Introduction Nous avons précédemment identifié une mutation non-sens dans TRMT10A, une putative méthyltransférase d'ARNt, comme responsable d'un nouveau syndrome du diabète juvénile et microcéphalie. ...Plusieurs nouvelles mutations perte-de-fonction en TRMT10A ont récemment été identifiées chez des patients avec un syndrome similaire. Des polymorphismes dans CDKAL1, une methylthio-transferase d'ARNt, ont été associés au diabète de type 2, suggérant que des altérations dans la modification des ARNt provoquent la défaillance des cellules bêta. Notre objectif était d'étudier le rôle de TRMT10A et les mécanismes pathogènes de la déficience de cette protéine. Matériels et Méthodes La méthylation d'ARNt a été examinée par HPLC-MS/MS dans des lymphoblastes de patients déficients en TRMT10A, et les substrats de TRMT10A identifiés par des essais d'extension d'amorce. L'expression de TRMT10A a été réduite par siRNA dans des cellules productrices d'insuline INS-1E (rat) et EndoC-βH1 (humain), seule ou combinée avec siRNA contre les proteins pro-apoptotiques Bim ou Bad. Les cellules ont été exposées au palmitate (0,5 mM) ou à la thapsigargine (1 μM) et la mort cellulaire étudiée par coloration nucléaire et blots pour caspases. Résultats L'HPLC-MS/MS a montré une diminution de la méthylation des guanosines dans l'ARNt de lymphoblastes de patients. Les essais d'extension d'amorces ont montré que TRMT10A méthyle tRNAGln et tRNA-Meth sur la guanosine en position 9. Le silençage de TRMT10A dans les cellules bêta de rat et humaines induit l'activation de la voie intrinsèque de l'apoptose, évaluée par clivage de caspase-9, et induit l'expression de Bim et Bad (≥ 50 % d'induction, p < 0,05, n = 4-6). Uniquement le silençage de Bim protège les cellules déficientes en TRMT10A de l'apoptose en condition basale et après traitement avec le palmitate ou la thapsigargine ( p < 0,05, n = 4-5). Conclusions Nous avons confirmé la fonction de TRMT10A comme une méthyltransférase d'ARNt, et identifiés tRNAGln et tRNAMeth comme ses substrats. Un déficit en méthylation pourrait affecter la charge et la stabilité des ARNt. La déficience en TRMT10A conduit à l'activation de la voie intrinsèque de l'apoptose dans les cellules bêta. Nous avons identifié la protéine pro-apoptotique Bim comme médiateur clé de ce processus.
Introduction Les régimes alimentaires riches en gras saturés contribuent à la défaillance de la cellule β dans le diabète de type 2 (DT2). Le palmitate induit le stress du réticulum endoplasmique ...(RE) et le dysfonctionnement/apoptose des cellules β. Afin d'identifier les médiateurs moléculaires de la lipotoxicité, nous avons effectué un séquençage-ARN d'îlots humains et une analyse protéomique de cellules INS-1E après un traitement au palmitate. Matériels et Méthodes Six préparations d'îlots humains ont été exposées au palmitate (0,5 mM) durant 48 h et l'ARN a été séquencé. Les cellules INS-1E ont été exposées au palmitate durant 4, 16 ou 24 h et le protéome a été profilé par iTRAQ ( n = 2). Les analyses des réseaux de régulations ont fait usage des bases de données IPA, DAVID et TRANSFAC. L'expression des gènes a été réduite par siRNA dans les cellules β clonales et primaires. La mort cellulaire a été examinée par coloration nucléaire. Résultats Des analyses comparatives du transcriptome et du protéome des cellules β traitées au palmitate ont identifié 93 gènes/protéines régulés positivement et 133 régulés négativement communs aux 2 analyses. La classification en catégories fonctionnelles montre une modification des gènes/protéines régulant le métabolisme d'acides gras, le stress du RE, l'apoptose et de plusieurs transporteurs d'acides aminés. Nos études fonctionnelles ont confirmé l'augmentation d'expression des transporteurs d'acides aminés Lat1 et Cat1 (>25 %, n = 4-5). La diminution de leur expression par siRNA, par contre, ne modifie pas le stress du RE lipotoxique. Un réseau de régulations inféré dévoile Arnt, FoxO1, APP, JunD et Bach1 comme des régulateurs clés de la lipotoxicité. Les rôles de FoxO1, Arnt et App dans la sécrétion d'insuline, le stress du RE et l'apoptose ont déjà été démontrés, accréditant l'hypothèse d'un possible rôle biologique dans le contexte de la lipotoxicité. Conclusions Cette étude est la première du genre à fournir un profilage transcriptomique et protéomique de cellules β exposées au palmitate. Nos données dévoilent de nouveaux médiateurs de lipotoxicité et des réseaux de régulation complexes potentiellement impliqués dans la défaillance de la cellule β dans le DT2.
Autophagy is the major mechanism involved in degradation and recycling of intracellular components, and its alterations have been proposed to cause beta cell dysfunction. In this study, we explored ...the effects of autophagy modulation in human islets under conditions associated to endoplasmic reticulum (ER) stress. Human pancreatic islets were isolated by enzymatic digestion and density gradient purification from pancreatic samples of non-diabetic (ND;
= 17; age 65 ± 21 years; gender: 5 M/12 F; BMI 23.4 ± 3.3 kg/m
) and T2D (
= 9; age 76 ± 6 years; 4 M/5 F; gender: BMI 25.4 ± 3.7 kg/m
) organ donors. Nine ND organ donors were treated for hypertension and 1 for both hypertension and hypercholesterolemia. T2D organ donors were treated with metformin (1), oral hypoglycemic agents (2), diet + oral hypoglycemic agents (3), insulin (3) or insulin plus metformin (3) as for antidiabetic therapy and, of these, 3 were treated also for hypertension and 6 for both hypertension and hypercholesterolemia. Two days after isolation, they were cultured for 1-5 days with 10 ng/ml rapamycin (autophagy inducer), 5 mM 3-methyladenine or 1.0 nM concanamycin-A (autophagy blockers), either in the presence or not of metabolic (0.5 mM palmitate) or chemical (0.1 ng/ml brefeldin A) ER stressors. In ND islets palmitate exposure induced a 4 to 5-fold increase of beta cell apoptosis, which was significantly prevented by rapamycin and exacerbated by 3-MA. Similar results were observed with brefeldin treatment. Glucose-stimulated insulin secretion from ND islets was reduced by palmitate (-40 to 50%) and brefeldin (-60 to 70%), and rapamycin counteracted palmitate, but not brefeldin, cytotoxic actions. Both palmitate and brefeldin induced PERK, CHOP and BiP gene expression, which was partially, but significantly prevented by rapamycin. With T2D islets, rapamycin alone reduced the amount of p62, an autophagy receptor that accumulates in cells when macroautophagy is inhibited. Compared to untreated T2D cells, rapamycin-exposed diabetic islets showed improved insulin secretion, reduced proportion of beta cells showing signs of apoptosis and better preserved insulin granules, mitochondria and ER ultrastructure; this was associated with significant reduction of PERK, CHOP and BiP gene expression. This study emphasizes the importance of autophagy modulation in human beta cell function and survival, particularly in situations of ER stress. Tuning autophagy could be a tool for beta cell protection.
Aims/hypothesis Pancreatic beta cells have highly developed endoplasmic reticulum (ER) due to their role in insulin secretion. Since ER stress has been associated with beta cell dysfunction, we ...studied several features of beta cell ER in human type 2 diabetes. Methods Pancreatic samples and/or isolated islets from non-diabetic controls (ND) and type 2 diabetes patients were evaluated for insulin secretion, apoptosis (electron microscopy and ELISA), morphometric ER assessment (electron microscopy), and expression of ER stress markers in beta cell prepared by laser capture microdissection and in isolated islets. Results Insulin release was lower and beta cell apoptosis higher in type 2 diabetes than ND islets. ER density volume was significantly increased in type 2 diabetes beta cells. Expression of alpha-mannosidase (also known as mannosidase, alpha, class 1A, member 1) and UDP-glucose glycoprotein glucosyl transferase like 2 (UGCGL2), assessed by microarray and/or real-time reverse transcriptase polymerase chain reaction (RT-PCR), differed between ND and type 2 diabetes beta cells. Expression of immunoglobulin heavy chain binding protein (BiP, also known as heat shock 70 kDa protein 5 glucose-regulated protein, 78 kDa HSPA5), X-box binding protein 1 (XBP-1, also known as XBP1) and C/EBP homologous protein (CHOP, also known as damage-inducible transcript 3 DDIT3) was not higher in type 2 diabetes beta cell or isolated islets cultured at 5.5 mmol/l glucose (microarray and real-time RT-PCR) than in ND samples. When islets were cultured for 24 h at 11.1 mmol/l glucose, there was induction of BiP and XBP-1 in type 2 diabetes islets but not in ND islets. Conclusions/interpretation Beta cell in type 2 diabetes showed modest signs of ER stress when studied in pancreatic samples or isolated islets maintained at physiological glucose concentration. However, exposure to increased glucose levels induced ER stress markers in type 2 diabetes islet cells, which therefore may be more susceptible to ER stress induced by metabolic perturbations.
Increased intra-abdominal fat is associated with insulin resistance and an atherogenic lipoprotein profile. Circulating concentrations of adiponectin, an adipocyte-derived protein, are decreased with ...insulin resistance. We investigated the relationships between adiponectin and leptin, body fat distribution, insulin sensitivity and lipoproteins.
We measured plasma adiponectin, leptin and lipid concentrations, intra-abdominal and subcutaneous fat areas by CT scan, and insulin sensitivity index (S(I)) in 182 subjects (76 M/106F).
Adiponectin concentrations were higher in women than in men (7.4+/-2.9 vs 5.4+/-2.3 micro g/ml, p<0.0001) as were leptin concentrations (19.1+/-13.7 vs 6.9+/-5.1 ng/ml, p<0.0001). Women were more insulin sensitive (S(I): 6.8+/-3.9 vs 5.9+/-4.4 x 10(-5) min(-1)/(pmol/l), p<0.01) and had more subcutaneous (240+/-133 vs 187+/-90 cm(2), p<0.01), but less intra-abdominal fat (82+/-57 vs 124+/-68 cm(2), p<0.0001). By simple regression, adiponectin was positively correlated with age ( r=0.227, p<0.01) and S(I) ( r=0.375, p<0.0001), and negatively correlated with BMI ( r=-0.333, p<0.0001), subcutaneous ( r=-0.168, p<0.05) and intra-abdominal fat ( r=-0.35, p<0.0001). Adiponectin was negatively correlated with triglycerides ( r=-0.281, p<0.001) and positively correlated with HDL cholesterol ( r=0.605, p<0.0001) and Rf, a measure of LDL particle buoyancy ( r=0.474, p<0.0001). By multiple regression analysis, adiponectin was related to age ( p<0.0001), sex ( p<0.005) and intra-abdominal fat ( p<0.01). S(I) was related to intra-abdominal fat ( p<0.0001) and adiponectin ( p<0.0005). Both intra-abdominal fat and adiponectin contributed independently to triglycerides, HDL cholesterol and Rf.
These data suggest that adiponectin concentrations are determined by intra-abdominal fat mass, with additional independent effects of age and sex. Adiponectin could link intra-abdominal fat with insulin resistance and an atherogenic lipoprotein profile.
Aims/hypothesis Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to ...diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. Methods LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. Results LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from ≥90% (<10 years) to ≥97% (>20 years) and remained constant thereafter. Conclusions/interpretation Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.
Longevity of human islet α- and β-cells Cnop, M.; Igoillo-Esteve, M.; Hughes, S. J. ...
Diabetes, obesity & metabolism,
October 2011, Letnik:
13, Številka:
s1
Journal Article
Odprti dostop
Pancreatic islet cell regeneration is considered to be important in the onset and progression of diabetes and as a potential cell therapy. Current hypotheses, largely based on rodent studies, ...indicate continuous turnover and plasticity of α‐ and β‐cells in adults; cell populations in rodents respond to increased secretory demand in obesity (30‐fold β‐cell increase) and pregnancy. Turnover and plasticity of islet cells decrease in mice within >1 year. In man, morphometric observations on postmortem pancreas have indicated that the cellular expansion is much smaller than the increased insulin secretion that accompanies obesity. Longevity of β‐cells in humans >20–30 years has been shown by 14C measurements and bromo‐deoxyuridine (BrdU) incorporation and there is an age‐related decline in the expression of proteins associated with cell division and regeneration including cyclin D3 and PDX‐1. Quantitative estimation and mathematical modelling of the longevity marker, cellular lipofuscin body content, in islets of subjects aged 1–84 years indicated an age‐related increase and that 97% of the human β‐cell population was established by the age of 20. New data show that human α‐cell lipofuscin content is less than that seen in β‐cells, but the age‐related accumulation is similar; lipofuscin‐positive (aged) cells form ≥95% of the population after 20 years. Increased turnover of cellular organelles such as mitochondria and endoplasmic reticulum could contribute to lipofuscin accumulation with age in long‐lived cells. Induction of regeneration of human islet cells will require understanding of the mechanisms associated with age‐related senescence.
Aims/hypothesis
Endoplasmic reticulum (ER) stress has been implicated in the development of type 2 diabetes, via effects on obesity, insulin resistance and pancreatic beta cell health. C/EBP ...homologous protein (CHOP) is induced by ER stress and has a central role in apoptotic execution pathways triggered by ER stress. The aim of this study was to characterise the role of CHOP in obesity and insulin resistance.
Methods
Metabolic studies were performed in
Chop
−/−
and wild-type C57Bl/6 mice, and included euglycaemic–hyperinsulinaemic clamps and indirect calorimetry. The inflammatory state of liver and adipose tissue was determined by quantitative RT-PCR, immunohistology and macrophage cultures. Viability and absence of ER stress in islets of Langerhans was determined by electron microscopy, islet culture and quantitative RT-PCR.
Results
Systemic deletion of
Chop
induced abdominal obesity and hepatic steatosis. Despite marked obesity,
Chop
−/−
mice had preserved normal glucose tolerance and insulin sensitivity. This discrepancy was accompanied by lower levels of pro-inflammatory cytokines and less infiltration of immune cells into fat and liver.
Conclusions/interpretation
These observations suggest that insulin resistance is not induced by fat accumulation per se, but rather by the inflammation induced by ectopic fat. CHOP may play a key role in the crosstalk between excessive fat deposition and induction of inflammation-mediated insulin resistance.
Aims/hypothesis Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of ...pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. Methods Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. Results Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate. Conclusions/interpretation Saturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.
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.
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