveness to glucose may be a key event in the link between catch-up growth, hyperinsulinemia and risks for later type 2 diabetes.
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http://dx.doi.org/10.1186/1743-7075-8-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033236PMC
January 2011

Role of mitochondria in beta-cell function and dysfunction.

Adv Exp Med Biol 2010 ;654:193-216

Department of Cell Physiology and Metabolism, University of Geneva Medical Centre, CH-1211 Geneva 4, Switzerland.

Pancreatic beta-cells are poised to sense glucose and other nutrient secretagogues to regulate insulin exocytosis, thereby maintaining glucose homeostasis. This process requires translation of metabolic substrates into intracellular messengers recognized by the exocytotic machinery. Central to this metabolism-secretion coupling, mitochondria integrate and generate metabolic signals, thereby connecting glucose recognition to insulin exocytosis. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin release. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. Mitochondrial defects, such as mutations and reactive oxygen species production, are discussed in the context of beta-cell failure that may participate to the etiology of diabetes.
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http://dx.doi.org/10.1007/978-90-481-3271-3_9DOI Listing
June 2010

Sclerocarya birrea (Anacardiaceae) stem-bark extract corrects glycaemia in diabetic rats and acts on beta-cells by enhancing glucose-stimulated insulin secretion.

J Endocrinol 2010 Apr 8;205(1):79-86. Epub 2010 Jan 8.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, Geneva 4, Switzerland.

Sclerocarya birrea is a plant widely used as traditional medication for the treatment of diabetes in sub-Saharan regions. However, the mechanism of action is unknown and only hypoglycaemic effects of S. birrea extract (SBE) in diabetic rats have been reported to date. Here, we tested aqueous extracts of S. birrea on insulin-secreting INS-1E cells and isolated rat islets. Following 24 h of treatment at 5 microg/ml, the extract markedly potentiated glucose-stimulated insulin secretion. Neither basal insulin release nor non-nutrient stimulation was affected. The potentiation of the secretory response at stimulatory glucose appeared after 12 h of treatment. No acute effects were observed and, at the effective concentration, SBE was safe regarding cell integrity and differentiation. The mechanism of action of the SBE was related to glucose metabolism as both ATP generation and glucose oxidation were enhanced following the 24-h treatment. In streptozotocin-induced diabetic rats, SBE administration corrected glycaemia and restored plasma insulin levels after 2 weeks of treatment. These data show direct action of S. birrea on insulin-secreting cells and favour further delineation for use of the plant in the management of diabetes.
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http://dx.doi.org/10.1677/JOE-09-0311DOI Listing
April 2010

Silencing of the mitochondrial NADH shuttle component aspartate-glutamate carrier AGC1/Aralar1 in INS-1E cells and rat islets.

Biochem J 2009 Dec 10;424(3):459-66. Epub 2009 Dec 10.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

Transfer of reducing equivalents between cytosolic compartments and the mitochondrial matrix is mediated by NADH shuttles. Among these, the malate-aspartate shuttle has been proposed to play a major role in beta-cells for the control of glucose-stimulated insulin secretion. AGC1 or Aralar1 (aspartate-glutamate carrier 1) is a key component of the malate-aspartate shuttle. Overexpression of AGC1 increases the capacity of the malate-aspartate shuttle, resulting in enhanced metabolism-secretion coupling, both in INS-1E cells and rat islets. In the present study, knockdown of AGC1 was achieved in the same beta-cell models, using adenovirus-mediated delivery of shRNA (small-hairpin RNA). Compared with control INS-1E cells, down-regulation of AGC1 blunted NADH formation (-57%; P<0.05), increased lactate production (+16%; P<0.001) and inhibited glucose oxidation (-22%; P<0.01). This correlated with a reduced secretory response at 15 mM glucose (-25%; P<0.05), while insulin release was unchanged at intermediate 7.5 mM and basal 2.5 mM glucose. In isolated rat islets, efficient AGC1 knockdown did not alter insulin exocytosis evoked by 16.7 mM glucose. However, 4 mM amino-oxyacetate, commonly used to block transaminases of the malate-aspartate shuttle, inhibited glucose-stimulated insulin secretion to similar extents in INS-1E cells (-66%; P<0.01) and rat islets (-56%; P<0.01). These results show that down-regulation of the key component of the malate-aspartate shuttle AGC1 reduced glucose-induced oxidative metabolism and insulin secretion in INS-1E cells, whereas similar AGC1 knockdown in rat islets did not affect their secretory response.
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http://dx.doi.org/10.1042/BJ20090729DOI Listing
December 2009

Mitochondrial glutamate carrier GC1 as a newly identified player in the control of glucose-stimulated insulin secretion.

J Biol Chem 2009 Sep 7;284(37):25004-14. Epub 2009 Jul 7.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland.

The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.
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http://dx.doi.org/10.1074/jbc.M109.015495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757205PMC
September 2009

Tissue specificity of mitochondrial glutamate pathways and the control of metabolic homeostasis.

Biochim Biophys Acta 2008 Jul-Aug;1777(7-8):965-72. Epub 2008 Apr 30.

Department of Cell Physiology and Metabolism, Geneva University Medical Centre, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

Glutamate is implicated in numerous metabolic and signalling functions that vary according to specific tissues. Glutamate metabolism is tightly controlled by activities of mitochondrial enzymes and transmembrane carriers, in particular glutamate dehydrogenase and mitochondrial glutamate carriers that have been identified in recent years. It is remarkable that, although glutamate-specific enzymes and transporters share similar properties in most tissues, their regulation varies greatly according to particular organs in order to achieve tissue specific functions. This is illustrated in this review when comparing glutamate handling in liver, brain, and pancreatic beta-cells. We describe the main cellular glutamate pathways and their specific functions in different tissues, ultimately contributing to the control of metabolic homeostasis at the organism level.
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http://dx.doi.org/10.1016/j.bbabio.2008.04.031DOI Listing
August 2008

Mitochondrial activation and the pyruvate paradox in a human cell line.

FEBS Lett 2004 Dec;578(3):224-8

Department of Cell Physiology and Metabolism, University Medical Centre, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland.

Pyruvate promotes hyperpolarization of the inner mitochondrial membrane. However, in isolated mitochondria, pyruvate could participate in a futile cycle leading to mitochondrial depolarization. Here, we investigated this paradox in intact human cells by measuring parameters reflecting mitochondrial activation in response to 1 mM pyruvate and 5 mM glucose. NAD(P)H levels were elevated similarly by both substrates. Conversely, pyruvate induced a first transient phase of mitochondrial depolarization before the establishment of the expected sustained hyperpolarization. This correlated with kinetics of cytosolic ATP levels exhibiting a first phase decrease followed by an increase. Therefore, pyruvate transiently depolarizes mitochondria and reduces ATP in intact cells.
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http://dx.doi.org/10.1016/j.febslet.2004.10.088DOI Listing
December 2004