Mature Beta Cells Research Articles

Transcriptional coactivator MED15 is required for beta cell maturation

Mediator, a co-regulator complex required for RNA Polymerase II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We observe reduced Mediator subunit MED15 expression in endocrine hormone-producing pancreatic islets isolated from people living with type 2 diabetes and sought to understand how MED15 and Mediator control gene expression programs important for the function of insulin-producing β-cells. Here we show that Med15 is expressed during mouse β-cell development and maturation. Knockout of Med15 in mouse β-cells causes defects in β-cell maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses found that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. In support of a conserved role during human development, human embryonic stem cell-derived β-like cells, genetically engineered to express high levels of MED15, express increased levels of maturation markers. We provide evidence of a conserved role for Mediator in β-cell maturation and demonstrate an additional layer of control that tunes β-cell transcription factor function.

12560 RIMOC1, A Novel Regulator Of Mitochondrial Function, And Its Role In Pancreatic Beta-cell Survival, Mitophagy, And Diabetogenesis

Abstract Disclosure: D. Filingeri: None. R. Liu: None. V. Negi: None. J. Lee: None. A. Kumar: None. V. Mandi: None. M. Moulik: None. V. Yechoor: None. Background: Literature supports preserving beta-cell function as an approach to slowing diabetes progression. Mitochondria emerge as an interesting area of focus, driving cellular respiration and glucose-stimulated insulin secretion (GSIS) within beta-cells in physiologic conditions, and leading to increased intracellular ROS generation when damaged. Damaged mitochondria are recycled via lysosomes by mitophagy, and defects in mitophagy have been linked to numerous disease states, with more recent data implicating impaired mitophagy with beta-cell death. Our previous work on the Hippo-TEAD pathway and beta-cell survival led to the isolation of the TEAD binding partner RIMOC1 (RAB7A-Interacting MON1-CCZ1 Complex Subunit 1), a protein highly expressed in pancreatic beta-cells, though its role in beta-cells remains unknown. The overarching hypothesis is that RIMOC1 is protective against the development of diabetes by preserving beta-cell mitophagy and mitochondrial function, and that impaired RIMOC1 function would lead to beta-cell failure and diabetes.Methods: We have generated both a stable INS2 RIMOC1 KO (knockout) cell line and an INS1-Cre RIMOC1 KO mouse model, that are beta-cell specific knockouts of RIMOC1. Mitochondrial membrane potentials were measured using TMRE and JC1 stains, and mitophagy was quantified using the mtKeima probe via flow cytometry. Protein expression was quantified via western blotting. Ongoing studies involving our mouse model include in vivo GTTs (glucose tolerance testing), ex vitro GSIS experiments to assess beta-cell function from mouse islets, and seahorse assays to quantify mitochondrial function. Results: Published GEO datasets show RIMOC1 expression is positively correlated with beta-cell maturity and negatively correlated with glucose load and HgbA1c. Our in vitro data demonstrates RIMOC1 knockout leads to decreased insulin mRNA expression, decreased insulin content, and increased beta-cell cycle inhibition. Early in vivo data shows worsened hyperglycemia with oral refeeding after fasting. In vitro mitophagy experiments demonstrate significantly diminished mitochondrial membrane potential in RIMOC1 KO INS2 cells. Conclusions: RIMOC1 is highly expressed in beta-cells and has a significant role in maintaining euglycemia through mitochondrial functioning, insulin processing, and altered mitophagy. This research highlights RIMOC1 as a promising target of further diabetes research. Presentation: 6/2/2024

Safeguarding genomic integrity in beta-cells: implications for beta-cell differentiation, growth, and dysfunction.

The maintenance of optimal glucose levels in the body requires a healthy reserve of the insulin producing pancreatic beta-cells. Depletion of this reserve due to beta-cell dysfunction and death results in development of diabetes. Recent findings highlight unresolved DNA damage as a key contributor to beta-cell defects in diabetes. Beta-cells face various stressors and metabolic challenges throughout life, rendering them susceptible to DNA breaks. The post-mitotic, long-lived phenotype of mature beta-cells further warrants robust maintenance of genomic integrity. Failure to resolve DNA damage during beta-cell development, therefore, can result in an unhealthy reserve of beta-cells and predispose to diabetes. Yet, the molecular mechanisms safeguarding beta-cell genomic integrity remain poorly understood. Here, we focus on the significance of DNA damage in beta-cell homeostasis and postulate how cellular expansion, epigenetic programming, and metabolic shifts during development may impact beta-cell genomic integrity and health. We discuss recent findings demonstrating a physiological role for DNA breaks in modulating transcriptional control in neurons, which share many developmental programs with beta-cells. Finally, we highlight key gaps in our understanding of beta-cell genomic integrity and discuss emerging areas of interest.

Effect of isoproterenol, a β-adrenergic agonist, on the differentiation of insulin-producing pancreatic β cells derived from human pluripotent stem cells

Research on islet replacement through the differentiation of functionally matured insulin-producing β-like cells for the treatment of diabetes presents a significant challenge. Neural signals in β cell differentiation significantly impact the pancreatic microenvironment in glucose metabolism, but they are not fully understood. In this study, isoproterenol, a β adrenoreceptor agonist, was introduced into pancreatic progenitor cells, derived from human pluripotent stem cells in vitro, undergoing endocrine differentiation, using 2-dimensional (2D) and 3-dimensional (3D) differentiation protocols. This resulted in increased insulin and C-peptide secretion, along with elevated expression of key pancreatic beta cell transcription factors, including PDX-1, NKX6.1, and MAFA, and improved function, demonstrated by increased responsiveness to glucose determined via a glucose-stimulated insulin secretion test. Moreover, RNA transcriptome analysis of isoproterenol-treated endocrine progenitors facilitated the identification of biological pathways and genes that contribute to mature beta cell differentiation efficiency correlated with neural signals, such as adrenoceptor beta 1, calcium/calmodulin dependent protein kinase II alpha, phospholipase C delta 4, and neurotrophic receptor tyrosine kinase 1. Among those genes, calcium/calmodulin dependent protein kinase II alpha was suggested as the most notable gene involved in the isoproterenol mechanism through inhibitor assays. This study illustrates that isoproterenol significantly enhances endocrine differentiation and underscores its effects on stem cell-derived beta cell maturation, emphasizing its therapeutic potential for the treatment of diabetes.

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function

Aims/hypothesisRegulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown.MethodsTo understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice.ResultsStem cell models of the homozygous variant RFX6−/− predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell–Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/− SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes.Conclusions/interpretationOur allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes.Data availabilityUltra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley (https://data.mendeley.com/datasets/g75drr3mgw/2).Graphical

Senescence of human pancreatic beta cells enhances functional maturation through chromatin reorganization and promotes interferon responsiveness.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1458.

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.

Sox9 regulates alternative splicing and pancreatic beta cell function

Despite significant research, mechanisms underlying the failure of islet beta cells that result in type 2 diabetes (T2D) are still under investigation. Here, we report that Sox9, a transcriptional regulator of pancreas development, also functions in mature beta cells. Our results show that Sox9-depleted rodent beta cells have defective insulin secretion, and aging animals develop glucose intolerance, mimicking the progressive degeneration observed in T2D. Using genome editing in human stem cells, we show that beta cells lacking SOX9 have stunted first-phase insulin secretion. In human and rodent cells, loss of Sox9 disrupts alternative splicing and triggers accumulation of non-functional isoforms of genes with key roles in beta cell function. Sox9 depletion reduces expression of protein-coding splice variants of the serine-rich splicing factor arginine SRSF5, a major splicing enhancer that regulates alternative splicing. Our data highlight the role of SOX9 as a regulator of alternative splicing in mature beta cell function.

NGF effects promote the maturation of rat pancreatic beta cells by regulating GLUT2 levels and distribution, and glucokinase activity.

The nerve growth factor (NGF) participates in cell survival and glucose-stimulated insulin secretion (GSIS) processes in rat adult beta cells. GSIS is a complex process in which metabolic events and ionic channel activity are finely coupled. GLUT2 and glucokinase (GK) play central roles in GSIS by regulating the rate of the glycolytic pathway. The biphasic release of insulin upon glucose stimulation characterizes mature adult beta cells. On the other hand, beta cells obtained from neonatal, suckling, and weaning rats are considered immature because they secrete low levels of insulin and do not increase insulin secretion in response to high glucose. The weaning of rats (at postnatal day 20 in laboratory conditions) involves a dietary transition from maternal milk to standard chow. It is characterized by increased basal plasma glucose levels and insulin levels, which we consider physiological insulin resistance. On the other hand, we have observed that incubating rat beta cells with NGF increases GSIS by increasing calcium currents in neonatal cells. In this work, we studied the effects of NGF on the regulation of cellular distribution and activity of GLUT2 and GK to explore its potential role in the maturation of GSIS in beta cells from P20 rats. Pancreatic islet cells from both adult and P20 rats were isolated and incubated with 5.6 mM or 15.6 mM glucose with and without NGF for 4 hours. Specific immunofluorescence assays were conducted following the incubation period to detect insulin and GLUT2. Additionally, we measured glucose uptake, glucokinase activity, and insulin secretion assays at 5.6 mM or 15.6 mM glucose concentrations. We observed an age-dependent variation in the distribution of GLUT2 in pancreatic beta cells and found that glucose plays a regulatory role in GLUT2 distribution independently of age. Moreover, NGF increases GLUT2 abundance, glucose uptake, and GSIS in P20 beta cells and GK activity in adult beta cells. Our results suggest that besides increasing calcium currents, NGF regulates metabolic components of the GSIS, thereby contributing to the maturation process of pancreatic beta cells.

Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates β-Cell Identity and Function.

As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of beta cell identity and function. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis

Aims/hypothesisRecovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids.MethodsIn zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids.ResultsAdjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment.Conclusions/interpretationAdjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states.Data availabilityRaw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398).Graphical

ZnT8 loss-of-function accelerates functional maturation of hESC-derived beta cells and resists metabolic stress in diabetes

ZnT8 loss-of-function accelerates functional maturation of hESC-derived beta cells and resists metabolic stress in diabetes

Beta cell dysfunction induced by bone morphogenetic protein (BMP)-2 is associated with histone modifications and decreased NeuroD1 chromatin binding

SummaryInsufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.

Obese mothers supplemented with melatonin during gestation and lactation ameliorate the male offspring's pancreatic islet cellular composition and beta-cell function.

Melatonin supplementation to obese mothers during gestation and lactation might benefit the pancreatic islet cellular composition and beta-cell function in male offspring adulthood. C57BL/6 females (mothers) were assigned to two groups (n = 20/each) based on their consumption in control (C 17% kJ as fat) or high-fat diet (HF 49% kJ as fat). Mothers were supplemented with melatonin (Mel) (10 mg/kg daily) during gestation and lactation, or vehicle, forming the groups (n = 10/each): C, CMel, HF, and HFMel. The male offspring were studied, considering they only received the C diet after weaning until three months old. The HF mothers and their offspring showed higher body weight, glucose intolerance, insulin resistance, and low insulin sensitivity than the C ones. However, HFMel mothers and their offspring showed improved glucose metabolism and weight loss than the HF ones. Also, the offspring's higher expressions of pro-inflammatory markers and endoplasmic reticulum (ER) stress were observed in HF but reduced in HFMel. Contrarily, antioxidant enzymes were less expressed in HF but improved in HFMel. In addition, HF showed increased beta-cell mass and hyperinsulinemia but diminished in HFMel. Besides, the beta-cell maturity and identity gene expressions diminished in HF but enhanced in HFMel. In conclusion, obese mothers supplemented with melatonin benefit their offspring's islet cell remodeling and function. In addition, improving pro-inflammatory markers, oxidative stress, and ER stress resulted in better glucose and insulin levels control. Consequently, pancreatic islets and functioning beta cells were preserved in the offspring of obese mothers supplemented with melatonin.

1737-P: Novel Interaction between Nkx6.1 and Pdx1 Is Needed for Proper Beta-Cell Function

The beta cell transcription factor Nkx6.1 is critical in pancreatic progenitor cells and mature beta cells. The pathways regulated by Nkx6.1 that results in these phenotypes are still being defined. We sought to define the transcriptional factors that interact with Nkx6.1 and may be harnessed to expand functional beta cell mass. INS-1 832/13 beta cells were transduced with AdCMV-Nkx6.1-BioID, AdCMV-GFP-BioID or left untreated. Biotinylated proteins were isolated and the Nkx6.1 interactome was defined using liquid chromatography-tandem mass spectrometry. We defined 176 biotinylated proteins in cells transduced with AdCMV-Nkx6.1-BioID that were not observed with AdCMV-GFP-BioID or naturally biotinylated proteins from untreated lysates. These potential Nkx6.1 interacting proteins are enriched in nucleotide binding, ribosomal proteins, and transcription factors. Among the enriched transcription factors, we validated that Pdx1 is biotinylated in response to transduction with AdCMV-Nkx6.1 using biotin-targeted immunoprecipitation and western blot. To examine if Pdx1 is likely to interact with Nkx6.1 on the genome, we analyzed the frequency of proximal binding sites for Nkx6.1 and Pdx1, and demonstrated that Pdx1 and Nkx6.1 binding sites in close proximity were statistically overrepresented in the genome. Given the role of Pdx1 on functional beta cell mass, we further characterized the interaction of endogenous Nkx6.1 and Pdx1 by co-immunoprecipitation (Co-IP). Our data demonstrates significant Pdx1 binding by Nkx6.1 Co-IP and significant Nkx6.1 binding by Pdx1 Co-IP, as compared to Co-IP with the IgG control. Finally, we demonstrate coregulation of Unc3 expression by Nkx6.1 and Pdx1 and demonstrate that CRISPR mediated deletion of either Nkx6.1 or Pdx1 is sufficient to impair INS-1 832/13 mediated expression of Unc3. These data define potential Nkx6.1 binding partners in INS-1 832/13 cells, as well as novel interaction between Nkx6.1 and Pdx1 in the regulation of Unc3. Disclosure J.S.Tessem: None. C.C.Littlefield: None. J.T.Hill: None.

375-OR: Epigenetic Control of Beta-Cell Stress Response and Senescence

Both type 1 and type 2 diabetes (T1D and T2D) result from chronic insulin insufficiency due to progressive dysfunction and loss of beta cells, driven by cellular stress. Beta cell failure in both forms of diabetes involves defective stress-response and the onset of premature senescence, an irreversible form of cell-cycle arrest induced by chronic stress. However, the mechanisms by which cellular stress triggers beta cell senescence are not clear. We now show that CTCF, a stress-sensitive epigenetic regulator, controls the beta cell stress-response and senescence programs during beta cell maturation process along with the cohesin Smc3. Chromatin immunoprecipitation data suggest that CTCF and Smc3 differentially occupy genes associated with stress and DNA damage responses, functional maturation, and senescence in neonatal versus adult beta cells. Our data show that CTCF and cohesin Smc3 dependent control protects replicating beta cells from DNA damage, and is especially critical during the neonatal growth phase marked by high rates of replication. Beta cell specific loss of CTCF triggers a p53/p21 dependent senescence program, followed by beta cell dysfunction and eventual beta cell failure with advancing age. We further demonstrate that reduced CTCF levels in beta cells lead to adaptive failure in response to a high fat diet challenge, and recapitulate a neonatal beta cell like epigenetic landscape. Notably, CTCF levels are reduced in beta cells in T1D and T2D in mice and humans, and lead to reduced occupancy of CTCF and Smc3 at genes involved in senescence, DNA damage response, and beta cell function. Our data show that CTCF and cohesin collectively protect against senescence and govern beta cell function and fitness through a program established in the neonatal maturation phase. Disclosure S.S.Varghese: None. N.Parveen: None. J.K.Wang: Employee; Pfizer Inc., Johnson & Johnson. A.G.Hernandez-de la pena: None. S.Dhawan: None. Funding National Institutes of Health (R01DK120253)

Ca2+-Mediated Signaling Pathways: A Promising Target for the Successful Generation of Mature and Functional Stem Cell-Derived Pancreatic Beta Cells In Vitro.

Diabetes mellitus is a chronic disease affecting over 500 million adults globally and is mainly categorized as type 1 diabetes mellitus (T1DM), where pancreatic beta cells are destroyed, and type 2 diabetes mellitus (T2DM), characterized by beta cell dysfunction. This review highlights the importance of the divalent cation calcium (Ca2+) and its associated signaling pathways in the proper functioning of beta cells and underlines the effects of Ca2+ dysfunction on beta cell function and its implications for the onset of diabetes. Great interest and promise are held by human pluripotent stem cell (hPSC) technology to generate functional pancreatic beta cells from diabetic patient-derived stem cells to replace the dysfunctional cells, thereby compensating for insulin deficiency and reducing the comorbidities of the disease and its associated financial and social burden on the patient and society. Beta-like cells generated by most current differentiation protocols have blunted functionality compared to their adult human counterparts. The Ca2+ dynamics in stem cell-derived beta-like cells and adult beta cells are summarized in this review, revealing the importance of proper Ca2+ homeostasis in beta-cell function. Consequently, the importance of targeting Ca2+ function in differentiation protocols is suggested to improve current strategies to use hPSCs to generate mature and functional beta-like cells with a comparable glucose-stimulated insulin secretion (GSIS) profile to adult beta cells.