Human beta cells become dysfunctional prior to type 1 diabetes diagnosis

Human beta cells become dysfunctional prior to type 1 diabetes diagnosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Human beta cells become dysfunctional prior to type 1 diabetes diagnosis Gladys Teitelman This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5285252/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Type 1 Diabetes is an autoimmune disease that leads to beta cell death. To test whether beta cell defects precede diagnosis we surveyed the expression of pCREB in human islet cells. pCREB is a transcription factor produced by islet alpha cells that is regulated by the incretin Glucagon-Like Peptide (GLP-1). Since levels of GLP-1 rise during diabetes development, we asked whether this increase would be deleterious to islet cells. This analysis indicated that while islet cells of control donors display pCREB in the nucleus, the transcription factor was also found in the cytoplasm of insulin cells of normoglycemic prediabetic donors and of beta and alpha cells of recently diagnosed donors. The translocation of pCREB, which decreases its activity, was correlated with reduced or absent expression of insulin and of a protease. These changes suggest an alteration in protein homeostasis. The cytoplasmic localization of pCREB is transient, since the transcription factor moves to the nuclei of insulin and glucagon cells of donors with longer standing disease. The present observations suggest a possible correlation between the known increase in GLP-1 during diabetes development, and early islet cell defects due to alteration in the cellular localization of pCREB. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Health sciences/Medical research Health sciences/Pathogenesis Type 1 diabetes pCREB Glucagon-like peptide- 1 Prediabetes insulin cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Type 1 diabetes is an autoimmune disease that results in beta cell death, which leads to high circulating glucose levels and the need for insulin therapy. The identity of the pathways that contribute to the loss of the insulin cells in humans is currently unknown. The traditional view of T1D development, mostly derived from studies in mutant mice, is one in which immune cells become reactive to beta cell proteins resulting in an inflammatory lesion termed insulitis. However, while islet insulitis characterizes islets of diabetic rodents, it is a rare condition in human islets 1 , 2 . Moreover, attempts to prevent progression of the disease by curtailing the immune destruction of the beta cells were met with little success 3 and led to a reassessment of the identity of the factors that initiate the disease. In particular, there is an emerging view that beta cells defects are culprits in T1D initiation, whether by the production of neoantigens elicited by ER stress 4 and/or by the presence of HLA hyperexpression, which makes beta cells visible to immune cells 5 , 6 . Recent studies also indicated that islet inflammation eventually triggers an invasion of immune cells into the islets 7 , which provided evidence that beta cell dysfunction can initiate the disorder. The current study sought to determine whether cellular processes known to lead to an inflammatory response appear in human islet cells prior to diagnosis. For this purpose, islets of prediabetic and T1D donors were assessed for the expression of the phosphorylated Cyclic AMP-Responsive Element-Binding Protein (pCREB), a general transcription activator that is phosphorylated by protein kinase A at serine 133 in response to diverse signaling pathways (reviewed in 8 ). The fact that pCREB controls the expression of transcription factors that are critical for beta cell differentiation and function, such as MafA, PDX-1and NeuroD 9 – 11 indicates that its function is critical for achieving a normal mature phenotype. It is known that pCREB activity is regulated by the glucagon-like peptide (GLP-1) 12 , 13 , an incretin produced by the islet glucagon cells that has a direct effect on islet function 14 – 17 . The report that the levels of GLP-1 levels increase during diabetes development 18 , raised to possibility that this surge will affect pCREB expression and disrupt normal islet cell traits. To test this hypothesis, we evaluated the expression of pCREB, insulin and a protease in sections of human pancreas of control, prediabetes and type 1 Diabetes donors . Results 1- Transient cytoplasmic translocation of pCREB in mature islet cells prior to type 1 Diabetes diagnosis. Comparison of pCREB localization in islets of control and of donors with one circulating autoantibody (Glutamic Acid Decarboxylase autoantibody, GADA) older than 16.7 years revealed significant differences in the cellular localization of pCREB among the two groups. Thus, while in controls pCREB was localized to the nucleus (Fig. 1 A), islets of two of the three GADA tissues displayed the transcription factor in both beta cell nuclei and cytoplasm (Fig. 1, B, C). In many instances, islets with altered subcellular localization of pCREB were located next to normal islets underscoring the presence of islet heterogeneity 19 . The cytoplasmic localization of the transcription factor persisted in insulin cells of all islets examined from donors with two circulating autoantibodies and in those from the two recent onset type 1 Diabetes donors examined (Fig. 1 D-E). However, the translocation of pCREB was more prominent in all islets in only one of the recent onset donors (donor #6362) than in the other (donor # 6520). pCREB expression was again nuclear in all beta cells of donors with long standing diabetes (Fig. 1, F). Glucagon cells of adult control donors also display nuclear pCREB expression (Fig. 2A) while those of one recent onset type 1 diabetes donor showed decrease nuclear localization of pCREB (donor #6520, Fig. 2B) and those of the second donor displayed significant cytoplasmic localization of the factor (Fig. 2C). However, islet glucagon cells of donors with long standing diabetes exhibited nuclear pCREB expression (Fig. 2D). Photomicrographs illustrate islet cells immunostained for glucagon (red) and pCREB (green). A- islet of donor control. B- Islet of donor # 6520 (0 time after diagnosis). Note that alpha cells lack nuclear pCREB expression. C- Islet of donor # 6362 (0 time after diagnosis). This photomicrograph documents the presence of alpha cells with overlapping glucagon (red) and pCREB (green) cytoplasmic labelling. D- islet of donor # 5000 (2 yrs after diagnosis). Note the nuclear localization of the transcription factor. The proposition that diabetes development involves a process of dedifferentiation 20 , suggested that the cytoplasmic translocation of pCREB in islet cells of adults reflects a transition to an immature state. To test this possibility, pCREB expression was examined in pancreata from control newborn to late adolescent donors. At postnatal day 0, islets of non-diabetic control donor displayed pCREB expression in the cytoplasm and nuclei of beta cells (Fig. 3, A-C) and maintain this distribution until late adolescent (age:> 16.7 years, Fig. 3 D-F). These findings raised the question of whether pCREB remains in a cytoplasmic location of islet cells of young Type 1 diabetic donors. Analysis of pancreas from this cohort revealed that pCREB expression remained in the cytoplasm of beta cells of some donors (Fig. 3, G-I) but was nuclear in others (Fig. 3, J-L). Pancreatic islets containing beta cells displaying nuclear pCREB were found in pancreas contained only few surviving islets, suggesting that this phenotype represented an extreme condition of the disease. 2- Increased cytoplasmic accumulation of pCREB in mature beta cells is correlated with reduced expression of insulin and caspase 3. To determine whether insulin cells of prediabetic and/or type 1 Diabetes donors undergo programmed cell death, pancreas sections were immunostained for insulin and activated caspase 3, a protease identified as a key mediator of apoptosis 21 , 22 . Surprisingly, activated caspase 3 immunoreactivity was specifically localized to the cytoplasm of beta cells but not to their nuclei or to alpha cells or to cells of the exocrine tissue (Fig. 4). Previous studies reported the expression of the protease only in nuclei of scattered beta cells of type 1 and type 2 diabetes donors 23 – 25 . However, the present study found cytoplasmic localization of the protease using antibodies to activated caspase 3 from the same source and batch number as that reported by others 24 , 25 . Moreover, the cytoplasmic localization of the enzyme was corroborated with antibodies from a second source (Chemicon) (Fig. 4). The reason/s for the discrepancy between these results and those from others remains to be elucidated. Photomicrograph illustrates islet of non-diabetic control (# 6134) immunostained for caspase (green), insulin (cyan) and glucagon (red). Note that the protease localizes to beta but not to alpha or exocrine cells. Photomicrographs B to F show caspase staining in islets of control # 6539 (B), #6535(C), #6548 (D), #6571(E ) and #6557(F). Islets shown in A to D were processed with antibodies from Cell Signalling while islets illustrated in photomicrographs E and F were immunostained with an antibody from Chemicon. Analysis of the expression of activated caspase 3 in the adult donor cohort during type 1 diabetes development revealed that the expression of the protease decreased significantly with disease progression. Thus, islets of GADA donors showed reduced caspase expression (Fig. 5B) when compared to that of control (Fig. 5A). In GADA donor #6314 the enzyme was localized not only to the cytoplasm (Fig. 5B) but also, in some islets, to the nuclei of beta cells (Fig. 5C). It should be noted that this is the only case found of all those examined that showed caspase localized to the nucleus. The decrease in the level of expression of the protease and hormone became more pronounced with disease progression. Thus, islets from recently diagnosed donor # 6250 were comprised of beta cells displaying a loss of activated caspase 3 expression (Fig. 5:D) that was concomitant with that of insulin (Fig. 5:E, F) while those of donor # 6362 expressed either caspase or insulin (Fig. 5: G, H). In contrast, insulin cells of donor # 6325, which had the disease for six years, were insulin + (Fig. 5 I) but caspase – (not shown). These observations indicate a decrease ability of beta cells to maintain the proper concentration of (at least) these two proteins and indicates a correlation, in islet cells of this cohort, between these defects and the accumulation of pCREB in their cytoplasm. However, there was significant heterogeneity in the expression of these traits between islets from the same donors. Moreover, one donor with long term type 1 Diabetes examined (# 6190) contained few islets populated by beta cells expressing insulin and caspase. The expression of cleaved caspase 3 in islets of some young type 1 Diabetes donors (Fig. 6: D-F) was similar to that of controls (Fig. 6: A-C) in one tissue examined but it decreased in beta cells of other donor (Fig. 6: G-I). In this donor, cleaved caspase 3 also labelled unidentified cells distributed within the islet and in the exocrine tissue. These cells were not seen in the pancreas of the young type 1 Diabetes donor showing normal caspase level (Fig. 6: D-F). Similarly, these caspase + insulin − cells were not found in islets from adult type 1 Diabetes donors. Presumably, these are effector T cells of the immune system which have been reported to have increased caspase 3 expression 26 . 3- pCREB and caspase in T2 Diabetic islets . The alteration in protein expression in islet cells with Type 1 diabetes progression raised the question of whether these observations are replicated in islets of type 2 Diabetic donors. Examination of pancreas from type 2 diabetic donors revealed significant cytoplasmic pCREB expression in most islets (Fig. 7). Interestingly, this location of the transcription factor was found in islets without (Fig. 7B) and with amyloid (Fig. 7E) deposition in the cytoplasm. It should be noted that the presence of amyloid was detected with DAPI, which localizes to the cytoplasm because the dye has higher affinity to protein aggregates than to DNA 27 . In contrast to islets from type 1diabetic donors, type 2 Diabetic islets do not show nuclear translocation of pCREB and express normal levels of caspase 3 (not shown). Discussion The main findings from this study are: 1- The transcription factor pCREB undergoes a movement from the nucleus to the cytoplasm of insulin cells of prediabetic autoantibody positive donors. 2- The presence of pCREB in the cytoplasm, which decreases its activity, is correlated with a reduction in the expression levels of insulin and caspase, a protease with apoptotic and non-apoptotic functions. 3- The displacement of the transcription factor to the cytoplasm is transient, since it translocates to the nucleus of islet cells from donors with long-standing disease. 4- pCREB is also relocated to the cytoplasm of beta cells from Type 2 Diabetes donors. This movement is not correlated with the presence of amyloid deposits, a known histopathological feature of the disease. The presence of pCREB in the cytoplasm of islet cells indicates an alteration in the normal distribution of this protein between subcellular compartments. The identity of the dysfunction/s that leads to pCREB presence in the cytoplasm of prediabetic and diabetic mature islet cells is currently unknown. The cytoplasmic location of the transcription factor is not, by itself, a reflection of a cellular defect since that is its place in normal beta cells from young controls. However, islet cells from these two groups have fundamental differences in their fate, since only those of adults are dysfunctional. This comparison indicates that the cytoplasmic location of the transcription factor in islet cells of prediabetic donors reflects the presence of additional cellular defects that prevents normal function. These observations concur with findings for other transcription factors during Type 2 diabetes development 28 , 29 indicating that the movement of nuclear factors to the cytoplasm in islet cells is a characteristic of both forms of the disease. The presence of pCREB in the cytoplasm of islet cells of prediabetic donors may be due to an abnormal autocrine and paracrine interaction between those cells. During maturation, beta cells become responsive to signals from other cell types (reviewed in 30 – 32 ). An important component of this interaction is the ability to respond to the incretin glucagon-like peptide-1 (GLP-1) produced by the alpha cells. It is known that expression of the GLP-1receptor by rodent beta cells starts after birth 33 , that alpha cells also express GLP-1 receptors 34 , and that the intra-islet levels of GLP-1, like those of glucagon, increase in Type 1 and Type 2 diabetes 15 , 16 , 18 . If GLP-1 regulates not only pCREB activity 12 , 13 but also its concentration, the cytoplasmic localization of the transcription factor in islet cells of diagnosed donors could be a consequence of excess incretin levels. If so, the presence of the transcription factor in islet cell cytoplasm of prediabetic donors would suggest that abnormal GLP-1 levels are present at this early stage. It is also possible that the cytoplasmic accumulation of pCREB is due to inhibition of nuclear import 35 – 37 and/or disruption of CREB activity in the mitochondria such as that found in neurodegenerative diseases 38 , 39 . Evidence from other cellular systems also suggest that the translocation of pCREB to the cytoplasm during disease progression in the adult is due to oxidative stress. The treatment of neurons with the oxidative neurotoxin 6-Hydroxydomanine (6-OHDA) resulted in CRE-mediated repression of CREB-regulated genes and accumulation of pCREB in the cytoplasm and its decrease in the nucleus. 35 , 40 . Oxidative stress in diabetes is believed to be due to elevated glucose concentration (glucose toxicity) 41 . However, oxidative stress is unlikely to play a role in the cytoplasmic translocation of pCREB in islet cells since the change in the cellular localization of the transcription factor occurs prior to diagnosis, when glucose levels are normal. The decreased activity of pCREB in cells of prediabetic donors is correlated with a gradual reduction in the expression of activated caspase 3 and insulin. The fall in levels of these two proteins became more pronounced in islets of recently diagnosed Type 1Diabetic adult donor and in those with long term disease. Since pCREB stimulates the expression of genes involved in a large number of cellular processes (reviewed in 11 , 42 , its translocation to the cytoplasm is likely to result in decreased levels of many other cellular proteins. Because caspases participate in a complex interaction with autophagy to maintain protein homeostasis 43 , its decreased expression during diabetes development may lead to the accumulation of intracellular debris and contribute to the ongoing dysregulation of protein homeostasis (or proteostasis). This abnormality, characteristic of human postmitotic cells such as neurons and islet cells, is known to contribute to the development of neurological and metabolic disorders 44 – 46 While pCREB is found in the cytoplasm of islet cells of recently diagnosed mature donors, the transcription factor has a nuclear localization in islet of adults that had the disease for at least one year. This movement, which leads to an activation of pCREB signalling, may be triggered by events that occur in the initial postdiagnosis period. One likely signal is the sterile inflammation induced by the abnormal protein homeostasis that islet cells display during that phase 47 , 48 . It has also been proposed that alteration in RNA processing during the initial post-diabetic period results in inflammation 49 . This inflammatory response may eventually lead to an autoimmune reaction during which the islet cells interact with cytotoxic cells of the immune system, contributing to the development of the disease 5 , 50 – 53 . It is postulated that the onset of sterile inflammation in patients with impaired proteastosis signify a point of no-return from which protein equilibrium cannot be rescued 50 , suggesting that appropriate therapy should be concentrated to resolve the initial defect. It is unclear whether islet cells from young donors go through a similar initial critical period. Recently diagnosed young Type 1 diabetic donors were not available for this study, and the analysis of tissues from donors with long term disease revealed that beta cells retained its cytoplasmic and nuclei localization of pCREB some cases, but it is expressed only in the nucleus of islet cells in others. Islets of type 2 Diabetic donors also showed cytoplasmic localization of pCREB in beta cells. This observation agrees with the report that beta cell dysfunction in Type 2 Diabetes is correlated with the inhibition of CREB signaling 10 . Notably, this localization was found in islets with and without amyloid deposits, a characteristic pathological feature of type 2 diabetes 54 , 55 , suggesting that the movement of pCREB between cellular compartments and the deposit of amyloid are caused by different signals. In summary, these observations suggest a model for T1D development in which pCREB is translocated to the cytoplasm during the normoglycemic stages of the disease. This movement results in an alteration in protein homeostasis and inflammation in islet cells, initiating a response that attracts cells of the immune system. Methods Paraffin sections of human pancreas were provided by the network of Pancreatic Organ Donors (nPOD). Immunohistochemical staining of sections was carried out essentially as previously described 56 . Guinea pig antibodies to insulin were purchased from Linco Res. (END Millipore, Burlington, MA). Rabbit antibodies to glucagon were purchased from Peninsula (BMA, Basel Switzerland ; Glu1-29, T-4359 -IHC 7165). Antibodies to pCREB were purchased from Cell Signalling, (pCREB #39561) and to activated caspase 3 were obtained from Cell Signalling (Danvers,MA; rabbit Mab #14013) and from Chemicon (Millipore Sigma, St. Louis, MO; Ab # 3623). Secondary antibodies: Anti-rabbit Alexa fluor 488 IgG, goat-anti-mouse Alexa fluor 594 were purchased from Molecular Probes (Eugene, Oregon) and donkey-anti guinea pig Cy5 IgG was purchased from Jackson ImmunoResearch (West Grove, PA). For immunostaining, 5 um paraffin sections were heated in 10 mmol/L citrate buffer, pH 6, for 30min in a water bath at 100 0 C and incubated overnight at 4 0 C with two primary antibodies, which were produced by different hosts. The following day, sections were incubated with the corresponding secondary antibodies for two hours at room temperature and cover slipped using mounting media with DAPI (Vector Labs). When paraffin sections of prediabetic and/or diabetic were processed for immunostaining, a control slide was always included in the procedure. Fluorescence microscopy- Images were capture with an Axio Observer 7/LSM 800 Confocal Carl Zeiss microscope. When possible, 6 islets from each stained section were captured although the number of islets examined was lower in pancreas from long standing Type 1 Diabetes. Confocal single plane images were acquired using the 405-488-555-640(frame) settings and a 20X lens (Zeiss). Images were scanned sequentially to prevent crosstalk between the fluorophores. The same confocal microscope setting for laser power, photomultiplier voltage gain, offset and pinhole value were used to collect all the signals. Images were analyzed using the bioanalysis platforms QuPath and Image J. Characteristics of the donors examined are presented in Tables 1 and 2. Declarations Author Contribution GT designed the study, prepared figures,wrote and reviewed the manuscript. Acknowledgements “This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by Breakthrough T1D and The Leona M. & Harry B. Helmsley Charitable Trust (Grant#3-SRA-2023-1417-S-B). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at https://npod.org/for-partners/npod-partners/ Data Availability Data availability: The data sets analyzed during the current study are available in the https://portal.jdrfnpod.org/ repository. References Campbell-Thompson, M. et al. Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes. Diabetes . 65 , 719–731 (2016). Pugliese, A. Insulitis in the pathogenesis of type 1 diabetes. Pediatr. Diabetes . 17 Suppl 22 , 31–36 (2016). Atkinson, M. 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Islet amyloid polypeptide (IAPP) and pro-IAPP immunoreactivity in human islets of Langerhans. Diabetes Res. Clin. Pract. 7 , 219–226 (1989). Teitelman, G. Human Islets Contain a Beta Cell Type That Expresses Proinsulin But Not the Enzyme That Converts the Precursor to Insulin. J. Histochem. Cytochem. 68 , 691–702 (2020). Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table12.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5285252","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":377307692,"identity":"76d4678f-12fa-4e36-910e-6941215eedda","order_by":0,"name":"Gladys Teitelman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie2NsWrDMBRFnzHIi4pXQSn5BRUvDSn4QzpUxpAs0e4hgyGgyZC1odD+Qkshs4TAk0tXDRkaDJmVrVOonUDpUIWOGXSWB4977gXweM4UCXDTnVCSX8/QnUedIqEPI3ZQ8DF9WoGjgun/lMH9XGlbkLv4sdqZnVinafShPqEYZaVDua4Rk7Ih/GH9/jZcim1W4Tyk0EzcisBUKkF4afjq8mKlGYYckUDoE0pspdoT/mym215JcdxGX8HerQwQBqlKwl/MFPVKUJEcQVC6FYrGVDY14a9mnAyXXXll2oSwepI4V+a6tcXslj+ZfGNso9NokW2snY2unCvyzzdzxA8rri6Px+Px/PAN3vVibXLEvjkAAAAASUVORK5CYII=","orcid":"","institution":"Downstate-Health Science University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gladys","middleName":"","lastName":"Teitelman","suffix":""}],"badges":[],"createdAt":"2024-10-17 21:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5285252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5285252/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68941900,"identity":"135aab92-ffa5-4143-8e81-570fd721c659","added_by":"auto","created_at":"2024-11-13 18:11:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":547362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNucleo-cytoplasmic translocation of pCREB in beta cells of mature donors during T1D development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.- Islet of donor control immunostained for pCREB (green) and insulin (red). Note clear nuclear localization of pCREB. B -Islet of prediabetic donor (#6123, GADA) stained for pCREB (green) and insulin (cyan). Area within the circle, enlarged in C, illustrates the presence of pCREB in the cytoplasm. \u0026nbsp;D-islet of T1D donor #6520 (0 time after diagnosis) immunostained for insulin and pCREB. E- illustrates area within the circle in D. Note the presence of cells lacking nuclear expression of the transcription factor in D and its presence in the cytoplasm in E. F- Islet of T1D #6477 (8 yrs duration) illustrates pCREB movement to the nucleus with time after diagnosis. Bar in B:20um. Bar in C: 5um.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/13022dfb09178797f49a6698.jpeg"},{"id":68941902,"identity":"4a280816-8785-4552-8274-90c2c2f6a08a","added_by":"auto","created_at":"2024-11-13 18:11:46","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":454529,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisplacement of pCREB to the cytoplasm of alpha cells of mature donors during T1D development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhotomicrographs illustrate islet cells immunostained for glucagon (red) and pCREB (green). A- islet of donor control. B- Islet of donor # 6520 (0 time after diagnosis). Note that alpha cells lack nuclear pCREB expression. C- Islet of donor # 6362 (0 time after diagnosis). This photomicrograph documents the presence of alpha cells with overlapping glucagon (red) and pCREB (green) cytoplasmic labelling. D- islet of donor # 5000 (2 yrs after diagnosis). Note the nuclear localization of the transcription factor.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/f5c9d88af10420da31b47eb7.jpeg"},{"id":68942325,"identity":"b96d1a17-cd77-4e93-8c91-c38e720a422f","added_by":"auto","created_at":"2024-11-13 18:19:46","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":930025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003epCREB expression in beta cells of control and T1D young donors\u003c/strong\u003e. A-B-Islet of non-diabetic control (#6214, 0 years) stained for pCREB (green, A) and insulin (red) + pCREB (B). D-E- Islet of control (# 6525, 16.7 years) stained for pCREB (D) and insulin+pCREB (E). Note that cytoplasmic localization of pCREB persists until late adolescence. G-H-Islet of T1D (# 6243, 13 years) stained for pCREB (G) and insulin+pCREB (H). J-K islet of T1D (#6084, 14 years), immunostained for pCREB (J), IN+pCREB (K). Photomicrographs C, F, I and L are high magnification images of areas within the circle in A, D, G and J respectively. Bar in A: 15 um; Bar in C: 4um.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/197519a52a309442c4fedc62.jpeg"},{"id":68941903,"identity":"2a6b62d2-244b-4350-aecb-5706d5a7f961","added_by":"auto","created_at":"2024-11-13 18:11:46","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":533068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivated caspase 3 localizes to the cytoplasm of beta cells\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003ePhotomicrograph illustrates islet of non-diabetic control (# 6134) immunostained for caspase (green), insulin (cyan) and glucagon (red). Note that the protease localizes to beta but not to alpha or exocrine cells. Photomicrographs B to F show caspase staining in islets of control # 6539 (B), #6535(C), #6548 (D), #6571(E ) and #6557(F). Islets shown in A to D were processed with antibodies from Cell Signalling while islets illustrated in photomicrographs E and F were immunostained with an antibody from Chemicon.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/d2f18f43696fb5ab3ad0b11b.jpeg"},{"id":68941906,"identity":"34a55fa8-beaf-4794-a866-c5a24c75b75e","added_by":"auto","created_at":"2024-11-13 18:11:46","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":902186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecreased expression of cleaved caspase 3 and insulin in mature islet cells during T1D development\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eA-Islet of non-diabetic donor immunostained for caspase (green). B- Islet of GADA donor # 6314 stained for caspase. Photomicrograph illustrates decreased expression of the enzyme in cytoplasm and its expression in a nucleus (arrow). C- islet of donor 6314 shows exclusive nuclear caspase expression. Islets in B and C are contiguous in the same region of the pancreas, documenting heterogeneity in the expression of the protease. Photomicrographs D-F show an islet of donor # 6520 (day 0 after diagnosis) stained for caspase(D), insulin (E) and caspase+insulin (F). Note significant reduction in the expression of the enzyme. G- islet of donor # 6362(day 0 after diagnosis) stained for caspase (green) and insulin (red). Dotted circle in G is enlarged in H to show beta cells that are either insulin\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e caspase\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e (cross) or insulin\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e caspase \u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e(asterisk). I- Islet of T1D donor is immune-positive for insulin but negative for caspase (not shown). Bar in A:15um; Bar in H: 5um.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/deb4e1b300bfd0cd010299c5.jpeg"},{"id":68941904,"identity":"799036b6-ebb8-4d70-850f-fee0f5e7342b","added_by":"auto","created_at":"2024-11-13 18:11:46","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":459134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential localization of caspase 3 in beta cells of young donors. \u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA-C-Islet of donor control (#6047, 7.8 yrs) show staining for caspase (A), IN (B) and IN+caspase (C). D-F- Islet of Type 1diabetic donor (#6406, 6.9 yrs) immunostained for caspase (D), IN (E) and IN +caspase (F). G-I-Islet of Type 1diabetic donor (# 6380,11.6 yrs) stained for caspase G, IN (H) and IN+ caspase(H). Note the significant decrease in both caspase and insulin levels in donor # 6380 and presence of cells that are insulin\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003ecaspase\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e within and surrounding the islet. These cells were not seen in donor #6406 or in islets of adult T1D donors. Bar: 15um.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/512d3e650c28e85d5eb8a8c6.jpeg"},{"id":68942324,"identity":"f27287e2-6e9a-4789-96bb-ac40c00bf33e","added_by":"auto","created_at":"2024-11-13 18:19:46","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":579992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytoplasmic pCREB expression in beta cells of T2D donors is not correlated with amyloid deposits.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIslets of T2D donor # 6203 immunostained for pCREB (A,D; green) and DAPI (B,E; blue). Photomicrographs C and F illustrate these islets labelled for insulin (red) +pCREB + DAPI. These two islets were located in the same section of pancreas, but only one (D-F) has amyloid deposit. Bar:15 um.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/0bdbb35869bc11e8b91563bb.jpeg"},{"id":70535068,"identity":"08f32eba-4b8f-4622-88b3-8be262e247ce","added_by":"auto","created_at":"2024-12-04 06:47:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4978610,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/c3cc332e-6c48-42f9-8d31-a1f56ee30f4d.pdf"},{"id":68941899,"identity":"6b0aa280-686d-4792-abb9-931c4c56d2b5","added_by":"auto","created_at":"2024-11-13 18:11:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14914,"visible":true,"origin":"","legend":"","description":"","filename":"Table12.docx","url":"https://assets-eu.researchsquare.com/files/rs-5285252/v1/af69be28fb0885fd033ee4e1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human beta cells become dysfunctional prior to type 1 diabetes diagnosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eType 1 diabetes is an autoimmune disease that results in beta cell death, which leads to high circulating glucose levels and the need for insulin therapy. The identity of the pathways that contribute to the loss of the insulin cells in humans is currently unknown. The traditional view of T1D development, mostly derived from studies in mutant mice, is one in which immune cells become reactive to beta cell proteins resulting in an inflammatory lesion termed insulitis. However, while islet insulitis characterizes islets of diabetic rodents, it is a rare condition in human islets \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Moreover, attempts to prevent progression of the disease by curtailing the immune destruction of the beta cells were met with little success \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and led to a reassessment of the identity of the factors that initiate the disease. In particular, there is an emerging view that beta cells defects are culprits in T1D initiation, whether by the production of neoantigens elicited by ER stress \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and/or by the presence of HLA hyperexpression, which makes beta cells visible to immune cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recent studies also indicated that islet inflammation eventually triggers an invasion of immune cells into the islets \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, which provided evidence that beta cell dysfunction can initiate the disorder.\u003c/p\u003e \u003cp\u003eThe current study sought to determine whether cellular processes known to lead to an inflammatory response appear in human islet cells prior to diagnosis. For this purpose, islets of prediabetic and T1D donors were assessed for the expression of the phosphorylated Cyclic AMP-Responsive Element-Binding Protein (pCREB), a general transcription activator that is phosphorylated by protein kinase A at serine 133 in response to diverse signaling pathways (reviewed in \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e). The fact that pCREB controls the expression of transcription factors that are critical for beta cell differentiation and function, such as MafA, PDX-1and NeuroD \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e indicates that its function is critical for achieving a normal mature phenotype. It is known that pCREB activity is regulated by the glucagon-like peptide (GLP-1) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, an incretin produced by the islet glucagon cells that has a direct effect on islet function\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The report that the levels of GLP-1 levels increase during diabetes development\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, raised to possibility that this surge will affect pCREB expression and disrupt normal islet cell traits. To test this hypothesis, we evaluated the expression of pCREB, insulin and a protease in sections of human pancreas of control, prediabetes and type 1 Diabetes donors .\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1- Transient cytoplasmic translocation of pCREB in mature islet cells prior to type 1 Diabetes diagnosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparison of pCREB localization in islets of control and of donors with one circulating autoantibody (Glutamic Acid Decarboxylase autoantibody, GADA) older than 16.7 years revealed significant differences in the cellular localization of pCREB among the two groups. Thus, while in controls pCREB was localized to the nucleus (Fig. 1 A), islets of two of the three GADA tissues displayed the transcription factor in both beta cell nuclei and cytoplasm (Fig. 1, B, C). In many instances, islets with altered subcellular localization of pCREB were located next to normal islets underscoring the presence of islet heterogeneity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The cytoplasmic localization of the transcription factor persisted in insulin cells of all islets examined from donors with two circulating autoantibodies and in those from the two recent onset type 1 Diabetes donors examined (Fig.\u0026nbsp;1 D-E). However, the translocation of pCREB was more prominent in all islets in only one of the recent onset donors (donor #6362) than in the other (donor # 6520). pCREB expression was again nuclear in all beta cells of donors with long standing diabetes (Fig.\u0026nbsp;1, F).\u003c/p\u003e\n\u003cp\u003eGlucagon cells of adult control donors also display nuclear pCREB expression (Fig. 2A) while those of one recent onset type 1 diabetes donor showed decrease nuclear localization of pCREB (donor #6520, Fig. 2B) and those of the second donor displayed significant cytoplasmic localization of the factor (Fig. 2C). However, islet glucagon cells of donors with long standing diabetes exhibited nuclear pCREB expression (Fig. 2D).\u003c/p\u003e\n\u003cp\u003ePhotomicrographs illustrate islet cells immunostained for glucagon (red) and pCREB (green). A- islet of donor control. B- Islet of donor # 6520 (0 time after diagnosis). Note that alpha cells lack nuclear pCREB expression. C- Islet of donor # 6362 (0 time after diagnosis). This photomicrograph documents the presence of alpha cells with overlapping glucagon (red) and pCREB (green) cytoplasmic labelling. D- islet of donor # 5000 (2 yrs after diagnosis). Note the nuclear localization of the transcription factor.\u003c/p\u003e\n\u003cp\u003eThe proposition that diabetes development involves a process of dedifferentiation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, suggested that the cytoplasmic translocation of pCREB in islet cells of adults reflects a transition to an immature state. To test this possibility, pCREB expression was examined in pancreata from control newborn to late adolescent donors. At postnatal day 0, islets of non-diabetic control donor displayed pCREB expression in the cytoplasm and nuclei of beta cells (Fig.\u0026nbsp;3, A-C) and maintain this distribution until late adolescent (age:\u0026gt; 16.7 years, Fig.\u0026nbsp;3 D-F).\u003c/p\u003e\n\u003cp\u003eThese findings raised the question of whether pCREB remains in a cytoplasmic location of islet cells of young Type 1 diabetic donors. Analysis of pancreas from this cohort revealed that pCREB expression remained in the cytoplasm of beta cells of some donors (Fig. 3, G-I) but was nuclear in others (Fig. 3, J-L). Pancreatic islets containing beta cells displaying nuclear pCREB were found in pancreas contained only few surviving islets, suggesting that this phenotype represented an extreme condition of the disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2- Increased cytoplasmic accumulation of pCREB in mature beta cells is correlated with reduced expression of insulin and caspase 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether insulin cells of prediabetic and/or type 1 Diabetes donors undergo programmed cell death, pancreas sections were immunostained for insulin and activated caspase 3, a protease identified as a key mediator of apoptosis \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Surprisingly, activated caspase 3 immunoreactivity was specifically localized to the cytoplasm of beta cells but not to their nuclei or to alpha cells or to cells of the exocrine tissue (Fig. 4). Previous studies reported the expression of the protease only in nuclei of scattered beta cells of type 1 and type 2 diabetes donors \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, the present study found cytoplasmic localization of the protease using antibodies to activated caspase 3 from the same source and batch number as that reported by others \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, the cytoplasmic localization of the enzyme was corroborated with antibodies from a second source (Chemicon) (Fig. 4). The reason/s for the discrepancy between these results and those from others remains to be elucidated.\u003c/p\u003e\n\u003cp\u003ePhotomicrograph illustrates islet of non-diabetic control (# 6134) immunostained for caspase (green), insulin (cyan) and glucagon (red). Note that the protease localizes to beta but not to alpha or exocrine cells. Photomicrographs B to F show caspase staining in islets of control # 6539 (B), #6535(C), #6548 (D), #6571(E ) and #6557(F). Islets shown in A to D were processed with antibodies from Cell Signalling while islets illustrated in photomicrographs E and F were immunostained with an antibody from Chemicon.\u003c/p\u003e\n\u003cp\u003eAnalysis of the expression of activated caspase 3 in the adult donor cohort during type 1 diabetes development revealed that the expression of the protease decreased significantly with disease progression. Thus, islets of GADA donors showed reduced caspase expression (Fig.\u0026nbsp;5B) when compared to that of control (Fig.\u0026nbsp;5A). In GADA donor #6314 the enzyme was localized not only to the cytoplasm (Fig.\u0026nbsp;5B) but also, in some islets, to the nuclei of beta cells (Fig.\u0026nbsp;5C). It should be noted that this is the only case found of all those examined that showed caspase localized to the nucleus.\u003c/p\u003e\n\u003cp\u003eThe decrease in the level of expression of the protease and hormone became more pronounced with disease progression. Thus, islets from recently diagnosed donor # 6250 were comprised of beta cells displaying a loss of activated caspase 3 expression (Fig.\u0026nbsp;5:D) that was concomitant with that of insulin (Fig.\u0026nbsp;5:E, F) while those of donor # 6362 expressed either caspase or insulin (Fig.\u0026nbsp;5: G, H). In contrast, insulin cells of donor # 6325, which had the disease for six years, were insulin\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e (Fig. 5 I) but caspase \u003csup\u003e\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e\u003c/sup\u003e (not shown). These observations indicate a decrease ability of beta cells to maintain the proper concentration of (at least) these two proteins and indicates a correlation, in islet cells of this cohort, between these defects and the accumulation of pCREB in their cytoplasm. However, there was significant heterogeneity in the expression of these traits between islets from the same donors. Moreover, one donor with long term type 1 Diabetes examined (# 6190) contained few islets populated by beta cells expressing insulin and caspase.\u003c/p\u003e\n\u003cp\u003eThe expression of cleaved caspase 3 in islets of some young type 1 Diabetes donors (Fig.\u0026nbsp;6: D-F) was similar to that of controls (Fig.\u0026nbsp;6: A-C) in one tissue examined but it decreased in beta cells of other donor (Fig.\u0026nbsp;6: G-I). In this donor, cleaved caspase 3 also labelled unidentified cells distributed within the islet and in the exocrine tissue. These cells were not seen in the pancreas of the young type 1 Diabetes donor showing normal caspase level (Fig.\u0026nbsp;6: D-F). Similarly, these caspase\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e insulin\u003csup\u003e\u003cstrong\u003e\u0026minus;\u003c/strong\u003e\u003c/sup\u003e cells were not found in islets from adult type 1 Diabetes donors. Presumably, these are effector T cells of the immune system which have been reported to have increased caspase 3 expression \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e3- pCREB and caspase in T2 Diabetic islets\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe alteration in protein expression in islet cells with Type 1 diabetes progression raised the question of whether these observations are replicated in islets of type 2 Diabetic donors. Examination of pancreas from type 2 diabetic donors revealed significant cytoplasmic pCREB expression in most islets (Fig. 7). Interestingly, this location of the transcription factor was found in islets without (Fig. 7B) and with amyloid (Fig. 7E) deposition in the cytoplasm. It should be noted that the presence of amyloid was detected with DAPI, which localizes to the cytoplasm because the dye has higher affinity to protein aggregates than to DNA \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In contrast to islets from type 1diabetic donors, type 2 Diabetic islets do not show nuclear translocation of pCREB and express normal levels of caspase 3 (not shown).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main findings from this study are: 1- The transcription factor pCREB undergoes a movement from the nucleus to the cytoplasm of insulin cells of prediabetic autoantibody positive donors. 2- The presence of pCREB in the cytoplasm, which decreases its activity, is correlated with a reduction in the expression levels of insulin and caspase, a protease with apoptotic and non-apoptotic functions. 3- The displacement of the transcription factor to the cytoplasm is transient, since it translocates to the nucleus of islet cells from donors with long-standing disease. 4- pCREB is also relocated to the cytoplasm of beta cells from Type 2 Diabetes donors. This movement is not correlated with the presence of amyloid deposits, a known histopathological feature of the disease.\u003c/p\u003e \u003cp\u003eThe presence of pCREB in the cytoplasm of islet cells indicates an alteration in the normal distribution of this protein between subcellular compartments. The identity of the dysfunction/s that leads to pCREB presence in the cytoplasm of prediabetic and diabetic mature islet cells is currently unknown. The cytoplasmic location of the transcription factor is not, by itself, a reflection of a cellular defect since that is its place in normal beta cells from young controls. However, islet cells from these two groups have fundamental differences in their fate, since only those of adults are dysfunctional. This comparison indicates that the cytoplasmic location of the transcription factor in islet cells of prediabetic donors reflects the presence of additional cellular defects that prevents normal function. These observations concur with findings for other transcription factors during Type 2 diabetes development \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e indicating that the movement of nuclear factors to the cytoplasm in islet cells is a characteristic of both forms of the disease.\u003c/p\u003e \u003cp\u003eThe presence of pCREB in the cytoplasm of islet cells of prediabetic donors may be due to an abnormal autocrine and paracrine interaction between those cells. During maturation, beta cells become responsive to signals from other cell types (reviewed in \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e). An important component of this interaction is the ability to respond to the incretin glucagon-like peptide-1 (GLP-1) produced by the alpha cells. It is known that expression of the GLP-1receptor by rodent beta cells starts after birth \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, that alpha cells also express GLP-1 receptors \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, and that the intra-islet levels of GLP-1, like those of glucagon, increase in Type 1 and Type 2 diabetes \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. If GLP-1 regulates not only pCREB activity \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e but also its concentration, the cytoplasmic localization of the transcription factor in islet cells of diagnosed donors could be a consequence of excess incretin levels. If so, the presence of the transcription factor in islet cell cytoplasm of prediabetic donors would suggest that abnormal GLP-1 levels are present at this early stage.\u003c/p\u003e \u003cp\u003eIt is also possible that the cytoplasmic accumulation of pCREB is due to inhibition of nuclear import \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and/or disruption of CREB activity in the mitochondria such as that found in neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Evidence from other cellular systems also suggest that the translocation of pCREB to the cytoplasm during disease progression in the adult is due to oxidative stress. The treatment of neurons with the oxidative neurotoxin 6-Hydroxydomanine (6-OHDA) resulted in CRE-mediated repression of CREB-regulated genes and accumulation of pCREB in the cytoplasm and its decrease in the nucleus. \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Oxidative stress in diabetes is believed to be due to elevated glucose concentration (glucose toxicity) \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, oxidative stress is unlikely to play a role in the cytoplasmic translocation of pCREB in islet cells since the change in the cellular localization of the transcription factor occurs prior to diagnosis, when glucose levels are normal.\u003c/p\u003e \u003cp\u003eThe decreased activity of pCREB in cells of prediabetic donors is correlated with a gradual reduction in the expression of activated caspase 3 and insulin. The fall in levels of these two proteins became more pronounced in islets of recently diagnosed Type 1Diabetic adult donor and in those with long term disease. Since pCREB stimulates the expression of genes involved in a large number of cellular processes (reviewed in \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, its translocation to the cytoplasm is likely to result in decreased levels of many other cellular proteins. Because caspases participate in a complex interaction with autophagy to maintain protein homeostasis \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, its decreased expression during diabetes development may lead to the accumulation of intracellular debris and contribute to the ongoing dysregulation of protein homeostasis (or proteostasis). This abnormality, characteristic of human postmitotic cells such as neurons and islet cells, is known to contribute to the development of neurological and metabolic disorders \u003csup\u003e\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile pCREB is found in the cytoplasm of islet cells of recently diagnosed mature donors, the transcription factor has a nuclear localization in islet of adults that had the disease for at least one year. This movement, which leads to an activation of pCREB signalling, may be triggered by events that occur in the initial postdiagnosis period. One likely signal is the sterile inflammation induced by the abnormal protein homeostasis that islet cells display during that phase \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. It has also been proposed that alteration in RNA processing during the initial post-diabetic period results in inflammation \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This inflammatory response may eventually lead to an autoimmune reaction during which the islet cells interact with cytotoxic cells of the immune system, contributing to the development of the disease \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. It is postulated that the onset of sterile inflammation in patients with impaired proteastosis signify a point of no-return from which protein equilibrium cannot be rescued \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, suggesting that appropriate therapy should be concentrated to resolve the initial defect. It is unclear whether islet cells from young donors go through a similar initial critical period. Recently diagnosed young Type 1 diabetic donors were not available for this study, and the analysis of tissues from donors with long term disease revealed that beta cells retained its cytoplasmic and nuclei localization of pCREB some cases, but it is expressed only in the nucleus of islet cells in others.\u003c/p\u003e \u003cp\u003eIslets of type 2 Diabetic donors also showed cytoplasmic localization of pCREB in beta cells. This observation agrees with the report that beta cell dysfunction in Type 2 Diabetes is correlated with the inhibition of CREB signaling \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Notably, this localization was found in islets with and without amyloid deposits, a characteristic pathological feature of type 2 diabetes \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, suggesting that the movement of pCREB between cellular compartments and the deposit of amyloid are caused by different signals.\u003c/p\u003e \u003cp\u003eIn summary, these observations suggest a model for T1D development in which pCREB is translocated to the cytoplasm during the normoglycemic stages of the disease. This movement results in an alteration in protein homeostasis and inflammation in islet cells, initiating a response that attracts cells of the immune system.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eParaffin sections of human pancreas were provided by the network of Pancreatic Organ Donors (nPOD). Immunohistochemical staining of sections was carried out essentially as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Guinea pig antibodies to insulin were purchased from Linco Res. (END Millipore, Burlington, MA). Rabbit antibodies to glucagon were purchased from Peninsula (BMA, Basel Switzerland ; Glu1-29, T-4359 -IHC 7165). Antibodies to pCREB were purchased from Cell Signalling, (pCREB #39561) and to activated caspase 3 were obtained from Cell Signalling (Danvers,MA; rabbit Mab #14013) and from Chemicon (Millipore Sigma, St. Louis, MO; Ab # 3623).\u003c/p\u003e\n\u003cp\u003eSecondary antibodies: Anti-rabbit Alexa fluor 488 IgG, goat-anti-mouse Alexa fluor 594 were purchased from Molecular Probes (Eugene, Oregon) and donkey-anti guinea pig Cy5 IgG was purchased from Jackson ImmunoResearch (West Grove, PA).\u003c/p\u003e\n\u003cp\u003eFor immunostaining, 5 um paraffin sections were heated in 10 mmol/L citrate buffer, pH 6, for 30min in a water bath at 100\u003csup\u003e0\u003c/sup\u003eC and incubated overnight at 4\u003csup\u003e0\u003c/sup\u003eC with two primary antibodies, which were produced by different hosts. The following day, sections were incubated with the corresponding secondary antibodies for two hours at room temperature and cover slipped using mounting media with DAPI (Vector Labs). When paraffin sections of prediabetic and/or diabetic were processed for immunostaining, a control slide was always included in the procedure.\u003c/p\u003e\n\u003cp\u003eFluorescence microscopy- Images were capture with an Axio Observer 7/LSM 800 Confocal Carl Zeiss microscope. When possible, 6 islets from each stained section were captured although the number of islets examined was lower in pancreas from long standing Type 1 Diabetes. Confocal single plane images were acquired using the 405-488-555-640(frame) settings and a 20X lens (Zeiss). Images were scanned sequentially to prevent crosstalk between the fluorophores. The same confocal microscope setting for laser power, photomultiplier voltage gain, offset and pinhole value were used to collect all the signals. Images were analyzed using the bioanalysis platforms QuPath and Image J. Characteristics of the donors examined are presented in Tables 1 and 2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGT designed the study, prepared figures,wrote and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e\u0026ldquo;This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by Breakthrough T1D and The Leona M. \u0026amp; Harry B. Helmsley Charitable Trust (Grant#3-SRA-2023-1417-S-B). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npod.org/for-partners/npod-partners/\u003c/span\u003e\u003cspan address=\"https://npod.org/for-partners/npod-partners/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData availability: The data sets analyzed during the current study are available in the https://portal.jdrfnpod.org/ repository.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCampbell-Thompson, M. et al. Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes. \u003cem\u003eDiabetes\u003c/em\u003e. \u003cb\u003e65\u003c/b\u003e, 719\u0026ndash;731 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePugliese, A. 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Cytochem.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e, 691\u0026ndash;702 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Type 1 diabetes, pCREB, Glucagon-like peptide- 1, Prediabetes, insulin cells","lastPublishedDoi":"10.21203/rs.3.rs-5285252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5285252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eType 1 Diabetes is an autoimmune disease that leads to beta cell death. To test whether beta cell defects precede diagnosis we surveyed the expression of pCREB in human islet cells. pCREB is a transcription factor produced by islet alpha cells that is regulated by the incretin Glucagon-Like Peptide (GLP-1). Since levels of GLP-1 rise during diabetes development, we asked whether this increase would be deleterious to islet cells. This analysis indicated that while islet cells of control donors display pCREB in the nucleus, the transcription factor was also found in the cytoplasm of insulin cells of normoglycemic prediabetic donors and of beta and alpha cells of recently diagnosed donors. The translocation of pCREB, which decreases its activity, was correlated with reduced or absent expression of insulin and of a protease. These changes suggest an alteration in protein homeostasis. The cytoplasmic localization of pCREB is transient, since the transcription factor moves to the nuclei of insulin and glucagon cells of donors with longer standing disease.\u003c/p\u003e \u003cp\u003eThe present observations suggest a possible correlation between the known increase in GLP-1 during diabetes development, and early islet cell defects due to alteration in the cellular localization of pCREB.\u003c/p\u003e","manuscriptTitle":"Human beta cells become dysfunctional prior to type 1 diabetes diagnosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 18:11:41","doi":"10.21203/rs.3.rs-5285252/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82138f81-9052-4c09-8760-5f6c93abffc8","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40169707,"name":"Biological sciences/Cell biology"},{"id":40169708,"name":"Health sciences/Diseases"},{"id":40169709,"name":"Health sciences/Endocrinology"},{"id":40169710,"name":"Health sciences/Medical research"},{"id":40169711,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2024-12-04T06:39:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 18:11:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5285252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5285252","identity":"rs-5285252","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}