Revisiting pancreatic islet isolation in murine models: A practical and effective technical protocol.

The endocrine pancreas is composed of clusters of cell groups called pancreatic islets. These cells are responsible for the synthesis and secretion of hormones crucial for glycemic homeostasis, such as insulin and glucagon. Therefore, these cells were the targets of many studies. One method to study and/or understand endocrine pancreatic physiology is the isolation of these islets and stimulation of hormone production using different concentrations of glucose, agonists, and/or antagonists of specific secretagogues and mimicking the stimulation of hormonal synthesis and secretion. Many researchers studied pancreatic physiology in murine models due to their ease of maintenance and rapid development. However, the isolation of pancreatic islets involves meticulous processes that may vary between rodent species. The present study describes a simple and effective technical protocol for isolating intact islets from mice and rats for use as a practical guide for researchers. The method involves digestion of the acinar parenchyma by intraductal collagenase. Isolated islets are suitable for in vitro endocrine secretion analyses, microscopy techniques, and biochemical analyses.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults.

The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of ß-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and ß-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell ß-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.

Acinar to ß-like cell conversion through inhibition of focal adhesion kinase.

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.

AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Illuminating the complete ß-cell mass of the human pancreas- signifying a new view on the islets of Langerhans.

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research.

Unraveling the significance of PPP1R1A gene in pancreatic ß-cell function: A study in INS-1 cells and human pancreatic islets.

BACKGROUND AND AIMS: The protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A) has been linked with insulin secretion and diabetes mellitus. Yet, its full significance in pancreatic ß-cell function remains unclear. This study aims to elucidate the role of the PPP1R1A gene in ß-cell biology using human pancreatic islets and rat INS-1 (832/13) cells. RESULTS: Disruption of Ppp1r1a in INS-1 cells was associated with reduced insulin secretion and impaired glucose uptake; however, cell viability, ROS, apoptosis or proliferation were intact. A significant downregulation of crucial ß-cell function genes such as Ins1, Ins2, Pcsk1, Cpe, Pdx1, Mafa, Isl1, Glut2, Snap25, Vamp2, Syt5, Cacna1a, Cacna1d and Cacnb3, was observed upon Ppp1r1a disruption. Furthermore, silencing Pdx1 in INS-1 cells altered PPP1R1A expression, indicating that PPP1R1A is a target gene for PDX1. Treatment with rosiglitazone increased Ppp1r1a expression, while metformin and insulin showed no effect. RNA-seq analysis of human islets revealed high PPP1R1A expression, with α-cells showing the highest levels compared to other endocrine cells. Muscle tissues exhibited greater PPP1R1A expression than pancreatic islets, liver, or adipose tissues. Co-expression analysis revealed significant correlations between PPP1R1A and genes associated with insulin biosynthesis, exocytosis machinery, and intracellular calcium transport. Overexpression of PPP1R1A in human islets augmented insulin secretion and upregulated protein expression of Insulin, MAFA, PDX1, and GLUT1, while silencing of PPP1R1A reduced Insulin, MAFA, and GLUT1 protein levels. CONCLUSION: This study provides valuable insights into the role of PPP1R1A in regulating ß-cell function and glucose homeostasis. PPP1R1A presents a promising opportunity for future therapeutic interventions.

Investigating the Impact of IL6 on Insulin Secretion: Evidence from INS-1 Cells, Human Pancreatic Islets, and Serum Analysis.

Interleukin-6 (IL6) is a pleiotropic cytokine implicated in metabolic disorders and inflammation, yet its precise influence on insulin secretion and glucose metabolism remains uncertain. This study examined IL6 expression in pancreatic islets from individuals with/without diabetes, alongside a series of functional experiments, including siRNA silencing; IL6 treatment; and assessments of glucose uptake, cell viability, apoptosis, and expression of key ß-cell genes, which were conducted in both INS-1 cells and human islets to elucidate the effect of IL6 on insulin secretion. Serum levels of IL6 from Emirati patients with type 2 diabetes (T2D) were measured, and the effect of antidiabetic drugs on IL6 levels was studied. The results revealed that IL6 mRNA expression was higher in islets from diabetic and older donors compared to healthy or young donors. IL6 expression correlated negatively with PDX1, MAFB, and NEUROD1 and positively with SOX4, HES1, and FOXA1. Silencing IL6 in INS-1 cells reduced insulin secretion and glucose uptake independently of apoptosis or oxidative stress. Reduced expression of IL6 was associated with the downregulation of Ins, Pdx1, Neurod1, and Glut2 in INS-1 cells. In contrast, IL6 treatment enhanced insulin secretion in INS-1 cells and human islets and upregulated insulin expression. Serum IL6 levels were elevated in patients with T2D and associated with higher glucose, HbA1c, and triglycerides, regardless of glucose-lowering medications. This study provides a new understanding of the role of IL6 in ß-cell function and the pathophysiology of T2D. Our data highlight differences in the response to IL6 between INS-1 cells and human islets, suggesting the presence of species-specific variations across different experimental models. Further research is warranted to unravel the precise mechanisms underlying the observed effects of IL-6 on insulin secretion.

Centella asiatica mitigates the detrimental effects of Bisphenol-A (BPA) on pancreatic islets.

Bisphenol-A (BPA) is widely used in food packaging and household products, leading to daily human exposure and potential health risks including metabolic diseases like type 2 diabetes mellitus (T2DM). Understanding BPA's mechanisms and developing intervention strategies is urgent. Centella asiatica, a traditional herbal medicine containing pentacyclic triterpenoids, shows promise due to its antioxidant and anti-inflammatory properties, utilized for centuries in Ayurvedic therapy. We investigated the effect of Centella asiatica (CA) ethanol extract on BPA-induced pancreatic islet toxicity in male Swiss albino mice. BPA administration (10 and 100 µg/kg body weight, twice daily) for 21 days caused glucose homeostasis disturbances, insulin resistance, and islet dysfunction, which were partially mitigated by CA supplementation (200 and 400 mg/kg body weight). Additionally, heightened oxidative stress, elevated levels of proinflammatory cytokines, loss of mitochondrial membrane potential (MMP), abnormal cell cycle, and increased apoptosis were implicated in the detrimental impact of BPA on the endocrine pancreas which were effectively counteracted by CA supplementation. In summary, CA demonstrated a significant ability to mitigate BPA-induced apoptosis, modulate redox homeostasis, alleviate inflammation, preserve MMP, and regulate the cell cycle. As a result, CA emerged as a potent agent in neutralizing the diabetogenic effects of BPA to a considerable extent.

Genetics and diet shape the relationship between islet function and whole body metabolism.

Despite the fact that genes and the environment are known to play a central role in islet function, our knowledge of how these parameters interact to modulate insulin secretory function remains relatively poor. Presently, we performed ex vivo glucose-stimulated insulin secretion and insulin content assays in islets of 213 mice from 13 inbred mouse strains on chow, Western diet (WD), and a high-fat, carbohydrate-free (KETO) diet. Strikingly, among these 13 strains, islets from the commonly used C57BL/6J mouse strain were the least glucose responsive. Using matched metabolic phenotyping data, we performed correlation analyses of isolated islet parameters and found a positive correlation between basal and glucose-stimulated insulin secretion, but no relationship between insulin secretion and insulin content. Using in vivo metabolic measures, we found that glucose tolerance determines the relationship between ex vivo islet insulin secretion and plasma insulin levels. Finally, we showed that islet glucose-stimulated insulin secretion decreased with KETO in almost all strains, concomitant with broader phenotypic changes, such as increased adiposity and glucose intolerance. This is an important finding as it should caution against the application of KETO diet for beta-cell health. Together these data offer key insights into the intersection of diet and genetic background on islet function and whole body glucose metabolism.NEW & NOTEWORTHY Thirteen strains of mice on chow, Western diet, and high-fat, carbohydrate-free (KETO), correlating whole body phenotypes to ex vivo pancreatic islet functional measurements, were used. The study finds a huge spectrum of functional islet responses and insulin phenotypes across all strains and diets, with the ubiquitous C57Bl/6J mouse exhibiting the lowest secretory response of all strains, highlighting the overall importance of considering genetic background when investigating islet function. Ex vivo basal and stimulated insulin secretion are correlated in the islet, and KETO imparts widescale downregulation of islet insulin secretion.

Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice.

Reducing ghrelin by ghrelin gene knockout (GKO), ghrelin-cell ablation, or high-fat diet feeding increases islet size and ß-cell mass in male mice. Here we determined if reducing ghrelin also enlarges islets in females and if pregnancy-associated changes in islet size are related to reduced ghrelin. Islet size and ß-cell mass were larger (P = .057 for ß-cell mass) in female GKO mice. Pregnancy was associated with reduced ghrelin and increased liver-expressed antimicrobial peptide-2 (LEAP2; a ghrelin receptor antagonist) in wild-type mice. Ghrelin deletion and pregnancy each increased islet size (by ∼19.9-30.2% and ∼34.9-46.4%, respectively), percentage of large islets (>25 µm2×103, by ∼21.8-42% and ∼21.2-41.2%, respectively), and ß-cell mass (by ∼15.7-23.8% and ∼65.2-76.8%, respectively). Neither islet cross-sectional area, ß-cell cross-sectional area, nor ß-cell mass correlated with plasma ghrelin, although all positively correlated with LEAP2 (P = .081 for islet cross-sectional area). In ad lib-fed mice, there was an effect of pregnancy, but not ghrelin deletion, to change (raise) plasma insulin without impacting blood glucose. Similarly, there was an effect of pregnancy, but not ghrelin deletion, to change (lower) blood glucose area under the curve during a glucose tolerance test. Thus, genetic deletion of ghrelin increases islet size and ß-cell cross-sectional area in female mice, similar to males. Yet, despite pregnancy-associated reductions in ghrelin, other factors appear to govern islet enlargement and changes to insulin sensitivity and glucose tolerance in the setting of pregnancy. In the case of islet size and ß-cell mass, one of those factors may be the pregnancy-associated increase in LEAP2.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing.

BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation. RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed. CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

FGF7 enhances the expression of ACE2 in human islet organoids aggravating SARS-CoV-2 infection.

The angiotensin-converting enzyme 2 (ACE2) is a primary cell surface viral binding receptor for SARS-CoV-2, so finding new regulatory molecules to modulate ACE2 expression levels is a promising strategy against COVID-19. In the current study, we utilized islet organoids derived from human embryonic stem cells (hESCs), animal models and COVID-19 patients to discover that fibroblast growth factor 7 (FGF7) enhances ACE2 expression within the islets, facilitating SARS-CoV-2 infection and resulting in impaired insulin secretion. Using hESC-derived islet organoids, we demonstrated that FGF7 interacts with FGF receptor 2 (FGFR2) and FGFR1 to upregulate ACE2 expression predominantly in ß cells. This upregulation increases both insulin secretion and susceptibility of ß cells to SARS-CoV-2 infection. Inhibiting FGFR counteracts the FGF7-induced ACE2 upregulation, subsequently reducing viral infection and replication in the islets. Furthermore, retrospective clinical data revealed that diabetic patients with severe COVID-19 symptoms exhibited elevated serum FGF7 levels compared to those with mild symptoms. Finally, animal experiments indicated that SARS-CoV-2 infection increased pancreatic FGF7 levels, resulting in a reduction of insulin concentrations in situ. Taken together, our research offers a potential regulatory strategy for ACE2 by controlling FGF7, thereby protecting islets from SARS-CoV-2 infection and preventing the progression of diabetes in the context of COVID-19.

Unveiling cell subpopulations in T1D mouse islets using single-cell RNA sequencing.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of beta cells by immune cells. The interactions among cells within the islets may be closely linked to the pathogenesis of T1D. In this study, we used single-cell RNA sequencing (scRNA-Seq) to analyze the cellular heterogeneity within the islets of a T1D mouse model. We established a T1D mouse model induced by streptozotocin and identified cell subpopulations using scRNA-Seq technology. Our results revealed 11 major cell types in the pancreatic islets of T1D mice, with heterogeneity observed in the alpha and beta cell subgroups, which may play a crucial role in the progression of T1D. Flow cytometry further confirmed a mature alpha and beta cell reduction in T1D mice. Overall, our scRNA-Seq analysis provided insights into the cellular heterogeneity of T1D islet tissue and highlighted the potential importance of alpha and beta cells in developing T1D.NEW & NOTEWORTHY In this study, we created a comprehensive single-cell atlas of pancreatic islets in a T1D mouse model using scRNA-Seq and identified 11 major cell types in the islets, highlighting the role of alpha and beta cells in T1D. This study revealed a significant reduction in the maturity alpha and beta cells in T1D mice through flow cytometry. It also demonstrated the heterogeneity of alpha and beta cells, potentially crucial for T1D progression. Overall, our scRNA-Seq analysis provided new insights for understanding and treating T1D by studying cell subtype changes and functions.

A practical and robust method to evaluate metabolic fluxes in primary pancreatic islets.

OBJECTIVE: Evaluation of mitochondrial oxygen consumption and ATP production is important to investigate pancreatic islet pathophysiology. Most studies use cell lines due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables, is expensive and poorly reproducible. Our aim was to establish a practical method to assess primary islet metabolic fluxes using standard commercial consumables. METHODS: Pancreatic islets were isolated from mice/rats, dispersed with trypsin, and adhered to pre-coated standard Seahorse or Resipher microplates. Oxygen consumption was evaluated using a Seahorse Extracellular Flux Analyzer or a Resipher Real-time Cell Analyzer. RESULTS: We provide a detailed protocol with all steps to optimize islet isolation with high yield and functionality. Our method requires a few islets per replicate; both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators and glucose. The technique was validated by other functional assays, which show these cells present conserved calcium influx and insulin secretion in response to glucose. We also show that our dispersed islets maintain robust basal respiration levels, in addition to maintaining up to 89% viability after five days in dispersed cultures. Furthermore, OCRs can be measured in Seahorse analyzers and in other plate respirometry systems, using standard materials. CONCLUSIONS: Overall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic ß-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

Tyrosine nitration of glucagon impairs its function: Extending the role of heme in T2D pathogenesis.

New studies raise the possibility that the higher glucagon (GCG) level present in type 2 diabetes (T2D) is a compensatory mechanism to enhance ß-cell function, rather than induce dysregulated glucose homeostasis, due to an important role for GCG that acts directly within the pancreas on insulin secretion by intra-islet GCG signaling. However, in states of poorly controlled T2D, pancreatic α cell mass increases (overproduced GCG) in response to insufficient insulin secretion, indicating decreased local GCG activity. The reason for this decrease is not clear. Recent evidence has uncovered a new role of heme in cellular signal transduction, and its mechanism involves reversible binding of heme to proteins. Considering that protein tyrosine nitration in diabetic islets increases and glucose-stimulated insulin secretion (GSIS) decreases, we speculated that heme modulates GSIS by transient interaction with GCG and catalyzing its tyrosine nitration, and the tyrosine nitration may impair GCG activity, leading to loss of intra-islet GCG signaling and markedly impaired insulin secretion. Data presented here elucidate a novel role for heme in disrupting local GCG signaling in diabetes. Heme bound to GCG and induced GCG tyrosine nitration. Two tyrosine residues in GCG were both sensitive to the nitrating species. Further, GCG was also demonstrated to be a preferred target peptide for tyrosine nitration by co-incubation with BSA. Tyrosine nitration impaired GCG stimulated cAMP-dependent signaling in islet ß cells and decreased insulin release. Our results provided a new role of heme for impaired GSIS in the pathological process of diabetes.

NPC1 is required for postnatal islet ß cell differentiation by maintaining mitochondria turnover.

Rationale: NPC1 is a protein localized on the lysosome membrane regulating intracellular cholesterol transportation and maintaining normal lysosome function. GWAS studies have found that NPC1 variants in T2D was a pancreatic islet expression quantitative trait locus, suggesting a potential role of NPC1 in T2D islet pathophysiology. Methods: Two-week-old Npc1-/- mice and wild type littermates were employed to examine pancreatic ß cell morphology and functional changes induced by loss of Npc1. Single cell RNA sequencing was conducted on primary islets. Npc1-/- Min6 cell line was generated using CRISPR/Cas9 gene editing. Seahorse XF24 was used to analyze primary islet and Min6 cell mitochondria respiration. Ultra-high-resolution cell imaging with Lattice SIM2 and electron microscope imaging were used to observe mitochondria and lysosome in primary islet ß and Min6 cells. Mitophagy Dye and mt-Keima were used to measure ß cell mitophagy. Results: In Npc1-/- mice, we found that ß cell survival and pancreatic ß cell mass expansion as well as islet glucose induced insulin secretion in 2-week-old mice were reduced. Npc1 loss retarded postnatal ß cell differentiation and growth as well as impaired mitochondria oxidative phosphorylation (OXPHOS) function to increase mitochondrial superoxide production, which might be attributed to impaired autophagy flux particularly mitochondria autophagy (mitophagy) induced by dysfunctional lysosome in Npc1 null ß cells. Conclusion: Our study revealed that NPC1 played an important role in maintaining normal lysosome function and mitochondria turnover, which ensured establishment of sufficient mitochondria OXPHOS for islet ß cells differentiation and maturation.

Heterozygous missense variant in GLI2 impairs human endocrine pancreas development.

Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic ß-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic ß-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.