A Phenotypic Screen Identifies Calcium Overload as a KeyMechanism of b-Cell GlucolipotoxicityJennifer 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 pancreaticb-cell mass and failure of the remaining b-cells to deliversufficient insulin to meet demand. b-Cell glucolipotox-icity (GLT), which refers to combined, deleterious effectsof elevated glucose and fatty acid levels on b-cell func-tion and survival, contributes to T2D-associated b-cellfailure. Drugs and mechanisms that protect b-cells fromGLT stress could potentially improvemetabolic control inpatients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected b-cellsfrom GLT, we identified compound A that selectivelyblocked GLT-induced apoptosis in rat insulinoma cells.Compound A and its optimized analogs also improvedviability and function in primary rat and human isletsunder GLT. We discovered that compound A analogsdecreased GLT-induced cytosolic calcium influx in isletcells, and all measured b-cell–protective effects corre-lated with this activity. Further studies revealed that theactive compound from this series largely reversed GLT-induced global transcriptional changes. Our results sug-gest that taming cytosolic calciumoverload in pancreaticislets can improve b-cell survival and function under GLTstress and thus could be an effective strategy for T2Dtreatment.

Diabetes is caused by inability of the pancreas to meetmetabolic demand for insulin due to a shortage of func-tional insulin-secreting b-cells (1). In type 2 diabetes (T2D),elevated levels of circulating glucose and fatty acids con-tribute to insulin resistance in peripheral tissues, which

leads to an increased demand for insulin as well as directeffects on theb-cell, such as stress and apoptosis. This conceptof toxic energy excess, termed glucolipotoxicity (GLT), hasbeen extensively studied in the last two decades (2,3).

GLT conditions are generally associated with increasedoxidative and endoplasmic reticulum (ER) stress, calciumdysregulation, and inflammasome activation (4–13). In thepancreas, GLT conditions result in increased b-cell death,low insulin content, and reduced glucose-stimulated in-sulin secretion (GSIS) (14–17). GLT conditions have beenshown to lead to reduced insulin gene expression viadysregulation of its upstream transcription factor PDX-1(3). Decreased PDX-1 mRNA levels and nuclear exclusionof PDX-1 were observed in b-cells under GLT conditionsand in islets from humans and rodents with diabetes(18–21). ER stress and ER calcium dysregulation have alsobeen implicated in GLT-induced b-cell dysfunction (22).Free fatty acids can induce calcium influx from the ERin b-cells via activation of Gq-coupled receptors such asG-protein–coupled receptor (GPR) 120, GPR40, and inosi-tol 1,4,5-trisphosphate receptor (IP3R). High glucose stim-ulation of b-cells results in membrane depolarization,leading to calcium influx through the voltage-gated L-typecalcium channels on the plasmamembrane and, as a result,calcium-induced calcium release from the ER via theryanodine receptor and IP3R. The influx of calcium intothe cytosol is necessary for the glucose-stimulated insulinrelease, but excess stimulation with high levels of glucoseand fatty acids depletes ER calcium stores, which can alsocontribute to the reduced insulin secretion under GLT (23).

1Gilead, Foster City, CA2Novartis Institutes for BioMedical Research, Cambridge, MA3Editas Medicine, Cambridge, MA4Rheos Medicines, Cambridge, MA5Sanofi, Cambridge, MA6Merck & Co., Kenilworth, NJ

Corresponding author: Alina Berdichevsky, alina.berdichevsky@novartis.com

Received 15 August 2019 and accepted 7 February 2020

This article contains Supplementary Data online at https://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0813/-/DC1.

© 2020 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at https://www.diabetesjournals.org/content/license.

1032 Diabetes Volume 69, May 2020

PHARMACOLOGY

AND

THERAPEUTIC

S

Moreover, several recent reports support a role for cytosoliccalcium overload in pathogenesis of diabetes. In mice express-ing a mutant form of Abcc8, a key component of the b-cellKATP channel showing constant depolarization similar to thatobserved with nutrient overload, intracellular calcium is con-stantly elevated, leading to diabetes (24). Furthermore, mouseislet cells exposed to diabetic serum show hyperactivation ofL-type calcium channels (25).

To date, there are no efficient therapies protecting patientswith diabetes from b-cell death and loss of b-cell mass.Glucagon-like peptide 1 (GLP-1) signaling has been reportedto exert direct b-cell–protective effects (26,27). However,despite many years of research, whether the beneficial effectsof GLP-1–based therapy on b-cell mass observed in animalstudies are applicable to humans is still unclear (28).

Here we report discovery of a chemical series thatprotects b-cells and islets from GLT-induced apoptosisand dysfunction. Mechanism of action (MOA) studiesrevealed that this series protects b-cells by inhibitingGLT-induced calcium influx. Our work suggests a centralrole for cytosolic calcium overload in b-cell GLT.

RESEARCH DESIGN AND METHODS

Cellular Apoptosis and Viability AssaysFor more details including cell culture, materials and screen-ing, please see Supplementary Methods. The GLT apoptosis

assay was performed in INS1E cells as follows. After over-night incubation in low-serum culture media (RPMI plus0.5% BSA), cells were preincubated with compounds for 1 hand then challenged with BSA-conjugated palmitate(800 mmol/L palmitate; palmitic acid [PA]:BSA ratio 6:1)(#P9767; Sigma-Aldrich). Apoptosis was measured bycaspase 3/7 Glo (CaspGlo, #G8091; Promega) 24 h afterthe palmitate challenge. Cell viability was measured byCell Titer Glo (#G7570; Promega) 48 h after the palmitatechallenge, according to the manufacturer’s instructions,and luminescence was detected by EnVision Plate Reader(Perkin Elmer). For both CaspGlo and Cell Titer Gloassays, the percentage reduction and percentage protec-tion, respectively, were calculated using relative luciferaseunits (RLU) as [(RLU of DMSO control 1 PA 2 RLU ofcompound 1 PA)/RLU of DMSO 1 PA]∗100%.

For TNF-related apoptosis-inducing ligand (TRAIL) ap-optosis assays, Jurkat cells (#TIB-152; ATCC) were treatedwith compounds for 1 h, followed by treatment withhuman recombinant TRAIL protein (100 mmol/L finalconcentration) (#375-TL-010; R&D Systems), and apo-ptosis was measured as described above using CaspGlo kit24 h after the TRAIL challenge.

For high-throughput screening, the final BSA-palmitateconcentration was 1 mmol/L (PA:BSA ratio was main-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 designand experimental flow, see the first section of RESULTS, Identification of a Molecule That Selectively Protects b-Cells From GLT-InducedApoptosis. B: Structure of the screening hit compound A. *Rac, chiral center on the racemic allene compound.

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Islet Apoptosis and GSIS AssaysRat pancreatic islets were isolated from Sprague Dawleyrats after perfusion with Liberase TL Research Grade(#5401020001; Sigma-Aldrich) as previously described(29). For apoptosis evaluation, islets were dispersed tosingle cells using a papain dissociation kit (#LK003150;Worthington) and plated in laminin-5–coated plates. After48 h recovery, islet cells were cotreated with GLT media(0.5% fatty acid-free BSA, 500 mmol/L palmitate in RPMI,PA:BSA ratio 6:1, 16 mmol/L glucose) and compounds ofinterest for 72 h. For determination of cell death, islet cellswere stained with 1:1,000Hoechst, 1:1,000 propidium iodide(PI), and 1:20 annexin V Alexa Fluor 488. The staining wasanalyzed using the Cellomics ArrayScan (Cellomics, Pitts-burgh, PA). See the Supplementary Data for additionaldetails and calculations of apoptosis protection.

Human islets were obtained from Prodo Laboratories(Aliso Viejo, CA), which provided donors’ demographic andclinical data, HbA1c level, and islet isolation parameters.Human islets were cultured for at least 24 h in PIM(S)human islet-specific medium (Prodo Laboratories), contain-ing 5% human AB serum and 5.8 mmol/L glucose beforeassay.

For GLT-GSIS assays, rat or human islets were incubatedin high-glucose, palmitate-containing media (300 mmol/LPA: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 bystereomicroscope observation. For each experiment, four toeight 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 toremove 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 determinedusing 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) withappropriate sample dilutions. All GSIS experiments wereperformed three times or more. For rat islets, pancreatafrom eight rats were pooled and combined for each isletisolation. For human islets, islets from two donors withoutdiabetes were used individually. For human islet assays,secreted insulin and insulin content were normalized byDNA content in human islets lysates (Cyquant DNA pro-liferation kit, #C7026; Invitrogen). We used GraphPadPrism 8.1.2 software for data analyses and ordinary one-way ANOVA for statistical significance assessment.

Sample Preparation for Quantitative PCR and RNASequencingIsolated islets from eight rats were divided into sevengroups (;1,000 islet equivalents/sample) as follows: twocontrol groups cultured in regular media (RPMI with

11 mmol/L glucose) with DMSO for 3 and 24 h, andtwo treated with GLT media (27 mmol/L glucose and300 mmol/L PA:BSA in RPMI) and DMSO, or GLT mediawith 300 nmol/L compound D or compound E for 3 and/or24 h. This experiment was performed three times withdifferent islet preparations to obtain three independentbiological repeats. Total RNA was prepared using theRNeasy kit (Qiagen). RNA quantity and quality wereassessed using an Agilent 2100 Bioanalyzer and 1 mg totalRNA was sent to the Beijing Genome Institute for reversetranscription, library preparation, and sequence analysis(Hi-Seq2000; Illumina). RNA sequencing (RNAseq) reads(100 base pairs) from paired-ends were mapped to the ratgenome (rn5) with TopHat2 (version 2.0.3) and Bowtie2(version 2.0.0) (30,31). Gene expression values weresummarized as raw counts by running high-throughputsequencing (HTSeq) software (32). The variance-stabilizingtransformation from DESEq software (33) was applied tothe raw sequencing count data before statistical analyses.See Supplementary Data for the statistical models toidentify significant gene expression changes. For PDX-1gene expression analyses by quantitative PCR we usedthe TaqMan probe set Rn01423448_m1 from ThermoFisher.

Figure 2—Compound (Cpd) A and its derivatives protect INS1E cellsand primary islet cells fromGLT-induced apoptosis.A: INS1E cells orJurkat cells were preincubated with compound A for 1 h, followed byapoptosis stimulus. INS1E cells were treated with GLT-conditionedmedia (800 mmol/L palmitate [PA:BSA, 6:1], 11 mmol/L glucose inRPMI). 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 analogE.C: Dissociated rat islet cells were cotreated with compound D andGLT media (RPMI supplemented with 15 mmol/L glucose and500 mmol/L PA:BSA). Apoptosis was detected after 72 h usingannexin-V Alexa Fluor 488 staining, and the percentage of apoptosisprotection was calculated as described in RESEARCH DESIGN AND

METHODS.

1034 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020

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 GLTmedia (RPMI with 300 mmol/L PA and 27mmol/L glucose) withindicated compounds for 24 h, and then GSIS assay was performed in the presence of the indicated compounds. After 2 h of preincubation inKRBB buffer with 2.8 mmol/L glucose, islets were stimulated with 16.7 mmol/L glucose in KRBB. Secreted insulin in media was measuredafter 1 h of stimulation (B), and the islets were then lysed to measure total insulin content (A). Islets from eight rats were pooled for eachexperiment. C and D: Human islet insulin secretion and content reduced by GLT are restored by compounds B and D. Human islets wereincubated with GLT media and the GLP-1R agonist exendin-4 (Ex-4; 100 nmol/L), compound B, or compound D (300 nmol/L) for 24 h,followed byGSIS and insulin contentmeasurement as for rat islets above. Compounds B andD preservedGSIS and insulin content in GLT ratislets, whereas exendin-4 increased GSIS without affecting insulin content. Experiments were repeated with islets from two donors withoutdiabetes. E: Compound D did not increase GSIS in nonstressed rat islets. Rat islets were preincubated with compound D in normal media(RPMImedia containing 11mmol/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.

diabetes.diabetesjournals.org Vogel and Associates 1035

Calcium Flux Assay in Islet CellsFor calcium flux assay, we used a Fluorescent Imaging PlateReader (FLIPR) Tetra High-Throughput Cellular ScreeningSystem. Papain-dissociated islets were plated on laminin-coated 384-well plates (354663; BD). After 48 h of recovery,islets were pretreated with compounds in GLT media(16 mmol/L glucose, 500 mmol/L PA:BSA in RPMI) for 1 hat 37°C in 5% CO2. FLIPR calcium dye 6 was then resus-pended in GLT media and added with appropriate com-pound dilutions for 2 h at 37°C in 5% CO2. Calcium influxwas measured for the next 10 min at 1-s intervals. Forchronic calcium load measurements, readings were takenevery minute over 10 consecutive hours after GLT treat-ment, and the cumulative calcium signal was calculated basedon the sum of individual measurements per assay condition.Analyses were done using Molecular Devices software, withthe amplitude of the response (Fig. 6A, DF/F maximum-minimum fluorescent signal) as the main reported readout.

Measurement of Recombinant L-Type CalciumChannel ActivityMeasurements of compound activities on L-type calciumchannel current was performed on the IonWorks Barra-cuda system (Molecular Devices, LLC, San Jose, CA) byChanTest Corporation (Cleveland, OH). Cav1.2 channel(cloned Cav1.2/b2/a2d1) was expressed in CHO cells (ATCC),and stable cell lines were generated. For Cav1.2 measure-ments, CHO cells stably expressing Cav1.2 were treated withcompounds 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-cuda at ambient temperature, before compound applicationto the cells (baseline) and 5 min after the application. Beforedigitization, the current records were low-pass filtered at one-fifth of the sampling frequency (10 kHz). Cav1.2 current wasmeasured using a stimulus voltage pattern consisting ofa depolarizing test pulse to 10 mV for 300 ms froma 290 mV holding potential. Peak current was measuredduring the step to 0 mV. Data acquisition and analyses wereperformed using the IonWorks Barracuda system operationsoftware (version 2.0.2). The decrease in current amplitudeafter test article application was used to calculate the per-centage block relative to control. Results for each compoundconcentration (n$ 2) were averaged, andmean and SD valueswere calculated and used to generate dose-response curves.

The blocking effect was calculated as: %Block 5 (1 2Icpd/IControl) 3 100%, where IControl and Icpd are the cur-rents measured before addition of compound and in thepresence of a compound, respectively. Concentration-response data for inhibitors were fit to an equation of thefollowing form: %Block 5 %VC 1 {(%PC 2 % VC)/[1 1(cpd/IC50)N]}, where cpd indicates compound concentra-tion, N is the Hill coefficient, %VC is the percentage of thecurrent run-down, and %Block is the mean value of thepercentage of ion channel current inhibited at each con-centration of compound. Nonlinear least squares fits were

solved with the XLfit add-in for Excel 2003 software(Microsoft, Redmond, WA).

Data and Resource AvailabilityThe RNAseq data reported in this study are available in theGene Expression Omnibus repository (GSE#134272). Noapplicable resources were generated or analyzed during thecurrent study.

RESULTS

Identification of a Molecule That Selectively Protectsb-Cells From GLT-Induced ApoptosisTo identify compounds that protect b-cells from GLT-induced apoptosis, we designed a low-molecular-weightcompound phenotypic screen (Fig. 1A). Caspase 3/7 acti-vation was measured in INS1E cells cultured in the pres-ence of glucose and palmitate (GLT conditions) to identifycompounds that reduced GLT-induced apoptosis. A totalof 312,000 compounds were screened in 1,536-well plateformat. The average Z9 for the primary high-throughputscreen was 0.59, and the average RZ9 was 0.72. To verifythat reduced caspase activation was not a result of celldeath, compounds from the primary screen were tested ina general cell viability assay that measured ATP levels inGLT-treated INS1E cells. Compounds that reduced caspaseactivation but did not cause general cytotoxicity werefurther profiled in an apoptosis assay in Jurkat cellsto eliminate general apoptosis inhibitors. Six scaffolds

Ctrl GLT GLT+cpdE

Ctrl GLT GLT+cpdD

3 hr GLT 24 hr GLT

GLT+cpd D

Figure 4—RNAseq analyses of GLT-stressed rat islets reveal thatmost of theGLT-induced gene expression changes are prevented bytreatment with compound (cpd) D. Total RNA was prepared from ratislets (1,000 islet equivalents/sample) treated with control or GLT-containing media (27 mmol/L glucose, 300 mmol/L PA:BSA) for 3 or24 h, with DMSO, cpd D, or cpd E, an inactive compoundwith similarchemical structure, at 300 nmol/L. For control (Ctrl) groups, isletswere collected after incubation in normal media (no PA, 11 mmol/Lglucose) and DMSO. The effects of cpd D and cpd E are shown onthe top 190 GLT-induced gene expression changes. Each columnrepresents an independent treatment/repeat. Red, increased ex-pression; green, decreased expression.

1036 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020

(chemical classes) that selectively protected b-cells fromGLT-induced apoptosis remained and were further profiledin a primary rat islet cell apoptosis assay to select com-pounds that protect primary islet cells. Multiple readoutswere used to measure apoptosis and general cell death inislets (annexin 5 and PI staining, respectively), to ensurespecificity of the observed effects. Finally, compounds weretested for prevention of GLT-induced reduction of GSIS.

Compound A (Fig. 1B), a racemic mixture, inhibitedGLT-induced caspase activation in INS1E cells but did notinhibit Jurkat cell apoptosis (Fig. 2A) and was selected forfurther characterization. The racemic compound A wasseparated into two enantiomers, more active B and lessactive C (Supplementary Fig. 1), and enantiomer B wasfurther optimized. As a result ofmedicinal chemistry efforts,compound D was developed; it protected pancreatic isletsfrom GLT-induced apoptosis at nmol/L concentrations(Fig. 2B and C). Further, a structurally related inactivetool, compound E (Fig. 2B), was used as a negative controlin follow-up MOA experiments.

To determine whether the islet cells that were protectedfrom apoptosis maintained their function, we testedwhether compounds B and D could promote insulin pro-duction and secretion in response to glucose in rat andhuman islets compromised by GLT. As previously reported(18), a significant reduction of insulin content and insulinsecretion after GLT treatment was observed in rat islets.Treatment with compounds B or D rescued both reductionin insulin content and GSIS in GLT-stressed rat islets (Fig.3A and B). Exendin-4, a clinically used GLP-1 receptoragonist and a known stimulator of insulin secretion, servedas the positive control in these experiments and increasedGSIS under GLT conditions (Fig. 3B). Interestingly, unlikecompounds B and D, exendin-4 did not alter islet insulincontent (Fig. 3A).

Compound D also protected human islets from GLT-induced dysfunction (i.e., increased insulin content andsecretion under GLT) (Fig. 3C and D). Notably, compoundD did not increase insulin secretion under normal cultureconditions, suggesting the effects on islet function werespecific to the GLT condition (Fig. 3E). Inhibition of insulinsecretion occurred with compound D treatment at mmol/Lconcentrations and was initially attributed to scaffold tox-icity (Fig. 3E). We then initiated studies to gain moreinsights into the bell-shaped effects of compound D onGSIS and to identify its MOA.

Compound D Targets a Central Regulator of Islet GLTTo determine which GLT-regulated genes might be affectedby compound D, we performed an RNAseq analysis of ratislets treated ex vivo, with or without GLT and compoundD,

Figure 5—L-type calcium channel blockers mimic many of theeffects of compound (Cpd) D in rat islets. A: Dissociated rat isletcells were cotreated with modulators of Ca21 channels and GLTmedia (RPMI supplemented with 15mmol/L glucose and 500mmol/LPA:BSA). Apoptosis was detected after 72 h using annexin-V AlexaFluor 488 staining, and the percentage of apoptosis protection wascalculated as described in RESEARCH DESIGN AND METHODS. L-typecalcium channel blockers but not ER calcium release inhibitorsreduced GLT-induced apoptosis in primary rat islets. B: Nifedipine(Nif) increased insulin content reduced by GLT, similar and non-additive to compound D, whereas the L-type Ca21 channel openerFPL1642 (Fpl) reversed compound D effects on islet insulin content.Rat islets (three islets/well, six to eight wells/condition) were in-cubated in GLT-containing media (RPMI with 300 mmol/L PA:BSAand 27mmol/L glucose) with compounds as indicated for 24 h. Totalinsulin content was measured as described in Fig. 3B. C: Rat isletswere cotreated with GLT media (RPMI with 300 mmol/L PA:BSA and27 mmol/L glucose) and nifedipine or compound D at 0.01, 0.1, and1 mmol/L for 24 h. Islet mRNA was then isolated, and Pdx-1expression measured by quantitative PCR. Similar to compoundD, nifedipine dose-dependently restored expression of Pdx-1 re-duced by 24-h GLT treatment. CON, control; Dnt, dantrolene(10 mmol/L); Ry, ryanodine (10 mmol/L); Tg, thapsigargin; Ver,

verapamil; Xs, xestospongin (10 mmol/L); -, DMSO. Unless statedotherwise, compounds were tested at 1 mmol/L. *P , 0.05 vs. GLTby one-way ANOVA.

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for 3 h and 24 h, respectively. The expected suppression ofb-cell function/specification induction genes, such as PDX-1and INS1, and induction of stress-response genes wasobserved after 24-h GLT treatment (Supplementary Tables1 and 2). Treatment with compound D prevented close to80% of the gene expression changes caused by a 24-h GLTtreatment (Fig. 4 and Supplementary Table 3), suggestingthat compound D protects islets by regulating a centralnode of GLT stress response. A closely related inactivecompound from the same scaffold, compound E (Fig. 2B),did not reverse gene expression changes caused by GLT(Fig. 4; see Supplementary Table 4 for all gene expressionchanges and statistical models used in this study).

Compound D Decreases Cytosolic Calcium OverloadInduced by GLTTo explore the MOA of compound D, we initially testedwhether compound Dmay target the GLP-1 receptor, whichhas been reported to protect b-cells and improve theirfunction; however, compound D failed to activate GLP-1receptor signaling (data not shown). Moreover, compoundD protected islets fromGLT-induced loss of insulin content,whereas exendin-4 did not (Fig. 3A), suggesting distinctmechanisms.

GLT depletes ER calcium stores and causes ER stress(13). We hypothesized that restoration of calcium homeo-stasis might be the potential MOA of compound D; there-fore, we tested whether calcium flux modulators couldphenocopy the effects of compound D in islets. We foundthat L-type calcium channel blockers nifedipine and verap-amil reduced GLT-triggered islet apoptosis similar to com-pound D, whereas the calcium channel opener FPL64176had the opposite effect, further boosting apoptosis underGLT conditions (Fig. 5A). We also tested whether modu-lators of ER calcium release and reuptake can mimic theeffects of compound D on apoptosis. We tested the inhib-itors of ryanodine receptor-mediated calcium release,ryanodine, dantrolene, and xestospongin, which inhibit IP3R-mediated calcium release. None of these three inhibitorshad any effect on apoptosis under our assay conditions (Fig.5A). Thapsigargin, which inhibits sarco-ER calcium ATPaseand calcium reuptake from the cytosol into the ER, greatlyincreased GLT-induced apoptosis, consistent with the pub-lished role of ER stress in GLT (Fig. 5A). The calcium channelblocker nifedipine also prevented GLT-induced reduction ininsulin content, and this effect was similar to and non-additive with that of compound D. Furthermore, the L-typecalcium 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 cellsincubatedwith cell-permeable fluorescent calciumdye 6 after treatment with GLTmedia (27mmol/L glucose, 500mmol/L PA:BSA). Readingswere 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 butwith readings taken everyminute for 10 h after glucose and lipid addition. Cumulative calcium in islets under normal or GLT conditions, with orwithout compound D at 300 nmol/L, was calculated by summing all readings over the course of 10 h.D: Potency of calcium flux inhibition wastightly associated with GLT-apoptosis protection potency (R2 5 0.87) by molecules from this chemical series (each point represents onecompound).

1038 Calcium Overload Mediates GLT in Islets Diabetes Volume 69, May 2020

effects of compound D, suggesting that compound D pro-tection requires functional L-type calcium channels (Fig.5B). Reduction in islet insulin content under GLT stress canbe partly due to the suppressed expression of PDX-1 that weand others have observed under GLT. Nifedipine treatmentpartially prevented the reduction in PDX-1 expression,again mimicking the compound D effect (Fig. 5C).

Compounds from this scaffold were then tested forability to directly regulate calcium flux in islet cells. Usinga fluorescent cell-permeable calcium dye, we monitoredcytosolic calcium in islet cells under GLT conditions (Fig.6A). Interestingly, the GLT-induced increase in calciuminflux into the cytosol was biphasic, with a sharp initialpeak within seconds of GLT media addition, followed bya second sustained plateau. To explore the nature of thetwo calcium peaks, we pretreated islet cells with calciummodulators with known MOA. L-type calcium channelmodulators and compound D dose-dependently reducedthe second calcium peak (Fig. 6B and Supplementary Fig.2), while depletion of sarco-ER calcium with thapsigargineliminated the first calcium peak (Supplementary Fig. 2).Although the above experiments measure acute response

to GLT, extended treatment of islets with GLTmedia led tosustained accumulation of cytosolic calcium over thecourse of 10 h, and compound D prevented this accumu-lation, reducing calcium to baseline levels (Fig. 6C). Theseresults suggest that acute inhibition of GLT-induced cal-cium influx by compound D translates into chronic sup-pression of GLT-induced calcium overload, and this effectmay be responsible for the protective effects of the scaf-fold. Indeed, using diverse molecules generated as part ofthe medicinal chemistry activities around this scaffold, weobserved a tight linear association of calcium flux inhibi-tion with apoptosis protection within the series (Fig. 6D),which suggests molecules from this series protect b-cellsfrom GLT by modulation of calcium influx.

To determine whether compound D is a bona fideL-type calcium channel blocker, its effect on recombinantlyexpressed L-type calcium channel Cav1.2 was examined inCHO cells. Nifedipine robustly blocked the Cav1.2- inducedresponse, whereas compound D had no effect (Fig. 7A). Inaddition, unlike compound D, which consistently increasedinsulin secretion under GLT conditions, enhanced GSISunder GLT conditions was not observed with nifedipine,which resulted in further worsening of GSIS (Fig. 7B). Thesedata imply that compound D regulates calcium influx bya mechanism that is distinct from that of nifedipine.

DISCUSSION

Here we report the design, execution, and analysis ofa phenotypic screen aimed to identifymolecules that protectpancreatic b-cells from nutrient overload-associated GLT.We used an in vitro INS1E GLT assay to screen a library ofcompounds for reduction of GLT-induced apoptosis. Our goalwas to identify compounds that specifically protect fromGLT-induced toxicity in b-cells. We used several counter-screens to eliminate general apoptosis inhibitors and toxicmolecules and identified a few selectiveb-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-typeCa21 channel. A: Inhibition of calcium current was measured on theIonWorks Barracuda system in CHO cells expressing recombinantCav1.2 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 afterthe application. Cav1.2 current was measured using a stimulusvoltage pattern consisting of a depolarizing test pulse to 10 mVfor 300 ms from a 290 mV holding potential. Peak current wasmeasured during the step to 0 mV. Data acquisition and analyseswere performed using the IonWorks Barracuda system operationsoftware. 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 inGLT-stressed rat islets. GLT-GSIS was performed as in Fig. 3. CON,control; Ex-4, exendin-4GLP-1 receptor agonist; -, DMSO. *P, 0.05vs. GLT.

Figure 8—A model for protective MOA of compound D. Ins, insulin.See DISCUSSION for details.

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despite increased b-cell mass, suggesting that b-cell massper se cannot always predict the functional outcome (18).We therefore assessed the effects of our hits on primaryislet function under GLT stress. Several observations in-dicated that the islet system recapitulates b-cell stressobserved in human diabetes. First, we observed a modestincrease in cell death, in line with an increased apoptoticindex reported for islets from humans with diabetes (34).Second, we observed the expected reduction of INS andPDX-1 gene expression and induction of ER stress genes(Fig. 4 and Supplementary Table 4). We also observedreduction of islet insulin content (Fig. 3), which is a featureof T2D (21,34,35). Finally, we observed a GLT-inducedreduction in insulin secretion that, as expected, could bereversed by a GLP-1 receptor agonist (Fig. 3), an approveddiabetes drug (27).

We identified a chemical scaffold that prevented apo-ptosis and improved insulin secretion in rodent and humanprimary islets under GLT stress.MOA studies pointed to thesuppression of cytosolic calcium influx as a primary mech-anism responsible for islet protection from GLT-inducedapoptosis. We generated a series of compounds structurallyrelated to our initial hit and observed a tight association ofcalcium flux modulation with apoptosis protection withinthe series. Unfortunately, the undesired pharmacokineticproperties prevented us from comprehensive testing ofthese molecules in vivo in a T2D animal model.

The discovery that our compounds inhibit calcium fluxwas unexpected. Although calcium dysregulation by excessfatty acids is well documented (13) and apoptosis pro-tection has been described for calcium channel blockers(36), the increased insulin secretion we observed wascontrary to the well-established inhibition of insulin se-cretion with calcium channel blockers. Indeed, we have notobserved inhibition of recombinant L-type channel withcompound D or improvement of insulin secretion with thecanonical calcium antagonist nifedipine. We hypothesizethat compound D regulates calcium influx by a mechanismthat is distinct from that of nifedipine and is a state-dependent L-type calcium channel antagonist or repre-sents a novel class of calcium antagonists.

The calcium antagonist described here could largely pre-vent gene expression changes induced by GLT, suggestingthat intracellular calcium overload is the central mechanismunderlying GLT-induced apoptosis and dysfunction. Severalrecent reports showed a link between calcium dysregulationand diabetes (37). Interestingly, we did not observe apopto-sis protection for ER calcium release modulators but didobserve protection with diverse L-type calcium channelblockers. The antidiabetic activity of verapamil was recentlyconfirmed in human studies, where patients on verapamilshowed decreased incidence of newly diagnosed T2D (38)and improvement in diabetes in a small clinical study (39).These human data support a role for calcium overload indiabetes pathology.

Our results are consistent with a model (Fig. 8) whereGLT conditions cause an increase in cytosolic calcium influx,

which can induce apoptosis as well as reduction in geneexpression of critical b-cell function genes, such as PDX-1and insulin, followed by reduction in islet insulin contentand GSIS. Molecules identified in the b-cell protectionscreen described here can reduce the excessive calciuminflux by restoring nearly optimal calcium levels and protectislets from all of the above detriments. Unlike the canonicalL-type calcium channel blocker nifedipine, these moleculesallow insulin secretion under conditions of calcium channeloverstimulation. Calcium antagonists with such propertiesmay prove to be a novel class of diabetes treatment andprevention drugs.

Acknowledgments. The authors thank Faye Zhao for uploading RNAseqdata into Gene Expression Omnibus, Yunshan Peng, David Parker, KatsumasaNakajima, Timothy Rasmusson, Toshio Kawanami, T.R. Vedananda, and YongjinGong for synthesizing analogs of compound D, Jeffrey A. Brown for insulinmeasurements, and Martin G. Waters and Jovita Marcinkeviciene (all from NovartisInstitutes for BioMedical Research at the time of this work) for discussions andcritical reading of the manuscript.Duality of Interest. All authors were employees of the Novartis Institutes forBioMedical Research at the time of the reported studies. No other potentialconflicts of interest relevant to this article were reported.Author Contributions. J.V., J.Y., L.S., S.X.W., R.Z., S.F., C.-H.C., A.C.W.,and B.D. performed experiments and analyzed data, A.C., F.Z., G.T., T.M.S, B.D.,D.M.R., X.C., and A.B. contributed to discussions and data analyses. H.X. analyzedRNAseq data. A.B. wrote the manuscript. All authors reviewed and edited themanuscript. A.B. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

References1. Alejandro EU, Gregg B, Blandino-Rosano M, Cras-Méneur C, Bernal-MizrachiE. Natural history of b-cell adaptation and failure in type 2 diabetes. Mol AspectsMed 2015;42:19–412. Poitout V, Amyot J, Semache M, Zarrouki B, Hagman D, Fontés G. Gluco-lipotoxicity of the pancreatic beta cell. Biochim Biophys Acta 2010;1801:289–2983. Rojas J, Bermudez V, Palmar J, et al. Pancreatic beta cell death: novelpotential mechanisms in diabetes therapy. J Diabetes Res 2018;2018:96018014. Back SH, Kaufman RJ. Endoplasmic reticulum stress and type 2 diabetes.Annu Rev Biochem 2012;81:767–7935. Bagnati M, Ogunkolade BW, Marshall C, et al. Glucolipotoxicity initiatespancreatic b-cell death through TNFR5/CD40-mediated STAT1 and NF-kB ac-tivation. Cell Death Dis 2016;7:e23296. Cunha DA, Hekerman P, Ladrière L, et al. Initiation and execution of lipotoxicER stress in pancreatic beta-cells. J Cell Sci 2008;121:2308–23187. El-Assaad W, Joly E, Barbeau A, et al. Glucolipotoxicity alters lipid partitioningand causes mitochondrial dysfunction, cholesterol, and ceramide deposition andreactive oxygen species production in INS832/13 ss-cells. Endocrinology 2010;151:3061–30738. Graciano MF, Valle MM, Kowluru A, Curi R, Carpinelli AR. Regulation of insulinsecretion and reactive oxygen species production by free fatty acids in pancreaticislets. Islets 2011;3:213–2239. Johnson JD, Luciani DS. Mechanisms of pancreatic beta-cell apoptosis indiabetes and its therapies. Adv Exp Med Biol 2010;654:447–46210. Laybutt DR, Preston AM, Akerfeldt MC, et al. Endoplasmic reticulumstress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia 2007;50:752–76311. Luciani DS, Gwiazda KS, Yang TL, et al. Roles of IP3R and RyR Ca21 channels inendoplasmic reticulum stress and beta-cell death. Diabetes 2009;58:422–432

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12. Lupi R, Dotta F, Marselli L, et al. Prolonged exposure to free fatty acids hascytostatic and pro-apoptotic effects on human pancreatic islets: evidence thatbeta-cell death is caspase mediated, partially dependent on ceramide pathway,and Bcl-2 regulated. Diabetes 2002;51:1437–144213. Ly LD, Xu S, Choi SK, et al. Oxidative stress and calcium dysregulation bypalmitate in type 2 diabetes. Exp Mol Med 2017;49:e29114. Barlow J, Jensen VH, Jastroch M, Affourtit C. Palmitate-induced impairmentof glucose-stimulated insulin secretion precedes mitochondrial dysfunction inmouse pancreatic islets. Biochem J 2016;473:487–49615. Giacca A, Xiao C, Oprescu AI, Carpentier AC, Lewis GF. Lipid-inducedpancreatic b-cell dysfunction: focus on in vivo studies. Am J Physiol EndocrinolMetab 2011;300:E255–E26216. Hasnain SZ, Prins JB, McGuckin MA. Oxidative and endoplasmic re-ticulum stress in b-cell dysfunction in diabetes. J Mol Endocrinol 2016;56:R33–R5417. Zhou YP, Grill VE. Long-term exposure of rat pancreatic islets to fatty acidsinhibits glucose-induced insulin secretion and biosynthesis through a glucose fattyacid cycle. J Clin Invest 1994;93:870–87618. Fontés G, Zarrouki B, Hagman DK, et al. Glucolipotoxicity age-dependentlyimpairs beta cell function in rats despite a marked increase in beta cell mass.Diabetologia 2010;53:2369–237919. Gremlich S, Bonny C, Waeber G, Thorens B. Fatty acids decrease IDX-1expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, andsomatostatin levels. J Biol Chem 1997;272:30261–3026920. Hagman DK, Hays LB, Parazzoli SD, Poitout V. Palmitate inhibits insulin geneexpression by altering PDX-1 nuclear localization and reducing MafA expression inisolated rat islets of Langerhans. J Biol Chem 2005;280:32413–3241821. Kelpe CL, Moore PC, Parazzoli SD, Wicksteed B, Rhodes CJ, Poitout V.Palmitate inhibition of insulin gene expression is mediated at the transcriptionallevel via ceramide synthesis. J Biol Chem 2003;278:30015–3002122. Zhang IX, Raghavan M, Satin LS. The endoplasmic reticulum and calciumhomeostasis in pancreatic beta cells. Endocrinology. 2020;161:bqz02823. Gwiazda KS, Yang TL, Lin Y, Johnson JD. Effects of palmitate on ER andcytosolic Ca21 homeostasis in beta-cells. Am J Physiol Endocrinol Metab 2009;296:E690–E70124. Stancill JS, Cartailler JP, Clayton HW, et al. Chronic b-cell depolarizationimpairs b-cell identity by disrupting a network of Ca21-regulated genes. Diabetes2017;66:2175–2187

25. Yang G, Shi Y, Yu J, et al. CaV1.2 and CaV1.3 channel hyperactivation inmouse islet b cells exposed to type 1 diabetic serum. Cell Mol Life Sci 2015;72:1197–120726. Yusta B, Baggio LL, Estall JL, et al. GLP-1 receptor activation improves betacell function and survival following induction of endoplasmic reticulum stress. CellMetab 2006;4:391–40627. Cunha DA, Ladrière L, Ortis F, et al. Glucagon-like peptide-1 agonists protectpancreatic beta-cells from lipotoxic endoplasmic reticulum stress through up-regulation of BiP and JunB. Diabetes 2009;58:2851–286228. Chon S, Gautier JF. An update on the effect of incretin-based therapies onb-cell function and mass. Diabetes Metab J 2016;40:99–11429. Yesil P, Michel M, Chwalek K, et al. A new collagenase blend increases thenumber of islets isolated from mouse pancreas. Islets 2009;1:185–19030. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2:accurate alignment of transcriptomes in the presence of insertions, deletions andgene fusions. Genome Biol 2013;14:R3631. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. NatMethods 2012;9:357–35932. Anders S, Pyl PT, Huber W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–16933. Anders S, Huber W. Differential expression analysis for sequence count data.Genome Biol 2010;11:R10634. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-celldeficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes2003;52:102–11035. Rechsteiner MP, Floros X, Boehm BO, et al. Automated assessment of b-cellarea and density per islet and patient using TMEM27 and BACE2 immunofluo-rescence staining in human pancreatic b-cells. PLoS One 2014;9:e9893236. Wang Y, Gao L, Li Y, Chen H, Sun Z. Nifedipine protects INS-1 b-cell fromhigh glucose-induced ER stress and apoptosis. Int J Mol Sci 2011;12:7569–758037. Xu G, Chen J, Jing G, Shalev A. Preventing b-cell loss and diabetes withcalcium channel blockers. Diabetes 2012;61:848–85638. Yin T, Kuo SC, Chang YY, Chen YT, Wang KK. Verapamil use is associatedwith reduction of newly diagnosed diabetes mellitus. J Clin Endocrinol Metab2017;102:2604–261039. Ovalle F, Grimes T, Xu G, et al. Verapamil and beta cell function in adults withrecent-onset type 1 diabetes. Nat Med 2018;24:1108–1112

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