Primary Rat Islets Research Articles

The mitochondrial tRNA-derived fragment, mt-tRF-LeuTAA, couples mitochondrial metabolism to insulin secretion

ObjectiveThe contribution of the mitochondrial electron transfer system to insulin secretion involves more than just energy provision. We identified a small RNA fragment (mt-tRF-LeuTAA) derived from the cleavage of a mitochondrially-encoded tRNA that is conserved between mice and humans. The role of mitochondrially-encoded tRNA-derived fragments remains unknown. This study aimed to characterize the impact of mt-tRF-LeuTAA, on mitochondrial metabolism and pancreatic islet functions. MethodsWe used antisense oligonucleotides to reduce mt-tRF-LeuTAA levels in primary rat and human islet cells, as well as in insulin-secreting cell lines. We performed a joint transcriptome and proteome analysis upon mt-tRF-LeuTAA inhibition. Additionally, we employed pull-down assays followed by mass spectrometry to identify direct interactors of the fragment. Finally, we characterized the impact of mt-tRF-LeuTAA silencing on the coupling between mitochondrial metabolism and insulin secretion using high-resolution respirometry and insulin secretion assays. ResultsOur study unveils a modulation of mt-tRF-LeuTAA levels in pancreatic islets in different Type 2 diabetes models and in response to changes in nutritional status. The level of the fragment is finely tuned by the mechanistic target of rapamycin complex 1. Located within mitochondria, mt-tRF-LeuTAA interacts with core subunits and assembly factors of respiratory complexes of the electron transfer system. Silencing of mt-tRF-LeuTAA in islet cells limits the inner mitochondrial membrane potential and impairs mitochondrial oxidative phosphorylation, predominantly by affecting the Succinate (via Complex II)-linked electron transfer pathway. Lowering mt-tRF-LeuTAA impairs insulin secretion of rat and human pancreatic β-cells. ConclusionsOur findings indicate that mt-tRF-LeuTAA interacts with electron transfer system complexes and is a pivotal regulator of mitochondrial oxidative phosphorylation and its coupling to insulin secretion.

CEBPA Overexpression Enhances β-Cell Proliferation and Survival.

A commonality between type 1 and type 2 diabetes is the decline in functional β-cell mass. The transcription factor Nkx6.1 regulates β-cell development and is integral for proper β-cell function. We have previously demonstrated that Nkx6.1 depends on c-Fos mediated upregulation and the nuclear hormone receptors Nr4a1 and Nr4a3 to increase β-cell insulin secretion, survival, and replication. Here, we demonstrate that Nkx6.1 overexpression results in upregulation of the bZip transcription factor CEBPA and that CEBPA expression is independent of c-Fos regulation. In turn, CEBPA overexpression is sufficient to enhance INS-1 832/13 β-cell and primary rat islet proliferation. CEBPA overexpression also increases the survival of β-cells treated with thapsigargin. We demonstrate that increased survival in response to ER stress corresponds with changes in expression of various genes involved in the unfolded protein response, including decreased Ire1a expression. These data show that CEBPA is sufficient to enhance functional β-cell mass by increasing β-cell proliferation and modulating the unfolded protein response.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

Naringenin protects pancreatic β cells in diabetic rat through activation of estrogen receptor β

Naringenin is a citrus flavonoid that potently improves metabolic parameters in animal models of metabolic disorders, such as type 2 diabetes. Estrogen receptor (ER) activation promotes β cell function and survival, thereby improving systemic glucose metabolism. In this study, we used a luciferase reporter assay, isolated rat islets and a diabetic rat model to investigate the effects of naringenin on ER signaling and the underlying mechanism of naringenin-mediated improvement of islet function in diabetes. Naringenin specifically activated ERβ without affecting the activity of ERα, G protein-coupled estrogen receptor (GPER) or estrogen-related receptor (ERR) α/β/γ. Additionally, treatment with naringenin enhanced glucose-stimulated insulin secretion in isolated rat islets. This effect was abrogated by PHTPP, an ERβ antagonist. Transcriptomic analysis revealed that naringenin upregulated the expression of genes, such as Pdx1 and Mafa, which are closely linked to improved β-cell function. In consistence, single administration of naringenin to normal rats elevated plasma insulin levels and improved glucose responses. These beneficial effects were blocked by PHTPP. In streptozocin-nicotinamide induced diabetic rats, treatment for 2 weeks with naringenin alone, but not in combination with PHTPP, significantly restored pancreatic β cell mass and improved glucose metabolism. Collectively, these data support that naringenin specifically activate ERβ to improve insulin secretion in the primary rat islets. Furthermore, naringenin administration also protected β cell function and reversed glucose dysregulation in diabetic rats. These beneficial effects are at least partially dependent on the ERβ pathway.

1728-P: Transcriptional Activation of Myc by Glucose Requires Recruitment of ChREBP to the Pvt1 Promoter Regulating Proliferation in ß-Cells

Myc and Carbohydrate Response Element (ChoRE) Binding Protein (ChREBP) are glucose-responsive transcriptional regulators of β-cell proliferation. Myc requires ChREBP to drive glucose-mediated proliferation in β-cells, yet no ChoREs are present in Myc’s promoter. In primates and rodents, the Myc gene and the long non-coding RNA, PVT1 gene are syntenic, separated by approximately 60 kbp. Mutations in this loci are associated with malignancies and complications of diabetes but are unexplored in β-cells. In INS-1 cells, Myc and PVT1 mRNAs were induced in response to glucose by 3.5±0.02and 2.9±0.1 fold, respectively, and Myc protein levels increased by 4-fold ±0.17. Silencing of ChREBPβ significantly blocked the glucose response of Myc and Pvt1. ChREBP ChIPseq revealed a single ChoRE located approximately 100 bp upstream of the Pvt1 transcription start site and is highly conserved in primates and rodents. By Chromosome Conformation Capture (3C), we demonstrated that high glucose increased interaction between the Pvt1 and Myc promoters by a factor of 3.5±0.4. Using adenoviral delivery of CRISPR-xCas9-sgRNA targeting the Pvt1 ChoRE, we specifically attenuated ChREBP recruitment by 2.8±0.8 fold, while other known ChoREs were unaffected. We demonstrated by 3C that the chromatin looping in response to glucose was abolished by the Pvt1 ChoRE disruptor. Both in primary rat islets and in INS-1 cells, disruption of Pvt1’s ChoRE resulted in complete and significant blockage of Pvt1 and Myc mRNA upregulation in response to glucose, with no effect on other ChoRE-containing genes. Finally, disruption of the ChoRE on the Pvt1 promoter eliminated glucose-mediated proliferation of primary rat β-cells (a decrease of 9.8±1.9 fold on Ki67+ Ins+ staining). In conclusion, we identify a novel mechanism driving the ChREBP-dependent activation of the Myc and Pvt1 genes and proliferation of β-cells. Disclosure L.S.Katz: None. P.Wang: None. G.Brill: None. P.Zhang: None. J.Haldeman: Employee; Janssen Research & Development, LLC. C.B.Newgard: Advisory Panel; Eli Lilly and Company, Novo Nordisk, AstraZeneca. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D.Scott: None. Funding National Institutes of Health (R01DK130300, P30DK020541)

The Proliferative and Functional Roles of CEBP/A in Ins-1 832/13 Cells and Primary Rat Islets

The beta cell transcription factor Nkx6.1, when overexpressed in primary rodent and human islets, is sufficient to enhance beta cell proliferation, increase insulin secretion and enhance cell survival. We have sought to define the transcriptional targets of Nkx6.1 that allow for its ability to increase functional beta cell mass. We have shown that Nkx6.1 induces expression of the transcription factor CEBP/A. CEBP/A overexpression is sufficient to induce proliferation of Ins-1 832/13 cells and primary rat islets. CEBP/A overexpression enhances glucose stimulated insulin secretion from primary rat islets, while decreasing total insulin content. Finally, CEBPa overexpression protects Ins-1 832/13 cells from thapsigargin and glucolipotoxicity induced cell death but fails to protect against etoposide and camptothecin induced cell death. These data suggest that CEBP/A may play a critical role in Nkx6.1 mediated expansion of functional beta cell mass through a endoplasmic reticulum stress-mediated pathway. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Waffle-inspired hydrogel-based macrodevice for spatially controlled distribution of encapsulated therapeutic microtissues and pro-angiogenic endothelial cells.

Macro-encapsulation systems for delivery of cellular therapeutics in diabetes treatment offer major advantages such as device retrievability and high cell packing density. However, microtissue aggregation and absence of vasculature have been implicated in the inadequate transfer of nutrients and oxygen to the transplanted cellular grafts. Herein, we develop a hydrogel-based macrodevice to encapsulate therapeutic microtissues positioned in homogeneous spatial distribution to mitigate their aggregation while concurrently supporting an organized intra-device network of vascular-inductive cells. Termed Waffle-inspired Interlocking Macro-encapsulation (WIM) device, this platform comprises two modules with complementary topography features that fit together in a lock-and-key configuration. The waffle-inspired grid-like micropattern of the "lock" component effectively entraps insulin-secreting microtissues in controlled locations while the interlocking design places them in a co-planar spatial arrangement with close proximity to vascular-inductive cells. The WIM device co-laden with INS-1E microtissues and human umbilical vascular endothelial cells (HUVECs) maintains desirable cellular viability in vitro with theencapsulated microtissues retaining their glucose-responsive insulin secretion while embedded HUVECs express pro-angiogenic markers. Furthermore, a subcutaneously implanted alginate-coated WIM device encapsulating primary rat islets achieves blood glucose control for 2 weeks in chemically induced diabetic mice. Overall, this macrodevice design lays foundation for a cell delivery platform, which has the potential to facilitate nutrients and oxygen transport to therapeutic grafts and thereby might lead to improved disease management outcome.

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 β-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions.

Serum accumulation of the gut microbial metabolite trimethylamine N-oxide (TMAO) is associated with high caloric intake and type 2 diabetes (T2D). Impaired pancreatic β-cell function is a hallmark of diet-induced T2D, which is linked to hyperglycemia and hyperlipidemia. While TMAO production via the gut microbiome-liver axis is well defined, its molecular effects on metabolic tissues are unclear, since studies in various tissues show deleterious and beneficial TMAO effects. We investigated the molecular effects of TMAO on functional β-cell mass. We hypothesized that TMAO may damage functional β-cell mass by inhibiting β-cell viability, survival, proliferation, or function to promote T2D pathogenesis. We treated INS-1 832/13 β-cells and primary rat islets with physiological TMAO concentrations and compared functional β-cell mass under healthy standard cell culture (SCC) and T2D-like glucolipotoxic (GLT) conditions. GLT significantly impeded β-cell mass and function by inducing oxidative and endoplasmic reticulum (ER) stress. TMAO normalized GLT-mediated damage in β-cells and primary islet function. Acute 40µM TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT-induced ER and oxidative stress. These novel results demonstrate that TMAO protects β-cell function and suggest that TMAO may play a beneficial molecular role in diet-induced T2D conditions.

Fucoidan Hydrogels Significantly Alleviate Oxidative Stress and Enhance the Endocrine Function of Encapsulated Beta Cells

AbstractMicroencapsulating pancreatic islets in immunoprotective alginate hydrogels is a promising strategy for treatment of type 1 diabetes. However, this strategy is limited by inflammation and hypoxia mediated oxidative stress, due to encapsulation and the hydrogel itself, leading to impaired insulin secretion and limited short and long term cell survival. Herein, the antioxidant effect of fucoidan, an algae derived polysaccharide, on beta cells, and its positive effects on encapsulated beta cell viability and function is presented. Fucoidan from Fucus vesiculosus (FF) exhibits a high total antioxidant capacity, and free radical scavenging activity, and is able to significantly alleviate intracellular oxidative stress in rat insolinoma beta cells (INS1E). In addition, FF significantly increases insulin secretion in a dose‐ and time‐dependent manner. When FF is incorporated in ultrapure alginate used for microencapsulation of primary rat islets, both viability and glucose responsiveness of rat islets in these socalled Fucogel microcapsules (Fucocaps) are found to be significantly higher compared to islets encapsulated in alginate alone. Similar results are obtained with INS1E pseudoislets and neonatal pig islets. Fucocaps can provide a redox‐modulatory niche and an immune barrier for islets and beta cells in the same time leading to significantly improved survival and endocrine function by mitigating oxidative stress.

Insulinoma-derived pseudo-islets for diabetes research.

The islets of Langerhans of the pancreas are the primary endocrine organ responsible for regulating whole body glucose homeostasis. The use of isolated primary islets for research development and training requires organ resection, careful digestion, and isolation of the islets from nonendocrine tissue. This process is time consuming, expensive, and requires substantial expertise. For these reasons, we sought to develop a more rapidly obtainable and consistent model system with characteristic islet morphology and function that could be employed to train personnel and better inform experiments prior to using isolated rodent and human islets. Immortalized β cell lines reflect several aspects of primary β cells, but cell propagation in monolayer cell culture limits their usefulness in several areas of research, which depend on islet morphology and/or functional assessment. In this manuscript, we describe the propagation and characterization of insulinoma pseudo-islets (IPIs) from a rat insulinoma cell line INS832/3. IPIs were generated with an average diameter of 200 μm, consistent with general islet morphology. The rates of oxygen consumption and mitochondrial oxidation-reduction changes in response to glucose and metabolic modulators were similar to isolated rat islets. In addition, the dynamic insulin secretory patterns of IPIs were similar to primary rat islets. Thus, INS832/3-derived IPIs provide a valuable and convenient model for accelerating islet and diabetes research.

1258-P: Il-1ß-Induced Transcription of Nf-kB Target Genes Is Repressed by Bet Bromodomain Inhibitors in Pancreatic ß-Cells

Autoimmune diabetes is characterized by chronic inflammation of pancreatic islets which ultimately destroys insulin-producing β-cells. In the islet, elevated proinflammatory cytokines lead to increased transcription of inducible nitric oxide synthase (iNOS) and its downstream product, nitric oxide (NO). Elevated levels of NO for prolonged periods lead to β-cell apoptosis. The exact causes of autoimmune diabetes are not well defined and, although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins indicates a role for epigenetic factors. Recent studies suggest that the bromodomain and extraterminal domain (BET) family of epigenetic regulatory proteins contribute to the onset and progression of autoimmune diabetes. BET proteins are epigenetic readers of sites of lysine acetylation found commonly on histones and have emerged as promising drug targets for a wide variety of diseases. We hypothesize that BET proteins increase proinflammatory cytokine-induced transcription in β-cells leading to β-cell damage and that selective inhibition of BET bromodomains with small molecules will prevent or reverse the progression of autoimmune diabetes. Here, we show that BET bromodomain inhibitors decrease the β-cell response to proinflammatory cytokines in an insulinoma β-cell line (INS832/13) and primary rat islets. BET inhibitors attenuated cytokine-induced transcription of inflammatory mediators, including iNOS. This decreased transcription is mediated through inhibition of the NF-κB signaling pathway by BET inhibitors. Using knockdown of individual BET proteins in a β-cell line, we identified the importance of individual BET family members in the altered transcriptional profile characteristic of autoimmune diabetes. This work uncovers important mechanisms of disease onset and progression of autoimmune diabetes, laying the groundwork for more targeted treatments with drug-like small molecules. Disclosure J. Nord: None. S. Wynia-smith: None. B. C. Smith: None. R. Jones lipinski: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institutes of Health (R01DK119359, T32HL134643)

BET bromodomain inhibitors diminish IL‐1B‐induced transcription of NF‐κB target genes

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of pancreatic β-cells, thought to be a result of chronic inflammation of pancreatic islets. Elevated proinflammatory cytokine levels in the islet results in increased nitric oxide production through increased transcription of inducible nitric oxide synthase (iNOS), reduced glucose responsiveness, and ultimately selective destruction of insulin-producing β-cells. The exact causes of T1D are poorly understood and although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins suggests an, as yet undetermined role for epigenetic factors. Recent studies have shown that epigenetic regulatory proteins contribute to the onset and progression of autoimmune diabetes. The bromodomain and extraterminal domain (BET) family of proteins (Brd2, Brd3, Brd4 and BrdT) have emerged as promising drug targets for a wide variety of diseases. Bromodomain-containing proteins are epigenetic “readers” of histone acetylation and we hypothesize they regulate proinflammatory cytokine induced transcription in β-cells. Uniquely, the BET family proteins contain two bromodomains (BD1 and BD2), which affords BET proteins the capacity to simultaneously bind two acetylated histones and/or other nuclear proteins such as DNA-binding factors to modulate transcription in a cell-type specific manner. Here, we show that BET bromodomain inhibitors decrease the β-cell response to proinflammatory cytokines in insulinoma β-cell line (INS832/13) and primary rat islets. We found that BET inhibitors mitigated cytokine-induced transcription of inflammatory mediators, including iNOS, which is known to damage β-cells. This decrease is mediated through inhibition of the NF-κB pathway by BET inhibitors. Furthermore, using knockdown of BET proteins in insulinoma β-cell lines, we identified key mechanisms through which this altered transcriptional profile occurs. This work provides important mechanistic insights into the disease onset and progression of autoimmune diabetes for which current therapies are ineffective.

Lin28a ameliorates glucotoxicity-induced β-cell dysfunction and apoptosis

An excessive and prolonged increase in glucose levels causes β-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicity-induced β-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced β-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in β-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirus-mediated Lin28a overexpression in β-cells reversed the glucotoxicity-induced reduction of insulin secretion and insulin mRNA expression via regulation of β-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic β-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway.

TRAIL induces proliferation in rodent pancreatic beta cells via AKT activation.

Strategies to increase functional pancreatic beta cell mass is of great interest in diabetes-related research. TNF-related apoptosis-inducing ligand (TRAIL) is well known to promote proliferation and survival in various cell types, including vascular smooth muscle and endothelial cells. Correlation between the protective nature of TRAIL on these cells and its proliferative effect is noteworthy. TRAIL's seemingly protective/therapeutic effect in diabetes prompted us to question whether it may act as an inducer of proliferation in pancreatic beta cells. We used rat primary islet cells and MIN6 mouse beta cell line to investigate TRAIL-induced proliferation. Cell viability and/or death was analyzed by MTT, WST-1, and Annexin-V/PI assays, while proliferation rates and pathways were assessed via immunocytochemical and Western blot analyses. Receptor neutralization antibodies identified the mediator receptors. Recombinant soluble TRAIL (sTRAIL) treatment led to 1.6-fold increased proliferation in insulin-positive cells in dispersed rat islets compared to the untreated group, while adenovirus-mediated overexpression of TRAIL increased the number of proliferating beta cells up to more than six-fold. sTRAIL or adenoviral vector-mediated TRAIL overexpression induced proliferation in MIN6 cells also. TRAIL's proliferative effect was mediated via AKT activation, which was suppressed upon specific inhibition. Neutralization of each TRAIL receptor reversed the proliferative effect to some degree, with the highest level of inhibition in death receptor 5 (DR5) blockage in MIN6 cells and in decoy receptor 1 (DcR1) blockage in primary rat beta cells. Thus, TRAIL induces proliferation in rodent pancreatic beta cells through activation of the AKT pathway.

Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion

Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine+ 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln+ BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln+ BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.

2117-P: BET Bromodomain Inhibitors Mitigate Cytokine-Induced Transcription in ß-Cells via Inhibition of Nf-κB

Type 1 diabetes is an autoimmune disease characterized by chronic inflammation of pancreatic islets, mediated by proinflammatory cytokines, leading to reduced response to glucose and ultimately selective destruction of insulin-producing β-cells. In common with many autoimmune diseases, the exact causes are poorly understood. Although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins suggest an, as yet undetermined, contribution of epigenetic factors. Recent studies have begun to explore the contributions of epigenetic regulatory proteins in onset and progression of autoimmune diabetes. An important class of epigenetic “readers” is the bromodomain and extraterminal domain (BET) family (Brd2, Brd3, Brd4, and Brdt), which has emerged as promising drug targets for a wide variety of diseases. A unique trait of the BET family is that these proteins contain two bromodomains (BD1 and BD2) that afford them the capacity to simultaneously bind two acetylated histones and/or non-histone proteins such as DNA-binding factors to modulate transcription in a cell-type specific manner. Here, we use an insulinoma β-cell line (INS832/13) and primary rat islets to investigate the ability of chemical inhibitors of BET bromodomain proteins to decrease the β-cell response to proinflammatory cytokines. We found that BET inhibitors mitigated cytokine-induced transcription of inflammatory mediators known to damage β-cells. This is mediated through the inhibition of the NF-κB pathway by BET inhibitors. Furthermore, using CRISPR/Cas9 knockout insulinoma β-cell lines of Brd2, Brd3, and Brd4 and BD1 vs. BD2 specific chemical inhibitors, we identified key mechanisms through which this altered transcriptional profile occurs. This work provides important mechanistic insights to disease onset and progression of autoimmune diabetes for which current therapies are ineffective. Disclosure J. Nord: None. S. Wynia-Smith: None. B.C. Smith: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK119359)

2131-P: BET Bromodomain Inhibition Upregulates SIRT1 In Pancreatic ß-Cells

Diabetes develops as a result of both genetic and environmental influences; however, low concordance rates of type 1 diabetes between monozygotic twins and increased penetrance of type 2 diabetes during aging suggests an, as yet, poorly understood epigenetic role in diabetes development and progression. The epigenetic code, of which lysine acetylation (Kac) is a major histone post-translational modification, regulates the structure and dynamics of chromatin leading to epigenetically-controlled changes in gene accessibility toward transcription factors. Histone acetylation is regulated by histone acetyltransferases and deacetylases which write and erase sites of histone acetylation, respectively. SIRT1 is a key protein deacetylase in the control of glucose homeostasis. Decreased SIRT1 can lead to impaired insulin signaling, therefore drugs that enhance either SIRT1 expression or enzymatic activity may treat insulin resistance. Bromodomains are epigenetic readers that interpret the complex pattern of histone acetylation by binding Kac residues on histones and other nuclear proteins. Of particular interest are the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins, which have recently emerged as a potential target to treat or prevent diabetes. Interestingly, we recently found that treatment of β-cells with BET bromodomain inhibitors leads to increased SIRT1 expression levels, although the underlying mechanism was unclear. Here, we use an insulinoma β-cell line (INS832/13) and primary rat islets to determine the mechanism through which BET proteins suppress SIRT1 expression. To modulate SIRT1 and BET protein levels and activity, we used a combination of small molecules that either inhibit or degrade BET proteins, in conjunction with CRISPR/Cas9 knockout β-cell lines of both SIRT1 and the individual BET proteins. In summary, BET inhibitors may provide an indirect means to increase SIRT1 activity in β-cells and improve β-cell function in the context of diabetes. Disclosure R.A. Jones Lipinski: None. S. Wynia-Smith: None. J. Nord: None. B.C. Smith: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK119359)

CXXC4 mediates glucose-induced β-cell proliferation.

CXXC finger protein 4 (CXXC4) is an identified negative regulator of the Wnt/β-catenin pathway, and it is involved in cancer cell proliferation. In this study, we sought to clarify whether CXXC4 is involved in glucose-stimulated β-cell proliferation. We investigated the biological function of CXXC4 in glucose-induced β-cell proliferation, and we investigated the underlying mechanism of this activity. First, we analyzed CXXC4 expression in established rat models treated for 24h with a high glucose infusion and in INS-1 cells and primary rat islets treated with different concentrations of glucose. Subsequently, we used an adenovirus to overexpress CXXC4 in INS-1 cells and primary islets. The proliferation rate of β-cells was evaluated by CCK-8 and EdU incorporation methods. Cell cycle analysis was performed by flow cytometry. Finally, the Wnt signaling pathway and its downstream genes were assessed by Western blot. CXXC4 mRNA levels were significantly lower in islets isolated from glucose-infused rats than they were in those isolated from saline-infused rats. Decreased expression of CXXC4 also correlated with high glucose treatment of INS-1 cells and primary rat β-cells. Furthermore, adenovirus-mediated overexpression of CXXC4 inhibited cell proliferation induced by the high glucose treatment in vitro, which was mechanistically mediated by Wnt signaling and a decrease in cyclin D2 expression. Glucose inhibits CXXC4 expression and hence promotes pancreatic β-cell proliferation. Our findings may provide a new potential target for the treatment of diabetes.

Why Does Nkx6.1 Fail to Increase Functional Beta‐cell Mass in Aged Islets?

Diabetes Mellitus is a major health problem across the world. The number of people diagnosed is predicted to reach 642 million by 2040. The major forms of diabetes mellitus, type 1 and type 2, have the shared characteristics of hyperglycemia, impaired insulin production, and decreased beta‐cell mass. A possible cure for diabetes mellitus is transplantation of the beta‐cell containing islets of Langerhans. A major constraint to greater use of islet transplantation is the relative scarcity of transplant ready islets. Understanding that molecular pathways that result in increased beta‐cell proliferation could be used to increasing the number of transplant ready islets. We have shown that the beta‐cell transcription factor Nkx6.1 is sufficient to enhance glucose stimulated insulin secretion and induce beta‐cell proliferation in primary rat islets. However, Nkx6.1 overexpression in aged rat islets is unable to drive expression of key target genes that result in increased glucose stimulated insulin secretion and proliferation. This dichotomy is extremely important, given that most islet donors are beyond adolescence. Therefore, understanding why aged beta‐cells are refractory to these Nkx6.1 mediated signals is essential. We hypothesize that Nkx6.1 is unable to drive expression of genes necessary for beta‐cell proliferation and insulin secretion in aged islets due to lack of accessibility to chromatin due to changes in Nkx6.1 binding partner expression. Here we present data demonstrating the difference in Nkx6.1 promoter localization and binding partners as a process of aging. These data begin to describe age dependent changes that leave the aged beta‐cell refractory to proliferative signals.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of β-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic β-cell mass and failure of the remaining β-cells to deliver sufficient insulin to meet demand. β-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on β-cell function and survival, contributes to T2D-associated β-cell failure. Drugs and mechanisms that protect β-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected β-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured β-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve β-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.