Apoptosis is likely to be the main form of beta-cell death in immune-mediated diabetes mellitus in rodents and possibly in humans. Clarification of the regulation of beta-cell death could indicate ...novel sites for therapeutic intervention in Type I (insulin-dependent) diabetes mellitus. We review the molecular effectors and signal transduction of immune-mediated beta-cell apoptosis. Data obtained on non-obese diabetic (NOD) mice suggest that macrophages and CD4+ T-cells are the main cellular effectors, whereas CD8+ T-cells are more important initiators of the immune process leading to beta-cell death. Perforin could be the effector molecule utilized by CD8+ T-cell initiation, whereas CD4+ mediated beta-cell destruction is mostly dependent on Fas/FasL and the cytokines IFNgamma and TNF-alpha. The macrophage cytokine IL-1beta in combination with IFN-gamma and TNF-alpha, plays an important role for beta-cell dysfunction and death. Signal transduction by these cytokines involves: (i) binding to specific receptors, (ii) signal transduction by cytosolic kinases (especially the so-called mitogen- and stress-activated protein kinases) and/or phosphatases, (iii) mobilization of diverse transcription factors - with nuclear factor kappaB (NF-kappaB), AP-1 and STAT-1 probably playing key roles for beta-cell apoptosis; (iv) up-regulation or down-regulation of gene transcription. Recent data obtained by microarray and proteomic analysis suggest that the process of beta-cell apoptosis depends on the parallel and/or sequential up-regulation and down-regulation of considerable numbers of genes, which can be grouped in gene modules or patterns according to their functions. A detailed characterization of these "gene modules", and of the signalling pathways and transcription factors regulating them could allow us to understand the ultimate mechanisms leading to beta-cell apoptosis.
Protein synthesis is increased by several-fold in stimulated pancreatic beta cells. Synthesis and folding of (pro)insulin takes place in the endoplasmic reticulum (ER), and beta cells trigger the ...unfolded protein response (UPR) to upgrade the functional capacity of the ER. Prolonged or excessive UPR activation contributes to beta cell dysfunction and death in type 2 diabetes, but there is another side of the UPR that may be of particular relevance for autoimmune type 1 diabetes, namely, the cross-talk between the UPR and innate immunity/inflammation. Recent evidence, discussed in this review, indicates that both saturated fats and inflammatory mediators such as cytokines trigger the UPR in pancreatic beta cells. The UPR potentiates activation of nuclear factor κB, a key regulator of inflammation. Two branches of the UPR, namely IRE1/XBP1s and PERK/ATF4/CHOP, mediate the UPR-induced sensitisation of pancreatic beta cells to the proinflammatory effects of cytokines. This can contribute to the upregulation of local inflammatory mechanisms and the aggravation of insulitis. The dialogue between the UPR and inflammation may provide an explanation for the parallel increase in the prevalence of childhood obesity and type 1 diabetes.
Inverse Relationship Between Cytotoxicity of Free Fatty Acids in Pancreatic Islet Cells and Cellular Triglyceride Accumulation
Miriam Cnop ,
Jean Claude Hannaert ,
Anne Hoorens ,
Décio L. Eizirik and
...Daniel G. Pipeleers
Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
Abstract
Studies in Zucker diabetic fatty rats have led to the concept that chronically elevated free fatty acid (FFA) levels can cause
apoptosis of triglyceride-laden pancreatic β-cells as a result of the formation of ceramides, which induce nitric oxide (NO)-dependent
cell death. This “lipotoxicity” hypothesis could explain development of type 2 diabetes in obesity. The present study examines
whether prolonged exposure to FFA affects survival of isolated normal rat β-cells and whether the outcome is related to the
occurrence of triglyceride accumulation. A dose-dependent cytotoxicity was detected at 5–100 nmol/l of unbound oleate and
palmitate, with necrosis occurring within 48 h and an additional apoptosis during the subsequent 6 days of culture. At equimolar
concentrations, the cytotoxicity of palmitate was higher than that of oleate but lower than that of its nonmetabolized analog
bromopalmitate. FFA cytotoxicity was not suppressed by etomoxir (an inhibitor of mitochondrial carnitine palmitoyltransferase
I) or by antioxidants; it was not associated with inducible NO synthase expression or NO formation. An inverse correlation
was observed between the percentage of dead β-cells on day 8 and their cellular triglyceride content on day 2. For equimolar
concentrations of the tested FFA, oleate caused the lowest β-cell toxicity and the highest cytoplasmic triglyceride accumulation.
On the other hand, oleate exerted the highest toxicity in islet non–β-cells, where no FFA-induced triglyceride accumulation
was detected. In conditions without triglyceride accumulation, the lower FFA concentrations caused primarily apoptosis, both
in islet β-cells and non–β-cells. It is concluded that FFAs can cause death of normal rat islet cells through an NO-independent
mechanism. The ability of normal β-cells to form and accumulate cytoplasmic triglycerides might serve as a cytoprotective
mechanism against FFA-induced apoptosis by preventing a cellular rise in toxic free fatty acyl moieties. It is conceivable
that this potential is lost or insufficient in cells with a prolonged triglyceride accumulation as may occur in vivo.
Footnotes
Address correspondence and reprint requests to Daniel Pipeleers, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan
103, 1090 Brussels, Belgium. E-mail: daniel.pipeleers{at}vub.ac.be .
Received for publication 5 October 2000 and accepted in revised form 1 May 2001.
FFA, free fatty acid; IL, interleukin; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PCR, polymerase chain reaction;
RT, reverse transcriptase.
Genome‐wide association studies (GWAS) have identified more than 50 loci associated with genetic risk of type 1 diabetes (T1D). Several T1D candidate genes have been suggested or identified within ...these regions, but the molecular mechanisms by which they contribute to insulitis and β‐cell destruction remain to be clarified. More than 60% of the T1D candidate genes are expressed in human pancreatic islets, suggesting that they contribute to T1D by regulating at least in part pathogenic mechanisms at the β‐cell level. Recent studies by our group indicate that important genetically regulated pathways in β‐cells include innate immunity and antiviral activity, involving RIG‐like receptors (particularly MDA5) and regulators of type I IFNs (i.e. PTPN2 and USP18), and genes related to β‐cell phenotype and susceptibility to pro‐apoptotic stimuli (i.e. GLIS3). These observations reinforce the concept that the early pathogenesis of T1D is characterized by a dialogue between the immune system and pancreatic β‐cells. This dialogue is probably influenced by polymorphisms in genes expressed at the β‐cell and/or immune system level, leading to inadequate responses to environmental cues such as viral infections. Further studies are needed to clarify how these disease‐associated variants affect pancreatic β‐cell responses to inflammation and the subsequent triggering of autoimmune responses and progressive β‐cell loss.
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.
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
Endoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets ...from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children).
Methods
Antibodies against the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) were validated using HeLa cells treated with the ER stressor thapsigargin. These antibodies were then used to stain serial sections of paraffin-embedded pancreas from type 1 diabetic and non-diabetic individuals; samples were also immunostained for CD45, insulin and glucagon. Immunostaining intensities of the ER stress markers were quantified using a software-based, unbiased quantitative approach.
Results
Islets from individuals with type 1 diabetes showed increased levels of CHOP and, at least for insulitis-positive and beta cell-containing islets, BIP. XBP-1 expression was not, however, increased.
Conclusions/interpretation
Islet cells from individuals with type 1 diabetes display a partial ER stress response, with evidence of the induction of some, but not all, components of the unfolded protein response.
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.
Aims/hypothesis Pro-inflammatory cytokines involved in the pathogenesis of type 1 diabetes deplete endoplasmic reticulum (ER) Ca²⁺ stores, leading to ER-stress and beta cell apoptosis. However, the ...cytokine-induced ER-stress response in beta cells is atypical and characterised by induction of the pro-apoptotic PKR-like ER kinase (PERK)-C/EBP homologous protein (CHOP) branch of the unfolded protein response, but defective X-box binding protein 1 (XBP1) splicing and activating transcription factor 6 activation. The purpose of this study was to overexpress spliced/active Xbp1 (XBP1s) to increase beta cell resistance to cytokine-induced ER-stress and apoptosis. Methods Xbp1s was overexpressed using adenoviruses and knocked down using small interference RNA in rat islet cells. In selected experiments, Xbp1 was also knocked down in FACS-purified rat beta cells and rat fibroblasts. Expression and production of XBP1s and key downstream genes and proteins was measured and beta cell function and viability were evaluated. Results Adenoviral-mediated overproduction of Xbp1s resulted in increased XBP1 activity and induction of several XBP1s target genes. Surprisingly, XBP1s overexpression impaired glucose-stimulated insulin secretion and increased beta cell apoptosis, whereas it protected fibroblasts against cell death induced by ER-stress. mRNA expression of Pdx1 and Mafa was inhibited in cells overproducing XBP1s, leading to decreased insulin expression. XBP1s knockdown partially restored cytokine/ER-stress-driven insulin and Pdx1 inhibition but had no effect on cytokine-induced ER-stress and apoptosis. Conclusions/interpretation XBP1 has a distinct inhibitory role in beta cell as compared with other cell types. Prolonged XBP1s production hampers beta cell function via inhibition of insulin, Pdx1 and Mafa expression, eventually leading to beta cell apoptosis.
Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. ...Isletinfiltrating leukocytes secrete cytokines such as IL-β and IFN-γ, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-κB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-κB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-κB protein inhibitor (ΔNIκBα), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the ΔNIκBα protein were resistant to the deleterious effects of IL-1β and IFN-γ, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-κB is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.
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