Human Β-cell Line Research Articles

9250 carRNA m6A methylation regulates transcription state and chromatin accessibility in human islets in normal physiology and type 2 diabetes

Abstract Disclosure: N. Shukla: None. C. Ju: None. H. Li: None. C. He: None. R. Kulkarni: Advisory Board Member; Self; Novo Nordisk, Biomea Fusion Inc, REDD Pharma. Grant Recipient; Self; Inversago. Chromatin-associated regulatory RNAs (carRNA), which include promoter- and enhancer-associated RNAs as well as several non-coding repeat elements have been established in the literature as important regulators of global gene transcription. However, the regulation of carRNAs themselves has been an enigma. Recent publications from the He Lab at UChicago have shown the role of RNA N6-methyladenosine (m6A) in regulating carRNA stability and function. m6A is among the most abundant mRNA modifications in mammals. Seminal work from our lab has reported that the “writers” of m6A (e.g. methyltransferase-like [METTL3] and METTL14) are significantly downregulated in islets of humans with type 2 diabetes (T2D). Consequently, the global m6A landscape in the islets segregates the T2D from the control group significantly better than the transcriptome (n=7 for Control and n=8 for T2D islets). Hypomethylation of key transcripts in the insulin/IGF-1-AKT-PDX1 pathway led to impaired insulin secretion and decreased β-cell proliferation. We now show this decreased m6A on RNA is not limited to the mRNA but also affects chromatin-associated regulatory RNAs (carRNA). Intriguingly, downregulation of METTL3 and METTL14 in human β-cell line EndoC-βH1 differentially regulates these regulatory versus protein-coding transcripts. We observe that while METTL14 KD causes global downregulation of transcripts belonging to pathways important for β-cell identity and function (including calcium signaling, PI3K-AKT/MAPK signaling pathway, pancreatic secretion etc.), it leads to an upregulation of pathways involved in RNA processing and global gene transcription (Transcription by RNA Pol II, metabolism and splicing of mRNA, chromatin organization and modification etc.) {false discovery rate (FDR) < 0.05}. Considering carRNAs have been previously reported to regulate global chromatin accessibility and transcription, our observations on their ability to get differentially regulated m6A writer proteins provide novel insights into the epigenetic regulation of human islet cells. These findings provide opportunities to develop therapeutic approaches to regulate β-cell identity and/or insulin secretion in T2D. Presentation: 6/3/2024

8052 carRNA m6A methylation regulates transcription state and chromatin accessibility in human islets in normal physiology and type 2 diabetes

Abstract Disclosure: N. Shukla: None. C. Ju: None. H. Li: None. C. He: None. R. Kulkarni: Advisory Board Member; Self; Novo Nordisk, Biomea Fusion Inc, REDD Pharma. Grant Recipient; Self; Inversago. Chromatin-associated regulatory RNAs (carRNA), which include promoter- and enhancer-associated RNAs as well as several non-coding repeat elements have been established in the literature as important regulators of global gene transcription. However, the regulation of carRNAs themselves has been an enigma. Recent publications from the He Lab at UChicago have shown the role of RNA N6-methyladenosine (m6A) in regulating carRNA stability and function. m6A is among the most abundant mRNA modifications in mammals. Seminal work from our lab has reported that the “writers” of m6A (e.g. methyltransferase-like [METTL3] and METTL14) are significantly downregulated in islets of humans with type 2 diabetes (T2D). Consequently, the global m6A landscape in the islets segregates the T2D from the control group significantly better than the transcriptome (n=7 for Control and n=8 for T2D islets). Hypomethylation of key transcripts in the insulin/IGF-1-AKT-PDX1 pathway led to impaired insulin secretion and decreased β-cell proliferation. We now show this decreased m6A on RNA is not limited to the mRNA but also affects chromatin-associated regulatory RNAs (carRNA). Intriguingly, downregulation of METTL3 and METTL14 in human β-cell line EndoC-βH1 differentially regulates these regulatory versus protein-coding transcripts. We observe that while METTL14 KD causes global downregulation of transcripts belonging to pathways important for β-cell identity and function (including calcium signaling, PI3K-AKT/MAPK signaling pathway, pancreatic secretion etc.), it leads to an upregulation of pathways involved in RNA processing and global gene transcription (Transcription by RNA Pol II, metabolism and splicing of mRNA, chromatin organization and modification etc.) {false discovery rate (FDR) < 0.05}. Considering carRNAs have been previously reported to regulate global chromatin accessibility and transcription, our observations on their ability to get differentially regulated m6A writer proteins provide novel insights into the epigenetic regulation of human islet cells. These findings provide opportunities to develop therapeutic approaches to regulate β-cell identity and/or insulin secretion in T2D. Presentation: 6/3/2024

EndoC-βH3 pseudoislets are suitable for intraportal transplantation in diabetic mice

ABSTRACT Background Islet or β-cell transplantation is a therapeutical approach to substitute the insulin-producing cells which are abolished in type 1 diabetes mellitus. The shortage of human islets as well as the complicated and costly isolation process limit the application of these techniques in daily clinical practice. EndoC-βH is a human β-cell line that readily forms aggregates termed pseudoislets, providing an alternative to primary human islets or β-cells. Methods EndoC-βH3 cells were seeded and incubated to form pseudoislets. Their insulin secretion was analyzed by ELISA and compared with cell monolayers. Pseudoislets were transplanted into streptozotocin-treated NMRi nu/nu mice. Blood glucose was monitored before and after transplantation and compared with wild types. Grafts were analyzed by immunohistology. Results This study shows that EndoC-βH cells are able to form pseudoislets by aggregation, leading to an enhanced glucose stimulated insulin secretion in vitro. These pseudoislets were then successfully transplanted into the livers of diabetic mice and produced insulin in vitro. Blood glucose levels of the streptozocin-treated recipient mice were significantly decreased when compared to pre-transplantation and matched the levels found in control mice. Conclusion We suggest pseudoislets aggregated from EndoC-βH cells as a valuable and promising model for islet transplantation research.

Extracellular electrophysiology on clonal human β-cell spheroids.

Pancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between β-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling. We have therefore asked whether 3D spheroids enhance clonal β-cell function such as electrical activity and hormone secretion using human EndoC-βH1, EndoC-βH5 and rodent INS-1 832/13 cells. Spheroids were formed either by hanging drop or proprietary devices. Extracellular electrophysiology was conducted using multi-electrode arrays with appropriate signal extraction and hormone secretion measured by ELISA. EndoC-βH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-βH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-βH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index. In conclusion, spheroid formation enhances physiological function of the human clonal β-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology.

Deucravacitinib, a tyrosine kinase 2 pseudokinase inhibitor, protects human EndoC-βH1 β-cells against proinflammatory insults.

Type 1 diabetes is characterized by pancreatic islet inflammation and autoimmune-driven pancreatic β-cell destruction. Interferon-α (IFNα) is a key player in early human type 1 diabetes pathogenesis. IFNα activates the tyrosine kinase 2 (TYK2)-signal transducer and activator of transcription (STAT) pathway, leading to inflammation, HLA class I overexpression, endoplasmic reticulum (ER) stress, and β-cell apoptosis (in synergy with IL-1β). As TYK2 inhibition has raised as a potential therapeutic target for the prevention or treatment of type 1 diabetes, we investigated whether the selective TYK2 inhibitor deucravacitinib could protect β-cells from the effects of IFNα and other proinflammatory cytokines (i.e., IFNγ and IL-1β). All experiments were performed in the human EndoC-βH1 β-cell line. HLA class I expression, inflammation, and ER stress were evaluated by real-time PCR, immunoblotting, and/or immunofluorescence. Apoptosis was assessed by the DNA-binding dyes Hoechst 33342 and propidium iodide or caspase 3/7 activity. The promoter activity was assessed by luciferase assay. Deucravacitinib prevented IFNα effects, such as STAT1 and STAT2 activation and MHC class I hyperexpression, in a dose-dependent manner without affecting β-cell survival and function. A comparison between deucravacitinib and two Janus kinase inhibitors, ruxolitinib and baricitinib, showed that deucravacitinib blocked IFNα- but not IFNγ-induced signaling pathway. Deucravacitinib protected β-cells from the effects of two different combinations of cytokines: IFNα + IL-1β and IFNγ + IL-1β. Moreover, this TYK2 inhibitor could partially reduce apoptosis and inflammation in cells pre-treated with IFNα + IL-1β or IFNγ + IL-1β. Our findings suggest that, by protecting β-cells against the deleterious effects of proinflammatory cytokines without affecting β-cell function and survival, deucravacitinib could be repurposed for the prevention or treatment of early type 1 diabetes.

Pharmacological HDAC inhibition impairs pancreatic β-cell function through an epigenome-wide reprogramming

Histone deacetylases enzymes (HDACs) are chromatin modifiers that regulate gene expression through deacetylation of lysine residues within specific histone and non-histone proteins. A cell-specific gene expression pattern defines the identity of insulin-producing pancreatic β cells, yet molecular networks driving this transcriptional specificity are not fully understood. Here, we investigated the HDAC-dependent molecular mechanisms controlling pancreatic β-cell identity and function using the pan-HDAC inhibitor trichostatin A through chromatin immunoprecipitation assays and RNA sequencing experiments. We observed that TSA alters insulin secretion associated with β-cell specific transcriptome programming in both mouse and human β-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative β-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers. Our data indicate that HDAC activity could be required to protect against loss of β-cell identity with unsuitable expression of genes associated with alternative cell fates.

Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells.

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human β-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.

The human batokine EPDR1 regulates β-cell metabolism and function

ObjectiveEpendymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic β-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in β-cells, the role of EPDR1 in β-cell metabolism and function has not been investigated. MethodsEPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-βH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis. ResultsEPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1 silencing in human islets and β-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K+-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells. ConclusionThese results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve β-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.

Loss of tetraspanin-7 expression reduces pancreatic β-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate β-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic β-cell function is not yet fully understood. Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. β-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. Tspan7 is expressed in insulin-containing granules of pancreatic β-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- β-cells, but β-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent β-cells, exocytotic Ca2+ sensitivity was reduced in a human β-cell line (EndoC-βH1) following Tspan7 KD. Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in β-cells. Its function is more significant in human β-cells than their rodent counterparts.

The Transcription Factor YY1 Is Essential for Normal DNA Repair and Cell Cycle in Human and Mouse β-Cells.

Identifying the mechanisms behind the β-cell adaptation to failure is important to develop strategies to manage type 2 diabetes (T2D). Using db/db mice at early stages of the disease process, we took advantage of unbiased RNA sequencing to identify genes/pathways regulated by insulin resistance in β-cells. We demonstrate herein that islets from 4-week-old nonobese and nondiabetic leptin receptor-deficient db/db mice exhibited downregulation of several genes involved in cell cycle regulation and DNA repair. We identified the transcription factor Yin Yang 1 (YY1) as a common gene between both pathways. The expression of YY1 and its targeted genes was decreased in the db/db islets. We confirmed the reduction in YY1 expression in β-cells from diabetic db/db mice, mice fed a high-fat diet (HFD), and individuals with T2D. Chromatin immunoprecipitation sequencing profiling in EndoC-βH1 cells, a human pancreatic β-cell line, indicated that YY1 binding regions regulate cell cycle control and DNA damage recognition and repair. We then generated mouse models with constitutive and inducible YY1 deficiency in β-cells. YY1-deficient mice developed diabetes early in life due to β-cell loss. β-Cells from these mice exhibited higher DNA damage, cell cycle arrest, and cell death as well as decreased maturation markers. Tamoxifen-induced YY1 deficiency in mature β-cells impaired β-cell function and induced DNA damage. In summary, we identified YY1 as a critical factor for β-cell DNA repair and cell cycle progression.

204-OR: The Chd4 Helicase Modulates ß-Cell Function In Vivo

During diabetes progression, a subset of failing β-cells incurs a loss of transcription factors (TFs) such as Pdx1, which drives expression of numerous β-cell functional genes including Ins2, MafA, and Slc2a2. Like other TFs, Pdx1 requires the recruitment of transcriptional coregulators to modulate its transcriptional activity. It has been established that the Pdx1-bound Swi/Snf complex plays a critical role in maintaining β-cell function in part by recruiting Pdx1 to the insulin gene and facilitating its transcription. Furthermore, interactions between Pdx1 and the Swi/Snf ATPase subunit Brg1 are reduced in type 2 diabetes (T2D) β-cells. Recently, through an IP-Mass Spec approach, Pdx1 has been shown to interact with numerous other coregulatory complexes, including the Chd4 ATPase subunit of the Nucleosome Remodeling and Deacetylase complex. Here, we confirmed Pdx1 and Chd4 interact in mouse and human β-cell lines and primary β-cells using co-immunoprecipitation and proximity ligation assays (PLA) . Tissue harvested from mice injected with glucose had more β-cell nuclei containing multiple PLA signals than tissue from fasted mice. Conversely, tissue from high-fat diet mice and T2D human donors had fewer β-cell nuclei containing multiple PLA signals than tissue from control mice and nondiabetes human donors. To investigate the role of Chd4 on mature β-cell function, we generated a tamoxifen inducible, β-cell-specific Chd4 knockout mouse model (Chd4 Δβ ) . Four weeks following Chd4 removal, Chd4 Δβ mutants were glucose intolerant and had elevated ad libitum fed blood glucose levels and impaired insulin secretion from isolated islets. Bulk mRNA-sequencing from flow-sorted lineage labeled Chd4Δβ β-cells identified 1093 upregulated genes (eg Adcy1 and Rapgef3) and 438 downregulated genes (eg MafA, Robo2, and Slc2a2) . Future efforts are focused on defining the mechanisms by which Chd4 controls gene expression in the β-cell, with a current focus on how Chd4 modulates chromatin accessibility and TF recruitment. Disclosure R.K.Davidson: None. N.Casey: None. S.Kanojia: None. J.Spaeth: None. Funding National Institutes of Health (K01DK115633) Showalter Trust Award

1447-P: The Snd1 Coregulator Controls ß-Cell Function

The main function of the pancreatic β-cell is glucose-stimulated insulin secretion (GSIS) which serves to maintain whole body glucose homeostasis. Under type 2 diabetic conditions, a subset of β-cells fail and lose expression of key transcription factors (TFs) required for GSIS. Among these TFs is Pdx1, which requires the recruitment of a distinct set of transcriptional coregulators to modulate its activity. Recently, Pdx1 has been shown, through and IP-Mass Spec approach, to interact with numerous coregulators, including Snd1 (Staphylococcal nuclease and Tudor domain-containing protein) , which has been shown to facilitate protein-protein interactions and transcriptional control through the recruitment of chromatin modifying enzymes. Here, we first confirmed Pdx1 and Snd1 interact in the rodent β-cell lines by Co-IP, and in primary mouse β-cells and human EndoC-βH1 β-cell lines using proximity ligation assay. Next, we utilized CRISPR-Cas9 technology to generate Snd1-deficient mouse β-cell lines (Snd1KO) . Bulk mRNA-sequencing performed on Control and Snd1KO β-cell lines identified numerous differentially expressed genes linked to insulin secretion and cell proliferation. WST-1 cell proliferation assays revealed impaired proliferation in Snd1KO cell lines and high-glucose stimulated insulin secretion was blunted in INS-1 832/13 cells following siRNA-mediated knockdown of Snd1. As Snd1 has been shown to facilitate protein-protein interactions in other tissues, we found Snd1 interacts with numerous nuclear enriched proteins in the β-cell by IP-Mass Spec, including Stat3, Pax6, Ruvbl2 and Trim28. Future efforts are underway to characterize how Snd1 interactions with these proteins impact gene expression in vitro. Moreover, the role of Snd1 in modulating gene expression and β-cell function in vivo is being evaluated in a novel β-cell-specific (Ins1-Cre) Snd1 knockout mouse model. Disclosure S.Kanojia: None. R.K.Davidson: None. J.Spaeth: None. N.Casey: None. Funding K01DK115633Wells Center/Pediatric startupCDMD Pilot feasibility grant

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic β-Cell Model.

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-βH1 β-cell line, the rat β-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as “unknowns”. Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a “new” diabetogenic agent. Our results indicate that the EndoC-βH1 cell line is a suitable human β-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.

Glucose-Dependent miR-125b Is a Negative Regulator of β-Cell Function.

Impaired pancreatic β-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of β cells. We have previously shown that β cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in β cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in β cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human β-cell line EndoCβ-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in β cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg β cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion.

Screening of Relevant Metabolism-Disrupting Chemicals on Pancreatic β-Cells: Evaluation of Murine and Human In Vitro Models.

Endocrine-disrupting chemicals (EDCs) are chemical substances that can interfere with the normal function of the endocrine system. EDCs are ubiquitous and can be found in a variety of consumer products such as food packaging materials, personal care and household products, plastic additives, and flame retardants. Over the last decade, the impact of EDCs on human health has been widely acknowledged as they have been associated with different endocrine diseases. Among them, a subset called metabolism-disrupting chemicals (MDCs) is able to promote metabolic changes that can lead to the development of metabolic disorders such as diabetes, obesity, hepatic steatosis, and metabolic syndrome, among others. Despite this, today, there are still no definitive and standardized in vitro tools to support the metabolic risk assessment of existing and emerging MDCs for regulatory purposes. Here, we evaluated the following two different pancreatic cell-based in vitro systems: the murine pancreatic β-cell line MIN6 as well as the human pancreatic β-cell line EndoC-βH1. Both were challenged with the following range of relevant concentrations of seven well-known EDCs: (bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), perfluorooctanesulfonic acid (PFOS), di(2-ethylhexyl) phthalate (DEHP), cadmium chloride (CdCl2), and dichlorodiphenyldichloroethylene (DDE)). The screening revealed that most of the tested chemicals have detectable, deleterious effects on glucose-stimulated insulin release, insulin content, electrical activity, gene expression, and/or viability. Our data provide new molecular information on the direct effects of the selected chemicals on key aspects of pancreatic β-cell function, such as the stimulus-secretion coupling and ion channel activity. In addition, we found that, in general, the sensitivity and responses were comparable to those from other in vivo studies reported in the literature. Overall, our results suggest that both systems can serve as effective tools for the rapid screening of potential MDC effects on pancreatic β-cell physiology as well as for deciphering and better understanding the molecular mechanisms that underlie their action.

Ribosomal biogenesis regulator DIMT1 controls β-cell protein synthesis, mitochondrial function, and insulin secretion

We previously reported that loss of mitochondrial transcription factor B1 (TFB1M) leads to mitochondrial dysfunction and is involved in the pathogenesis of type 2 diabetes (T2D). Whether defects in ribosomal processing impact mitochondrial function and could play a pathogenetic role in β-cells and T2D is not known. To this end, we explored expression and the functional role of dimethyladenosine transferase 1 homolog (DIMT1), a homolog of TFB1M and a ribosomal RNA (rRNA) methyltransferase implicated in the control of rRNA. Expression of DIMT1 was increased in human islets from T2D donors and correlated positively with expression of insulin mRNA, but negatively with insulin secretion. We show that silencing of DIMT1 in insulin-secreting cells impacted mitochondrial function, leading to lower expression of mitochondrial OXPHOS proteins, reduced oxygen consumption rate, dissipated mitochondrial membrane potential, and a slower rate of ATP production. In addition, the rate of protein synthesis was retarded upon DIMT1 deficiency. Consequently, we found that DIMT1 deficiency led to perturbed insulin secretion in rodent cell lines and islets, as well as in a human β-cell line. We observed defects in rRNA processing and reduced interactions between NIN1 (RPN12) binding protein 1 homolog (NOB-1) and pescadillo ribosomal biogenesis factor 1 (PES-1), critical ribosomal subunit RNA proteins, the dysfunction of which may play a part in disturbing protein synthesis in β-cells. In conclusion, DIMT1 deficiency perturbs protein synthesis, resulting in mitochondrial dysfunction and disrupted insulin secretion, both potential pathogenetic processes in T2D.

Intracellular Toxic Advanced Glycation End-Products in 1.4E7 Cell Line Induce Death with Reduction of Microtubule-Associated Protein 1 Light Chain 3 and p62.

Background: The death of pancreatic islet β-cells (β-cells), which are the insulin-producing cells, promote the pathology in both Type 1 and Type 2 diabetes mellitus (DM) (T1DM and T2DM), and they are protected by autophagy which is one of the mechanisms of cell survival. Recently, that some advanced glycation end-products (AGEs), such as methylglyoxial-derived AGEs and Nε-carboxymethyllysine, induced the death of β-cells were revealed. In contrast, we had reported AGEs derived from glyceraldehyde (GA, the metabolism intermediate of glucose and fructose) are considered to be toxic AGEs (TAGE) due to their cytotoxicity and role in the pathogenesis of T2DM. More, serum levels of TAGE are elevated in patients with T1 and T2DM, where they exert cytotoxicity. Aim: We researched the cytotoxicity of intracellular and extracellular TAGE in β-cells and the possibility that intracellular TAGE were associated with autophagy. Methods: 1.4E7 cells (a human β-cell line) were treated with GA, and analyzed viability, quantity of TAGE, microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, and p62. We also examined the viability of 1.4E7 cells treated with TAGE-modified bovine serum albumin, a model of TAGE in the blood. Results: Intracellular TAGE induced death of 1.4E7 cells, decrease of LC3-I, LC3-II, and p62. Extracellular TAGE didn’t show cytotoxicity in the physiological concentration. Conclusion: Intracellular TAGE induced death of β-cells more strongly than extracellular TAGE, and may suppress autophagy via reduction of LC3-I, LC3-II, and p62 to inhibit the degradation of them.

Large-Scale Functional Genomics Screen to Identify Modulators of Human β-Cell Insulin Secretion.

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic β-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-βH1 cells, a human β-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-βH1 cells for large-scale siRNA screening to identify candidate genes regulating β-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional β-cell mass occurring in T2D.

Palmitoylated small GTPase ARL15 is translocated within Golgi network during adipogenesis.

ABSTRACTThe small GTPase ARF family member ARL15 gene locus is associated in population studies with increased risk of type 2 diabetes, lower adiponectin and higher fasting insulin levels. Previously, loss of ARL15 was shown to reduce insulin secretion in a human β-cell line and loss-of-function mutations are found in some lipodystrophy patients. We set out to understand the role of ARL15 in adipogenesis and showed that endogenous ARL15 palmitoylated and localised in the Golgi of mouse liver. Adipocyte overexpression of palmitoylation-deficient ARL15 resulted in redistribution to the cytoplasm and a mild reduction in expression of some adipogenesis-related genes. Further investigation of the localisation of ARL15 during differentiation of a human white adipocyte cell line showed that ARL15 was predominantly co-localised with a marker of the cis face of Golgi at the preadipocyte stage and then translocated to other Golgi compartments after differentiation was induced. Finally, co-immunoprecipitation and mass spectrometry identified potential interacting partners of ARL15, including the ER-localised protein ARL6IP5. Together, these results suggest a palmitoylation dependent trafficking-related role of ARL15 as a regulator of adipocyte differentiation via ARL6IP5 interaction.This article has an associated First Person interview with the first author of the paper.

1243-P: The Chd4 Helicase Regulates Pdx1-Controlled Genes Involved in ß-Cell Function

Diabetes progression is partly characterized by a loss of key transcription factors (TFs) in a subset of failing β-cells. Among these TFs is Pdx1, which coordinates the expression of numerous genes related to β-cell function (MafA, Ins2, Ucn3), mitochondrial function (Tfam), and calcium signaling (Cacna1d, Camkk2, Atp2a2). Pdx1 requires the recruitment of transcriptional coregulators to modulate its activity and has recently been shown to interact with the Chd4 ATPase subunit of the Nucleosome Remodeling and Deacetylase complex. In other cellular contexts, Chd4 is required to maintain cellular identity and mitochondrial function; however, the contribution of Chd4 in controlling Pdx1-target genes in the β-cell has yet to be explored. Here, we confirmed the Pdx1:Chd4 interaction using co-IP in mouse β-cell lines and proximity ligation assay in mouse and human β-cell lines and tissue. We found, by ChIP-Sequencing, that Chd4 binds to 4796 genes in β-cell lines, 4463 of which are also bound by Pdx1, including MafA, Cacna1d, and Atp2a2. Transient siRNA knockdown of Chd4 in the βTC-3 murine insulinoma cell line led to a reduction of MafA at the transcript (41% ± 3, p < 0.05) and protein level, as well as reduced expression of mitochondrial TF Tfam (39% ± 8, p <0.05) and calcium regulators Camkk2 (53% ± 6, p <0.05) and Serca2b (51% ± 13, p <0.05). Furthermore, glucose-stimulated insulin secretion in the INS1 832/13 rat insulinoma cell line was impaired (p = 0.0504) in Chd4 knockdown cells. We next generated a tamoxifen-inducible, β-cell-specific (MIP-CreERT) mouse knockout line of Chd4 and found significant impairment in glucose tolerance in male mice 2 weeks post-tamoxifen. Together, these data indicate Chd4 is required for the expression of numerous Pdx1-controlled genes important for β-cell function and whole-body glucose homeostasis. Future efforts are focused on defining the contributions of Chd4 in regulating gene expression and chromatin accessibility in β-cells using our β-cell-specific Chd4 knockout model. Disclosure R. K. Davidson: None. N. Casey: None. S. Kanojia: None. J. Spaeth: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K01DK115633)