MondoA Is an Essential Glucose-ResponsiveTranscription Factor in Human Pancreatic b-CellsPaul Richards,1,2,3 Latif Rachdi,1,2,3 Masaya Oshima,1,2,3 Piero Marchetti,4 Marco Bugliani,4

Mathieu Armanet,5 Catherine Postic,1,2,3 Sandra Guilmeau,1,2,3 and Raphael Scharfmann1,2,3

Diabetes 2018;67:461–472 | https://doi.org/10.2337/db17-0595

Although the mechanisms by which glucose regulatesinsulin secretion from pancreatic b-cells are now well de-scribed, the way glucose modulates gene expression insuch cells needs more understanding. Here, we demon-strate that MondoA, but not its paralog carbohydrate-responsive element–binding protein, is the predominantglucose-responsive transcription factor in human pan-creatic b-EndoC-bH1 cells and in human islets. In high-glucose conditions, MondoA shuttles to the nucleus whereit is required for the induction of the glucose-responsivegenes arrestin domain–containing protein 4 (ARRDC4)and thioredoxin interacting protein (TXNIP), the latter beinga protein strongly linked to b-cell dysfunction and diabe-tes. Importantly, increasing cAMP signaling in humanb-cells, using forskolin or the glucagon-like peptide 1mimetic Exendin-4, inhibits the shuttling of MondoA andpotently inhibits TXNIP and ARRDC4 expression. Further-more, we demonstrate that silencing MondoA expressionimproves glucose uptake in EndoC-bH1 cells. These re-sults highlightMondoA as a novel target inb-cells that coor-dinates transcriptional response to elevated glucose levels.

In eukaryotic cells, glucose uptake and metabolism representa major source of energy, but are also a strong regulator ofgene expression and cellular function. Pancreatic b-cellsrepresent a model system to dissect these processes, becausethey are responsible for orchestrating the response of thebody to rising postprandial glucose levels by secreting insulinto avoid excessive hyperglycemia. Glucose enters b-cells viaGLUTs and is first metabolized through the high-Km gluco-kinase (GK; hexokinase IV), which is considered to be “glu-cose sensor” of the b-cell (1). After this, insulin secretion

occurs through a process of cellular depolarization via ATP-sensitive potassium channels, calcium entry, vesicle dock-ing, and exocytosis (2). The incretin hormones glucagon-likepeptide 1 (GLP-1) and gastric inhibitory polypeptide furtheramplify insulin secretion. Both hormones act directly onb-cells to elevate intracellular cAMP levels and promotesecretion downstream of glucose sensing. Both hormonesalso activate the transcription factor cAMP-responsiveelement–binding protein and thereby influence the b-celltranscriptome (3).

Although the critical role of glucose on insulin secretionis now well described both in rodent and human b-cells (2),the effect of glucose on the b-cell transcriptome has beenless explored. Long-term hyperglycemic conditions havebeen shown to be detrimental to b-cell function, leadingto decreased insulin transcription, synthesis, and secretiongiving rise to the concept of glucolipotoxicity (4). However,there is a limited understanding of the shorter-term effectsof glucose on the b-cell transcriptome, particularly in humanmodels.

Carbohydrate-responsive transcription factors have emergedas major mediators of glucose action on gene expression.Adipocytes and hepatocytes express the carbohydrate-responsive element–binding protein (ChREBP), also namedMondoB, whereas skeletal muscle cells express its paralogMondoA (5,6). Both transcription factors reside in the cy-toplasm in low-glucose conditions and undergo nucleartranslocation in high-glucose conditions. They belong tothe same family, with ChREBP encoded by the MLX inter-acting protein–like (MLXIPL) gene and MondoA encoded bythe MLX interacting protein (MLXIP). ChREBP and MondoAare multidomain proteins with highly homologous N-terminal

1INSERM U1016, Cochin Institute, Paris, France2CNRS UMR 8104, Paris, France3University of Paris Descartes, Sorbonne Paris Cité, Paris, France4Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy5Cell Therapy Unit Hospital Saint-Louis and University Paris-Diderot, Paris, France

Corresponding author: Raphael Scharfmann, raphael.scharfmann@inserm.fr.

Received 22 May 2017 and accepted 15 December 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0595/-/DC1.

© 2017 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 http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, March 2018 461

ISLETSTUDIES

and COOH-terminal regions. They contain a bHLHZ (basichelix-loop-helix leucine zipper) region and a COOH-terminaldimerization domain mediating DNA binding and hetero-dimerization with a common binding protein named MLX.Both ChREBP and MondoA contain transcriptional activationdomains, whereas MLX is much shorter and lacks intrinsictransactivation capacity (7,8). The two complexes bind thecarbohydrate response element consensus sequence in pro-moter regions of specific target genes (7). In hepatocytes,ChREBP shifts the cellular state to maximize glucose storageas lipids by upregulating glycolytic and lipogenic genes (9).In skeletal muscle, the activation of MondoA, which is pre-dominant in this cell type, suppresses glucose uptake viaincreased expression of thioredoxin interacting protein (TXNIP)and the arrestin domain–containing protein 4 (ARRDC4)(10,11).

Previous work focusing on the immediate effects of glucoseon the b-cell transcriptome has mainly used rodent models.This research has indicated a role for ChREBP in long-termcellular deterioration via lipotoxicity (12,13) or in promot-ing cellular proliferation (14). Recently, the effect of glucosein rat pancreatic cells was shown to occur via two transcrip-tional programs, one directly dependent on ChREBP andthe other downstream of target genes of ChREBP (15).Furthermore, only limited insights have been obtained usinghuman models. An early study (16) analyzing the transcrip-tional change of human islets exposed to high glucose levelsfor 24 h demonstrated TXNIP as the most highly upregu-lated gene. Importantly, the contribution of paralog tran-scription factor of ChREBP, MondoA, to glucose sensing inpancreatic cells has not been investigated.

Here, we probed the effect of glucose using the recentlydeveloped glucose-responsive EndoC-bH1 human cell line(17) and human islets. In contrast to previous reports inrodents, we found no role for ChREBP in upregulating gly-colytic or lipogenic genes in response to short-term high-glucose treatment (1–24 h). In contrast to ChREBP, weobserved that MondoA was responsive to high glucose con-centrations in EndoC-bH1 cells and islets, leading to sub-sequent upregulation of TXNIP and ARRDC4 expression,thereby reducing cellular glucose uptake. Taken together,we propose that MondoA is an essential glucose-responsivetranscription factor in human b-cells.

RESEARCH DESIGN AND METHODS

Culture of Human b-Cell LineEndoC-bH1 cells (17) were cultured in low-glucose (5.6mmol/L) DMEM (Sigma-Aldrich) with 2% BSA fraction V(RocheDiagnostics), 50mmol/L 2-mercaptoethanol, 10mmol/Lnicotinamide (Calbiochem), 5.5 mg/mL transferrin (Sigma-Aldrich), 6.7 ng/mL selenite (Sigma-Aldrich), 100 units/mLpenicillin, and 100 mg/mL streptomycin. Cells were seededat a 40% confluence on plates coated with Matrigel (1%;Sigma-Aldrich)/fibronectin (2 mg/mL; Sigma-Aldrich). Cellswere cultured at 37°C and 5% CO2 in an incubator andpassaged once a week when they were 90–95% confluent.

The glucose, forskolin, GLP-1, mannoheptulose, H89, U0126,and PD98059 used in the experiment were from Sigma-Aldrich. cAMP-dependent protein kinase (PKA) inhibitor14–22 amide (PKAi) was from Calbiochem.

Human IsletsHuman islets were obtained from seven donors (mean age55.8 6 7.5 years; BMI 27.5 6 1.5 kg/m2). Up to 100handpicked islets were deposited in each well of a 12-wellplate and cultured in the same culture medium as used forEndoC-bH1 cells.

Mouse IsletsChREBP2/2 mice were previously described (18). Wild-typeand homozygous ChREBP knockout (ChREBP2/2) mice wereused in accordance with the guidelines of the French AnimalCare Committee. The mice were bred on a genetic C57BL/6J background and raised in a 12-h light/dark cycle. Theywere fed a standard laboratory chow diet. Islets were isolat-ed from 12-week-old mice by collagenase digestion (Sigma-Aldrich) followed by direct handpicking. After overnightculture in DMEM containing 0.5 mmol/L glucose, groups of50 islets in triplicate were preincubated for 8 h in DMEMcontaining 0.5 or 20 mmol/L glucose.

Small Interfering RNA Transfection of EndoC-bH1 Cellsand Human IsletsEndoC-bH1 cells were passaged and transfected usingLipofectamine RNAiMAX (Life Technologies) 24 h later.ON-TARGETplus small interfering RNA (siRNA) SMARTpoolfor human MLXIPL, MLXIP, or MLX, or ON-TARGETplusnontargeting control pool (siN) were used (Dharmacon).Briefly, siRNA and Lipofectamine RNAiMAX were combinedin OptiMEM and applied to the cells. Three hours later,medium was changed for fresh culture medium. Cells wereharvested 4–5 days post-transfection, with preliminary ex-periments showing that siRNA knockdown was consistentlysustained for .7 days post-transfection.

On the day of receiving the samples, ;100 human isletswere handpicked for each condition, washed in PBS, andtreated with 1 mL of Accutase (PAA Laboratories) for 5 min.Partial dissociation of the human islets was achieved withslow pipetting and Accutase was removed after a centrifuga-tion step. The cell clusters were gently suspended in Opti-MEM, and siN, siMlxip, or siMlxipl Lipofectamine RNAiMaxcomplexes were added. Cell clusters were plated on platescoated with Matrigel (1%)/fibronectin (2 mg/mL). After4–5 h, an equal amount of EndoC-bH1 cell culture mediumwas added. Three days later, the medium was changed tolow-glucose (1 mmol/L) culture medium for 5 h, and thenglucose (to 20 mmol/L) was added to half of the wells. After16 h, the cells were harvested.

RNA Isolation, Reverse Transcription, and QuantitativePCRRNeasy Micro Kit (Qiagen) was used to extract total RNAfrom EndoC-bH1 cells and human islets. A First StrandcDNA Kit (Thermo Fisher Scientific) was used to synthesizecDNA. Quantitative RT-PCR was performed using Power

462 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

SYBR Green mix (Applied Biosystems) with a QuantStudioanalyzer. Custom primers were designed with Primer-Questonline software (IDT), and the efficiency was determinedfor each with a serial dilution of cDNA samples fromEndoC-bH1 cells or human islets. Cyclophilin-A transcriptlevels were used for the normalization of each sample.

Transcriptome Analysis and Access to Raw DataTranscriptomic profiles were obtained using GeneChip Hu-man Gene 2.0 ST Array (Affymetrix), following the manufac-turer instructions. Microarray data and all experimentaldetails are available in the Gene Expression Omnibus (GEO)database (accession GSE98501).

ImmunostainingEndoC-bH1 cells were cultured on Matrigel/fibronectin-coated glass coverslips. Cells were starved in low-glucose(0.5 mmol/L) culture medium overnight and exposed tolow-glucose (0.5 mmol/L) or high-glucose (20 mmol/L) culturemedium for 3 h the following day. Cells were fixed using 4%paraformaldehyde for 30 min and processed for immunostain-ing by blocking in 3% BSA, 2% serum, and 0.1% Tween-20for 30 min. The cells were exposed to a primary antibodyagainst MondoA raised in rabbit (ProteinTech) over-night at 4°C, washed three times, then exposed to a fluo-rescent anti-rabbit antibody for 2 h. Nuclei were stainedwith Hoechst 33342 fluorescent stain (Life Technologies).Images were acquired with a Leica Leitz Fluorescent Micro-scope equipped with cooled three-chip charge-coupled de-vice camera (model C5810; Hamamatsu) and processedusing ImageJ software.

ImmunoblottingEndoC-bH1 cells at 80% confluence were starved in low-glucose (0.5 mmol/L) culture medium overnight and exposedto different test compounds the following day for 3 h. Thenuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific), and protein concentrations were quantifiedusing a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).Proteins (40 mg) were resolved by SDS-PAGE and trans-ferred to a membrane using an iBlot2 Gel Transfer Device(Thermo Fisher Scientific). Membranes were immunoblot-ted using antibodies against PDX1 (1:1,000) (19), tubulin(1:2,000; Sigma-Aldrich), ChREBP (1:1,000; Novus Bio-logicals), or MondoA (1:500; ProteinTech). After washing,membranes were incubated with species-specific horse-radish peroxidase–linked secondary antibodies (1:10,000),washed again, and visualized after enhanced chemilumines-cence exposure.

Glucose Uptake AssayWe transfected EndoC-bH1 cells with siRNA SMARTpoolagainst human MLXIPL, MXIP, or siN. Five days later, cellswere starved in low-glucose medium (0.5 mmol/L) for 6 h.Next, cells were cultured overnight in media containing20 mmol/L glucose. Glucose uptake was measured usingthe calorimetric Glucose Uptake Assay Kit (Abcam) asper the instruction manual.

StatisticsData were analyzed using GraphPad Prism 6 software andare presented as the mean 6 SD. For comparison betweentwo mean values, statistical significance was estimated us-ing a two-tailed Student t test. For comparison among threeor more values, one-way ANOVA was used with Bonferronipost hoc test (repeated-measures).

RESULTS

Glucose Upregulates a Limited Number of Genesin Human b-CellsWe searched for genes whose expression was induced byglucose in EndoC-bH1. Microarray analysis was used tocompare transcriptional profiles of EndoC-bH1 cells after8 h of exposure to low or high glucose, with or withoutforskolin. Expression profiles are depicted in Fig. 1 as scat-ter plots, which show the relative intensities of all probesrepresented on the microarrays. High glucose exposureupregulated a limited number of genes, and the most prom-inent was TXNIP (Fig. 1A). The scarce number of upregu-lated genes observed was apparent when compared withthe effects observed after forskolin treatment (Fig. 1B)(6 vs. 75 genes greater than twofold upregulated in eachcondition, respectively). Surprisingly, analyses of microarraydata indicated that in EndoC-bH1 cells, glucose did notsignificantly upregulate the vast majority of known ChREBPtargets (Fig. 1C). Specifically, although glucose treatmentincreased TXNIP mRNA levels, it did not modulate theexpression of ACACA, MLXIPL, PFKL, PKLR, SCD, RGS16,HBEGF, GPD1, and RORC (Fig. 1C).

The Majority of ChREBP Target Genes Are NotSignificantly Affected by Glucose in Human b-Cell Linesand in Human IsletsTo further investigate the upregulation of glucose-dependentgenes, EndoC-bH1 cells were exposed to different concen-trations of glucose (1, 5, 15, or 20 mmol/L) for 8 h. Quanti-tative PCR (qPCR) analyses indicated that 15 and 20 mmol/Lglucose caused a robust induction in TXNIP gene expression,whereas 5 mmol/L did not (Fig. 2A). The glucose-dependentstimulation of TXNIP gene expression has been attributedto ChREBP in rodent b-cells (20). qPCR analyses indicatedthat, with the exception of TXNIP, all other ChREBP targetgenes tested (ACACA,MLXIPL, PFKL, PKLR, RGS16, HBEGF,GPD1, and RORC) were not significantly upregulated inEndoC-bH1 cells exposed to 20 mmol/L glucose for 1, 4,8, or 24 h (Fig. 2B and C and Supplementary Fig. 1). To testwhether this was also the case in primary human cells,human islets from donors were exposed to media contain-ing 1 or 20 mmol/L glucose, and, consistently, only TXNIPwas found to be significantly upregulated (Fig. 2D and Eand Supplementary Fig. 2).

TXNIP Expression in Human b-Cell Lines and in HumanIsletsTo elucidate whether the glucose-dependent activation ofTXNIP required glucose metabolism, EndoC-bH1 cells andhuman islets were preincubated with 25 mmol/L of GK

diabetes.diabetesjournals.org Richards and Associates 463

inhibitor mannoheptulose. This prevented the glucose-dependent upregulation of TXNIP (Supplementary Fig. 3Aand B). Furthermore, forskolin and the GLP-1 mimeticExendin-4, which both activate the cAMP pathway, de-creased glucose-induced TXNIP expression in both EndoC-bH1 cells and human islets (Supplementary Fig. 3C and D).Taken together, glucose upregulates TXNIP expression similarlyin both human b-cell lines and human islets, and this effectis dependent on glucose metabolism and cAMP signaling.

The Glucose-Dependent Upregulation of TXNIP Is NotBlunted in Islets From ChREBP2/2 MiceTo establish the necessity of ChREBP in glucose-induced TXNIPexpression, islets from wild-type and ChREBP2/2 micewere isolated and exposed to either low (0.5 mmol/L) orhigh (20 mmol/L) glucose for 8 h. qPCR analyses indicatedthat txnip induction by glucose was similar in both groups(Fig. 3), demonstrating that ChREBP is not required todrive txnip expression upon glucose stimulation in rodentislets.

MondoA Is Robustly Expressed in Rodent and HumanIslet Cells and in EndoC-bH1 CellsTo the best of our knowledge, the expression and functionof the ChREBP paralog MondoA has not been evaluated in

detail in pancreatic b-cells. qPCR indicated that humanEndoC-bH1 cells and human islets expressed high levelsof ChREBP (MLXIPL) and MondoA (MLXIP) as well as theirobligatory binding partner MLX (Fig. 4A and B). Moreover,data from GeneChip (Affymetrix) analyses indicated thatARRDC4, a key target of MondoA in skeletal muscles (10),was significantly upregulated by high glucose concentra-tions in EndoC-bH1 cells (Fig. 1C). This upregulation wasvalidated by qPCR both in EndoC-bH1 cells (Fig. 4C) and inhuman islets (Fig. 4D), and was prevented by GK inhibitionand cAMP activation (Fig. 4E and F) in a similar fashion toTXNIP (Supplementary Fig. 3).

High Glucose Causes MondoA Nuclear Translocationin EndoC-bH1 CellsBoth MondoA and ChREBP are localized to the cytoplasmin an inactive state, and a glucose stimulus causes theirtranslocation to the nucleus (7). To determine how glucosesignals in b-cells, we studied the cytoplasmic and nuclearlocalization of MondoA and ChREBP in EndoC-bH1 cellsafter glucose treatment. Immunoblotting for MondoArevealed that 3 h of 20 mmol/L glucose exposure causedMondoA nuclear translocation (Fig. 5A), which was furtherconfirmed by immunocytochemistry (Fig. 5B and C for

Figure 1—Glucose upregulates a limited number of genes in human b-cells. Microarray expression profiles of EndoC-bH1 cells exposed for 8 hto low (0.5 mmol/L) vs. high (20 mmol/L) glucose (A) or high glucose with or without forskolin (25 mmol/L) (B) are presented as a scatter plot.Robust multi-array average intensities of each microarray probe are plotted on a log(2) scale. Dashed lines represent a twofold difference.C: Glucose-responsive genes from the transcriptomic analyses are shown, ranked by the fold change. n = 3.

464 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

quantification). Of note, the nuclear translocation of Mon-doA was prevented by forskolin, consistent with the in-hibition of ARRDC4 and TXNIP transcription by thecompound (Fig. 5A). The effects of forskolin were mediatedby PKA. Indeed, PKA inhibitors (PKAi and H89) butnot mitogen-activated protein kinase/extracellular signal–

regulated kinase (ERK) kinase (U0126) or ERK (PD98059)inhibitors blunted the effects of forskolin on glucose-induced TXNIP (Fig. 5D). Surprisingly, we did not observeChREBP nuclear translocation upon glucose exposure (Fig.5E), indicating that MondoA, but not ChREBP, is sensitiveto short-term glucose in EndoC-bH1 cells.

Figure 2—Glucose upregulates TXNIP but no other ChREBP target genes in EndoC-bH1 cells and human islets. A: EndoC-bH1 cells wereexposed to different glucose concentrations for 8 h, and TXNIP expression was quantified by qPCR. B–E: A variety of these genes was analyzedby qPCR after different lengths of glucose exposure in EndoC-bH1 cells (B and C) and human islets (D and E). n = 3–5. *P , 0.05; **P , 0.01;***P , 0.001.

Figure 3—Islets from ChREBP2/2 (KO) mice still have robust glucose-dependent upregulation of txnip. qPCR of ChREBP (mlxipl) (A) and txnip(B) in wild-type (WT) or ChREBP2/2 islets cultured ex vivo in high or low glucose for 8 h. n = 9. ***P , 0.001.

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MondoA Regulates Glucose-Dependent GeneExpression and Glucose Uptake in EndoC-bH1 Cells

To establish whether MondoA is the primary glucose-responsive transcription factor in human b-cells, itsencoding mRNA, or that of ChREBP or their obligatorybinding partner MLX, were knocked down in EndoC-bH1

cells using siRNA (Fig. 6A–C). ChREBP silencing had noeffect on the glucose-induced upregulation of TXNIP orARRDC4 (Fig. 6D and E). On the other hand, knockingdownMondoA or MLX significantly compromised the effectof glucose on both ARRDC4 and TXNIP upregulation (Fig.6D and E).

Figure 4—ChREBP, MondoA, and ARRDC4 expression in human b-cells. The expressions of ChREBP (MLXIPL), MondoA (MLXIP), and MLXwere analyzed by qPCR in EndoC-bH1 cells (A) and human islets (B) exposed to different glucose concentrations. ARRDC4 expression wasanalyzed by qPCR in EndoC-bH1 cells (C) and human islets (D–F) exposed to glucose (C and D) or glucose plus mannoheptulose (E) or forskolin(F). n = 3–8. *P , 0.05; ***P , 0.01; ****P , 0.0001.

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Figure 5—MondoA, but not ChREBP, localizes in the nucleus under high-glucose conditions in EndoC-bH1 cells. A: Western blot of cytoplas-mic and nuclear fractions of EndoC-bH1 cells exposed to low (0.5 mmol/L) or high (20 mmol/L) glucose with forskolin for 3 h. The membraneswere hybridized with antibodies against tubulin, PDX1, and MondoA. B: Immunocytochemistry of EndoC-bH1 cells exposed to low or highglucose for 3 h, stained with an antibody against MondoA (green) and Hoechst stain (blue). Scale bars, 20 mm. C: Quantification of EndoC-bH1cells with a MondoA nuclear staining after 3 h of incubation at low or high glucose levels. n = 3. ***P , 0.001. D: The effect of PKA,mitogen-activated protein kinase/ERK kinase, or ERK inhibitors on the expression of TXNIP was analyzed by qPCR in EndoC-bH1 cells treatedfor 8 h with 0.5 or 20 mmol/L glucose with or without forskolin. n = 3. ***P , 0.001. E: Western blot of cytoplasmic and nuclear fractions ofEndoC-bH1 cells exposed to low or high glucose plus forskolin for 3 h. The membranes were hybridized with antibodies against tubulin, PDX1,and ChREBP.

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ARRDC4 and TXNIP both control glucose uptake inskeletal muscle cells (21,22). To determine whether Mon-doA regulates glucose uptake in human b-cells, glucoseuptake was measured in EndoC-bH1 cells. MondoAknockdown selectively suppressed TXNIP and ARRDC4gene expression in EndoC-bH1 cells, whereas ChREBP si-lencing had no effect on these genes (Supplementary Fig. 4).Consistent with this observation, glucose uptake was

unaltered in ChREBP-silenced cells but was signifi-cantly higher in cells with reduced MondoA expression(Fig. 6F).

MondoA Regulates Glucose-Dependent Transcription inPrimary Human IsletsFinally, we asked whether the glucose-dependent regula-tion of ARRDC4 and TXNIP is also MondoA dependent in

Figure 6—MondoA is necessary for the upregulation of TXNIP and ARRDC4 in response to glucose in human b-cells. A–E: ChREBP (MLXIPL),MondoA (MLXIP), or MLX genes were knocked down in EndoC-bH1 cells using siRNA. Four days post-transfection the cells were starvedovernight in low glucose (0.5 mmol/L) medium and the following day they were stimulated with high (20 mmol/L) glucose for 8 h. The expressionof ChREBP (MLXIPL) (A), MondoA (MLXIP) (B), MLX (C), TXNIP (D), and ARRDC4 (E) was measured by qPCR. F: A glucose uptake assay wasused to measure glucose transport 5 days after siRNA knockdown of ChREBP or MondoA in EndoC-bH1 cells. n = 3–6. **P , 0.01; ***P ,0.001; ****P , 0.0001.

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primary human b-cells. For this purpose, we performedsiRNA-mediated knockdown of MondoA and ChREBP inhuman islets. ChREBP siRNA induced a nearly 80% de-crease in ChREBP RNA steady-state level, whereas MondoAsiRNA induced a nearly 70% decrease in MondoA RNAsteady-state levels without any impact on ChREBP mRNAlevels (Fig. 7A and B). Importantly, and consistent withdata obtained in EndoC-bH1 cells, ChREBP knockdowndid not alter glucose-induced ARRDC4 and TXNIP upregu-lation, whereas such inductions were blunted after MondoAknockdown (Fig. 7C and D).

DISCUSSION

There have been limited observations of the effects of glucoseon the human b-cell transcriptome. Here, using EndoC-bH1cells and islets from organ donors, we demonstrated thatthe glucose-responsive transcription factor MondoA is ro-bustly expressed in pancreatic b-cells. MondoA undergoesnuclear translocation in high-glucose conditions where itupregulates the expression of a limited number of genesincluding TXNIP and ARRDC4, both of which are involvedin glucose uptake inhibition. Therefore, we conclude thatMondoA is the glucose-responsive transcription factor inhuman pancreatic b-cells.

Studies have previously analyzed glucose-regulated geneexpression in rodent b-cells using either b-cell lines or isletpreparations, whereas fewer data have been generated us-ing human b-cells (15). However, improving knowledge of

human b-cell physiology is crucial, as, despite many simi-larities, rodent and human b-cells differ on various specificpoints, including disparities in b-cell function (23,24). Thelimited number of studies using human b-cells mainlystems from the difficulty in accessing primary human isletpreparations that derive from deceased donors. Availablehuman islets often come from transplantation rejectionand therefore are not in an optimal state for furtherex vivo studies. Moreover, they contain b-cells in differentproportions from one preparation to another, giving riseto data variability (25). The difficulty of generating func-tional human b-cell lines has also represented a major lim-itation for decades (26). The present work mainly focuseson glucose-regulated gene expression in pancreatic b-cells ina human context, using both human islets from donors andthe recently developed functional human b-cell line EndoC-bH1 (17). In these two models, we observed a very limitednumber of genes that were efficiently affected by short-term high-glucose treatment. This is in accordance witha previous microarray analysis (16) reporting only 14 genessignificantly upregulated in human islets upon 24-h expo-sure to high-glucose exposure. Of note, only five of thesegenes were induced more than twofold, with TXNIP beingthe most upregulated gene. In contrast, it was recentlyreported that .2,000 genes are significantly induced byglucose after a 12-h treatment in the rat b-cell line INS-1(15). Although improvements in the sensitivity of recenttranscriptomic tools can account for some increase in the

Figure 7—Human islets with knockdown of MondoA have a compromised glucose-dependent induction of TXNIP and ARRDC4. ChREBP(MLXIPL) or MondoA (MLXIP) was knocked down in human islets using siRNA. Three days post-transfection, the cells were starved for 6 h inlow-glucose (1 mmol/L) medium and stimulated overnight with high (20 mmol/L) glucose. The expressions of ChREBP (A), MondoA (B), TXNIP(C), and ARRDC4 (D) were measured by qPCR. n = 3. *P , 0.05; **P , 0.01; ***P , 0.001; ****P , 0.0001.

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number of detectable genes, a clear underlying differencebetween the two-species transcriptional response to glucoseappears to exist. Among known glucose-related species dif-ferences, glucose transport in rodent b-cells is mainly de-pendent on GLUT2 (SLC2A2), a low-Km GLUT, whereas thehigh-Km GLUT1 (SLC2A1) plays a major role in humanb-cells (27); Moreover, the panel of voltage-gated ion chan-nels differs between rodent and human b-cells (28), anddifferent set points for glucose-stimulated insulin secretionhave been reported in these two species (28). Regardingglucose-induced cell cycle entry, glucose efficiently activatesa large set of cell cycle–related genes that activate b-cellproliferation in rodent b-cells (29), a process that is lessefficient in the case of human b-cells (30). Altogether, thesedata suggest that human b-cells could be quantitatively lesssensitive to glucose in terms of the regulation of gene ex-pression than rodent b-cells.

Historically, it was first thought that in pancreatic b-cellsthe upstream stimulatory factors regulated glucose stimu-lation of gene expression (31). ChREBP was next proposedas the b-cell carbohydrate-responsive transcription factorbased on a series of arguments. ChREBP was first reportedas expressed in pancreatic islets and in b-cell lines (32) andits overexpression in INS-1 cells using the Tetracycline-Onsystem improved the glucose-dependent induction of itstarget gene L-pyruvate kinase (LPK) (32). Moreover, over-expression of a constitutively active mutant of ChREBP inINS-1 cells led to the induction of several ChREBP targetgenes, including TXNIP (33). Finally, glucose was shown toregulate gene expression in pancreatic b-cells through thecarbohydrate response element consensus sequence thatChREBP binds (34). However, the direct evidence thatChREBP acts as the primary glucose transcription factorhas not always been consistent. Indeed, although studieshave reported that chrebp knockdown in INS-1 cells altersglucose-stimulated txnip expression (34), others have foundeither no or limited effects in both INS-1 cells and rat islets(35). Here, we demonstrate that islets from ChREBP2/2

animals have a similar upregulation of txnip comparedwith islets from wild-type littermates. Although this resultdoes not preclude a role for ChREBP in pancreatic b-cells, itdirectly demonstrates that ChREBP is not necessary forglucose-stimulated txnip expression in mouse pancreatic is-let cells. Other functions for ChREBP in human islets mayyet be discovered, which could relate to its described nucleartranslocation and activation under periods of endoplasmicreticulum stress induced by thapsigargin (36).

A key finding of the present work is that MondoA playsa major role in glucose-stimulated gene expression inhuman pancreatic b-cells. We observed that MondoA isexpressed in b-cells. Moreover, glucose induced MondoAnuclear translocation and activated the expression of itstarget genes, as is the case in muscle cells (10). It waspreviously postulated, based on data from HEK293Tcells expressing different forms of hexokinase, thatMondoA activity requires glucose metabolism. We dem-onstrate here that this is also the case in human b-cells

as MondoA activity was inhibited by mannoheptulose, aninhibitor of the GK enzyme, the hexokinase that catalyzesthe first reaction in the glycolytic pathway in pancreaticb-cells.

Our experiments performed with either forskolin or theGLP-1 receptor agonist Exendin-4 demonstrated that thetranslocation of MondoA as well as its transcriptionalactivity were inhibited by cAMP signaling. Although thistype of inhibitory effect has been observed for theregulation of ChREBP activity in the liver (37), cAMP ef-fects of MondoA translocation have not been previouslydescribed. ChREBP is phosphorylated by PKA at Ser196 inresponse to glucagon, leading to a cytoplasmic localizationthat involves the 14–3–3 protein (37). This specific PKAphosphorylation site has not been described in the MON-DOA sequence, and our results may suggest another PKAphosphorylation site that needs to be characterized.

Interestingly, previous data indicated that Exendin-4acts as an antiapoptotic agent on b-cells by decreasingtxnip expression, though the authors did not elucidatethe mechanism by which Exendin-4 exerted this inhibitoryeffect (38). Our data would suggest that this antiapoptoticeffect of Exendin-4 occurs through MondoA inhibition. Re-cently, an inhibitor of MondoA has been reported to havebeneficial effects on insulin and glucose handling in high fat–fed mice (39). Based on our findings of MondoA actions inb-cells, it would be interesting to explore whether its spe-cific inhibition here could be in part responsible for theobserved improved glucose handling. Of note, glucose,which induces insulin secretion, also activates the expres-sion of TXNIP and ARRDC4, both of which are involved inglucose uptake inhibition. These new data suggest that glu-cose entrance in human b-cells induces a transcriptionalresponse that triggers a negative regulatory feedback loopon glucose uptake, which might contribute to the transitionalglucose signal in these cells.

Our data indicate that MondoA plays an essential role inglucose-stimulated gene expression in human pancreaticb-cells. However, its functional role in b-cells is not yetelucidated. It has been demonstrated that its paralogChREBP is required for glucose-stimulated b-cell prolif-eration (14). Indeed, the loss of ChREBP decreases glucose-induced BrdU incorporation in isolated human b-cells andin b-cells isolated from ChREBP-deficient mice. Given theevidence for the involvement of MondoA in cancer cell pro-liferation through its transcriptional control of TXNIP andsubsequent effects on glucose uptake (40), future studiesusing MondoA-deficient mice will be useful to determinewhether MondoA is implicated in glucose-mediated b-cellproliferation in physiological conditions, but also after b-cellinjury.

Until now, studies on the regulation of gene expres-sion by glucose in pancreatic b-cells has mainly focused onChREBP because of the many similarities between hepato-cytes and b-cells, including endodermal origins and meta-bolic functions. However, our study notably demonstratesfor the first time that its paralog, MondoA, is an essential

470 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

glucose-responsive transcription factor in human pancreaticb-cells.

Acknowledgments. The authors thank Dr. B.B. Kahn (Harvard MedicalSchool, Boston, MA) for sharing ChREBP2/2 mice and Dr. D.E. Ayer (Universityof Utah, Salt Lake City, UT) for suggestions on MondoA antibodies. The authorsalso thank the transcriptomic platform from the Cochin Institute for performingarray hybridizations and N. Glaser (INSERM U1016) for help in further dataanalyses.Funding. P.R. was supported by a postdoctoral grant from Agence Nationalede la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73). This study was funded by the Cochin Internal Program PIC (to L.R. andS.G.). The R.S. laboratory is supported by Agence Nationale de la Recherche (ANR-10-LABX-73) and the Bettencourt Schueller Foundation. The C.P. and R.S. researchgroups belong to the Département Hospitalo Universitaire (DHU). This project re-ceived funding from the Innovative Medicines Initiative 2 Joint Undertaking undergrant agreement number 115881 (RHAPSODY). This Joint Undertaking receivessupport from the European Union’s Horizon 2020 research and innovation pro-gramme and EFPIA. This work is supported by the Swiss State Secretariat forEducation Research and Innovation (SERI) under contract number 16.0097.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. P.R. conceptualized the work, performed inves-tigations, and wrote the manuscript. L.R. and S.G. conceptualized the work,performed investigations, and reviewed and edited the manuscript. M.O. performedinvestigations. P.M., M.B., and M.A. provided human islets. C.P. conceptualized thework and reviewed and edited the manuscript. R.S. conceptualized the work, wrotethe manuscript, and supervised the work. R.S. is the guarantor of this work and, assuch, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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