Aims/hypothesisInflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate... Show moreAims/hypothesisInflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage.MethodsWe used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-βH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells.ResultsAPOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)−signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus.Conclusions/interpretationAPOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes. Show less
Blanchi, B.; Taurand, M.; Colace, C.; Thomaidou, S.; Audeoud, C.; Fantuzzi, F.; ... ; Ravassard, P. 2023
ObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new... Show moreObjectivesReadily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-βH5 cells, the latest in the EndoC-βH cell family.MethodsEndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed transcriptome, immunological and extensive functional assays.ResultsReady to use EndoC-βH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-βH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-βH5 cells elicit specific A2-alloreactive CD8 T cell activation.ConclusionsEndoC-βH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models. Show less
Thomaidou, S.; Garcia, A.M.; Lange, S. de; Gan, J.; Slik, A.R. van der; Hoeben, R.C.; ... ; Zaldumbide, A. 2023
Aims/hypothesisThe inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell... Show moreAims/hypothesisThe inflammatory milieu characteristic of insulitis affects translation fidelity and generates defective ribosomal products (DRiPs) that participate in autoimmune beta cell destruction in type 1 diabetes. Here, we studied the role of early innate cytokines (IFNα) and late immune adaptive events (IFNɣ) in insulin DRiP-derived peptide presentation to diabetogenic CD8+ T cells.MethodsSingle-cell transcriptomics of human pancreatic islets was used to study the composition of the (immuno)proteasome. Specific inhibition of the immunoproteasome catalytic subunits was achieved using siRNA, and antigenic peptide presentation at the cell surface of the human beta cell line EndoC-βH1 was monitored using peptide-specific CD8 T cells.ResultsWe found that IFNγ induces the expression of the PSMB10 transcript encoding the β2i catalytic subunit of the immunoproteasome in endocrine beta cells, revealing a critical role in insulin DRiP-derived peptide presentation to T cells. Moreover, we showed that PSMB10 is upregulated in a beta cell subset that is preferentially destroyed in the pancreases of individuals with type 1 diabetes.Conclusions/interpretationOur data highlight the role of the degradation machinery in beta cell immunogenicity and emphasise the need for evaluation of targeted immunoproteasome inhibitors to limit beta cell destruction in type 1 diabetes. Show less
Tienhoven, R. van; Kracht, M.J.L.; Slik, A.R. van der; Thomaidou, S.; Wolters, A.H.G.; Giepmans, B.G.; ... ; Roep, B.O. 2023
Aims/hypothesis Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Methods:... Show moreAims/hypothesis Transcriptome analyses revealed insulin-gene-derived transcripts in non-beta endocrine islet cells. We studied alternative splicing of human INS mRNA in pancreatic islets. Methods: Alternative splicing of insulin pre-mRNA was determined by PCR analysis performed on human islet RNA and single-cell RNA-seq analysis. Antisera were generated to detect insulin variants in human pancreatic tissue using immunohistochemistry, electron microscopy and single-cell western blot to confirm the expression of insulin variants. Cytotoxic T lymphocyte (CTL) activation was determined by MIP-1 beta release. Results: We identified an alternatively spliced INS product. This variant encodes the complete insulin signal peptide and B chain and an alternative C-terminus that largely overlaps with a previously identified defective ribosomal product of INS. Immunohistochemical analysis revealed that the translation product of this INS-derived splice transcript was detectable in somatostatin-producing delta cells but not in beta cells; this was confirmed by light and electron microscopy. Expression of this alternatively spliced INS product activated preproinsulin-specific CTLs in vitro. The exclusive presence of this alternatively spliced INS product in delta cells may be explained by its clearance from beta cells by insulin-degrading enzyme capturing its insulin B chain fragment and a lack of insulin-degrading enzyme expression in delta cells. Conclusions/interpretation: Our data demonstrate that delta cells can express an INS product derived from alternative splicing, containing both the diabetogenic insulin signal peptide and B chain, in their secretory granules. We propose that this alternative INS product may play a role in islet autoimmunity and pathology, as well as endocrine or paracrine function or islet development and endocrine destiny, and transdifferentiation between endocrine cells. INS promoter activity is not confined to beta cells and should be used with care when assigning beta cell identity and selectivity. Show less
Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. Accumulating evidence suggests the engagement of cellular stress during... Show moreBeta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. Accumulating evidence suggests the engagement of cellular stress during the initial stage of the disease, preceding destruction and triggering immune cell infiltration. While the role of the endoplasmic reticulum (ER) in this process has been largely described, the participation of the other cellular organelles, particularly the mitochondria which are central mediator for beta-cell survival and function, remains poorly investigated. Here, we have explored the contribution of ER stress, in activating type-I interferon signaling and innate immune cell recruitment. Using human beta-cell line EndoC-beta H1 exposed to thapsigargin, we demonstrate that induction of cellular stress correlates with mitochondria dysfunction and a significant accumulation of cytosolic mitochondrial DNA (mtDNA) that triggers neutrophils migration by an IL8-dependent mechanism. These results provide a novel mechanistic insight on how ER stress can cause insulitis and may ultimately facilitate the identification of potential targets to protect beta-cells against immune infiltration. Show less
Saitoski, K.; Ryaboshapkina, M.; Hamza, G.M.; Jarnuczak, A.F.; Berthault, C.; Carlotti, F.; ... ; Scharfmann, R. 2022
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low... Show moreProprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-beta H1. We demonstrate that PCSK9 is expressed and secreted by EndoC-beta H1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLAABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells. Show less
Noordstra, I.; Berg, C.M. van den; Boot, F.W.J.; Katrukha, E.A.; Yu, K.L.; Tas, R.P.; ... ; Akhmanova, A. 2022
Insulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these... Show moreInsulin secretion in pancreatic beta-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5 beta (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5 beta or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchangewere stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release. Show less
One hundred years after the discovery of insulin, Kieffer and colleagues (Ramzy et al., 2021) and Foyt and colleagues (Shapiro et al., 2021) report interim results from a multicenter clinical trial... Show moreOne hundred years after the discovery of insulin, Kieffer and colleagues (Ramzy et al., 2021) and Foyt and colleagues (Shapiro et al., 2021) report interim results from a multicenter clinical trial showing insulin secretion from engrafted pluripotent stem cell-derived endocrine progenitor cells in patients with type 1 diabetes. Show less
Groen, N.; Leenders, F.; Mahfouz, A.; Munoz-Garcia, A.; Muraro, M.J.; Graaf, N. de; ... ; Carlotti, F. 2021
The maintenance of pancreatic islet architecture is crucial for proper beta-cell function. We previously reported that disruption of human islet integrity could result in altered beta-cell identity... Show moreThe maintenance of pancreatic islet architecture is crucial for proper beta-cell function. We previously reported that disruption of human islet integrity could result in altered beta-cell identity. Here we combine beta-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of beta-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and beta-cell phenotype, and strengthen the concept of altered beta-cell identity as a mechanism underlying the loss of functional beta-cell mass. Show less
Leenders, F.; Groen, N.; Graaf, N. de; Engelse, M.A.; Rabelink, T.J.; Koning, E.J.P. de; Carlotti, F. 2021
Pancreatic beta-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. beta-cells have low anti-oxidant capacity,... Show morePancreatic beta-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. beta-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human beta-cells. We show that primary human beta-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of beta-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced beta-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional beta-cell mass in early stages of development of T1D. Show less
Thomaidou, S.; Slieker, R.C.; Slik, A.R. van der; Boom, J.; Mulder, F.; Munoz-Garcia, A.; ... ; Zaldumbide, A. 2021
Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of the insulin-producing pancreatic beta -cells. Increasing evidence suggest that the beta ... Show moreType 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of the insulin-producing pancreatic beta -cells. Increasing evidence suggest that the beta -cells themselves contribute to their own destruction by generating neoantigens through the production of aberrant or modified proteins that escape central tolerance. We recently demonstrated that ribosomal infidelity amplified by stress could lead to the generation of neoantigens in human beta -cells, emphasizing the participation of nonconventional translation events in autoimmunity, as occurring in cancer or virus-infected tissues. Using a transcriptome-wide profiling approach to map translation initiation start sites in human beta -cells under standard and inflammatory conditions, we identify a completely new set of polypeptides derived from noncanonical start sites and translation initiation within long noncoding RNA. Our data underline the extreme diversity of the beta -cell translatome and may reveal new functional biomarkers for beta -cell distress, disease prediction and progression, and therapeutic intervention in T1D. Show less
Heparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. While the role of heparanase in sustaining the pathology of autoimmune... Show moreHeparanase is the predominant enzyme that cleaves heparan sulfate, the main polysaccharide in the extracellular matrix. While the role of heparanase in sustaining the pathology of autoimmune diabetes is well documented, its association with metabolic syndrome/type 2 diabetes attracted less attention. Our research was undertaken to elucidate the significance of heparanase in impaired glucose metabolism in metabolic syndrome and early type 2 diabetes. Here, we report that heparanase exerts opposite effects in insulin-producing (i.e., islets) vs. insulin-target (i.e., skeletal muscle) compartments, sustaining or hampering proper regulation of glucose homeostasis depending on the site of action. We observed that the enzyme promotes macrophage infiltration into islets in a murine model of metabolic syndrome, and fosters beta-cell-damaging properties of macrophages activated in vitro by components of diabetogenic/obese milieu (i.e., fatty acids). On the other hand, in skeletal muscle (prototypic insulin-target tissue), heparanase is essential to ensure insulin sensitivity. Thus, despite a deleterious effect of heparanase on macrophage infiltration in islets, the enzyme appears to have beneficial role in glucose homeostasis in metabolic syndrome. The dichotomic action of the enzyme in the maintenance of glycemic control should be taken into account when considering heparanase-targeting strategies for the treatment of diabetes. Show less
Thomaidou, S.; Kracht, M.J.L.; Slik, A. van der; Laban, S.; Koning, E.J. de; Carlotti, F.; ... ; Zaldumbide, A. 2020
The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated beta -cell destruction in type 1 diabetes (T1D). Among them, the... Show moreThe signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated beta -cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human beta -cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1 alpha /miR-17-5p axis and demonstrate that inhibition of the IRE1 alpha activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of beta -cells to the immune system and position the IRE1 alpha /miR-17 pathway as a central component in beta -cell destruction processes and as a potential target for the treatment of autoimmune T1D. Show less
Trinanes, J.; Dijke, P. ten; Groen, N.; Hanegraaf, M.; Porrini, E.; Rodriguez-Rodriguez, A.E.; ... ; Vries, A.P.J. de 2020
Active maintenance of beta-cell identity through fine-tuned regulation of key transcription factors ensures beta-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of... Show moreActive maintenance of beta-cell identity through fine-tuned regulation of key transcription factors ensures beta-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of diabetes after organ transplantation, but underlying molecular mechanisms are unclear. Here we show that tacrolimus induces loss of human beta-cell maturity and beta-cell failure through activation of the BMP/SMAD signaling pathway when administered under mild metabolic stress conditions. Tacrolimus-induced phosphorylated SMAD1/5 acts in synergy with metabolic stress-activated FOXO1 through formation of a complex. This interaction is associated with reduced expression of the key beta-cell transcription factor MAFA and abolished insulin secretion, both in vitro in primary human islets and in vivo in human islets transplanted into high-fat diet-fed mice. Pharmacological inhibition of BMP signaling protects human beta-cells from tacrolimus-induced beta-cell dysfunction in vitro. Furthermore, we confirm that BMP/SMAD signaling is activated in protocol pancreas allograft biopsies from recipients on tacrolimus. To conclude, we propose a novel mechanism underlying the diabetogenicity of tacrolimus in primary human beta-cells. This insight could lead to new treatment strategies for new-onset diabetes and may have implications for other forms of diabetes. Show less
Balak, J.R.A.; Juksar, J.; Carlotti, F.; Nigro, A. lo; Koning, E.J.P. de 2019
Purpose of Review Novel 3D organoid culture techniques have enabled long-term expansion of pancreatic tissue. This review comprehensively summarizes and evaluates the applications of primary tissue... Show morePurpose of Review Novel 3D organoid culture techniques have enabled long-term expansion of pancreatic tissue. This review comprehensively summarizes and evaluates the applications of primary tissue-derived pancreatic organoids in regenerative studies, disease modelling, and personalized medicine.Recent Findings Organoids derived from human fetal and adult pancreatic tissue have been used to study pancreas development and repair. Generated adult human pancreatic organoids harbor the capacity for clonal expansion and endocrine cell formation. In addition, organoids have been generated from human pancreatic ductal adenocarcinoma in order to study tumor behavior and assess drug responses.Summary Pancreatic organoids constitute an important translational bridge between in vitro and in vivo models, enhancing our understanding of pancreatic cell biology. Current applications for pancreatic organoid technology include studies on tissue regeneration, disease modelling, and drug screening. Show less
Early-phase insulin secretion is a determinant of postprandial glucose homeostasis. In this study, we aimed to identify novel genetic variants associated with the early-phase insulin response to a... Show moreEarly-phase insulin secretion is a determinant of postprandial glucose homeostasis. In this study, we aimed to identify novel genetic variants associated with the early-phase insulin response to a liquid mixed meal by a genome-wide association study using a discovery and replication design embedded in the Netherlands Epidemiology of Obesity (NEO) study. The early-phase insulin response was defined as the difference between the natural logarithm-transformed insulin concentrations of the postprandial state at 30 min after a meal challenge and the fasting state (Delta insulin). After Bonferroni correction, rs505922 (beta: -6.5% [minor allele frequency (MAF) 0.32, P = 3.3 x 10(-8)]) located in the ABO gene reached genome-wide significant level (P < 5 x 10(-8)) and was also replicated successfully (beta: -7.8% [MAF 0.32, P = 7.2 x 10(-5)]). The function of the ABO gene was assessed using in vitro shRNA-mediated knockdown of gene expression in the murine pancreatic beta-cell line MIN6. Knocking down the ABO gene led to decreased insulin secretion in the murine pancreatic beta-cell line. These data indicate that the previously identified elevated risk of type 2 diabetes for carriers of the ABO rs505922:C allele may be caused by decreased early-phase insulin secretion. Show less
The lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration.... Show moreThe lack of efficient gene transfer methods into primary human pancreatic exocrine cells hampers studies on the plasticity of these cells and their possible role in beta cell regeneration. Therefore, improved gene transfer protocols are needed. Lentiviral vectors are widely used to drive ectopic gene expression in mammalian cells, including primary human islet cells. Here we aimed to optimize gene transfer into primary human exocrine cells using modified lentiviral vectors or transduction conditions. We evaluated different promoters, viral envelopes, medium composition and transduction adjuvants. Transduction efficiency of a reporter vector was evaluated by fluorescence microscopy and flow cytometry. We show that protamine sulfate-assisted transduction of a VSV-G-pseudotyped vector expressing eGFP under the control of a CMV promoter in a serum-free environment resulted in the best transduction efficiency of exocrine cells, reaching up to 90% of GFP-positive cells 5 days after transduction. Our findings will enable further studies on pancreas (patho)physiology that require gene transfer such as gene overexpression, gene knockdown or lineage tracing studies. Show less
Loomans, C.J.M.; Giuliani, N.W.; Balak, J.; Ringnalda, F.; Gurp, L. van; Huch, M.; ... ; Koning, E.J.P. de 2018
