Primary Pancreatic Islets Research Articles

High-Yield Generation of Glucose-Responsive Pseudoislets From Murine Insulinoma Cells for In Vitro Studies and Longitudinal Monitoring of Graft Survival In Vivo

Compared to primary pancreatic islets, insulinoma cell-derived 3D pseudoislets offer a more accessible, consistent, renewable, and widely applicable model system for optimization and mechanistic studies in type 1 diabetes (T1D). Here, we report a simple and efficient method for generating 3D pseudoislets from MIN6 and NIT-1 murine insulinoma cells. These pseudoislets are homogeneous in size and morphology (~150 µm), exhibit functional glucose-stimulated insulin secretion (GSIS) up to 18 days (NIT-1) enabling long-term studies, are produced in high yield [>35,000 Islet Equivalence from 30 ml culture], and are suitable for both in vitro and in vivo studies, including for encapsulation studies. To enable non-invasive longitudinal monitoring of graft survival in vivo, we transduced NIT-1 cells with green fluorescent protein-luciferase and confirmed comparable morphology, viability, and GSIS to untransduced cells in vitro. After subcutaneous implantation, we show capability to monitor graft survival in immunodeficient mice, recurrence of autoimmunity in non-obese diabetic mice, and allorejection in C57BL/6 mice. Overall, this platform provides an accessible protocol for generating high yields of 3D pseudoislets and non-invasive longitudinal monitoring of graft survival in different models offer advantages over primary islets for optimization and mechanistic studies of β cell biology, drug discovery, T1D pathogenesis and prevention, and β cell transplantation.

NMDA Suppresses Pancreatic ABCA1 Expression through the MEK/ERK/LXR Pathway in Pancreatic Beta Cells.

Dysfunction or loss of pancreatic β cells can cause insulin deficiency and impaired glucose regulation, resulting in conditions like type 2 diabetes. The ATP-binding cassette transporter A1 (ABCA1) plays a key role in the reverse cholesterol transport system, and its decreased expression is associated with pancreatic β cell lipotoxicity, resulting in abnormal insulin synthesis and secretion. Increased glutamate release can cause glucotoxicity in β cells, though the detailed mechanisms remain unclear. This study investigated the effect of N-methyl-D-aspartic acid (NMDA) on ABCA1 expression in INS-1 cells and primary pancreatic islets to elucidate the signaling mechanisms that suppress insulin secretion. Using Western blotting, microscopy, and biochemical analyses, we found that NMDA activated the mitogen-activated protein kinase (MEK)-dependent pathway, suppressing ABCA1 protein and mRNA expression. The MEK-specific inhibitor PD98059 restored ABCA1 promoter activity, indicating the involvement of the extracellular signal-regulated kinase (MEK/ERK) pathway. Furthermore, we identified the liver X receptor (LXR) as an effector transcription factor in NMDA regulation of ABCA1 transcription. NMDA treatment increased cholesterol and triglyceride levels while decreasing insulin secretion, even under high-glucose conditions. These effects were abrogated by treatment with PD98059. This study reveals that NMDA suppresses ABCA1 expression via the MEK/ERK/LXR pathway, providing new insights into the pathological suppression of insulin secretion in pancreatic β cells and emphasizing the importance of investigating the role of NMDA in β cell dysfunction.

Coxsackievirus B infection invokes unique cell-type specific responses in primary human pancreatic islets.

Coxsackievirus B (CVB) infection has long been considered an environmental factor precipitating Type 1 diabetes (T1D), an autoimmune disease marked by loss of insulin-producing β cells within pancreatic islets. Previous studies have shown CVB infection negatively impacts islet function and viability but do not report on how virus infection individually affects the multiple cell types present in human primary islets. Therefore, we hypothesized that the various islet cell populations have unique transcriptional responses to CVB infection. Here, we performed single-cell RNA sequencing on human cadaveric islets treated with either CVB or poly(I:C), a viral mimic, for 24 and 48 hours. Our global analysis reveals CVB differentially induces dynamic transcriptional changes associated with multiple cell processes and functions over time whereas poly(I:C) promotes an immune response that progressively increases with treatment duration. At the single-cell resolution, we find CVB infects all islet cell types at similar rates yet induces unique cell-type specific transcriptional responses with β, α, and ductal cells having the strongest response. Sequencing and functional data suggest that CVB negatively impacts mitochondrial respiration and morphology in distinct ways in β and α cells, while also promoting the generation of reactive oxygen species. We also observe an increase in the expression of the long-noncoding RNA MIR7-3HG in β cells with high viral titers and reveal its knockdown reduces gene expression of viral proteins as well as apoptosis in stem cell-derived islets. Together, these findings demonstrate a cell-specific transcriptional, temporal, and functional response to CVB infection and provide new insights into the relationship between CVB infection and T1D.

RIPK1 is dispensable for cell death regulation in β-cells during hyperglycemia

ObjectiveReceptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear. MethodsHere, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents. ResultsInhibition of RIPK1 kinase activity (Ripk1D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition. ConclusionsUnlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.

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

ObjectiveEvaluation 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. MethodsPancreatic 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. ResultsWe 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. ConclusionsOverall, we established a practical and robust method to assess islet metabolic fluxes and oxidative phosphorylation, a valuable tool to uncover basic β-cell metabolic mechanisms as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols.

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling.

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.NEW & NOTEWORTHY Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields.

Apolipoprotein L genes are novel mediators of inflammation in beta cells

Aims/hypothesisInflammation induces beta cell dysfunction and demise but underlying molecular mechanisms remain unclear. The apolipoprotein L (APOL) family of genes has been associated with innate immunity and apoptosis in non-pancreatic cell types, but also with metabolic syndrome and type 2 diabetes mellitus. Here, we hypothesised that APOL genes play a role in inflammation-induced beta cell damage.MethodsWe used single-cell transcriptomics datasets of primary human pancreatic islet cells to study the expression of APOL genes upon specific stress conditions. Validation of the findings was carried out in EndoC-βH1 cells and primary human islets. Finally, we performed loss- and gain-of-function experiments to investigate the role of APOL genes in beta cells.ResultsAPOL genes are expressed in primary human beta cells and APOL1, 2 and 6 are strongly upregulated upon inflammation via the Janus kinase (JAK)−signal transducer and activator of transcription (STAT) pathway. APOL1 overexpression increases endoplasmic reticulum stress while APOL1 knockdown prevents cytokine-induced beta cell death and interferon-associated response. Furthermore, we found that APOL genes are upregulated in beta cells from donors with type 2 diabetes compared with donors without diabetes mellitus.Conclusions/interpretationAPOLs are novel regulators of islet inflammation and may contribute to beta cell damage during the development of diabetes.Data availabilityscRNAseq data generated by our laboratory and used in this study are available in the Gene Expression Omnibus (GEO; www.ncbi.nlm.nih.gov/geo/), accession number GSE218316.Graphical

Identification and validation of core genes for type 2 diabetes mellitus by integrated analysis of single-cell and bulk RNA-sequencing

BackgroundThe exact mechanisms of type 2 diabetes mellitus (T2DM) remain largely unknown. We intended to authenticate critical genes linked to T2DM progression by tandem single-cell sequencing and general transcriptome sequencing data.MethodsT2DM single-cell RNA-sequencing data were submitted by the Gene Expression Omnibus (GEO) database and ArrayExpress (EBI), from which gene expression matrices were retrieved. The common cell clusters and representative marker genes were ascertained by principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE), CellMarker, and FindMarkers in two datasets (GSE86469 and GSE81608). T2DM-related differentially expressed marker genes were defined by intersection analysis of marker genes and GSE86468-differentially expressed genes. Receiver operating characteristic (ROC) curves were utilized to assign representative marker genes with diagnostic values by GSE86468, GSE29226 and external validation GSE29221, and their prospective target compounds were forecasted by PubChem. Besides, the R package clusterProfiler-based functional annotation was designed to unveil the intrinsic mechanisms of the target genes. At last, western blot was used to validate the alternation of CDKN1C and DLK1 expression in primary pancreatic islet cells cultured with or without 30mM glucose.ResultsThree common cell clusters were authenticated in two independent T2DM single-cell sequencing data, covering neurons, epithelial cells, and smooth muscle cells. Functional ensemble analysis disclosed an intimate association of these cell clusters with peptide/insulin secretion and pancreatic development. Pseudo-temporal trajectory analysis indicated that almost all epithelial and smooth muscle cells were of neuron origin. We characterized CDKN1C and DLK1, which were notably upregulated in T2DM samples, with satisfactory availability in recognizing three representative marker genes in non-diabetic and T2DM samples, and they were also robustly interlinked with the clinical characteristics of patients. Western blot also demonstrated that, compared with control group, the expression of CDKN1C and DLK1 were increased in primary pancreatic islet cells cultured with 30 mM glucose for 48 h. Additionally, PubChem projected 11 and 21 potential compounds for CDKN1C and DLK1, respectively.ConclusionIt is desirable that the emergence of the 2 critical genes indicated (CDKN1C and DLK1) could be catalysts for the investigation of the mechanisms of T2DM progression and the exploitation of innovative therapies.

Tryptophan metabolism promotes immune evasion in human pancreatic β cells

To resist the autoimmune attack characteristic of type 1 diabetes, insulin producing pancreatic β cells need to evade T-cell recognition. Such escape mechanisms may be conferred by low HLA class I (HLA-I) expression and upregulation of immune inhibitory molecules such as Programmed cell Death Ligand 1 (PD-L1). The expression of PD-L1, HLA-I and CXCL10 was evaluated in the human β cell line, ECN90, and in primary human and mouse pancreatic islets. Most genes were determined by real-time RT-PCR, flow cytometry and Western blot. Activator and inhibitor of the AKT signaling were used to modulate PD-L1 induction. Key results were validated by monitoring activity of CD8+ Jurkat T cells presenting β cell specific T-cell receptor and transduced with reporter genes in contact culture with the human β cell line, ECN90. In this study, we identify tryptophan (TRP) as an agonist of PD-L1 induction through the AKT signaling pathway. TRP also synergistically enhanced PD-L1 expression on β cells exposed to interferon-γ. Conversely, interferon-γ-mediated induction of HLA-I and CXCL10 genes was down-regulated upon TRP treatment. Finally, TRP and its derivatives inhibited the activation of islet-reactive CD8+ T cells by β cells. Collectively, our findings indicate that TRP could induce immune tolerance to β cells by promoting their immune evasion through HLA-I downregulation and PD-L1 upregulation. Dutch Diabetes Research Foundation, DON Foundation, the Laboratoire d'Excellence consortium Revive (ANR-10-LABX-0073), Agence Nationale de la Recherche (ANR-19-CE15-0014-01), Fondation pour la Recherche Médicale (EQ U201903007793-EQU20193007831), Innovative Medicines InitiativeINNODIA and INNODIA HARVEST, Aides aux Jeunes Diabetiques (AJD) and Juvenile Diabetes Research Foundation Ltd (JDRF).

Inferring regulators of cell identity in the human adult pancreas.

Cellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity and cell states in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. BHLHE41 depletion induced apoptosis in primary pancreatic islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity and cell states in the human adult pancreas.

Strong ubiquitous micro-promoters for recombinant adeno-associated viral vectors

Significant progress has been made in developing recombinant adeno-associated virus (rAAV) for clinical gene therapy. While rAAV is a versatile gene delivery platform, its packaging limit of 4.7 kb limits the diseases it can target. Here, we report two unusually small promoters that enable the expression of larger transgenes than standard promoters. These micro-promoters are only 84 (MP-84) and 135bp (MP-135) in size but have activity in most cells and tissues comparable to the CAG promoter, the strongest ubiquitous promoter to date. MP-84- and MP-135-based rAAV constructs displayed robust activity in cultured cells from the three different germ-layer lineages. In addition, reporter gene expression was documented in human primary hepatocytes and pancreatic islets and in multiple mouse tissues invivo, including brain and skeletal muscle. MP-84 and MP-135 will enable the therapeutic expression of transgenes currently too large for rAAV vectors.

Human hypoimmune primary pancreatic islets avoid rejection and autoimmunity and alleviate diabetes in allogeneic humanized mice.

Transplantation of allogeneic pancreatic donor islets has successfully been performed in selected patients with difficult-to-control insulin-dependent diabetes and impaired awareness of hypoglycemia (IAH). However, the required systemic immunosuppression associated with this procedure prevents this cell replacement therapy from more widespread adoption in larger patient populations. We report the editing of primary human islet cells to the hypoimmune HLA class I- and class II-negative and CD47-overexpressing phenotype and their reaggregation into human HIP pseudoislets (p-islets). Human HIP p-islets were shown to survive, engraft, and ameliorate diabetes in immunocompetent, allogeneic, diabetic humanized mice. HIP p-islet cells were further shown to avoid autoimmune killing in autologous, diabetic humanized autoimmune mice. The survival and endocrine function of HIP p-islet cells were not impaired by contamination of unedited or partially edited cells within the p-islets. HIP p-islet cells were eliminated quickly and reliably in this model using a CD47-targeting antibody, thus providing a safety strategy in case HIP cells exert toxicity in a future clinical setting. Transplantation of human HIP p-islets for which no immunosuppression is required has the potential to lead to wider adoption of this therapy and help more diabetes patients with IAH and history of severe hypoglycemic events to achieve insulin independence.

Discovery of IHMT-MST1-39 as a novel MST1 kinase inhibitor and AMPK activator for the treatment of diabetes mellitus

Insulin-producing pancreatic β cell death is the fundamental cause of type 1 diabetes (T1D) and a contributing factor to type 2 diabetes (T2D). Moreover, metabolic disorder is another hallmark of T2D. Mammalian sterile 20-like kinase 1 (MST1) contributes to the progression of diabetes mellitus through apoptosis induction and acceleration of pancreatic β cell dysfunction. AMP-activated protein kinase (AMPK) is an energy sensing kinase and its activation has been suggested as a treatment option for metabolic diseases. Thus, pharmacological inhibition of MST1 and activation of AMPK simultaneously represents a promising approach for diabetes therapy. Here, we discovered a novel selective MST1 kinase inhibitor IHMT-MST1-39, which exhibits anti-apoptosis efficacy and improves the survival of pancreatic β cells under diabetogenic conditions, as well as primary pancreatic islets in an ex vivo disease model. Mechanistically, IHMT-MST1-39 activated AMPK signaling pathway in hepatocytes in vitro, combination of IHMT-MST1-39 and metformin synergistically prevented hyperglycemia and significantly ameliorated glucose tolerance and insulin resistance in diabetic mice. Taken together, IHMT-MST1-39 is a promising anti-diabetic candidate as a single agent or in combination therapy for both T1D and T2D.

Nuclear factor-Y mediates pancreatic β-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress.

Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear. In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress. Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment. Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.

A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing

SummaryThe seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans-membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3′end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

Intermittent Hypoxia Interferes with Autocrine Effects of GABA on Insulin Secretion in Postnatal Rodents-Implications for Pediatric Obstructive Sleep Apnea.

Gamma-amino butyric acid (GABA) is well known to help elevate pancreatic β cell vitality and insulin levels in blood. GABA works via a coupling with GABA receptors; thus, the concentration of GABAA receptors on the plasma membrane of β cells appears to be critical for insulin regulation. Various medical conditions, such as pediatric and adult obstructive sleep apnea (OSA), show high levels of Type 2 diabetes; such patients also are exposed to intermittent hypoxia (IH), which modifies the GABA levels. To evaluate the potential therapeutic roles of GABA for diabetic patients with OSA, we studied the interactions of IH with GABA and GABAA receptors in young rats. Using rat pups and primary pancreatic islets, we evaluated the roles of GABA in insulin secretion. We show that GABA effectively increased the insulin secretion of pancreatic islets under normal ambient oxygen levels, as well as in culture medium with a glucose level of 2 mM. GABA also increased islet insulin secretion conditioned under IH in a 16 mM glucose medium. When islets were IH-treated, insulin secretion decreased due to lower intracellular chloride levels in accordance with the increased KCC2 levels. The results show that IH challenges down-regulate the GABAA receptor levels in pancreatic islets, which decreases GABA–GABAA receptor coupling action, as well as membrane depolarization for insulin secretion. The findings have the potential to suggest novel interventions for insulin regulation during IH of disordered breathing, including OSA.

Nrf2a dependent and independent effects of early life exposure to 3,3'-dichlorobiphenyl (PCB-11) in zebrafish (Danio rerio).

The environmental pollutant 3,3'-dichlorobiphenyl (PCB-11) is a lower-chlorinated polychlorinated biphenyl (PCB) congener present in air and water samples. Both PCB-11 and its metabolite, 4-PCB-11-Sulfate, are detected in humans, including in pregnant women. Previous research in zebrafish (Danio rerio) has shown that 0.2μM exposures to 4-PCB-11-Sulfate starting at 1 day post fertilization (dpf) increase hepatic neutral lipid accumulation in larvae at 15 dpf. Here, we explored whether nuclear factor erythroid 2-related factor 2 (Nrf2), known as the master-regulator of the adaptive response to oxidative stress, contributes to metabolic impacts of 4-PCB-11-Sulfate. For this work, embryos were collected from homozygous wildtype or Nrf2a mutant adult zebrafish that also express GFP in pancreatic β-cells, rendering Tg(ins:GFP;nrf2afh318+/+) and Tg(ins:GFP;nrf2afh318-/-) lines. Exposures were conducted from 1-15 dpf to either 0.05% DMSO or DMSO-matched 0.2µM 4-PCB-11-Sulfate, and at 15 dpf subsets of larvae were imaged for overall morphology, primary pancreatic islet area, and collected for fatty acid profiling and RNAseq. At 15 dpf, independent of genotype, fish exposed to 4-PCB-11-Sulfate survived significantly more at 80-85% compared to 65-73% survival for unexposed fish, and had primary pancreatic islets 8% larger compared to unexposed fish. Fish growth at 15 dpf was dependent on genotype, with Nrf2a mutant fish a significant 3-5% shorter than wildtype fish, and an interaction effect was observed where Nrf2a mutant fish exposed to 4-PCB-11-Sulfate experienced a significant 29% decrease in the omega-3 fatty acid DHA compared to unexposed mutant fish. RNAseq revealed 308 differentially expressed genes, most of which were dependent on genotype. These findings suggest that Nrf2a plays an important role in growth as well as for DHA production in the presence of 4-PCB-11-Sulfate. Further research would be beneficial to understand the importance of Nrf2a throughout the lifecourse, especially in the context of toxicant exposures.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line βlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

302-OR: Hypoimmune Human Primary Pancreatic Islets Survive and Function in Humanized Diabetic Mice

Treatment of type 1 diabetes mellitus (T1DM) via allogeneic donor transplant has limited success due to morbidities from immunosuppression (IS) and a gradual loss of engrafted pancreatic islet function. We set out to engineer human primary pancreatic islets (PI) using our hypoimmune strategy that could engraft and evade host immune detection in the absence of IS. Human cadaveric PIs were engineered to knock-out function of HLA class I and II and overexpress CD47 (HIP PI) . Unedited PIs were cultured identically (Wt PI) as controls. NSG-SGM3 mice were reconstituted with human CD34+ cells and had >45% engraftment of human CD45+ cells. Mice were rendered diabetic (streptozotocin) . A total of 300 HIP PI or Wt PI were transplanted IM. After 6 days, splenocytes and serum were obtained for immune analyses. HIP PI showed no T cell recognition (ELISpot) , no graft-specific antibodies (Abs) , and were protected from NK cell and macrophage killing. Wt PI had a strong adaptive immune response with high ELISpot frequencies and an increase in graft-directed Abs. Serial BLI showed survival of all HIP PI grafts but rapid rejection of Wt PI, confirmed by histopathology at 4 weeks. Glucose levels gradually decreased after HIP PI transplantation and remained stable around 200 mg/dl after 14 days. These data suggest that hypoimmune engineering may generate HIP PI capable of persisting and functioning in diabetic patients without IS. Disclosure X.Hu: None. C.Gattis: None. C.Young: Employee; Sana Biotechnology Inc. A.G.Olroyd: None. E.Y.L.Chu: None. K.White: None. A.Friera: None. S.Schrepfer: Other Relationship; Sana Biotechnology Inc.

The cultivation conditions affect the aggregation and functionality of β-cell lines alone and in coculture with mesenchymal stromal/stem cells.

The manufacturing of viable and functional β-cell spheroids is required for diabetes cell therapy and drug testing. Mesenchymal stromal/stem cells (MSCs) are known to improve β-cell viability and functionality. We therefore investigated the aggregation behavior of three different β-cell lines (rat insulinoma-1 cell line [INS-1], mouse insulinoma-6 cell line [MIN6], and a cell line formed by the electrofusion of primary human pancreatic islets and PANC-1 cells [1.1B4]), two MSC types, and mixtures of β-cells and MSCs under different conditions. We screened several static systems to produce uniform β-cell and MSC spheroids, finding cell-repellent plates the most suitable. The three different β-cell lines differed in their aggregation behavior, spheroid size, and growth in the same static environment. We found no major differences in spheroid formation between primary MSCs and an immortalized MSC line, although both differed with regard to the aggregation behavior of the β-cell lines. All spheroids showed a reduced viability due to mass transfer limitations under static conditions. We therefore investigated three dynamic systems (shaking multi-well plates, spinner flasks, and shaking flasks). In shaking flasks, there were no β-cell-line-dependent differences in aggregation behavior, resulting in uniform and highly viable spheroids. We found that the aggregation behavior of the β-cell lines changed in a static coculture with MSCs. The β-cell/MSC coculture conditions must be refined to avoid a rapid segregation into distinct populations under dynamic conditions.