In type 2 diabetes (T2D), molecular pathways driving β cell failure are difficult to resolve with standard single cell analysis. Here we developed an interpretable, supervised machine learning framework that couples sparse rule-based classification (SnakeClassifier), pathway constrained modelling (BlackSwanClassifier), and β cell mitochondrial fitness stratification (Kolmogorov-Arnold Neural Networks KANN), linking and integrating them into disease mechanisms in single cell RNA sequencing (scRNA-seq) from 52 human donors. SnakeClassifier trained on 50 genes accurately predicted T2D at single cell resolution, outperforming classical ensemble machine learning classifier models, and yielded donor level diabetes scores that correlated with chronic hyperglycemia. The clustering of β cell populations (β1-4) revealed a resilient non-diabetic (ND) β1 subtype characterized by preserved β cell identity genes and lower disease risk, whereas T2D β2-4 subtypes exhibited upregulation of genes involved in cellular and mitochondrial stress and suppression of genes promoting oxidative phosphorylation and insulin secretion. Mitophagy emerged as the dominant program linked to T2D and a mitophagy focused BlackSwanClassifier nominated PINK1, BNIP3, and FUNDC1 as key regulators. PINK1 was enriched in ND β1, decreased with T2D disease score and connected sex stratified mitophagy. We generated a KANN derived mitochondrial fitness index (MFI) integrating mitophagy, mitochondrial proteostasis, biogenesis and oxidative phosphorylation into a single interpretable score (R2 = 0.934 vs module-based mitochondria quality index), which identified mitophagy PINK1, SQSTM1, PRKN and BNIP3 as top contributors to T2D progression. These transparent models unify prediction with T2D disease mechanism and identify the mitophagy receptor PINK1 as a central determinant of β cell metabolic fitness.

Pancreatic islets of Langerhans are central to the pathogenesis of all major forms of diabetes. The ability to study human islets ex vivo has advanced our understanding of diabetes and aided in the development of novel therapeutics. However, for decades, very few laboratories had access to this critical resource and experiments on human islets were typically underpowered. More recently, multiple consortia around the world have started to enable islet biology at scale, enriching our understanding of the intra-individual variability of islet function and disease mechanisms. This article reviews and compares existing large-scale human islet tissue and data resources, offering suggestions for their improvement and for developing new resources.

Abstract A main mechanism of β-cell dysfunction in diabetes is loss of identity, controlled by transcription factors that induce identity gene expression and disallowed gene repression. How transcription factors facilitate simultaneous expression and repression is not fully understood, representing a knowledge gap in diabetes research. We identify the transcriptional co-factors transducin β-like 1 x-linked (TBL1X) and its homolog TBL1X-related (TBL1XR1, together TBL/R1) as crucial regulators of β-cell identity and determinants of diabetes development and progression. β-cell specific TBL/R1 knockout in mice leads to progressive hypoinsulinemia and hyperglycemia. scRNA-sequencing reveals loss of β-cells, emergence of polyhormonal cells, and reduced β-cell maturity upon TBL/R1 knockout. Interactome screens and chromatin immunoprecipitation show TBL/R1 directly regulate insulin promoter activity through a PAX6-HDAC3 gene regulatory network, evident also in human models. TBL/R1 associates with diabetes in humans, thus our study uncovers an additional regulatory layer maintaining β-cell identity crucial for diabetes development and progression.

The ability to generate stem cell-derived pancreatic β (SCβ) cells and stem cell-derived islets has opened new opportunities for type 1 diabetes mellitus therapies and disease modelling. Carefully assessing how closely SCβ cells resemble their primary counterparts is crucial to the support of ongoing clinical trials and human-centric studies on diabetes pathophysiology. Observed differences between SCβ cells and primary human β cells are generally attributed to the incomplete maturation of SCβ cells in vitro. However, even cells further matured in vivo can differ considerably from mature primary islet cells. In this Review, we examine key features of human β cells and islets, and summarize our current knowledge of these features in stem cell-derived products. We highlight knowledge gaps and discuss the requirements for comparative studies, emphasizing the importance of tissue quality, harmonizing approaches, integrating datasets and data sharing.

ABSTRACT Stem-cell-derived β-like-cells (SCβ-cells) provide a promising platform for diabetes modelling and cell replacement therapy, but their incomplete functional maturation remains a challenge. Here, we compared immature SCβ-cells to human primary β-cells utilizing a multi-omic, single-cell framework integrating patch-clamp electrophysiology with scRNA sequencing (patch-seq), regulatory network inference, and functional phenotyping. Despite low insulin secretion and reduced insulin content, SCβ-cells displayed larger Na + and Ca 2+ currents and depolarization-induced exocytosis. Ultrastructural and metabolic profiling revealed immature insulin granules, altered mitochondrial morphology, elevated basal respiration and proton leak, and diminished spare respiratory capacity and glucose-responsive metabolism. Patch-seq linked exocytotic activity in SCβ-cells to oxidative phosphorylation and MYC target programs, consistent with incomplete terminal differentiation, whereas SCβ-cells expressing higher levels of mature identity markers showed reduced ion channel hyperactivity. Multi-omics profiling showed that electrophysiological features in SCβ-cells were embedded in transcriptional programs distinct from those of primary β-cells and other endocrine cells. Network control theory nominated SREBP1, an endoplasmic reticulum tethered transcription factor regulating cholesterol and lipid homeostasis, as a promising candidate involved in this immature state. Inhibition of cholesterol trafficking increased SREBF1 expression and shifted metabolic and transcriptional features towards a more mature β-like state. These data identify potential targets and pathways that can be leveraged to improve SCβ-cell maturation and validate cholesterol and lipid homeostasis through the SREBP1 axis as one such candidate.

Abstract Insulin production is a cardinal feature of pancreatic β cells. Studies in rodents show that β cells can switch between low and high insulin gene activity states and that elevated insulin production makes β cells more vulnerable to stresses associated with diabetes. In people, genetically elevated insulin production increases the risk of type 1 diabetes. Via effects on obesity, hyperinsulinemia contributes to the pathogenesis of type 2 diabetes. Here, we characterize β cells in low and high INS gene activity states sorted from primary human islets transduced with INS -GFP adenovirus and differentiated INS -EGFP knock-in embryonic stem cells (SCβ cells). We profile β cell function, protein synthesis, resilience to diabetes associated stress, single β cell transcriptomes and their co-activity networks, and purified β cell proteomes. We show that human β cells transition between distinct states. High INS cells have elevated maturity marker mRNAs and proteins, increased protein translation, are larger, but also more susceptible to cell death when exposed to diabetes-relevant stresses. We also catalogue thousands of differences in proteins in high INS stem cell-derived β cells compared directly with high INS primary β cells. Our study improves our understanding of the delicate balance between insulin production and β cell resilience and guides the engineering of better β cells. Blurb: transcriptional, proteomic, and functional analyses of insulin gene expression states in human β cells from donor islets and stem cells Key findings Functional, transcriptomic, and proteomic analyses identify similarities and differences between high and low INS gene activity states in primary and stem cell-derived β cells. We characterize the relationship between insulin production and fragility, demonstrating that increased insulin production comes at a cost of reduced resilience to multiple stresses. We report a comprehensive side-by-side proteomic analysis of purified primary and stem cell-derived β cells in the high INS state and identify differences in protein production and secretion machinery, providing a roadmap for making better β cells. Proteomic analyses of unfolded protein response markers and cell death effectors reveal key differences in how primary and stem cell-derived β cells respond to the stress of high insulin production.

Epidemiological studies consistently report associations between circulating concentrations of persistent organic pollutants and increased type 2 diabetes risk. Measures of pollutant concentrations in pancreas are limited; given the role of the endocrine pancreas in diabetes pathogenesis, this is an important gap in the literature. Additionally, no studies have correlated pollutant concentrations with direct measures of human beta cell function. We measure concentrations from 3 pollutant classes-dioxins/furans, polychlorinated biphenyls, and organochlorine pesticides-in pancreas and peripancreatic adipose tissue biopsies obtained from 31 human organ donors. We show that pollutants are consistently detected in human pancreas, and for some analytes, at higher concentrations than in adipose. We next assess correlations between pollutant concentrations and systemic indicators of diabetes risk (body mass index, age, and haemoglobin A1c) and direct measures of beta cell function in isolated islets from the same 31 donors. Pancreas polychlorinated biphenyls and organochlorine pesticides positively correlate with body mass index, age and basal insulin secretion but negatively correlate with stimulation index (ratio of insulin secretion under high glucose / low glucose conditions). Pancreas dioxins/furans positively correlate with fatty acid- and amino acid-stimulated insulin secretion. These data indicate that lipophilic pollutants accumulate in human pancreas and positively correlate with surrogate markers of diabetes risk.

Cancer survivors have an increased risk of developing Type 2 diabetes compared to the general population. Patients treated with cisplatin, a common chemotherapeutic agent, are more likely to develop metabolic syndrome and Type 2 diabetes than age- and sex-matched controls. Surprisingly, the impact of cisplatin on pancreatic islets has not been reported. Our study aimed to determine if mouse islet function is adversely affected by systemic (in vivo) or direct (in vitro) exposure to cisplatin. In vivo cisplatin exposure led to deficits in glucose-stimulated plasma insulin levels in both male and female mice, despite no differences in glucose tolerance. In vitro cisplatin exposure to mouse islets dysregulated insulin release and reduced oxygen consumption in a non-sex specific manner. When shifting our focus to male mouse islets, cisplatin altered the expression of genes related to insulin production, oxidative stress, and the Bcl-2 family as early as 6-hours post-exposure. Genome-wide expression analysis confirmed the pronounced downregulation of genes within the insulin secretion pathway in cisplatin-exposed mouse islets. Data from 3 human organ donors confirms that the detrimental effects of cisplatin on insulin secretion and gene expression are reproduced in human islets. Our findings indicate that cisplatin exposure causes significant defects in insulin secretion and may have lasting effects on islet health.

Pancreatic islets are highly specialized tissue compartments that regulate metabolism, and their dysfunction contributes to diseases such as prediabetes and diabetes. Characterizing islet morphology and cell plasticity is essential for understanding these pathophysiological states, yet high-resolution spatial proteomics remains challenging due to the islets’ small size and cellular complexity. Here, we present multiplexed deep visual proteomics (mxDVP), an approach that integrates high-plex imaging with ultra-sensitive mass spectrometry to achieve deep spatial proteome profiling of defined cell types in tissue sections. Enhanced segmentation, semi- automated annotation, and optimized laser microdissection enable the enrichment of rare endocrine populations that are often overlooked in single-cell analyses. mxDVP achieves deep proteome coverage of >6,000 proteins from as few as 100 cells, including low-abundance transcription factors critical for endocrine cell fate determination. By profiling over 864,000 human pancreatic cells, we identify 12 endocrine subtypes, including polyhormonal hybrids, revealing previously unrecognized islet heterogeneity, metabolic regulation, and cellular adaptability. ### Competing Interest Statement E.L. is an advisor for the Chan-Zuckerberg Initiative Foundation, Element Biosciences, Cartography Biosciences, Pfizer, Moleculent AB, and Pixelgen Technologies. The terms of these arrangements have been reviewed and approved by Stanford University in accordance with its conflict of interest policies. M.M. is an indirect shareholder in EvoSep Biosystems.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing β-cells, along with dysregulated glucagon secretion from pancreatic α-cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors to investigate the mechanisms underlying their dysfunction. Surviving β-cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In α-cells, we observed hyper-responsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to α-cell dysfunction, including MHC class I which were closely linked with α-cell dysfunction. Our data provide novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual β-cell function and modulate α-cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES:Glycine acts in an autocrine positive feedback loop in human β cells through its ionotropic receptors (GlyRs). In type 2 diabetes (T2D), islet GlyR activity is impaired by unknown mechanisms. We sought to investigate if the GlyR dysfunction in T2D is replicated by hyperglycemia per se, and to further characterize its action in β cells and islets. METHODS:GlyR-mediated currents were measured using whole-cell patch-clamp in human β cells from donors with or without T2D, or after high glucose (15 mM) culture. We also correlated glycine-induced current amplitude with transcript expression levels through patch-seq. The expression of the GlyR α1, α3, and β subunit mRNA splice variants was compared between islets from donors with and without T2D, and after high glucose culture. Insulin secretion from human islets was measured in the presence or absence of the GlyR antagonist strychnine. RESULTS:Although gene expression of GlyRs was decreased in T2D islets, and β cell GlyR-mediated currents were smaller, we found no evidence for a shift in GlyR subunit splicing. Glycine-induced currents are also reduced after 48 h culture of islets from donors without diabetes in high glucose, where we also find the reduction of the α1 subunit expression, but an increase in the α3 subunit. We discovered that glycine-evoked currents are highly heterogeneous amongst β cells, inversely correlate with donor HbA1c, and are significantly correlated to the expression of 92 different transcripts and gene regulatory networks (GRNs) that include CREB3(+), RREB1(+) and ZNF697(+). Finally, glucose-stimulated insulin secretion is decreased in the presence of the GlyR antagonist strychnine. CONCLUSIONS:We demonstrate that glucose can modulate GlyR expression, and that the current decrease in T2D is likely due to the receptor gene expression downregulation, and not a change in transcript splicing. Moreover, we define a previously unknown set of genes and regulons that are correlated to GlyR-mediated currents and could be involved in GlyR downregulation in T2D. Among those we validate the negative impact of EIF4EBP1 expression on GlyR activity.

Islet-resident macrophages contribute to hypoxia-induced islet cell death during pancreatic islet transplantation. However, their specific role during this process remains elusive. Here, we report that interleukin-1α (IL-1α) and IL-1β are released by islet-resident macrophages, resulting in the suppression of insulin secretion. This may be due to a decreased inflammation-driven expression of pancreatic and duodenal homeobox 1 (PDX-1) and MafA in β cells. Islet-resident macrophages release significantly less IL-1α when compared to IL-1β. However, both cytokines inhibit insulin expression and secretion to a comparable extent. We identified heparan sulfate on the islet surface, which acts as a "molecular glue" potentiating the inhibitory action of IL-1α on insulin expression via specific binding to IL-1 receptor (IL-1R). In vivo analyses revealed that the loss of IL-1 signaling in isolated islets accelerates their revascularization and, thus, enhances their endocrine function. These findings indicate that heparan sulfate fine-tuned IL-1 signaling crucially determines the outcome of islet transplantation.

HumanIslets.com supports diabetes research by offering easy access to islet phenotyping data, analysis tools, and data download. It includes molecular omics, islet and cellular function assays, tissue processing metadata, and phenotypes from 547 donors. As it expands, the resource aims to improve human islet data quality, usability, and accessibility.

Pancreatic and duodenal homeobox protein (PDX)1 is a major transcription factor for the regulation of insulin, glucagon and somatostatin (SST) expression. PDX1 is phosphorylated by CK2 and inhibition of this kinase results in an increased insulin and decreased glucagon secretion. Therefore, we speculated in this study that CK2 also affects SST expression. To test this, we analyzed the effects of the two CK2 inhibitors CX-4945 and SGC as well as of PDX1 overexpression on SST expression and secretion in RIN14B cells by qRT-PCR, luciferase assays, Western blot and ELISA. SST expression and secretion were additionally assessed in isolated murine and human islets exposed to the CK2 inhibitors. Moreover, we determined the expression and secretion of the pancreatic endocrine hormones in CX-4945-treated mice. We found a suppressed SST expression in RIN14B cells due to a methylated SST promoter, which could be abolished by DNA demethylation. Under these conditions, we showed that CK2 inhibition increases SST gene expression and secretion. Additional experiments with overexpression of a CK2-phosphorylation mutant of PDX1 verified that SST expression is regulated by CK2. The exposure of isolated murine and human islets to CX-4945 or SGC as well as the treatment of mice with CX-4945 revealed that CK2 also regulates SST expression under physiological conditions. Taken together, these findings not only demonstrate that CK2 controls SST expression in pancreatic δ-cells but also emphasize the crucial role of this kinase in regulating the main hormones of the endocrine pancreas.

AIMS/HYPOTHESIS:Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet alpha cells, although the underlying mechanisms regulating glucagon secretion and alpha cell dysfunction remain unclear. While insulin secretion from pancreatic beta cells has long been known to be controlled partly by intracellular phospholipid signalling, very little is known about the role of phospholipids in glucagon secretion. Using patch-clamp electrophysiology and single-cell RNA sequencing, we previously found that expression of PIP4P2 (encoding TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate [PIP2] to phosphatidylinositol-5-phosphate [PI5P]) correlates with alpha cell function. We hypothesise that TMEM55A is involved in glucagon secretion and aim to validate the role of TMEM55A and its potential signalling molecules in alpha cell function and glucagon secretion. METHODS:Correlation analysis was generated from the data in www.humanislets.com . Human islets were isolated at the Alberta Diabetes Institute IsletCore. Electrical recordings were performed on dispersed human or mouse islets with scrambled siRNA or si-PIP4P2 (si-Pip4p2 for mouse) transfection. Glucagon secretion was measured using an islet perfusion system with intact mouse islets. TMEM55A activity was measured using an in vitro on-beads phosphatase assay and live-cell imaging. GTPase activity was measured using an active GTPase pull-down assay. Confocal microscopy was used to quantify F-actin intensity using primary alpha cells and alphaTC1-9 cell lines after chemical treatment. RESULTS:TMEM55A regulated alpha cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse alpha cells reduced exocytosis at low glucose levels and this was rescued by the direct reintroduction of PI5P. PI5P, instead of PIP2 increased the glucagon secretion using intact mouse islets. This did not occur through an effect on Ca2+ channel activity but through a remodelling of cortical F-actin dependent on TMEM55A lipid phosphatase activity, which occurred in response to oxidative stress. TMEM55A- and PI5P-induced F-actin remodelling depends on the inactivation of GTPase and RhoA, instead of Ras-related C3 botulinum toxin substrate 1 or CDC42. CONCLUSIONS/INTERPRETATION:We reveal a novel pathway by which TMEM55A regulates alpha cell exocytosis by controlling intracellular PI5P and the F-actin network.

Epidemiological studies consistently report associations between circulating concentrations of persistent organic pollutants (POPs) and increased type 2 diabetes risk. Measures of POP concentrations in pancreas are limited; given the role of the endocrine pancreas in diabetes pathogenesis, this is an important gap in the literature. Additionally, no studies have correlated POP concentrations with direct measures of beta cell function in humans. We hypothesized that lipophilic POPs accumulate in human pancreas and correlate with markers of diabetes risk. To test this hypothesis, we measured POP concentration from 3 chemical classes - dioxins/furans, polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) - in pancreas and peripancreatic adipose tissue biopsies obtained from 31 human organ donors via the Alberta Diabetes Institute IsletCore. Indeed, POPs were consistently detected in human pancreas, and for some pollutants, at higher concentrations than in adipose. We next assessed correlations between POP concentrations and systemic indicators of diabetes risk (BMI, age, and %HbA1c) and direct measures of beta cell function. To this end, we measured insulin secretion in response to numerous secretagogues (i.e. glucose, fatty acids, amino acids, exendin-4, or KCl) in isolated islets from the same 31 donors. Pancreas PCBs and OCPs positively correlated with BMI, age and basal insulin secretion but negatively correlated with stimulation index (ratio of insulin secretion under high glucose / low glucose conditions). In contrast, pancreas dioxins/furans positively correlated with fatty acid- and amino acid-stimulated insulin secretion. These data confirm that lipophilic pollutants accumulate in human pancreas and positively correlate with markers of diabetes risk. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by a Canadian Institutes of Health Research (CIHR) Project Grant (#PJT-2018-159590). J.E.B. is supported by an Early Researcher Award from the Ontario Government. E.E.M is supported by a Diabetes Canada End Diabetes Award (OG-3-24-5818-EM). M.P.H. was supported by a CIHR CGS-D award. M.E.A.C. was supported by an NSERC CGS-M and NSERC CGS-D award. J.P. was supported by the Guiding interdisciplinary Research on Womens and girls health and Wellbeing (GROWW) scholarship and Ontario Graduate Scholarship. This work includes data from HumanIslets.com funded by the Canadian Institutes of Health Research, JDRF Canada, and Diabetes Canada (5-SRA-2021-1149-S-B/TG 179092) with data from islets isolated by the Alberta Diabetes Institute IsletCore with the support of the Human Organ Procurement and Exchange program, Trillium Gift of Life Network, BC Transplant, Quebec Transplant, and other Canadian organ procurement organizations. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: the Research Ethics Board at Carleton University (#106701) and the University of Alberta (Pro00013094) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

BACKGROUND:Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. METHODS:Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. RESULTS:Differentiation was capable of generating 90.4% PDX1 + /NKX6.1 + pancreatic progenitors and 100% C-peptide + /NKX6.1 + iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19 + ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 10 4 cells/cm 2 (total 8.3 × 10 6 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 10 6 cells/mL (total 105.0 × 10 6 cells), reducing costs by 88.8%. CONCLUSIONS:This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing β cells, along with dysregulated glucagon secretion from pancreatic α cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors. The few surviving β cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In α cells, we observed hyperresponsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization, and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to α cell dysfunction, including MHC class I, which were closely linked with α cell dysfunction. Our data provide what we believe are novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual β cell function and modulate α cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D.