Search

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro .Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo ,beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Centronuclear myopathies (CNMs) are a group of rare and early-onset genetic muscle disorders for which there is no curative treatment. This fatal disease is known to be caused by mutations in several genes, including myotubularin (MTM1) and amphiphysin 2 (BIN1). Mutations in the MTM1 gene cause X-linked myotubular myopathy (XLCNM), which is the most common and severe form of CNM. In addition, mutations in the BIN1 gene have been associated with autosomal dominant and recessive CNM, a late-onset myopathy that is usually less severe. Several defects at the cellular level have been observed in CNM, such as abnormal triad structure and impaired excitation-contraction coupling, which is critical for skeletal muscle contraction.In our laboratory, we generated mtm1 zebrafish knockout mutants (KO) using CRISPR/Cas9 technology. These mtm1KO mutant show progressive degeneration of fin folds, defective motor behavior, and early mortality. However, no defects in muscular ultrastructure were observed. Recent studies have shown that mutant mRNA can trigger genetic compensation. To avoid this compensation and any active transcription of the mutant mRNA, we generated mtm1 mutant that has a large deletion encompassing the entire promoter of the mtm1 gene. Surprisingly, this promoterless (PL) mutant shows a similar phenotype to the KO mutants, including the lack of a muscle phenotype. This result suggests that no compensation was induced in the KO mutant. We also generated a bin1b mutant lacking a promoter (bin1bPL), in which we found no differences from WT larvae and adult fish. These two mutants challenge the roles of mtm1 and bin1b in zebrafish muscle. Finally, we generated an mtm1-bin1b mutant lacking a double promoter (DPL). This zebrafish mutant not only shows the same features as the mtm1PL mutants, but also exhibits severe muscle defects at both the structural and functional levels that recapitulate human CNM.In summary, we have generated a zebrafish mutant that exhibits a st, Les myopathies centronucléaires (CNM) définissent un groupe de maladies musculaires génétiques rares et à apparition précoce pour lesquelles aucun traitement curatif n'est disponible actuellement. Des mutations dans plusieurs gènes, notamment la myotubularine (MTM1) et l'amphiphysine 2 (BIN1) causent ces maladies létales. Les mutations du gène MTM1 sont à l'origine de la myopathie myotubulaire liée à l'X (XLCNM), qui est la forme la plus fréquente et la plus grave de CNM. D’autre part, des mutations dans le gène BIN1 ont été associées à des CNM autosomiques dominantes et récessives qui sont généralement moins graves et se manifestent plus tardivement chez les patients. Dans la CNM, plusieurs défauts ont été observés au niveau cellulaire, comme une structure anormale des triades et un dysfonctionnement du couplage excitation-contraction, crucial pour la contraction des muscles squelettiques.Dans notre laboratoire, nous avons généré un mutant mtm1 knockout (KO) de poisson zèbre en utilisant la technologie CRISPR/Cas9. Ce mutant mtm1KO présente une dégénérescence progressive des nageoires, une diminution du comportement moteur et une mortalité précoce. Cependant, aucun défaut dans l'ultrastructure musculaire n'a été observé chez ce mutant, contrairement à ce qui était attendu. Des études récentes ont montré, dans différents modèles, que l’expression d’ARNm mutés peut déclencher une compensation génétique et une réduction de la gravité ou une absence du phénotype attendu. Pour éviter cette compensation et toute transcription d’ARNm muté, nous avons généré des mutants mtm1 portant de larges délétions englobant la totalité du promoteur du gène mtm1. De manière surprenante, ce mutant sans promoteur (« promoterless mutant », PL) présente un phénotype similaire au mutant KO, y compris par l'absence de phénotype musculaire. Ce résultat suggère qu'aucune compensation n'a été déclenchée chez le mutant KO. N, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome., info:eu-repo/semantics/published

Deficient as well as excessive/prolonged endoplasmic reticulum (ER) stress signaling can lead to pancreatic β cell failure and the development of diabetes. Saturated free fatty acids (FFAs) such as palmitate induce lipotoxic ER stress in pancreatic β cells. One of the main ER stress response pathways is under the control of the protein kinase R-like endoplasmic reticulum kinase (PERK), leading to phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α). The antihypertensive drug guanabenz has been shown to inhibit eIF2α dephosphorylation and protect cells from ER stress. Here we examined whether guanabenz protects pancreatic β cells from lipotoxicity. Guanabenz induced β cell dysfunction in vitro and in vivo in rodents and led to impaired glucose tolerance. The drug significantly potentiated FFA-induced cell death in clonal rat β cells and in rat and human islets. Guanabenz enhanced FFA-induced eIF2α phosphorylation and expression of the downstream proapoptotic gene C/EBP homologous protein (CHOP), which mediated the sensitization to lipotoxicity. Thus, guanabenz does not protect β cells from ER stress; instead, it potentiates lipotoxic ER stress through PERK/eIF2α/CHOP signaling. These data demonstrate the crucial importance of the tight regulation of eIF2α phosphorylation for the normal function and survival of pancreatic β cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Pancreatic β-cell lipotoxicity is a central feature of the pathogenesis of type 2 diabetes. To study the mechanism by which fatty acids cause β-cell death and develop novel approaches to prevent it, a high-throughput screen on the β-cell line INS1 was carried out. The cells were exposed to palmitate to induce cell death and compounds that reversed palmitate-induced cytotoxicity were ascertained. Hits from the screen were analyzed by an increasingly more stringent testing funnel, ending with studies on primary human islets treated with palmitate. MAP4K4 inhibitors, which were not part of the screening libraries but were ascertained by a bioinformatics analysis, and the endocannabinoid anandamide were effective at inhibiting palmitate-induced apoptosis in INS1 cells as well as primary rat and human islets. These targets could serve as the starting point for the development of therapeutics for type 2 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, andhumanβ-cells exposed to cytokines or thapsigargininduced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure ofβ-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published

Pancreatic β-cell dysfunction and death are central in the pathogenesis of type 2 diabetes. Saturated fatty acids cause β-cell failure and contribute to diabetes development in genetically predisposed individuals.Here we used RNA-sequencing to map transcripts expressed in five palmitate-treated human islet preparations, observing 1,325 modified genes. Palmitate induced fatty acid metabolism and endoplasmic reticulum (ER) stress. Functional studies identified novel mediators of adaptive ER stress signaling. Palmitate modified genes regulating ubiquitin and proteasome function, autophagy and apoptosis. Inhibition of autophagic flux and lysosome function contributed to lipotoxicity. Palmitate inhibited transcription factors controlling β-cell phenotype including PAX4 and GATA6. 59 type 2 diabetes candidate genes were expressed in human islets, and 11 were modified by palmitate. Palmitate modified expression of 17 splicing factors and shifted alternative splicing of 3,525 transcripts. Ingenuity Pathway Analysis of modified transcripts and genes confirmed that top changed functions related to cell death. DAVID analysis of transcription binding sites in palmitate-modified transcripts revealed a role for PAX4, GATA and the ER stress response regulators XBP1 and ATF6.This human islet transcriptome study identified novel mechanisms of palmitate-induced β-cell dysfunction and death. The data point to crosstalk between metabolic stress and candidate genes at the β-cell level., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Scope: A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established. Methods and results: Mice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals. Conclusions: We identified an antidiabetic phytochemical that protects pancreatic beta cells from ER stress and apoptosis induced by high-fat diet/lipotoxicity. At the tissue level, this led to a tripling of beta cell mass. At the molecular level, the protective effect of the phytochemical was mediated by increasing BCL2 expression in beta cells., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

The prevalence of obesity and type 1 diabetes in children is increasing worldwide. Insulin resistance and augmented circulating free fatty acids associated with obesity may cause pancreatic β-cell endoplasmic reticulum (ER) stress. We tested the hypothesis that mild ER stress predisposes β-cells to an exacerbated inflammatory response when exposed to IL-1β or TNF-α, cytokines that contribute to the pathogenesis of type 1 diabetes. INS-1E cells or primary rat β-cells were exposed to a low dose of the ER stressor cyclopiazonic acid (CPA) or free fatty acids, followed by low-dose IL-1β or TNF-α. ER stress signaling was inhibited by small interfering RNA. Cells were evaluated for proinflammatory gene expression by RT-PCR and ELISA, gene reporter activity, p65 activation by immunofluorescence, and apoptosis. CPA pretreatment enhanced IL-1β- induced, but not TNF-α-induced, expression of chemokine (C-C motif) ligand 2, chemokine (C-X-C motif) ligand 1, inducible nitric oxide synthase, and Fas via augmented nuclear factor κB (NF-κB) activation. X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1, but not CCAAT/enhancer binding protein homologous protein, knockdown prevented the CPA-induced exacerbation of NF-κB-dependent genes and decreased IL-1β-induced NF-κB promoter activity. XBP1 modulated NF-κB activity via forkhead box O1 inhibition. In conclusion, rat β-cells facing mild ER stress are sensitized to IL-1β, generating a more intense and protracted inflammatory response through inositol-requiring enzyme 1/XBP1 activation. These observations link β-cell ER stress to the triggering of exacerbated local inflammation., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Objective: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused in almost all cases by homozygosity for a GAA trinucleotide repeat expansion in the frataxin gene. Frataxin is a mitochondrial protein involved in iron homeostasis. FRDA patients have a high prevalence of diabetes, the pathogenesis of which is not known. We aimed to evaluate the relative contribution of insulin resistance and β-cell failure and the pathogenic mechanisms involved in FRDA diabetes. Methods: Forty-one FRDA patients, 26 heterozygous carriers of a GAA expansion, and 53 controls underwent oral and intravenous glucose tolerance tests. β-Cell proportion was quantified in postmortem pancreas sections from 9 unrelated FRDA patients. Using an in vitro disease model, we studied how frataxin deficiency affects β-cell function and survival. Results: FRDA patients had increased abdominal fat and were insulin resistant. This was not compensated for by increased insulin secretion, resulting in a markedly reduced disposition index, indicative of pancreatic β-cell failure. Loss of glucose tolerance was driven by β-cell dysfunction, which correlated with abdominal fatness. In postmortem pancreas sections, pancreatic islets of FRDA patients had a lower β-cell content. RNA interference–mediated frataxin knockdown impaired glucose-stimulated insulin secretion and induced apoptosis in rat β cells and human islets. Frataxin deficiency sensitized β cells to oleate-induced and endoplasmic reticulum stress-induced apoptosis, which could be prevented by the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide. Interpretation: Pancreatic β-cell dysfunction is central to diabetes development in FRDA as a result of mitochondrial dysfunction and higher sensitivity to metabolic and endoplasmic reticulum stress-induced β-cell death. ANN NEUROL 2012;72:971–982, info:eu-repo/semantics/published

Second generation n3-PUFA-depleted rats represent a good animal model of metabolic syndrome as they display several features of the disease such as liver steatosis, visceral obesity and insulin resistance. The goal of our study was to investigate the influence of n3-PUFA deficiency on hepatic glycerol metabolism. Aquaglyceroporin 9 (AQP9) allows hepatic glycerol transport and consequently contributes to neoglucogenesis. AQP9 knockout mice display hypertriacyl-glycerolemia, one of the hallmarks of the metabolic syndrome. Our data show reduced AQP9 expression at the protein level in n3-PUFA-depleted rats, without any changes at the mRNA levels. [U-¹⁴C]glycerol uptake was increased in hepatocytes from n3-PUFA-depleted animal cells. The apparent discrepancy between decreased AQP9 protein expression, and increased [U-¹⁴C]glycerol uptake could be explained by an observed increase in glycerol kinase activity., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

info:eu-repo/semantics/nonPublished

Pancreatic β-cell dysfunction is central to the pathogenesis of type 2 diabetes, and the loss of functional β-cell mass in type 2 diabetes is at least in part secondary to increased β-cell apoptosis. Accumulating evidence suggests that endoplasmic reticulum (ER) stress is present in β-cells in type 2 diabetes. Free fatty acids (FFAs) cause ER stress and are putative mediators of β-cell dysfunction and death. In this review, we discuss the molecular mechanisms underlying ER stress induced by saturated and unsaturated FFAs. Oleate and palmitate trigger ER stress through ER Ca2+ depletion and build-up of unfolded proteins in the secretory pathway. Saturated and unsaturated FFAs elicit a differential signal transduction in the three branches of the ER stress response, resulting in different survival/apoptosis outcomes. The protection of β-cells against FFAs through the interference with ER stress signalling has opened novel therapeutic perspectives for type 2 diabetes. Chemical chaperones, salubrinal and glucagon-like peptide-1 (GLP-1) analogues have been used to protect β-cells from lipotoxic ER stress. Importantly, the pro- and antiapoptotic effects of these compounds are cell and context dependent. © 2010 Blackwell Publishing Ltd., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published