Identification of unique cell type responses in pancreatic islets to stress.

Diabetes involves the death or dysfunction of pancreatic ß-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response, we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types, we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ß-, α-, and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1, which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics.

Scavengers in islets fuel diabetic autoimmunity.

Autoreactive lymphocytes that infiltrate the pancreatic islet environment and target ß cells are primary drivers of type 1 diabetes. In this issue of Immunity, Srivastava et al.1 examine the role of the islet microenvironment in autoimmunity and find that the scavenging receptor CXCL16 on islet-resident macrophages uptakes oxidized low-density lipoproteins and promotes the differentiation and survival of infiltrating pathogenic CD8+ T cells.

Single Cell RNAseq Analysis of Cytokine-Treated Human Islets: Association of Cellular Stress with Impaired Cytokine Responsiveness.

Pancreatic ß-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1ß) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent ß-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1ß-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.

CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets.

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

Decreased islet amyloid polypeptide staining in the islets of insulinoma patients.

Islet amyloid polypeptide (IAPP) is a factor that regulates food intake and is secreted from both pancreatic islets and insulinoma cells. Here, we aimed to evaluate IAPP immunohistochemically in islets or insulinoma cells in association with clinical characteristics. We recruited six insulinoma patients and six body mass index-matched control patients with pancreatic diseases other than insulinoma whose glucose tolerance was confirmed to be normal preoperatively. IAPP and IAPP-insulin double staining were performed on pancreatic surgical specimens. We observed that the IAPP staining level and percentage of IAPP-positive beta cells tended to be lower (p = 0.1699) in the islets of insulinoma patients than in those of control patients, which might represent a novel IAPP expression pattern under persistent hyperinsulinemia and hypoglycemia.

PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system.

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

Scalable Generation of 3D Pancreatic Islet Organoids from Human Pluripotent Stem Cells in Suspension Bioreactors.

We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population-level variation and mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized. We defined prototypical insulin secretion responses to three macronutrients in islets from 140 cadaveric donors, including those with type 2 diabetes. The majority of donors' islets exhibited the highest insulin response to glucose, moderate response to amino acid, and minimal response to fatty acid. However, 9% of donors' islets had amino acid responses, and 8% had fatty acid responses that were larger than their glucose-stimulated insulin responses. We leveraged this heterogeneity and used multi-omics to identify molecular correlates of nutrient responsiveness, as well as proteins and mRNAs altered in type 2 diabetes. We also examined nutrient-stimulated insulin release from stem cell-derived islets and observed responsiveness to fat but not carbohydrate or protein-potentially a hallmark of immaturity. Understanding the diversity of insulin responses to carbohydrate, protein, and fat lays the groundwork for personalized nutrition.

Hypertonicity during a rapid rise in D-glucose mediates first-phase insulin secretion.

Introduction: Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Methods: Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Results: Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. Discussion: The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.

Predicting type 2 diabetes via machine learning integration of multiple omics from human pancreatic islets.

Type 2 diabetes (T2D) is the fastest growing non-infectious disease worldwide. Impaired insulin secretion from pancreatic beta-cells is a hallmark of T2D, but the mechanisms behind this defect are insufficiently characterized. Integrating multiple layers of biomedical information, such as different Omics, may allow more accurate understanding of complex diseases such as T2D. Our aim was to explore and use Machine Learning to integrate multiple sources of biological/molecular information (multiOmics), in our case RNA-sequening, DNA methylation, SNP and phenotypic data from islet donors with T2D and non-diabetic controls. We exploited Machine Learning to perform multiOmics integration of DNA methylation, expression, SNPs, and phenotypes from pancreatic islets of 110 individuals, with ~ 30% being T2D cases. DNA methylation was analyzed using Infinium MethylationEPIC array, expression was analyzed using RNA-sequencing, and SNPs were analyzed using HumanOmniExpress arrays. Supervised linear multiOmics integration via DIABLO based on Partial Least Squares (PLS) achieved an accuracy of 91 ± 15% of T2D prediction with an area under the curve of 0.96 ± 0.08 on the test dataset after cross-validation. Biomarkers identified by this multiOmics integration, including SACS and TXNIP DNA methylation, OPRD1 and RHOT1 expression and a SNP annotated to ANO1, provide novel insights into the interplay between different biological mechanisms contributing to T2D. This Machine Learning approach of multiOmics cross-sectional data from human pancreatic islets achieved a promising accuracy of T2D prediction, which may potentially find broad applications in clinical diagnostics. In addition, it delivered novel candidate biomarkers for T2D and links between them across the different Omics.

Molecular profiling of NOD mouse islets reveals a novel regulator of insulitis onset.

Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.

Machine-learning-guided recognition of α and ß cells from label-free infrared micrographs of living human islets of Langerhans.

Human islets of Langerhans are composed mostly of glucagon-secreting α cells and insulin-secreting ß cells closely intermingled one another. Current methods for identifying α and ß cells involve either fixing islets and using immunostaining or disaggregating islets and employing flow cytometry for classifying α and ß cells based on their size and autofluorescence. Neither approach, however, allows investigating the dynamic behavior of α and ß cells in a living and intact islet. To tackle this issue, we present a machine-learning-based strategy for identification α and ß cells in label-free infrared micrographs of living human islets without immunostaining. Intrinsic autofluorescence is stimulated by infrared light and collected both in intensity and lifetime in the visible range, dominated by NAD(P)H and lipofuscin signals. Descriptive parameters are derived from micrographs for ~ 103 cells. These parameters are used as input for a boosted decision-tree model (XGBoost) pre-trained with immunofluorescence-derived cell-type information. The model displays an optimized-metrics performance of 0.86 (i.e. area under a ROC curve), with an associated precision of 0.94 for the recognition of ß cells and 0.75 for α cells. This tool promises to enable longitudinal studies on the dynamic behavior of individual cell types at single-cell resolution within the intact tissue.

Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Phase-changing citrate macromolecule combats oxidative pancreatic islet damage, enables islet engraftment and function in the omentum.

Clinical outcomes for total-pancreatectomy followed by intraportal islet autotransplantation (TP-IAT) to treat chronic pancreatitis (CP) are suboptimal due to pancreas inflammation, oxidative stress during islet isolation, and harsh engraftment conditions in the liver's vasculature. We describe a thermoresponsive, antioxidant macromolecule poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to protect islet redox status and function and to enable extrahepatic omentum islet engraftment. PPCN solution transitions from a liquid to a hydrogel at body temperature. Islets entrapped in PPCN and exposed to oxidative stress remain functional and support long-term euglycemia, in contrast to islets entrapped in a plasma-thrombin biologic scaffold. In the nonhuman primate (NHP) omentum, PPCN is well-tolerated and mostly resorbed without fibrosis at 3 months after implantation. In NHPs, autologous omentum islet transplantation using PPCN restores normoglycemia with minimal exogenous insulin requirements for >100 days. This preclinical study supports TP-IAT with PPCN in patients with CP and highlights antioxidant properties as a mechanism for islet function preservation.

Ameliorating and refining islet organoids to illuminate treatment and pathogenesis of diabetes mellitus.

Diabetes mellitus, a significant global public health challenge, severely impacts human health worldwide. The organoid, an innovative in vitro three-dimensional (3D) culture model, closely mimics tissues or organs in vivo. Insulin-secreting islet organoid, derived from stem cells induced in vitro with 3D structures, has emerged as a potential alternative for islet transplantation and as a possible disease model that mirrors the human body's in vivo environment, eliminating species difference. This technology has gained considerable attention for its potential in diabetes treatment. Despite advances, the process of stem cell differentiation into islet organoid and its cultivation demonstrates deficiencies, prompting ongoing efforts to develop more efficient differentiation protocols and 3D biomimetic materials. At present, the constructed islet organoid exhibit limitations in their composition, structure, and functionality when compared to natural islets. Consequently, further research is imperative to achieve a multi-tissue system composition and improved insulin secretion functionality in islet organoid, while addressing transplantation-related safety concerns, such as tumorigenicity, immune rejection, infection, and thrombosis. This review delves into the methodologies and strategies for constructing the islet organoid, its application in diabetes treatment, and the pivotal scientific challenges within organoid research, offering fresh perspectives for a deeper understanding of diabetes pathogenesis and the development of therapeutic interventions.

Regulation of endocrine cell alternative splicing revealed by single-cell RNA sequencing in type 2 diabetes pathogenesis.

The prevalent RNA alternative splicing (AS) contributes to molecular diversity, which has been demonstrated in cellular function regulation and disease pathogenesis. However, the contribution of AS in pancreatic islets during diabetes progression remains unclear. Here, we reanalyze the full-length single-cell RNA sequencing data from the deposited database to investigate AS regulation across human pancreatic endocrine cell types in non-diabetic (ND) and type 2 diabetic (T2D) individuals. Our analysis demonstrates the significant association between transcriptomic AS profiles and cell-type-specificity, which could be applied to distinguish the clustering of major endocrine cell types. Moreover, AS profiles are enabled to clearly define the mature subset of ß-cells in healthy controls, which is completely lost in T2D. Further analysis reveals that RNA-binding proteins (RBPs), heterogeneous nuclear ribonucleoproteins (hnRNPs) and FXR1 family proteins are predicted to induce the functional impairment of ß-cells through regulating AS profiles. Finally, trajectory analysis of endocrine cells suggests the ß-cell identity shift through dedifferentiation and transdifferentiation of ß-cells during the progression of T2D. Together, our study provides a mechanism for regulating ß-cell functions and suggests the significant contribution of AS program during diabetes pathogenesis.

Single-housing-induced islet epigenomic changes are related to polymorphisms in diabetic KK mice.

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance.

Advancing Diabetes Research: A Novel Islet Isolation Method from Living Donors.

Pancreatic islet isolation is critical for type 2 diabetes research. Although -omics approaches have shed light on islet molecular profiles, inconsistencies persist; on the other hand, functional studies are essential, but they require reliable and standardized isolation methods. Here, we propose a simplified protocol applied to very small-sized samples collected from partially pancreatectomized living donors. Islet isolation was performed by digesting tissue specimens collected during surgery within a collagenase P solution, followed by a Lympholyte density gradient separation; finally, functional assays and staining with dithizone were carried out. Isolated pancreatic islets exhibited functional responses to glucose and arginine stimulation mirroring donors' metabolic profiles, with insulin secretion significantly decreasing in diabetic islets compared to non-diabetic islets; conversely, proinsulin secretion showed an increasing trend from non-diabetic to diabetic islets. This novel islet isolation method from living patients undergoing partial pancreatectomy offers a valuable opportunity for targeted study of islet physiology, with the primary advantage of being time-effective and successfully preserving islet viability and functionality. It enables the generation of islet preparations that closely reflect donors' clinical profiles, simplifying the isolation process and eliminating the need for a Ricordi chamber. Thus, this method holds promises for advancing our understanding of diabetes and for new personalized pharmacological approaches.

Regulatory Role of Fatty Acid Metabolism on Glucose-Induced Changes in Insulin and Glucagon Secretion by Pancreatic Islet Cells.

A detailed study of palmitate metabolism in pancreatic islets subject to different experimental conditions, like varying concentrations of glucose, as well as fed or starved conditions, has allowed us to explore the interaction between the two main plasma nutrients and its consequences on hormone secretion. Palmitate potentiates glucose-induced insulin secretion in a concentration-dependent manner, in a physiological range of both palmitate (0-2 mM) and glucose (6-20 mM) concentrations; at glucose concentrations lower than 6 mM, no metabolic interaction with palmitate was apparent. Starvation (48 h) increased islet palmitate oxidation two-fold, and the effect was resistant to its inhibition by glucose (6-20 mM). Consequently, labelled palmitate and glucose incorporation into complex lipids were strongly suppressed, as well as glucose-induced insulin secretion and its potentiation by palmitate. 2-bromostearate, a palmitate oxidation inhibitor, fully recovered the synthesis of complex lipids and insulin secretion. We concluded that palmitate potentiation of the insulin response to glucose is not attributable to its catabolic mitochondrial oxidation but to its anabolism to complex lipids: islet lipid biosynthesis is dependent on the uptake of plasma fatty acids and the supply of α-glycerol phosphate from glycolysis. Islet secretion of glucagon and somatostatin showed a similar dependence on palmitate anabolism as insulin. The possible mechanisms implicated in the metabolic coupling between glucose and palmitate were commented on. Moreover, possible mechanisms responsible for islet gluco- or lipotoxicity after a long-term stimulation of insulin secretion were also discussed. Our own data on the simultaneous stimulation of insulin, glucagon, and somatostatin by glucose, as well as their modification by 2-bromostearate in perifused rat islets, give support to the conclusion that increased FFA anabolism, rather than its mitochondrial oxidation, results in a potentiation of their stimulated release. Starvation, besides suppressing glucose stimulation of insulin secretion, also blocks the inhibitory effect of glucose on glucagon secretion: this suggests that glucagon inhibition might be an indirect or direct effect of insulin, but not of glucose. In summary, there seems to exist three mechanisms of glucagon secretion stimulation: 1. glucagon stimulation through the same secretion coupling mechanism as insulin, but in a different range of glucose concentrations (0 to 5 mM). 2. Direct or indirect inhibition by secreted insulin in response to glucose (5-20 mM). 3. Stimulation by increased FFA anabolism in glucose intolerance or diabetes in the context of hyperlipidemia, hyperglycemia, and hypo-insulinemia. These conclusions were discussed and compared with previous published data in the literature. Specially, we discussed the mechanism for inhibition of glucagon release by glucose, which was apparently contradictory with the secretion coupling mechanism of its stimulation.

Dapagliflozin administration to a mouse model of type 2 diabetes induces DNA methylation and gene expression changes in pancreatic islets.

Decreased pancreatic ß-cell volume is a serious problem in patients with type 2 diabetes mellitus, and there is a need to establish appropriate treatments. Increasingly, sodium/glucose cotransporter 2 (SGLT2) inhibitors, which have a protective effect on pancreatic ß-cells, are being prescribed to treat diabetes; however, the underlying mechanism is not well understood. We previously administered SGLT2 inhibitor dapagliflozin to a mouse model of type 2 diabetes and found significant changes in gene expression in the early-treated group, which led us to hypothesize that epigenetic regulation was a possible mechanism of these changes. Therefore, we performed comprehensive DNA methylation analysis by methylated DNA immunoprecipitation using isolated pancreatic islets after dapagliflozin administration to diabetic model mice. As a result, we identified 31 genes with changes in expression due to DNA methylation changes. Upon immunostaining, cystic fibrosis transmembrane conductance regulator and cadherin 24 were found to be upregulated in islets in the dapagliflozin-treated group. These molecules may contribute to the maintenance of islet morphology and insulin secretory capacity, suggesting that SGLT2 inhibitors' protective effect on pancreatic ß-cells is accompanied by DNA methylation changes, and that the effect is long-term and not temporary. In future diabetes care, SGLT2 inhibitors may be expected to have positive therapeutic effects, including pancreatic ß-cell protection.