INS-1 Β-cells Research Articles

314-OR: Synaptotagmin-9 Regulates Early Phase Insulin Secretion by Regulating Tomosyn-1

Insulin secretion from pancreatic β-cells occurs in a biphasic manner. The loss of insulin secretion, entailing the inability of insulin granules to fuse with the plasma membrane (PM) via the formation of the SNARE complex, is a key determinant in impaired glucose tolerance and development of type 2 diabetes (T2D) . Synaptotagmins (Syt) are calcium sensors that regulate insulin-containing granule fusion to the PM for insulin secretion. Syt9 is highly expressed in β-cells; however, its role in insulin secretion is not well-characterized. Our goal is to determine how Syt9 affects insulin secretion from pancreatic β-cells. Mice lacking Syt9 (Syt9-/-) have improved glucose clearance with no change in insulin action than wild-type control mice. Interestingly, Syt9-/- mice showed increased plasma insulin levels at 5 min and 15 min post-glucose challenge. Similarly, islets isolated from Syt9-/- mice demonstrate increased stimulation of insulin secretion in response to the early phase insulin secretagogues (KCl and tolbutamide) compared to WT control islets. Similarly, siRNA-mediated knockdown of Syt9 in INS1 cells increased KCL-stimulated insulin secretion at 5 min and min. Altogether, these data suggest that Syt9 regulates the early phase of insulin secretion from β-cells. Strikingly, Syt9-/- mouse islets have reduced Tomosyn-1 levels (∼50%) without altering the levels of other key SNARE proteins (Stx1A, Stx4A, SNAP25) . Tomosyn-1, an inhibitor of insulin secretion whose increased protein abundance is associated with reduced insulin secretion, binds, cofractionates, and colocalizes with Syt9 and Stx1A. The knockdown of Tomosyn-1 in INS1 β-cells treated with KCL prevents the localization of Syt9 with Stx1A. Further, the knockdown of Syt9 or Tomosyn-1 increases the SNARE complex formation. In sum, these data demonstrate the identification of a novel Stx1A-Tomosyn-1-Syt9 molecular complex that regulates the fusion of insulin granules in the early phase. Disclosure M.Rahman: None. H.Alsharif: n/a. S.Bhatnagar: None. Funding R01DK120684-01

Pancreatic INS-1 β-Cell Response to Thapsigargin and Rotenone: A Comparative Proteomics Analysis Uncovers Key Pathways of β-Cell Dysfunction.

Insulin-secreting β-cells in the pancreatic islets are exposed to various endogenous and exogenous stressing conditions, which may lead to β-cell dysfunction or apoptosis and ultimately to diabetes mellitus. However, the detailed molecular mechanisms underlying β-cell's inability to survive under severe stresses remain to be explored. This study used two common chemical stressors, thapsigargin and rotenone, to induce endoplasmic reticulum (ER) and mitochondria stress in a rat insuloma INS-1 832/13 β-cell line, mimicking the conditions experienced by dysfunctional β-cells. Proteomic changes of cells upon treatment with stressors at IC50 were profiled with TMT-based quantitative proteomics and further verified using label-free quantitive proteomics. The differentially expressed proteins under stress conditions were selected for in-depth bioinformatic analysis. Thapsigargin treatment specifically perturbed unfolded protein response (UPR) related pathways; in addition, 58 proteins not previously linked to the UPR related pathways were identified with consistent upregulation under stress induced by thapsigargin. Conversely, rotenone treatment resulted in significant proteome changes in key mitochondria regulatory pathways such as fatty acid β-oxidation, cellular respiration, citric acid cycle, and respiratory electron transport. Our data also demonstrated that both stressors increased reactive oxygen species production and depleted adenosine triphosphate synthesis, resulting in significant dysregulation of oxidative phosphorylation signaling pathways. These novel dysregulated proteins may suggest an alternative mechanism of action in β-cell dysfunction and provide potential targets for probing ER- and mitochondria stress-induced β-cell death.

Large-Scale Production of Size-Adjusted β-Cell Spheroids in a Fully Controlled Stirred-Tank Reactor

For β-cell replacement therapies, one challenge is the manufacturing of enough β-cells (Edmonton protocol for islet transplantation requires 0.5–1 × 106 islet equivalents). To maintain their functionality, β-cells should be manufactured as 3D constructs, known as spheroids. In this study, we investigated whether β-cell spheroid manufacturing can be addressed by a stirred-tank bioreactor (STR) process. STRs are fully controlled bioreactor systems, which allow the establishment of robust, larger-scale manufacturing processes. Using the INS-1 β-cell line as a model for process development, we investigated the dynamic agglomeration of β-cells to determine minimal seeding densities, spheroid strength, and the influence of turbulent shear stress. We established a correlation to exploit shear forces within the turbulent flow regime, in order to generate spheroids of a defined size, and to predict the spheroid size in an STR by using the determined spheroid strength. Finally, we transferred the dynamic agglomeration process from shaking flasks to a fully controlled and monitored STR, and tested the influence of three different stirrer types on spheroid formation. We achieved the shear stress-guided production of up to 22 × 106 ± 2 × 106 viable and functional β-cell spheroids per liter of culture medium, which is sufficient for β-cell therapy applications.

Carnosic Acid Protects INS-1 β-Cells against Streptozotocin-Induced Damage by Inhibiting Apoptosis and Improving Insulin Secretion and Glucose Uptake.

Carnosic acid (CA), a natural polyphenolic diterpene derived from Rosmarinus officinalis, has been proven to possess a broad spectrum of medicinal properties. Nevertheless, no studies on its impact on pancreatic β-cells have been conducted to date. Herein, clonal rat INS-1 (832/13) cells were pretreated with CA for 24 h and then incubated with streptozotocin (STZ) for 3 h. Several functional experiments were performed to determine the effect of CA on STZ-induced pancreatic β-cell damage, including cell viability assay, apoptosis analysis, and measurement of the level of insulin secretion, glucose uptake, malondialdehyde (MDA), reactive oxygen species (ROS), and proteins expression. STZ treatment decreased cell survival, insulin secretion, glucose uptake, and increased apoptosis, MDA, and ROS production in INS-1 cells. Furthermore, protein expression/phosphorylation analysis showed significant down-regulation in insulin, PDX-1, PI3K, AKT/p-AKT, and Bcl2. On the other hand, expression of BAX and BAD and cleaved PARP were significantly increased. Interestingly, preincubation with CA reversed the adverse impact of STZ at the cellular and protein expression levels. In conclusion, the data indicate that CA protects β-cells against STZ-induced damage, presumably through its modulatory effect on the different pathways, including the Pi3K/AKT/PDX-1/insulin pathway and mitochondria-mediated apoptosis.

In Vitro Evaluation of Nanoerythrosome Cytotoxicity and Uptake in Pancreatic Endothelial Cells: Implications for β-Cell Imaging Applications

Biomolecule-targeted imaging represents one of the most difficult challenges in medicine. Nanoerythrosomes (NERs) are nanovesicles obtained after lysis of red blood cells, and they are promising tools for drug delivery and imaging. In this work, a formulation based on NERs functionalized with 7-amino-3-methylcoumarin via cross-linking was tested on rat INS-1E and mouse MIN6 β-cells and endothelial MSI cell lines. First, the morphology, size, ζ-potentials, and spectroscopic properties of the aggregates were investigated, highlighting that the functionalization did not significantly affect the nanoparticles' physicochemical features. In vitro, the nanoparticles did not significantly affect the proliferation and function of INS-1E and MIN6 β-cells at different concentrations. Only at the highest concentration tested on the MSI cell line, the formulation inhibited proliferation. Furthermore, NER aggregates were not internalized in both INS-1E and MIN6 cell lines, while a diffuse fluorescence was noticed in the cytosol of the MSI cell line at the highest concentrations. These findings proved that NER formulations might represent a new nanotool for β-cell imaging as a part of a strategy aimed to prevent any intracellular accumulation, thus reducing/avoiding side effects.

Circular RNA PIP5K1A act as microRNA-552-3p sponge to regulates inflammation, oxidative damage in glucolipotoxicity-induced pancreatic INS-1 β-cells via Janus kinase 1

ABSTRACT Elevated level of glucolipotoxicity induces the loss of pancreatic β-cells functions and plays an important role in the development of type 2 diabetes (T2DM). Previous studies have indicated the importance of developing therapies against T2DM, while circular RNA (circRNA) has gained attraction as a modulator of pancreatic β-cell function. In the present study role of circPIP5K1A in dysfunctional β cells and mouse pancreas was comprehensively analyzed. INS-1E, as it has close similarity with naïve pancreatic β-cells, and clinical samples of T2DM patients were used to investigate the effect of circPIP5K1A, miR-552-3p, and Janus kinase 1 (JAK1). While, INS-1E cells were exposed to PAHG conditions (0.5 mM palmitic acid and 28 mM glucose) as studies have suggested that increased level of fatty acid and glucose resulted in autophagy activation of pancreatic β-cells that leads to T2DM. Key player of JAK1-STAT3 pathway and the level of Reactive Oxygen Species, inflammatory factors, and insulin secretion was detected to analyze the of the active association of circPIP5K1A, miR-552-3p with JAK1pathway. Our study has revealed the elevated level ofcircPIP5K1A and JAK1, but reduced level of miR-552-3pin the serum of T2DM patients. Furthermore, we also found that reduced expression ofcircPIP5K1A leads to decreased rate of inflammation, oxidative damage and apoptosisinINS-1E cells induced by glucolipotoxicity. CircPIP5K1A was available to competitively combine with miR-552-3p, while whose direct target was JAK1. In conclusion, our study suggested a novel involvement of circPIP5K1A in a cross talk between miR5523p/JAK1/STAT3 pathways in β-cells as a new therapeutic target for T2DM.

Pleiotrophin Expression and Actions in Pancreatic β-Cells.

Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic β-cells in young vs. adult animals, and its effects on DNA synthesis, β-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse β-cells, especially within immature β-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-β/ζ, were also expressed in the proliferative INS1E β-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with β-cells. The percentage of β-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during β-cell regeneration in young mice following depletion of β-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse β-cells in young and adult life and could potentially contribute to adaptive increases in β-cell mass during early life or pregnancy.

ATP synthase inhibitory factor subunit 1 regulates islet β-cell function via repression of mitochondrial homeostasis

AbstractMitochondrial homeostasis is crucial for the function of pancreatic β-cells. ATP synthase inhibitory factor subunit 1 (IF1) is a mitochondrial protein interacting with ATP synthase to inhibit its enzyme activity. IF1 may also play a role in maintaining ATP synthase oligomerization and mitochondrial inner membrane formation. A recent study confirmed IF1 expresses in β-cells. IF1 knockdown in cultured INS-1E β-cells enhances glucose-induced insulin release. However, the role of IF1 in islet β-cells remains little known. The present study investigates islets freshly isolated from mouse lines with global IF1 knockout (IF1−/−) and overexpression (OE). The glucose-stimulated insulin secretion was increased in islets from IF1−/− mice but decreased in islets from IF1 OE mice. Transmitted Electronic Microscopic assessment of isolated islets revealed that the number of matured insulin granules (with dense core) was relatively higher in IF1−/−, but fewer in IF1 OE islets than those of controlled islets. The mitochondrial ultrastructure within β-cells of IF1 overexpressed islets was comparable with those of wild-type mice, whereas those in IF1−/− β-cells showed increased mitochondrial mass. Mitochondrial network analysis in cultured INS-1 β-cells showed a similar pattern with an increased mitochondrial network in IF1 knockdown cells. IF1 overexpressed INS-1 β-cells showed a compromised rate of mitochondrial oxidative phosphorylation with attenuated cellular ATP content. In contrast, INS-1 cells with IF1 knockdown showed markedly increased cellular respiration with improved ATP production. These results support that IF1 is a negative regulator of insulin production and secretion via inhibiting mitochondrial mass and respiration in β-cells. Therefore, inhibiting IF1 to improve β-cell function in patients can be a novel therapeutic strategy to treat diabetes.

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1.

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca2+-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca2+ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca2+ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca2+ entry were investigated in Fura-2 Ca2+-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca2+ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S1172A mutant displayed enhanced Ca2+ entry. The TRPM3-mediated Ca2+ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.

Cathepsin C Regulates Cytokine-Induced Apoptosis in β-Cell Model Systems.

Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic β cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates β-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in β-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E β-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of β-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated β-cell destruction in T1D.

SetD7 (Set7/9) is a novel target of PPARγ that promotes the adaptive pancreatic β-cell glycemic response

Loss of functional pancreatic β-cell mass leads to type 2 diabetes (T2D), attributable to modified β-cell-dependent adaptive gene expression patterns. SetD7 is a histone methyltransferase enriched in pancreatic islets that mono- and dimethylates histone-3-lysine-4 (H3K4), promoting euchromatin modifications, and also maintains the regulation of key β-cell function and survival genes. However, the transcriptional regulation of this important epigenetic modifier is unresolved. Here we identified the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPARγ) as a major transcriptional regulator of SetD7 and provide evidence for direct binding and functionality of PPARγ in the SetD7 promoter region. Furthermore, constitutive shRNA-mediated PPARγ knockdown in INS-1 β-cells or pancreas-specific PPARγ deletion in mice led to downregulation of SetD7 expression as well as its nuclear enrichment. The relevance of the SetD7-PPARγ interaction in β-cell adaptation was tested in normoglycemic 60% partial pancreatectomy (Px) and hyperglycemic 90% Px rat models. Whereas a synergistic increase in islet PPARγ and SetD7 expression was observed upon glycemic adaptation post-60% Px, in hyperglycemic 90% Px rats, islet PPARγ, and PPARγ targets SetD7 and Pdx1 were downregulated. PPARγ agonist pioglitazone treatment in 90% Px rats partially restored glucose homeostasis and β-cell mass and enhanced expression of SetD7 and Pdx1. Collectively, these data provide evidence that the SetD7-PPARγ interaction serves as an important element of the adaptive β-cell response.

The role of mitochondrial apoptotic pathway in islet amyloid-induced β-cell death

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to β-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated β-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet β-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in β-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak βDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic β-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced β-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced β-cell death and a potential strategy to reduce intracellular amyloid β-cell toxicity by blocking cytochrome c apoptotic function.

TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell.

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs β-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 β-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the β-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving β-cell survival.

294-OR: RIPK3-Mediated Necroptosis Is an Alternative Form of TNFa-Induced ß-Cell Death

Necroptosis is an inflammatory and immunogenic form of lytic cell death that can occur downstream of TNFα signaling. Receptor interacting protein kinase 3 (RIPK3) mediates TNFα-induced necroptosis when caspase activity is repressed, but this pathway has not been examined in insulin-producing β-cells. As TNFα contributes to β-cell death in the setting of diabetes and since we found RIPK3 is expressed in INS-1 β-cells and mouse islets, we hypothesized that β-cells are susceptible to necroptosis. To test this hypothesis, INS-1 cells were treated with TNFα (40 ng/ml) in the absence or presence of a synthetic pan-caspase inhibitor (zVAD, 50 μM) for 24 h, cell death was monitored in real-time, and caspase activity was determined. TNFα increased INS-1 cell death (1.63 ± 0.13 vs. 2.44 ± 0.13 fold, n=7, p<0.001) and caspase activity, consistent with apoptosis. When caspases were inhibited with zVAD, TNFα no longer increased caspase activity, but induced a similar degree of cell death (1.63 ± .013 vs. 2.82 ± 0.23 fold, n=7, p<0.001), suggesting an alternate mechanism under these conditions. Moreover, when TNFα-induced cell death was amplified by the SMAC/DIABLO mimetic BV6 (5 μM), we again observed similar levels of cell death with or without zVAD. To further investigate the mechanism of cell death under caspase inhibition, we used the RIPK3 inhibitor GSK ‘872 (5 μM). GSK ‘872 had no effect on TNFα+BV6-induced cell death when caspases were activated (3.35 ± 0.61 vs. 3.48 ± 1.21 fold, n=3, p=0.93), but reduced cell death when caspases were inhibited (4.69 ± 0.57 vs. 1.81 ± 0.57 fold, n=3, p=0.04). These data indicate that TNFα induces β-cell death independent of caspase activity level. However, the mechanism by which TNFα elicits β-cell death does depend on caspase activity, with apoptosis occurring when caspases are activated, and RIPK3-mediated necroptosis occurring when caspases are inhibited. Additional studies are required to understand the factors that may promote β-cell necroptosis in the setting of human diabetes. Disclosure C. J. Contreras: None. L. Lin: None. M. F. Hogan: None. A. Oberst: None. S. E. Kahn: Advisory Panel; Self; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk, Pfizer Inc. A. T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659, I01BX001060)

1256-P: Insights into Acetyl-Coa-Carboxylase (ACC1) Phospho-Regulation in Pancreatic Beta Cells

Aims: ACC1 couples glucose metabolism with de novo lipogenesis in lipogenic tissues, by converting acetyl-CoA to malonyl-CoA. In β-cells, ACC1 plays a critical role in β-cell growth and insulin secretion. However, very little is known about ACC1 regulation in β-cell. The aims of this study were to 1) assess the role of ACC1S79 in β-cell function and whole body glucose homeostasis; and 2) screen for novel phosphosites that may regulate ACC1 activity in β-cell. Methods: 1) ACC1 single knock-in mouse model, with ACC1S79 mutated to alanine ACC1S79A, was obtained by crossing ACC1 double knock-in mice with wild type mice to isolate the ACC1S79A point mutation. 2) ACC1 protein was purified from INS1 β-cell following culture with different glucose concentrations. Quantitative phosphoproteomics was performed to characterise ACC1 phosphosites using a Q-Executive MS. Data analysed using MaxQuant and Perseus. Results: 1) Female ACC1S79A mice exhibited no alternations in glucose homeostasis. In male mice we found no differences in serum insulin, GTT or ITT, although a small but significant reduction in fasting blood glucose was recorded in ACC1S79A mice compared to littermate controls. There was no significant change in GSIS from isolated ACC1S79A islets at 2mM glucose or in response to 7.5 and 20mM glucose, suggesting that ACC1S79 phosphorylation does not play a role in insulin secretion under physiological conditions.2) Using phosphoproteomics, we identified twenty phosphosites on ACC1 protein in β-cells. ACC1S1215 was highly phosphorylated at 2mM glucose, and showed a significant reduction in phosphorylation in response to 15mM glucose. In contrast, phosphorylation of ACC1S25 was lower at basal glucose and increased upon glucose stimulation. Conclusions: Our data show that ACC1S79 phosphorylation does not play a major role as a glucose-regulated phosphosite in β-cells. However, other phosphosites show greater dynamic regulation by glucose and may play a role in regulating β-cell ACC1 activity. Disclosure R. Bany bakar: None. S. Liberatori: Employee; Self; LONZA. A. Veprik: None. N. N. Tebeka: None. B. E. Kemp: None. S. Mohammed: None. J. Cantley: Other Relationship; Self; Oxstem Beta.

125-OR: Synaptotagmin 9 Regulates Tomosyn-2 Protein Abundance to Affect Early Phase of Insulin Secretion

Insulin secretion from pancreatic β-cells occurs in a biphasic manner. The loss of the early phase of insulin secretion, which occurs due to the inability of insulin granules to fuse with the plasma membrane (PM) via the formation of the SNARE complex, acts as a key determinant in impaired glucose tolerance and development of type 2 diabetes (T2D). Thus, it is critical to elucidate molecular mechanisms underlying early-phase insulin secretion. Tomosyn-2 binds to Stx1A, a key component of the SNARE complex, and functions as an endogenous inhibitor of insulin secretion. The mechanism by which Tomosyn-2 regulates the fusion of insulin granules is not completely understood. Our preliminary data show that Tomosyn-2 binds, colocalizes, and co-fractionates with synaptotagmin-9 (Syt9) at high glucose; Syt9 protein is present in the insulin granule membrane, and its role in insulin secretion remains poorly defined. Our data show that mice lacking Syt9 (Syt9-/-) have improved glucose clearance, increased in vivo early-phase insulin secretion at 5 min and 15 min post-glucose challenge with no change in insulin tolerance vs. the wild type control mice, suggesting that Syt9 regulates the early phase of insulin secretion from β-cells. Strikingly, Syt9-/- mouse islets have reduced Tomosyn-2 levels (∼50%) without altering the levels of other key SNARE proteins. The knockdown of Tomosyn in INS1 β-cells treated with KCL prevents the localization of Syt9 with Stx1A. These results show that Syt9 binding stabilizes Tomosyn-2 protein in a non-fusogenic complex by potentially blocking insulin granules’ access to the PM-Stx1A for membrane fusion and insulin secretion. In summary, our data reveal a novel molecular complex in Stx1A-Tomosyn-2-Syt-9 to regulate the early-phase insulin secretion. Disclosure A. Pathak: None. H. Alsharif: None. J. E. Trombley: None. M. Rahman: None. S. Bhatnagar: None. Funding National Institutes of Health (R01DK120684-01)

Cellulose-based scaffolds enhance pseudoislets formation and functionality

In vitro research for the study of type 2 diabetes (T2D) is frequently limited by the availability of a functional model for islets of Langerhans. To overcome the limitations of obtaining pancreatic islets from different sources, such as animal models or human donors, immortalized cell lines as the insulin-producing INS1E β-cells have appeared as a valid alternative to model insulin-related diseases. However, immortalized cell lines are mainly used in flat surfaces or monolayer distributions, not resembling the spheroid-like architecture of the pancreatic islets. To generate islet-like structures, the use of scaffolds appeared as a valid tool to promote cell aggregations. Traditionally-used hydrogel encapsulation methods do not accomplish all the requisites for pancreatic tissue engineering, as its poor nutrient and oxygen diffusion induces cell death. Here, we use cryogelation technology to develop a more resemblance scaffold with the mechanical and physical properties needed to engineer pancreatic tissue. This study shows that carboxymethyl cellulose (CMC) cryogels prompted cells to generate β-cell clusters in comparison to gelatin-based scaffolds, that did not induce this cell organization. Moreover, the high porosity achieved with CMC cryogels allowed us to create specific range pseudoislets. Pseudoislets formed within CMC-scaffolds showed cell viability for up to 7 d and a better response to glucose over conventional monolayer cultures. Overall, our results demonstrate that CMC-scaffolds can be used to control the organization and function of insulin-producing β-cells, representing a suitable technique to generate β-cell clusters to study pancreatic islet function.

Candidate plasticity gene 16 and jun dimerization protein 2 are involved in the suppression of insulin gene expression in rat pancreatic INS-1 β-cells

Chronic hyperglycemia causes pancreatic β-cell dysfunction, impaired insulin secretion and the suppression of insulin gene expression. This phenomenon is referred to as glucotoxicity, and is a critical component of the pathogenesis of type 2 diabetes. We previously reported that the expression of candidate plasticity gene 16 (CPG16) was higher in rat pancreatic INS-1 β-cells under glucotoxic conditions and CPG16 suppressed insulin promoter activity. However, the molecular mechanisms of the CPG16-mediated suppression of insulin gene expression are unclear. In this study, we found that CPG16 directly bound and phosphorylated jun dimerization protein 2 (JDP2), an AP-1 family transcription factor. CPG16 co-localized with JDP2 in the nucleus of INS-1 cells. JDP2 bound to the G1 element of the insulin promoter and up-regulated promoter activity. Finally, CPG16 suppressed the up-regulation of insulin promoter activity by JDP2 in a kinase activity-dependent manner. These results suggest that CPG16 suppresses insulin promoter activity by phosphorylating JDP2.

68722 Role of ER calcium in beta cell senescence and diabetes pathophysiology

ABSTRACT IMPACT: The proposed study has the potential to inform new paradigms of type 1 diabetes prevention and therapy with the overall goal of improving β cell health during autoimmunity. OBJECTIVES/GOALS: Type 1 diabetes (T1D) results from immune-mediated destruction of pancreatic βcells. Recent data suggest that activation of senescence and acquisition of a senescence associated secretory phenotype (SASP) by βcells may contribute to T1D pathogenesis. However, the molecular mechanisms responsible for this phenotype are not well understood. METHODS/STUDY POPULATION: We hypothesize that loss of endoplasmic reticulum (ER) Ca2+ induces βcell senescence, SASP as well as mitochondrial dysfunction which drive T1D development. The current study utilizes SERCA2 KO INS-1 βcells (S2KO) exhibiting loss of ER Ca2+ and a SERCA2 haploinsufficient mice on a non-obese diabetic background (NOD-S2+/-) to test the role of ER Ca2+ loss during T1D development. Senescence associated βgalactosidase staining (SA-βgal), expression of senescence markers (RT-qPCR), mitochondrial function (Seahorse, TMRM) and mitochondrial copy number (qPCR) were all measured in S2KO versus WT βcells and are currently being measured in the NOD-S2+/- mouse model at 6, 8, 12, 14, and 16wks of age. RESULTS/ANTICIPATED RESULTS: RT-qPCR assays detecting senescence markers cdkn1a and cdkn2a and mitochondrial specific genes cox1 and nd1 were developed and validated in both INS-1 βcells and mouse islets. Mitochondrial function assay (Seahorse) was optimized for use in INS-1 βcells and is currently under development for use in intact mouse islets. S2KO βcells displayed increased SA- βgal staining as well as increased mitochondrial coupling efficiency (p=0.0146) and baseline mitochondrial copy number (p=0.0053) compared to WT βcells, suggesting a senescence phenotype and altered mitochondrial function. NOD-S2+/- mice exhibited increased expression of the senescence marker cdkn2a in the islet at 12wks (p=0.0117) compared to control mice, whereas cdkn1a remained unchanged across all timepoints tested. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results suggest that loss of SERCA2 and reduced ER Ca2+ alter βcell mitochondrial function and are associated with features of senescence. Future studies will test whether SERCA2 activation and/or senolytic/senomorphic drugs are able to prevent or delay diabetes onset in NOD-S2+/- mice.

Discovery of a Novel Series of Pyridone-Based EP3 Antagonists for the Treatment of Type 2 Diabetes.

A novel series of pyridones were discovered as potent EP3 antagonists. Optimization guided by EP3 binding and functional assays as well as by eADME and PK profiling led to multiple compounds with good physical properties, excellent oral bioavailability, and a clean in vitro safety profile. Compound 13 was identified as a lead compound as evidenced by the reversal of sulprostone-induced suppression of glucose-stimulated insulin secretion in INS 1E β-cells in vitro and in a rat ivGTT model in vivo. A glutathione adduction liability was eliminated by replacing the naphthalene of structure 13 with the indazole ring of structure 43.