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. 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 (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. For PDX-1 ing 5% human AB serum and 5.8 mmol/L glucose before gene expression analyses by quantitative PCR we used assay. the TaqMan probe set Rn01423448_m1 from Thermo For GLT-GSIS assays, rat or human islets were incubated Fisher. in high-glucose, palmitate-containing media (300 mmol/L PA:BSA, 27 mmol/L glucose) overnight at 37°C in 5% CO2. Islets were washed with Krebs-Ringer bicarbonate buffer (KRBB) containing 2.8 mmol/L glucose, and three size- matched islets per well were picked into a 96-well plate by stereomicroscope observation. For each experiment, four to eight wells (three islets each) per condition were assayed. After 2 h of incubation in KRBB at low glucose (2.8 mmol/L) condition, islets were stimulated with high glucose (16.7 mmol/L) for 1 h. The culture media were filtered to remove cell debris and stored at 220°C for analysis. In- tracellular insulin was extracted overnight in lysis buffer (Cyquant kit, #C7026; Invitrogen) at 220°C. Insulin con- centration in culture media and islet lysates was determined using the Cisbio insulin ultrasensitive assay kit (62IN2PEG; Cisbio) and/or the Mercodia insulin ELISA kits (rat: Mer- codia #10-1,124-10; human: Mercodia #10-1,113-01) with appropriate sample dilutions. All GSIS experiments were performed three times or more. For rat islets, pancreata from eight rats were pooled and combined for each islet isolation. For human islets, islets from two donors without diabetes were used individually. For human islet assays, secreted insulin and insulin content were normalized by Figure 2—Compound (Cpd) A and its derivatives protect INS1E cells and primary islet cells from GLT-induced apoptosis. A: INS1E cells or DNA content in human islets lysates (Cyquant DNA pro- Jurkat cells were preincubated with compound A for 1 h, followed by liferation kit, #C7026; Invitrogen). We used GraphPad apoptosis stimulus. INS1E cells were treated with GLT-conditioned Prism 8.1.2 software for data analyses and ordinary one- media (800 mmol/L palmitate [PA:BSA, 6:1], 11 mmol/L glucose in way ANOVA for statistical significance assessment. RPMI). Jurkat cell apoptosis was induced with 100 mmol/L TRAIL. Apoptosis was measured in both cell lines after 24 h (n 5 4–8 wells/ condition). B: Structure of compounds B, D, and the inactive analog Sample Preparation for Quantitative PCR and RNA E. C: Dissociated rat islet cells were cotreated with compound D and Sequencing GLT media (RPMI supplemented with 15 mmol/L glucose and m Isolated islets from eight rats were divided into seven 500 mol/L PA:BSA). Apoptosis was detected after 72 h using ; annexin-V Alexa Fluor 488 staining, and the percentage of apoptosis groups ( 1,000 islet equivalents/sample) as follows: two protection was calculated as described in RESEARCH DESIGN AND control groups cultured in regular media (RPMI with METHODS. diabetes.diabetesjournals.org Vogel and Associates 1035

Figure 3—Compound (Cpd) B and compound D increased insulin content and GSIS in rat and human islets under GLT conditions. A and B: Rat islets (three islets/well, six to eight wells/condition) were incubated in GLT media (RPMI with 300 mmol/L PA and 27 mmol/L glucose) with indicated compounds for 24 h, and then GSIS assay was performed in the presence of the indicated compounds. After 2 h of preincubation in KRBB buffer with 2.8 mmol/L glucose, islets were stimulated with 16.7 mmol/L glucose in KRBB. Secreted insulin in media was measured after 1 h of stimulation (B), and the islets were then lysed to measure total insulin content (A). Islets from eight rats were pooled for each experiment. C and D: Human islet insulin secretion and content reduced by GLT are restored by compounds B and D. Human islets were incubated with GLT media and the GLP-1R exendin-4 (Ex-4; 100 nmol/L), compound B, or compound D (300 nmol/L) for 24 h, followed by GSIS and insulin content measurement as for rat islets above. Compounds B and D preserved GSIS and insulin content in GLT rat islets, whereas exendin-4 increased GSIS without affecting insulin content. Experiments were repeated with islets from two donors without diabetes. E: Compound D did not increase GSIS in nonstressed rat islets. Rat islets were preincubated with compound D in normal media (RPMI media containing 11 mmol/L glucose and no PA) for 24 h and then stimulated with glucose for GSIS assay, followed by GSIS as above. *P , 0.05 vs. GLT. #P , 0.05 vs. control (CON), by one-way ANOVA. 1036 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020

Calcium Flux Assay in Islet Cells solved with the XLfit add-in for Excel 2003 software For calcium flux assay, we used a Fluorescent Imaging Plate (Microsoft, Redmond, WA). Reader (FLIPR) Tetra High-Throughput Cellular Screening System. Papain-dissociated islets were plated on laminin- Data and Resource Availability coated 384-well plates (354663; BD). After 48 h of recovery, The RNAseq data reported in this study are available in the islets were pretreated with compounds in GLT media Gene Expression Omnibus repository (GSE#134272). No (16 mmol/L glucose, 500 mmol/L PA:BSA in RPMI) for 1 h applicable resources were generated or analyzed during the at 37°C in 5% CO2. FLIPR calcium dye 6 was then resus- current study. pended in GLT media and added with appropriate com- fl RESULTS pound dilutions for 2 h at 37°C in 5% CO2. Calcium in ux was measured for the next 10 min at 1-s intervals. For Identification of a Molecule That Selectively Protects chronic calcium load measurements, readings were taken b-Cells From GLT-Induced Apoptosis every minute over 10 consecutive hours after GLT treat- To identify compounds that protect b-cells from GLT- ment, and the cumulative calcium signal was calculated based induced apoptosis, we designed a low-molecular-weight on the sum of individual measurements per assay condition. compound phenotypic screen (Fig. 1A). Caspase 3/7 acti- Analyses were done using Molecular Devices software, with vation was measured in INS1E cells cultured in the pres- the amplitude of the response (Fig. 6A, DF/F maximum- ence of glucose and palmitate (GLT conditions) to identify minimum fluorescent signal) as the main reported readout. compounds that reduced GLT-induced apoptosis. A total of 312,000 compounds were screened in 1,536-well plate 9 Measurement of Recombinant L-Type Calcium format. The average Z for the primary high-throughput Channel Activity screen was 0.59, and the average RZ9 was 0.72. To verify Measurements of compound activities on L-type calcium that reduced caspase activation was not a result of cell channel current was performed on the IonWorks Barra- death, compounds from the primary screen were tested in cuda system (Molecular Devices, LLC, San Jose, CA) by a general cell viability assay that measured ATP levels in ChanTest Corporation (Cleveland, OH). Cav1.2 channel GLT-treated INS1E cells. Compounds that reduced caspase (cloned Cav1.2/b2/a2d1) was expressed in CHO cells (ATCC), activation but did not cause general cytotoxicity were and stable cell lines were generated. For Cav1.2 measure- further profiledinanapoptosisassayinJurkatcells ments, CHO cells stably expressing Cav1.2 were treated with to eliminate general apoptosis inhibitors. Six scaffolds compounds diluted in a calcium-free HEPES-buffered phys- iological saline (composition in mmol/L): NaCl, 137; KCl, 4.0; MgCl2, 2.8; HEPES, 10; glucose, 10; and BaCl, 5.0 (pH 7.4). The current recordings were performed on IonWorks Barra- 3 hr GLT 24 hr GLT cuda at ambient temperature, before compound application to the cells (baseline) and 5 min after the application. Before digitization, the current records were low-pass filtered at one- fifth of the sampling frequency (10 kHz). Cav1.2 current was measured using a stimulus voltage pattern consisting of a depolarizing test pulse to 10 mV for 300 ms from a 290 mV holding potential. Peak current was measured during the step to 0 mV. Data acquisition and analyses were performed using the IonWorks Barracuda system operation software (version 2.0.2). The decrease in current amplitude after test article application was used to calculate the per- centage block relative to control. Results for each compound concentration (n $ 2) were averaged, and mean and SD values were calculated and used to generate dose-response curves. Ctrl GLT GLT+cpd D Ctrl GLT GLT+ GLT+ The blocking effect was calculated as: %Block 5 (1 2 cpdD cpdE 3 Icpd/IControl) 100%, where IControl and Icpd are the cur- Figure 4—RNAseq analyses of GLT-stressed rat islets reveal that rents measured before addition of compound and in the most of the GLT-induced gene expression changes are prevented by presence of a compound, respectively. Concentration- treatment with compound (cpd) D. Total RNA was prepared from rat fi islets (1,000 islet equivalents/sample) treated with control or GLT- response data for inhibitors were ttoanequationofthe m 5 1 2 1 containing media (27 mmol/L glucose, 300 mol/L PA:BSA) for 3 or following form: %Block %VC {(%PC %VC)/[1 24 h, with DMSO, cpd D, or cpd E, an inactive compound with similar (cpd/IC50)N]}, where cpd indicates compound concentra- chemical structure, at 300 nmol/L. For control (Ctrl) groups, islets tion, N is the Hill coefficient, %VC is the percentage of the were collected after incubation in normal media (no PA, 11 mmol/L current run-down, and %Block is the mean value of the glucose) and DMSO. The effects of cpd D and cpd E are shown on the top 190 GLT-induced gene expression changes. Each column percentage of current inhibited at each con- represents an independent treatment/repeat. Red, increased ex- centration of compound. Nonlinear least squares fits were pression; green, decreased expression. diabetes.diabetesjournals.org Vogel and Associates 1037

(chemical classes) that selectively protected b-cells from GLT-induced apoptosis remained and were further profiled in a primary rat islet cell apoptosis assay to select com- pounds that protect primary islet cells. Multiple readouts were used to measure apoptosis and general cell death in islets (annexin 5 and PI staining, respectively), to ensure specificity of the observed effects. Finally, compounds were tested for prevention of GLT-induced reduction of GSIS. Compound A (Fig. 1B), a racemic mixture, inhibited GLT-induced caspase activation in INS1E cells but did not inhibit Jurkat cell apoptosis (Fig. 2A) and was selected for further characterization. The racemic compound A was separated into two enantiomers, more active B and less active C (Supplementary Fig. 1), and enantiomer B was further optimized. As a result of medicinal chemistry efforts, compound D was developed; it protected pancreatic islets from GLT-induced apoptosis at nmol/L concentrations (Fig. 2B and C). Further, a structurally related inactive tool, compound E (Fig. 2B),wasusedasanegativecontrol in follow-up MOA experiments. To determine whether the islet cells that were protected from apoptosis maintained their function, we tested whether compounds B and D could promote insulin pro- duction and secretion in response to glucose in rat and human islets compromised by GLT. As previously reported (18), a significant reduction of insulin content and insulin secretion after GLT treatment was observed in rat islets. Treatment with compounds B or D rescued both reduction in insulin content and GSIS in GLT-stressed rat islets (Fig. 3A and B). Exendin-4, a clinically used GLP-1 receptor agonist and a known stimulator of insulin secretion, served as the positive control in these experiments and increased GSIS under GLT conditions (Fig. 3B). Interestingly, unlike compounds B and D, exendin-4 did not alter islet insulin content (Fig. 3A). Compound D also protected human islets from GLT- induced dysfunction (i.e., increased insulin content and secretion under GLT) (Fig. 3C and D). Notably, compound D did not increase insulin secretion under normal culture Figure 5—L-type blockers mimic many of the effects of compound (Cpd) D in rat islets. A: Dissociated rat islet conditions, suggesting the effects on islet function were cells were cotreated with modulators of Ca21 channels and GLT specific to the GLT condition (Fig. 3E). Inhibition of insulin media (RPMI supplemented with 15 mmol/L glucose and 500 mmol/L secretion occurred with compound D treatment at mmol/L PA:BSA). Apoptosis was detected after 72 h using annexin-V Alexa concentrations and was initially attributed to scaffold tox- Fluor 488 staining, and the percentage of apoptosis protection was E calculated as described in RESEARCH DESIGN AND METHODS. L-type icity (Fig. 3 ). We then initiated studies to gain more calcium channel blockers but not ER calcium release inhibitors insights into the bell-shaped effects of compound D on reduced GLT-induced apoptosis in primary rat islets. B: GSIS and to identify its MOA. (Nif) increased insulin content reduced by GLT, similar and non- additive to compound D, whereas the L-type Ca21 Compound D Targets a Central Regulator of Islet GLT FPL1642 (Fpl) reversed compound D effects on islet insulin content. Rat islets (three islets/well, six to eight wells/condition) were in- To determine which GLT-regulated genes might be affected cubated in GLT-containing media (RPMI with 300 mmol/L PA:BSA by compound D, we performed an RNAseq analysis of rat and 27 mmol/L glucose) with compounds as indicated for 24 h. Total islets treated ex vivo, with or without GLT and compound D, insulin content was measured as described in Fig. 3B. C: Rat islets were cotreated with GLT media (RPMI with 300 mmol/L PA:BSA and 27 mmol/L glucose) and nifedipine or compound D at 0.01, 0.1, and 1 mmol/L for 24 h. Islet mRNA was then isolated, and Pdx-1 ; Xs, xestospongin (10 mmol/L); -, DMSO. Unless stated expression measured by quantitative PCR. Similar to compound otherwise, compounds were tested at 1 mmol/L. *P , 0.05 vs. GLT D, nifedipine dose-dependently restored expression of Pdx-1 re- by one-way ANOVA. duced by 24-h GLT treatment. CON, control; Dnt, dantrolene (10 mmol/L); Ry, ryanodine (10 mmol/L); Tg, thapsigargin; Ver, 1038 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020 for 3 h and 24 h, respectively. The expected suppression of GLT depletes ER calcium stores and causes ER stress b-cell function/specification induction genes, such as PDX-1 (13). We hypothesized that restoration of calcium homeo- and INS1, and induction of stress-response genes was stasis might be the potential MOA of compound D; there- observed after 24-h GLT treatment (Supplementary Tables fore, we tested whether calcium flux modulators could 1 and 2). Treatment with compound D prevented close to phenocopy the effects of compound D in islets. We found 80% of the gene expression changes caused by a 24-h GLT that L-type calcium channel blockers nifedipine and verap- treatment (Fig. 4 and Supplementary Table 3), suggesting amil reduced GLT-triggered islet apoptosis similar to com- that compound D protects islets by regulating a central pound D, whereas the calcium channel opener FPL64176 node of GLT stress response. A closely related inactive had the opposite effect, further boosting apoptosis under compound from the same scaffold, compound E (Fig. 2B), GLT conditions (Fig. 5A). We also tested whether modu- did not reverse gene expression changes caused by GLT lators of ER calcium release and can mimic the (Fig. 4; see Supplementary Table 4 for all gene expression effects of compound D on apoptosis. We tested the inhib- changes and statistical models used in this study). itors of ryanodine receptor-mediated calcium release, ryanodine, dantrolene, and xestospongin, which inhibit IP3R- Compound D Decreases Cytosolic Calcium Overload mediated calcium release. None of these three inhibitors Induced by GLT had any effect on apoptosis under our assay conditions (Fig. To explore the MOA of compound D, we initially tested 5A). Thapsigargin, which inhibits sarco-ER calcium ATPase whether compound D may target the GLP-1 receptor, which and calcium reuptake from the cytosol into the ER, greatly has been reported to protect b-cells and improve their increased GLT-induced apoptosis, consistent with the pub- function; however, compound D failed to activate GLP-1 lished role of ER stress in GLT (Fig. 5A). The calcium channel receptor signaling (data not shown). Moreover, compound blocker nifedipine also prevented GLT-induced reduction in D protected islets from GLT-induced loss of insulin content, insulin content, and this effect was similar to and non- whereas exendin-4 did not (Fig. 3A), suggesting distinct additive with that of compound D. Furthermore, the L-type mechanisms. calcium channel opener FPL64176 blocked the protective

Figure 6—Compound D and its analogs protect islets by reducing calcium influx. A: Calcium influx was measured in dispersed islet cells incubated with cell-permeable fluorescent calcium dye 6 after treatment with GLT media (27 mmol/L glucose, 500 mmol/L PA:BSA). Readings were taken every 2 s for 10 min after GLT addition. Maximum amplitude of the second peak (DF/F max-min) is indicated by the arrow. B: Calcium influx (DF/F max-min of the second peak) was measured in dispersed rat islet cells treated with compound (Cpd) D or nifedipine (Nif) at increasing concentrations (1 nmol/L–10 mmol/L) for 10 min after glucose and lipid addition. C: Calcium influx was measured as above but with readings taken every minute for 10 h after glucose and lipid addition. Cumulative calcium in islets under normal or GLT conditions, with or without compound D at 300 nmol/L, was calculated by summing all readings over the course of 10 h. D: Potency of calcium flux inhibition was tightly associated with GLT-apoptosis protection potency (R2 5 0.87) by molecules from this chemical series (each point represents one compound). diabetes.diabetesjournals.org Vogel and Associates 1039 effects of compound D, suggesting that compound D pro- to GLT, extended treatment of islets with GLT media led to tection requires functional L-type calcium channels (Fig. sustained accumulation of cytosolic calcium over the 5B). Reduction in islet insulin content under GLT stress can course of 10 h, and compound D prevented this accumu- be partly due to the suppressed expression of PDX-1 that we lation, reducing calcium to baseline levels (Fig. 6C). These and others have observed under GLT. Nifedipine treatment results suggest that acute inhibition of GLT-induced cal- partially prevented the reduction in PDX-1 expression, cium influx by compound D translates into chronic sup- again mimicking the compound D effect (Fig. 5C). pression of GLT-induced calcium overload, and this effect Compounds from this scaffold were then tested for may be responsible for the protective effects of the scaf- ability to directly regulate calcium flux in islet cells. Using fold. Indeed, using diverse molecules generated as part of a fluorescent cell-permeable calcium dye, we monitored the medicinal chemistry activities around this scaffold, we cytosolic calcium in islet cells under GLT conditions (Fig. observed a tight linear association of calcium flux inhibi- 6A). Interestingly, the GLT-induced increase in calcium tion with apoptosis protection within the series (Fig. 6D), influx into the cytosol was biphasic, with a sharp initial which suggests molecules from this series protect b-cells peak within seconds of GLT media addition, followed by from GLT by modulation of calcium influx. a second sustained plateau. To explore the nature of the To determine whether compound D is a bona fide two calcium peaks, we pretreated islet cells with calcium L-type calcium , its effect on recombinantly modulators with known MOA. L-type calcium channel expressed L-type calcium channel Cav1.2 was examined in modulators and compound D dose-dependently reduced CHO cells. Nifedipine robustly blocked the Cav1.2- induced the second calcium peak (Fig. 6B and Supplementary Fig. response, whereas compound D had no effect (Fig. 7A). In 2), while depletion of sarco-ER calcium with thapsigargin addition, unlike compound D, which consistently increased eliminated the first calcium peak (Supplementary Fig. 2). insulin secretion under GLT conditions, enhanced GSIS Although the above experiments measure acute response under GLT conditions was not observed with nifedipine, which resulted in further worsening of GSIS (Fig. 7B). These data imply that compound D regulates calcium influx by a mechanism that is distinct from that of nifedipine.

DISCUSSION Here we report the design, execution, and analysis of a phenotypic screen aimed to identify molecules that protect pancreatic b-cells from nutrient overload-associated GLT. We used an in vitro INS1E GLT assay to screen a library of compounds for reduction of GLT-induced apoptosis. Our goal was to identify compounds that specifically protect from GLT-induced toxicity in b-cells. We used several counter- screens to eliminate general apoptosis inhibitors and toxic molecules and identified a few selective b-cell–protective hits. GLT is associated with reduced insulin gene expression, reduced islet insulin content, and reduced insulin secretion (2). In rats, glucose and intralipid infusion decreases in- sulin biosynthesis, GSIS, and expression of b-cell genes

Figure 7—Compound D does not block the recombinant L-type Ca21 channel. A: Inhibition of calcium current was measured on the IonWorks Barracuda system in CHO cells expressing recombinant Cav1.2 after treatment with nifedipine (Nif) and compound (Cpd) D. The current recordings were performed at ambient temperature, before compound application to the cells (baseline) and 5 min after the application. Cav1.2 current was measured using a stimulus voltage pattern consisting of a depolarizing test pulse to 10 mV for 300 ms from a 290 mV holding potential. Peak current was measured during the step to 0 mV. Data acquisition and analyses were performed using the IonWorks Barracuda system operation software. The decrease in current amplitude after test article appli- cation was used to calculate the percentage block relative to control. B: Unlike scaffold compounds, nifedipine did not restore GSIS in GLT-stressed rat islets. GLT-GSIS was performed as in Fig. 3. CON, control; Ex-4, exendin-4 GLP-1 receptor agonist; -, DMSO. *P , 0.05 Figure 8—A model for protective MOA of compound D. Ins, insulin. vs. GLT. See DISCUSSION for details. 1040 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020 despite increased b-cell mass, suggesting that b-cell mass which can induce apoptosis as well as reduction in gene per se cannot always predict the functional outcome (18). expression of critical b-cell function genes, such as PDX-1 We therefore assessed the effects of our hits on primary and insulin, followed by reduction in islet insulin content islet function under GLT stress. Several observations in- and GSIS. Molecules identified in the b-cell protection dicated that the islet system recapitulates b-cell stress screen described here can reduce the excessive calcium observed in human diabetes. First, we observed a modest influx by restoring nearly optimal calcium levels and protect increase in cell death, in line with an increased apoptotic islets from all of the above detriments. Unlike the canonical index reported for islets from humans with diabetes (34). L-type nifedipine, these molecules Second, we observed the expected reduction of INS and allow insulin secretion under conditions of calcium channel PDX-1 gene expression and induction of ER stress genes overstimulation. Calcium antagonists with such properties (Fig. 4 and Supplementary Table 4). We also observed may prove to be a novel class of diabetes treatment and reduction of islet insulin content (Fig. 3), which is a feature prevention drugs. of T2D (21,34,35). Finally, we observed a GLT-induced reduction in insulin secretion that, as expected, could be reversed by a GLP-1 receptor agonist (Fig. 3), an approved Acknowledgments. The authors thank Faye Zhao for uploading RNAseq diabetes (27). data into Gene Expression Omnibus, Yunshan Peng, David Parker, Katsumasa We identified a chemical scaffold that prevented apo- Nakajima, Timothy Rasmusson, Toshio Kawanami, T.R. Vedananda, and Yongjin ptosis and improved insulin secretion in rodent and human Gong for synthesizing analogs of compound D, Jeffrey A. Brown for insulin measurements, and Martin G. Waters and Jovita Marcinkeviciene (all from Novartis primary islets under GLT stress. MOA studies pointed to the fl Institutes for BioMedical Research at the time of this work) for discussions and suppression of cytosolic calcium in ux as a primary mech- critical reading of the manuscript. anism responsible for islet protection from GLT-induced Duality of Interest. All authors were employees of the Novartis Institutes for apoptosis. We generated a series of compounds structurally BioMedical Research at the time of the reported studies. No other potential related to our initial hit and observed a tight association of conflicts of interest relevant to this article were reported. calcium flux modulation with apoptosis protection within Author Contributions. J.V., J.Y., L.S., S.X.W., R.Z., S.F., C.-H.C., A.C.W., the series. Unfortunately, the undesired pharmacokinetic and B.D. performed experiments and analyzed data, A.C., F.Z., G.T., T.M.S, B.D., properties prevented us from comprehensive testing of D.M.R., X.C., and A.B. contributed to discussions and data analyses. H.X. analyzed these molecules in vivo in a T2D animal model. RNAseq data. A.B. wrote the manuscript. All authors reviewed and edited the The discovery that our compounds inhibit calcium flux manuscript. A.B. is the guarantor of this work and, as such, had full access to all was unexpected. 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