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).
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.
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.
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.
Components of the unfolded protein response (a cellular response to stress of the endoplasmic reticulum [ER]) act as beneficial regulators under physiological conditions or as inducers of beta cell dysfunction and apoptosis under chronic stress. In vitro studies showed that proinflammatory cytokines (IL-1β and IFN-γ) can induce beta cell ER stress [1, 2] suggesting that ER stress might contribute to beta cell loss in type 1 diabetes. These cytokines activate some, but not all, branches of the ER stress pathway and hamper beta cell defences by inhibiting ER chaperones [1, 2]. In the context of type 1 diabetes, ER stress might amplify proapoptotic pathways, augment inflammation and increase the presentation of beta cell antigens to the immune system.
There is evidence of enhanced ER stress in beta cells from both humans with type 2 diabetes and from rodent models of the disease [1, 3, 4]. Furthermore, in patients or in mouse models with mutations in key components of the unfolded protein response or in proinsulin, beta cell loss and diabetes develop . However, it remains to be established whether ER stress occurs in the beta cells of humans with type 1 diabetes. To address this question, we have evaluated the presence of the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) in pancreatic sections from type 1 diabetic and control individuals. Particular care was taken to correlate insulitis with levels of beta cell ER stress markers and to analyse the data in an unbiased and quantitative way.
Human pancreatic tissue
Pancreases from type 1 diabetic, adult and paediatric non-diabetic individuals from two different collections—UK autopsy cases [5, 6] and the Juvenile Diabetes Research Foundation-sponsored Network for Pancreatic Organ Donors with Diabetes (nPOD) programme, USA—were studied, with ethical permission. The series consisted of 13 patients with type 1 diabetes (seven female and six male); eight with recent-onset type 1 diabetes (age [mean±SD] 7.9 ± 8.7 years, range 1.5–27 years, time since diagnosis <3 months) and five cases of long-standing type 1 diabetes (age 24.6 ± 14.8 years, range 12–50 years, mean time since diagnosis 9.8 ± 7.1 years). Fifteen pancreases from non-diabetic individuals—seven children (four females and three males, mean age 5.5 ± 3.7 years, range 0.5–10 years) and eight adults (five females and three males, mean age 55.4 ± 15.7 years, range 30–72 years)–served as controls.
The pancreases had been fixed in formalin buffer and embedded in paraffin. Most of the type 1 diabetic pancreases retained some insulin-containing islets, and insulitis was also present in some of these (electronic supplementary material [ESM] Table 1). As positive controls, HeLa cells were treated with the ER stressor thapsigargin (1 μmol/l) for 24 h and then embedded in paraffin for subsequent immunostaining, or retrieved for evaluation of mRNA expression of selected ER stress markers .
Serial sections of 4 μm thickness were processed and labelled for CHOP, BIP, XBP-1, CD45, insulin and glucagon using a standard immunoperoxidase and double immunofluorescence methods. Immunostaining was carried out as described in ESM Supplementary Methods.
Immunohistochemical quantification of ER stress markers
To quantify the immunostaining in an unbiased way, images of each slide were acquired at 20× magnification using a Hamamatsu NanoZoomer HT2.0 whole-slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) as previously described . The islets were manually selected using the annotation tool within NDP-view software (Hamamatsu Photonics), allowing superposition of the same islet in successive sections. A dedicated in-house tool was used to automatically extract each selected islet and to export it as a standard bitmap image file . A routine was developed for Definiens Developer XD 1.2.1 (Definiens AG, Munich, Germany) to detect the selected islets from extracted bitmap images. Staining was quantified using Definiens TissueMap 3.0 (Definiens AG), which integrates with Definiens Developer XD-1.2.1. The ‘quick-score’ feature , defined as the ratio of the sum of stained pixel intensities over the islet area, was then calculated. Positive pixels were detected by a TissueMap 3.0 routine using a constant threshold manually optimised across the different slides.
The quantification results of immunostaining intensities from the UK and nPOD pancreas cases were initially examined and evaluated separately (data not shown). Since the results from each were similar, they are presented together.
Validation of the CHOP, BIP and XBP-1 antibodies in ER-stressed HeLa cells
HeLa cells treated with thapsigargin (1 μmol/l for 24 h) were used as a positive control (ESM Fig. 1a). Compared with untreated HeLa cells, CHOP, BIP and XBP-1 levels were increased in response to thapsigargin. The increase in protein expression was paralleled by an increase in mRNA expression for each marker (ESM Fig. 1b).
Evaluation of CHOP, BIP and XBP-1 levels in human type 1 diabetic and non-diabetic pancreas
Immunostaining for insulin and CD45 (a marker of immune cells) in type 1 diabetic pancreases revealed three types of islet: insulin-containing islets (ICIs) without detectable inflammation (Fig. 1g), ICIs with immune cell infiltration (insulitis; defined as more than ten CD45-positive cells within the islet periphery [Fig. 1m, r]) and insulin-deficient islets (IDIs; Fig. 1s). No IDIs or insulitis-positive islets were detected in non-diabetic pancreases.
Weak cytoplasmic immunostaining for CHOP was observed in the majority of islet cells in all samples. However, the intensity of this staining was increased in type 1 diabetic islets (with or without insulitis) in comparison with control islets (Fig. 1; compare h, n and t against b).
Immunostaining for BIP was heterogeneous, with some islet cells displaying strong cytoplasmic staining while others were immunonegative. This pattern was similar in control islets (Fig. 1c) and in insulitis-negative islets in type 1 diabetes (whether ICI or IDI; Fig. 1i, u). In contrast, insulitis-positive islets of type 1 diabetic patients displayed an enhanced intensity of BIP expression, and the protein was present in a larger proportion of cells (panel o).
XBP-1 immunostaining was mainly cytoplasmic or perinuclear and was present at equal intensity in the majority of endocrine cells in islets from both control and type 1 diabetic samples (Fig. 1d, j, p, v).
Unbiased quantification of immunostaining intensity revealed that CHOP staining was significantly elevated in type 1 diabetic islets compared with controls (Fig. 2a, b). The intensity of BIP immunostaining also tended to be greater in type 1 diabetic islets, but this achieved statistical significance only for insulitis-positive islets (Fig. 2c, d). No significant differences were observed in XBP-1 immunostaining intensity between type 1 diabetic and non-diabetic pancreases (Fig. 2e, f). The patterns of expression and intensity of staining of the three ER stress markers was similar in adult and paediatric non-diabetic pancreases (Fig. 2).
Double staining for CHOP or BIP and insulin or glucagon in selected cases indicated co-localisation between CHOP or BIP and insulin in many but not all cells (ESM Fig. 2).
The role of ER stress in mediating beta cell loss in type 1 diabetes remains unclear, and few studies have addressed whether ER stress markers are present in islets from individuals with type 1 diabetes. Evidence of enhanced expression of activating transcription factor 3 in type 1 diabetic beta cells has been presented , whereas CHOP was not found . Conversely, studies on pancreases from patients with type 2 diabetes revealed increased levels of CHOP, BIP and activating transcription factor ATF-3 [3, 4, 8] as well as ER dilation .
Such differences in islet expression of ER stress markers between type 2 and type 1 diabetes might reflect differences in the islet milieu in each condition. Thus, glucolipotoxicity is likely to underlie the changes in type 2 diabetes, whereas proinflammatory cytokines may play a role in type 1 diabetes . If proinflammatory cytokines are responsible for mediating ER stress in islet cells in type 1 diabetes, it is probable that the expression of ER stress markers will be heterogeneous, with the highest levels of induction found in inflamed islets. These considerations, coupled with the scarcity of adequate samples for histology from individuals in the pre-clinical stages of type 1 diabetes and the difficulty of finding reliable antibodies for ER stress markers (as our own unpublished observations [I. Marhfour and D.L. Eizirik] and a study by Haataja et al.  show) may explain the conflicting results on the presence of ER stress markers in islets from patients with type 1 diabetes.
We have now studied pancreases from 13 individuals with type 1 diabetes (plus non-diabetic controls) obtained from two independent sources, and have performed an unbiased and quantitative evaluation of immunostained samples. The results indicate that islets from type 1 diabetic individuals display increased levels of CHOP and, in the case of insulitis-positive islets, BIP. No increase in the level of XBP-1 was detected in type 1 diabetes, although this marker was present in all the islets examined. These results suggest that islet cells from individuals with type 1 diabetes show a partial ER stress response, which is reminiscent of that described previously in purified beta cells exposed to proinflammatory cytokines in vitro . Interestingly, increased CHOP expression is not limited to beta cells, since it is also present in IDIs.
These observations, taken together with the recent findings that islets isolated from prediabetic NOD mice show markedly increased levels of ER stress markers , suggest that ER stress may be a contributory factor for beta cell dysfunction/death and insulitis in early type 1 diabetes.
Immunoglobulin heavy chain
C/EBP homologous protein
Network for Pancreatic Organ Donors with Diabetes
X-box binding protein 1
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We thank F. Morel from the Pathology Department (Erasme Hospital, Brussels, Belgium) for technical help and G. S. Hotamisligil (Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USA) for helpful discussions.
This work was supported by grants from the Juvenile Diabetes Research Foundation (JDRF Grant 17-2009-106), Diabetes Research and Wellness Foundation, European Union (project Naimit, in the Framework Programme 7 of the European Community) and Network for Pancreatic Organ Donors with Diabetes (nPOD), a collaborative research project on type 1 diabetes sponsored by the JDRF. Organ procurement organisations partnering with nPOD to provide research resources are listed at www.jdrfnpod.org/our-partners.php. The Center for Microscopy and Molecular Imaging is supported by the European Regional Development Fund and the Walloon Region. Xavier Moles Lopez is supported by the Télévie programme of the Fond National de la Recherche Scientifique.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
DLE, FA and IM contributed to the study concept and design; IM, IS, FA, SJR and NGM acquired the data; XML, IM, IS and DL performed image and statistical analysis; DLE and NGM supervised the study; IM and DLE drafted the manuscript; FA, SJR and NGM reviewed the manuscript for important intellectual content. All authors revised the article and approved the final version to be published.
Electronic supplementary material
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Specificity of the ER stress markers CHOP, Bip and XBP-1 immunodetections a: Immunodetection of CHOP, Bip and XBP-1 (brown deposit) in HeLa cells non-treated (control) or treated with thapsigargin (1 μM for 24 h). In control HeLa cells, CHOP immunostaining is negative, while Bip is expressed in the majority of cells. After thapsigargin treatment, CHOP is induced and Bip and XBP-1 immunostaining are increased as illustrated by the positive immunostaining in the cytoplasm of the majority of thapsigargin treated HeLa cells and also in the nucleus of some cells (arrows). Images were captured at ×400 magnification (scale bar: 100 μm). b: mRNA expression of Chop, Bip, total Xbp-1 and spliced Xbp-1 in HeLa cells treated with thapsigargin for 24 h (red bars) and in non-treated cells (control; blue bars). mRNA expression of the above described genes was significantly increased after treatment with thapsigargin (n = 5; t-test: **p < 0.01; ***p < 0.001) (PDF 232 kb)
Localization of ER stress markers in human islet cells by double immunofluorescence. Serial pancreatic sections from control (a-c, g-i, m-o, s-v) and type 1 diabetic (d-f, j-l, p-r, w-y) pancreases were double immunostained for CHOP (a-l) or Bip (m-y) and insulin (b, e, n, q) or glucagon (h, k, t, x). CHOP and Bip (red fluorescence) are co-localized with some insulin-positive cells (green fluorescence) as illustrated by the merged images in c, f, o and r (arrows). CHOP is present in a few glucagon-positive cells (i & l; arrow heads), while no co-localization of Bip and glucagon was observed (v & y). Images were captured at ×400 magnification (scale bar: 40 μm) (PDF 216 kb)
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Marhfour, I., Lopez, X.M., Lefkaditis, D. et al. Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes. Diabetologia 55, 2417–2420 (2012). https://doi.org/10.1007/s00125-012-2604-3
- Diabetes mellitus
- ER stress
- Pancreatic beta cells
- Pancreatic islets
- Type 1 diabetes