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A Phenotypic Screen Identifies Calcium Overload As a Key 1032 Diabetes Volume 69, May 2020 A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of b-Cell Glucolipotoxicity Jennifer Vogel,1 Jianning Yin,2 Liansheng Su,2 Sharon X. Wang,2 Richard Zessis,2 Sena Fowler,2 Chun-Hao Chiu,2 Aaron C. Wilson,3 Amy Chen,2 Frederic Zecri,2 Gordon Turner,2 Thomas M. Smith,2 Brian DeChristopher,4 Heming Xing,5 Deborah M. Rothman,6 Xinming Cai,5 and Alina Berdichevsky2 Diabetes 2020;69:1032–1041 | https://doi.org/10.2337/db19-0813 Type 2 diabetes (T2D) is caused by loss of pancreatic leads to an increased demand for insulin as well as direct b-cell mass and failure of the remaining b-cells to deliver effects on the b-cell, such as stress and apoptosis. This concept sufficient insulin to meet demand. b-Cell glucolipotox- of toxic energy excess, termed glucolipotoxicity (GLT), has icity (GLT), which refers to combined, deleterious effects been extensively studied in the last two decades (2,3). of elevated glucose and fatty acid levels on b-cell func- GLT conditions are generally associated with increased tion and survival, contributes to T2D-associated b-cell oxidative and endoplasmic reticulum (ER) stress, calcium b failure. Drugs and mechanisms that protect -cells from dysregulation, and inflammasome activation (4–13). In the GLT stress could potentially improve metabolic control in pancreas, GLT conditions result in increased b-cell death, patients with T2D. In a phenotypic screen seeking low- low insulin content, and reduced glucose-stimulated in- b molecular-weight compounds that protected -cells sulin secretion (GSIS) (14–17). GLT conditions have been from GLT, we identified compound A that selectively shown to lead to reduced insulin gene expression via blocked GLT-induced apoptosis in rat insulinoma cells. dysregulation of its upstream transcription factor PDX-1 Compound A and its optimized analogs also improved (3). Decreased PDX-1 mRNA levels and nuclear exclusion viability and function in primary rat and human islets b under GLT. We discovered that compound A analogs of PDX-1 were observed in -cells under GLT conditions decreased GLT-induced cytosolic calcium influx in islet and in islets from humans and rodents with diabetes – cells, and all measured b-cell–protective effects corre- (18 21). ER stress and ER calcium dysregulation have also b lated with this activity. Further studies revealed that the been implicated in GLT-induced -cell dysfunction (22). PHARMACOLOGY AND THERAPEUTICS fl active compound from this series largely reversed GLT- Free fatty acids can induce calcium in ux from the ER induced global transcriptional changes. Our results sug- in b-cells via activation of Gq-coupled receptors such as gest that taming cytosolic calcium overload in pancreatic G-protein–coupled receptor (GPR) 120, GPR40, and inosi- islets can improve b-cell survival and function under GLT tol 1,4,5-trisphosphate receptor (IP3R). High glucose stim- stress and thus could be an effective strategy for T2D ulation of b-cells results in membrane depolarization, treatment. leading to calcium influx through the voltage-gated L-type calcium channels on the plasma membrane and, as a result, calcium-induced calcium release from the ER via the Diabetes is caused by inability of the pancreas to meet ryanodine receptor and IP3R. The influx of calcium into metabolic demand for insulin due to a shortage of func- the cytosol is necessary for the glucose-stimulated insulin tional insulin-secreting b-cells (1). In type 2 diabetes (T2D), release, but excess stimulation with high levels of glucose elevated levels of circulating glucose and fatty acids con- and fatty acids depletes ER calcium stores, which can also tribute to insulin resistance in peripheral tissues, which contribute to the reduced insulin secretion under GLT (23). 1Gilead, Foster City, CA This article contains Supplementary Data online at https://diabetes.diabetesjournals.org/ 2Novartis Institutes for BioMedical Research, Cambridge, MA lookup/suppl/doi:10.2337/db19-0813/-/DC1. 3 Editas Medicine, Cambridge, MA © 2020 by the American Diabetes Association. Readers may use this article as 4 Rheos Medicines, Cambridge, MA long as the work is properly cited, the use is educational and not for profit, and the 5 fi Sano , Cambridge, MA work is not altered. More information is available at https://www.diabetesjournals 6 Merck & Co., Kenilworth, NJ .org/content/license. Corresponding author: Alina Berdichevsky, [email protected] Received 15 August 2019 and accepted 7 February 2020 diabetes.diabetesjournals.org Vogel and Associates 1033 Moreover, several recent reports support a role for cytosolic assay was performed in INS1E cells as follows. After over- calcium overload in pathogenesis of diabetes. In mice express- night incubation in low-serum culture media (RPMI plus ing a mutant form of Abcc8, a key component of the b-cell 0.5% BSA), cells were preincubated with compounds for 1 h KATP channel showing constant depolarization similar to that and then challenged with BSA-conjugated palmitate observed with nutrient overload, intracellular calcium is con- (800 mmol/L palmitate; palmitic acid [PA]:BSA ratio 6:1) stantly elevated, leading to diabetes (24). Furthermore, mouse (#P9767; Sigma-Aldrich). Apoptosis was measured by islet cells exposed to diabetic serum show hyperactivation of caspase 3/7 Glo (CaspGlo, #G8091; Promega) 24 h after L-type calcium channels (25). the palmitate challenge. Cell viability was measured by To date, there are no efficient therapies protecting patients Cell Titer Glo (#G7570; Promega) 48 h after the palmitate with diabetes from b-cell death and loss of b-cell mass. challenge, according to the manufacturer’s instructions, Glucagon-like peptide 1 (GLP-1) signaling has been reported and luminescence was detected by EnVision Plate Reader to exert direct b-cell–protective effects (26,27). However, (Perkin Elmer). For both CaspGlo and Cell Titer Glo despite many years of research, whether the beneficial effects assays, the percentage reduction and percentage protec- of GLP-1–based therapy on b-cell mass observed in animal tion, respectively, were calculated using relative luciferase studies are applicable to humans is still unclear (28). units(RLU)as[(RLUofDMSOcontrol1 PA 2 RLU of Here we report discovery of a chemical series that compound 1 PA)/RLU of DMSO 1 PA]∗100%. b protects -cells and islets from GLT-induced apoptosis For TNF-related apoptosis-inducing ligand (TRAIL) ap- and dysfunction. Mechanism of action (MOA) studies optosis assays, Jurkat cells (#TIB-152; ATCC) were treated b revealed that this series protects -cells by inhibiting with compounds for 1 h, followed by treatment with fl GLT-induced calcium in ux. Our work suggests a central human recombinant TRAIL protein (100 mmol/L final b role for cytosolic calcium overload in -cell GLT. concentration) (#375-TL-010; R&D Systems), and apo- ptosis was measured as described above using CaspGlo kit RESEARCH DESIGN AND METHODS 24 h after the TRAIL challenge. Cellular Apoptosis and Viability Assays For high-throughput screening, the final BSA-palmitate For more details including cell culture, materials and screen- concentration was 1 mmol/L (PA:BSA ratio was main- ing, please see Supplementary Methods. The GLT apoptosis tained at 6:1). Figure 1—An unbiased chemical screen for b-cell protection from GLT identifies compound (Cpd) A. A: Screen flowchart. For screen design and experimental flow, see the first section of RESULTS, Identification of a Molecule That Selectively Protects b-Cells From GLT-Induced Apoptosis. B: Structure of the screening hit compound A. *Rac, chiral center on the racemic allene compound. 1034 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020 Islet Apoptosis and GSIS Assays 11 mmol/L glucose) with DMSO for 3 and 24 h, and Rat pancreatic islets were isolated from Sprague Dawley two treated with GLT media (27 mmol/L glucose and rats after perfusion with Liberase TL Research Grade 300 mmol/LPA:BSAinRPMI)andDMSO,orGLTmedia (#5401020001; Sigma-Aldrich) as previously described with 300 nmol/L compound D or compound E for 3 and/or (29). For apoptosis evaluation, islets were dispersed to 24 h. This experiment was performed three times with single cells using a papain dissociation kit (#LK003150; different islet preparations to obtain three independent Worthington) and plated in laminin-5–coated plates. After biological repeats. Total RNA was prepared using the 48 h recovery, islet cells were cotreated with GLT media RNeasy kit (Qiagen). RNA quantity and quality were (0.5% fatty acid-free BSA, 500 mmol/L palmitate in RPMI, assessed using an Agilent 2100 Bioanalyzer and 1 mgtotal PA:BSA ratio 6:1, 16 mmol/L glucose) and compounds of RNA was sent to the Beijing Genome Institute for reverse interest for 72 h. For determination of cell death, islet cells transcription, library preparation, and sequence analysis were stained with 1:1,000 Hoechst, 1:1,000 propidium iodide (Hi-Seq2000; Illumina). RNA sequencing (RNAseq) reads (PI), and 1:20 annexin V Alexa Fluor 488. The staining was (100 base pairs) from paired-ends were mapped to the rat analyzed using the Cellomics ArrayScan (Cellomics, Pitts- genome (rn5) with TopHat2 (version 2.0.3) and Bowtie2 burgh, PA). See the Supplementary Data for additional (version 2.0.0) (30,31). Gene expression values were details and calculations of apoptosis protection. summarized as raw counts by running high-throughput Human islets were obtained from Prodo Laboratories sequencing (HTSeq) software (32). The variance-stabilizing (Aliso Viejo, CA), which provided donors’ demographic and transformation from DESEq software (33) was applied to clinical data, HbA1c level, and islet isolation parameters. the raw sequencing count data before statistical analyses. Human islets were cultured for at least 24 h in PIM(S) See Supplementary Data for the statistical models to human islet-specific medium (Prodo Laboratories), contain- identify significant gene expression changes.
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