Mondoa Is an Essential Glucose-Responsive Transcription Factor in Human Pancreatic B-Cells

Diabetes Volume 67, March 2018 461

MondoA Is an Essential Glucose-Responsive Transcription Factor in Human Pancreatic b-Cells

Paul 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 regulates occurs through a process of cellular depolarization via ATP- insulin secretion from pancreatic b-cells are now well de- sensitive potassium channels, calcium entry, vesicle dock- scribed, the way glucose modulates gene expression in ing, and exocytosis (2). The incretin hormones glucagon-like such cells needs more understanding. Here, we demon- peptide 1 (GLP-1) and gastric inhibitory polypeptide further strate that MondoA, but not its paralog carbohydrate- amplify insulin secretion. Both hormones act directly on responsive element–binding protein, is the predominant b-cells to elevate intracellular cAMP levels and promote glucose-responsive transcription factor in human pan- secretion downstream of glucose sensing. Both hormones b b creatic -EndoC- H1 cells and in human islets. In high- also activate the transcription factor cAMP-responsive SE STUDIES ISLET glucose conditions, MondoA shuttles to the nucleus where element–binding protein and thereby inﬂuence the b-cell it is required for the induction of the glucose-responsive transcriptome (3). genes arrestin domain–containing protein 4 (ARRDC4) Although the critical role of glucose on insulin secretion and thioredoxin interacting protein (TXNIP), the latter being is now well described both in rodent and human b-cells (2), a protein strongly linked to b-cell dysfunction and diabe- b tes. Importantly, increasing cAMP signaling in human the effect of glucose on the -cell transcriptome has been b-cells, using forskolin or the glucagon-like peptide 1 less explored. Long-term hyperglycemic conditions have mimetic Exendin-4, inhibits the shuttling of MondoA and been shown to be detrimental to b-cell function, leading potently inhibits TXNIP and ARRDC4 expression. Further- to decreased insulin transcription, synthesis, and secretion more, we demonstrate that silencing MondoA expression giving rise to the concept of glucolipotoxicity (4). However, improves glucose uptake in EndoC-bH1 cells. These re- there is a limited understanding of the shorter-term effects sults highlight MondoA as a novel target in b-cells that coor- of glucose on the b-cell transcriptome, particularly in human dinates transcriptional response to elevated glucose levels. models. Carbohydrate-responsive transcription factors have emerged as major mediators of glucose action on gene expression. In eukaryotic cells, glucose uptake and metabolism represent Adipocytes and hepatocytes express the carbohydrate- a major source of energy, but are also a strong regulator of responsive element–binding protein (ChREBP), also named gene expression and cellular function. Pancreatic b-cells MondoB, whereas skeletal muscle cells express its paralog represent a model system to dissect these processes, because MondoA (5,6). Both transcription factors reside in the cy- they are responsible for orchestrating the response of the toplasm in low-glucose conditions and undergo nuclear body to rising postprandial glucose levels by secreting insulin translocation in high-glucose conditions. They belong to to avoid excessive hyperglycemia. Glucose enters b-cells via the same family, with ChREBP encoded by the MLX inter- GLUTs and is ﬁrst metabolized through the high-Km gluco- acting protein–like (MLXIPL) gene and MondoA encoded by kinase (GK; hexokinase IV), which is considered to be “glu- the MLX interacting protein (MLXIP). ChREBP and MondoA cose sensor” of the b-cell (1). After this, insulin secretion are multidomain proteins with highly homologous N-terminal

1INSERM U1016, Cochin Institute, Paris, France This article contains Supplementary Data online at http://diabetes 2CNRS UMR 8104, Paris, France .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0595/-/DC1. 3 University of Paris Descartes, Sorbonne Paris Cité, Paris, France © 2017 by the American Diabetes Association. Readers may use this article as 4 Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy long as the work is properly cited, the use is educational and not for profit, and the 5 Cell Therapy Unit Hospital Saint-Louis and University Paris-Diderot, Paris, France work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Raphael Scharfmann, [email protected]. .org/content/license. Received 22 May 2017 and accepted 15 December 2017. 462 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018 and COOH-terminal regions. They contain a bHLHZ (basic The glucose, forskolin, GLP-1, mannoheptulose, H89, U0126, helix-loop-helix leucine zipper) region and a COOH-terminal and PD98059 used in the experiment were from Sigma- dimerization domain mediating DNA binding and hetero- Aldrich. cAMP-dependent protein kinase (PKA) inhibitor dimerization with a common binding protein named MLX. 14–22 amide (PKAi) was from Calbiochem. Both ChREBP and MondoA contain transcriptional activation Human Islets domains, whereas MLX is much shorter and lacks intrinsic Human islets were obtained from seven donors (mean age transactivation capacity (7,8). The two complexes bind the 2 55.8 6 7.5 years; BMI 27.5 6 1.5 kg/m ). Up to 100 carbohydrate response element consensus sequence in pro- fi handpicked islets were deposited in each well of a 12-well moter regions of speci c target genes (7). In hepatocytes, plate and cultured in the same culture medium as used for ChREBP shifts the cellular state to maximize glucose storage EndoC-bH1 cells. as lipids by upregulating glycolytic and lipogenic genes (9). In skeletal muscle, the activation of MondoA, which is pre- Mouse Islets 2/2 dominant in this cell type, suppresses glucose uptake via ChREBP mice were previously described (18). Wild-type 2/2 increased expression of thioredoxin interacting protein (TXNIP) and homozygous ChREBP knockout (ChREBP )micewere and the arrestin domain–containing protein 4 (ARRDC4) used in accordance with the guidelines of the French Animal (10,11). Care Committee. The mice were bred on a genetic C57BL/ Previous work focusing on the immediate effects of glucose 6J background and raised in a 12-h light/dark cycle. They on the b-cell transcriptome has mainly used rodent models. were fed a standard laboratory chow diet. Islets were isolat- This research has indicated a role for ChREBP in long-term ed from 12-week-old mice by collagenase digestion (Sigma- cellular deterioration via lipotoxicity (12,13) or in promot- Aldrich) followed by direct handpicking. After overnight ing cellular proliferation (14). Recently, the effect of glucose culture in DMEM containing 0.5 mmol/L glucose, groups of in rat pancreatic cells was shown to occur via two transcrip- 50 islets in triplicate were preincubated for 8 h in DMEM tional programs, one directly dependent on ChREBP and containing 0.5 or 20 mmol/L glucose. the other downstream of target genes of ChREBP (15). Small Interfering RNA Transfection of EndoC-bH1 Cells Furthermore, only limited insights have been obtained using and Human Islets human models. An early study (16) analyzing the transcrip- EndoC-bH1 cells were passaged and transfected using tional change of human islets exposed to high glucose levels Lipofectamine RNAiMAX (Life Technologies) 24 h later. for 24 h demonstrated TXNIP as the most highly upregu- ON-TARGETplus small interfering RNA (siRNA) SMARTpool lated gene. Importantly, the contribution of paralog tran- for human MLXIPL, MLXIP, or MLX, or ON-TARGETplus scription factor of ChREBP, MondoA, to glucose sensing in nontargeting control pool (siN) were used (Dharmacon). pancreatic cells has not been investigated. Briefly, siRNA and Lipofectamine RNAiMAX were combined Here, we probed the effect of glucose using the recently in OptiMEM and applied to the cells. Three hours later, developed glucose-responsive EndoC-bH1 human cell line medium was changed for fresh culture medium. Cells were (17) and human islets. In contrast to previous reports in harvested 4–5 days post-transfection, with preliminary ex- rodents, we found no role for ChREBP in upregulating gly- periments showing that siRNA knockdown was consistently colytic or lipogenic genes in response to short-term high- sustained for .7 days post-transfection. glucose treatment (1–24 h). In contrast to ChREBP, we On the day of receiving the samples, ;100 human islets observed that MondoA was responsive to high glucose con- werehandpickedforeachcondition,washedinPBS,and centrations in EndoC-bH1 cells and islets, leading to sub- treated with 1 mL of Accutase (PAA Laboratories) for 5 min. sequent upregulation of TXNIP and ARRDC4 expression, Partial dissociation of the human islets was achieved with thereby reducing cellular glucose uptake. Taken together, slow pipetting and Accutase was removed after a centrifuga- we propose that MondoA is an essential glucose-responsive tion step. The cell clusters were gently suspended in Opti- transcription factor in human b-cells. MEM, and siN, siMlxip, or siMlxipl Lipofectamine RNAiMax complexes were added. Cell clusters were plated on plates RESEARCH DESIGN AND METHODS coated with Matrigel (1%)/fibronectin (2 mg/mL). After – Culture of Human b-Cell Line 4 5h,anequalamountofEndoC-bH1 cell culture medium EndoC-bH1 cells (17) were cultured in low-glucose (5.6 was added. Three days later, the medium was changed to mmol/L) DMEM (Sigma-Aldrich) with 2% BSA fraction V low-glucose (1 mmol/L) culture medium for 5 h, and then (Roche Diagnostics), 50 mmol/L 2-mercaptoethanol, 10 mmol/L glucose (to 20 mmol/L) was added to half of the wells. After nicotinamide (Calbiochem), 5.5 mg/mL transferrin (Sigma- 16 h, the cells were harvested. Aldrich), 6.7 ng/mL selenite (Sigma-Aldrich), 100 units/mL RNA Isolation, Reverse Transcription, and Quantitative penicillin, and 100 mg/mL streptomycin. Cells were seeded PCR at a 40% confluence on plates coated with Matrigel (1%; RNeasy Micro Kit (Qiagen) was used to extract total RNA Sigma-Aldrich)/fibronectin (2 mg/mL; Sigma-Aldrich). Cells from EndoC-bH1 cells and human islets. A First Strand were cultured at 37°C and 5% CO2 in an incubator and cDNA Kit (Thermo Fisher Scientific) was used to synthesize passaged once a week when they were 90–95% confluent. cDNA. Quantitative RT-PCR was performed using Power diabetes.diabetesjournals.org Richards and Associates 463

SYBR Green mix (Applied Biosystems) with a QuantStudio Statistics analyzer. Custom primers were designed with Primer-Quest Data were analyzed using GraphPad Prism 6 software and online software (IDT), and the efficiency was determined are presented as the mean 6 SD. For comparison between for each with a serial dilution of cDNA samples from two mean values, statistical significance was estimated us- EndoC-bH1 cells or human islets. Cyclophilin-A transcript ing a two-tailed Student t test. For comparison among three levels were used for the normalization of each sample. or more values, one-way ANOVA was used with Bonferroni post hoc test (repeated-measures). Transcriptome Analysis and Access to Raw Data Transcriptomic profiles were obtained using GeneChip Hu- man Gene 2.0 ST Array (Affymetrix), following the manufac- RESULTS turer instructions. Microarray data and all experimental Glucose Upregulates a Limited Number of Genes details are available in the Gene Expression Omnibus (GEO) in Human b-Cells database (accession GSE98501). We searched for genes whose expression was induced by glucose in EndoC-bH1. Microarray analysis was used to Immunostaining compare transcriptional profiles of EndoC-bH1 cells after b fi EndoC- H1 cells were cultured on Matrigel/ bronectin- 8 h of exposure to low or high glucose, with or without coated glass coverslips. Cells were starved in low-glucose forskolin. Expression profiles are depicted in Fig. 1 as scat- (0.5 mmol/L) culture medium overnight and exposed to ter plots, which show the relative intensities of all probes low-glucose (0.5 mmol/L) or high-glucose (20 mmol/L) culture fi represented on the microarrays. High glucose exposure medium for 3 h the following day. Cells were xed using 4% upregulated a limited number of genes, and the most prom- paraformaldehyde for 30 min and processed for immunostain- inent was TXNIP (Fig. 1A). The scarce number of upregu- ing by blocking in 3% BSA, 2% serum, and 0.1% Tween-20 lated genes observed was apparent when compared with for 30 min. The cells were exposed to a primary antibody the effects observed after forskolin treatment (Fig. 1B) against MondoA raised in rabbit (ProteinTech) over- fl (6 vs. 75 genes greater than twofold upregulated in each night at 4°C, washed three times, then exposed to a uo- condition, respectively). Surprisingly, analyses of microarray rescent anti-rabbit antibody for 2 h. Nuclei were stained b fl data indicated that in EndoC- H1 cells, glucose did not with Hoechst 33342 uorescent stain (Life Technologies). significantly upregulate the vast majority of known ChREBP Images were acquired with a Leica Leitz Fluorescent Micro- targets (Fig. 1C). Specifically, although glucose treatment scopeequippedwithcooledthree-chipcharge-coupledde- increased TXNIP mRNAlevels,itdidnotmodulatethe vice camera (model C5810; Hamamatsu) and processed expression of ACACA, MLXIPL, PFKL, PKLR, SCD, RGS16, using ImageJ software. HBEGF, GPD1,andRORC (Fig. 1C). Immunoblotting The Majority of ChREBP Target Genes Are Not fl EndoC-bH1 cells at 80% con uence were starved in low- Significantly Affected by Glucose in Human b-Cell Lines glucose (0.5 mmol/L) culture medium overnight and exposed and in Human Islets to different test compounds the following day for 3 h. The To further investigate the upregulation of glucose-dependent nuclear and cytoplasmic proteins were extracted using NE- genes, EndoC-bH1 cells were exposed to different concen- PER Nuclear and Cytoplasmic Extraction Reagents (Thermo trations of glucose (1, 5, 15, or 20 mmol/L) for 8 h. Quanti- Fisher Scientific), and protein concentrations were quantified tative PCR (qPCR) analyses indicated that 15 and 20 mmol/L using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). glucose caused a robust induction in TXNIP gene expression, Proteins (40 mg) were resolved by SDS-PAGE and trans- whereas 5 mmol/L did not (Fig. 2A). The glucose-dependent ferred to a membrane using an iBlot2 Gel Transfer Device stimulation of TXNIP gene expression has been attributed (Thermo Fisher Scientific). Membranes were immunoblot- to ChREBP in rodent b-cells (20). qPCR analyses indicated ted using antibodies against PDX1 (1:1,000) (19), tubulin that, with the exception of TXNIP, all other ChREBP target (1:2,000; Sigma-Aldrich), ChREBP (1:1,000; Novus Bio- genes tested (ACACA, MLXIPL, PFKL, PKLR, RGS16, HBEGF, logicals), or MondoA (1:500; ProteinTech). After washing, GPD1,andRORC) were not significantly upregulated in membranes were incubated with species-specific horse- EndoC-bH1 cells exposed to 20 mmol/L glucose for 1, 4, radish peroxidase–linked secondary antibodies (1:10,000), 8, or 24 h (Fig. 2B and C and Supplementary Fig. 1). To test washed again, and visualized after enhanced chemilumines- whether this was also the case in primary human cells, cence exposure. human islets from donors were exposed to media contain- TXNIP Glucose Uptake Assay ing 1 or 20 mmol/L glucose, and, consistently, only fi D E We transfected EndoC-bH1 cells with siRNA SMARTpool was found to be signi cantly upregulated (Fig. 2 and against human MLXIPL, MXIP, or siN. Five days later, cells and Supplementary Fig. 2). were starved in low-glucose medium (0.5 mmol/L) for 6 h. TXNIP Expression in Human b-Cell Lines and in Human Next, cells were cultured overnight in media containing Islets 20 mmol/L glucose. Glucose uptake was measured using To elucidate whether the glucose-dependent activation of the calorimetric Glucose Uptake Assay Kit (Abcam) as TXNIP required glucose metabolism, EndoC-bH1 cells and per the instruction manual. human islets were preincubated with 25 mmol/L of GK 464 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

Figure 1—Glucose upregulates a limited number of genes in human b-cells. Microarray expression proﬁles of EndoC-bH1 cells exposed for 8 h to 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.

inhibitor mannoheptulose. This prevented the glucose- detail in pancreatic b-cells. qPCR indicated that human dependent upregulation of TXNIP (Supplementary Fig. 3A EndoC-bH1 cells and human islets expressed high levels and B). Furthermore, forskolin and the GLP-1 mimetic of ChREBP (MLXIPL) and MondoA (MLXIP)aswellastheir Exendin-4, which both activate the cAMP pathway, de- obligatory binding partner MLX (Fig. 4A and B). Moreover, creased glucose-induced TXNIP expression in both EndoC- data from GeneChip (Affymetrix) analyses indicated that bH1 cells and human islets (Supplementary Fig. 3C and D). ARRDC4, a key target of MondoA in skeletal muscles (10), Taken together, glucose upregulates TXNIP expression similarly was signiﬁcantly upregulated by high glucose concentra- in both human b-cell lines and human islets, and this effect tions in EndoC-bH1 cells (Fig. 1C). This upregulation was is dependent on glucose metabolism and cAMP signaling. validated by qPCR both in EndoC-bH1 cells (Fig. 4C)andin D The Glucose-Dependent Upregulation of TXNIP Is Not human islets (Fig. 4 ), and was prevented by GK inhibition 2 2 E F Blunted in Islets From ChREBP / Mice and cAMP activation (Fig. 4 and )inasimilarfashionto TXNIP To establish the necessity of ChREBP in glucose-induced TXNIP (Supplementary Fig. 3). 2/2 expression, islets from wild-type and ChREBP mice High Glucose Causes MondoA Nuclear Translocation were isolated and exposed to either low (0.5 mmol/L) or in EndoC-bH1 Cells high (20 mmol/L) glucose for 8 h. qPCR analyses indicated Both MondoA and ChREBP are localized to the cytoplasm txnip that induction by glucose was similar in both groups in an inactive state, and a glucose stimulus causes their (Fig. 3), demonstrating that ChREBP is not required to translocation to the nucleus (7). To determine how glucose txnip drive expression upon glucose stimulation in rodent signals in b-cells, we studied the cytoplasmic and nuclear islets. localization of MondoA and ChREBP in EndoC-bH1 cells MondoA Is Robustly Expressed in Rodent and Human after glucose treatment. Immunoblotting for MondoA Islet Cells and in EndoC-bH1 Cells revealed that 3 h of 20 mmol/L glucose exposure caused To the best of our knowledge, the expression and function MondoA nuclear translocation (Fig. 5A), which was further of the ChREBP paralog MondoA has not been evaluated in conﬁrmed by immunocytochemistry (Fig. 5B and C for diabetes.diabetesjournals.org Richards and Associates 465

Figure 2—Glucose upregulates TXNIP but no other ChREBP target genes in EndoC-bH1 cells and human islets. A:EndoC-bH1 cells were exposed to different glucose concentrations for 8 h, and TXNIP expression was quantiﬁed by qPCR. B–E: A variety of these genes was analyzed by 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. quantiﬁcation). Of note, the nuclear translocation of Mon- regulated kinase (ERK) kinase (U0126) or ERK (PD98059) doA was prevented by forskolin, consistent with the in- inhibitors blunted the effects of forskolin on glucose- hibition of ARRDC4 and TXNIP transcription by the induced TXNIP (Fig. 5D). Surprisingly, we did not observe compound (Fig. 5A). The effects of forskolin were mediated ChREBP nuclear translocation upon glucose exposure (Fig. by PKA. Indeed, PKA inhibitors (PKAi and H89) but 5E), indicating that MondoA, but not ChREBP, is sensitive not mitogen-activated protein kinase/extracellular signal– to short-term glucose in EndoC-bH1 cells.

Figure 3—Islets from ChREBP2/2 (KO) mice still have robust glucose-dependent upregulation of txnip.qPCRofChREBP(mlxipl)(A)andtxnip (B)inwild-type(WT)orChREBP2/2 islets cultured ex vivo in high or low glucose for 8 h. n =9.***P , 0.001. 466 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

Figure 4—ChREBP, MondoA,andARRDC4 expression in human b-cells. The expressions of ChREBP (MLXIPL), MondoA (MLXIP), and MLX were analyzed by qPCR in EndoC-bH1 cells (A)andhumanislets(B) exposed to different glucose concentrations. ARRDC4 expression was analyzed by qPCR in EndoC-bH1 cells (C)andhumanislets(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.

MondoA Regulates Glucose-Dependent Gene cells using siRNA (Fig. 6A–C). ChREBP silencing had no Expression and Glucose Uptake in EndoC-bH1 Cells effect on the glucose-induced upregulation of TXNIP or To establish whether MondoA is the primary glucose- ARRDC4 (Fig. 6D and E). On the other hand, knocking responsive transcription factor in human b-cells, its down MondoA or MLX signiﬁcantly compromised the effect encoding mRNA, or that of ChREBP or their obligatory of glucose on both ARRDC4 and TXNIP upregulation (Fig. binding partner MLX, were knocked down in EndoC-bH1 6D and E). diabetes.diabetesjournals.org Richards and Associates 467

Figure 5—MondoA, but not ChREBP, localizes in the nucleus under high-glucose conditions in EndoC-bH1 cells. A: Western blot of cytoplasmic 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 membranes were hybridized with antibodies against tubulin, PDX1, and MondoA. B: Immunocytochemistry of EndoC-bH1 cells exposed to low or high glucose for 3 h, stained with an antibody against MondoA (green) and Hoechst stain (blue). Scale bars, 20 mm. C: Quantiﬁcation of EndoC-bH1 cells 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 treated for 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 of EndoC-bH1 cells exposed to low or high glucose plus forskolin for 3 h. The membranes were hybridized with antibodies against tubulin, PDX1, and ChREBP. 468 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018

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 starved overnight 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 expression of ChREBP (MLXIPL)(A), MondoA (MLXIP)(B), MLX (C), TXNIP (D), and ARRDC4 (E) was measured by qPCR. F: A glucose uptake assay was used 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.

ARRDC4 and TXNIP both control glucose uptake in unaltered in ChREBP-silenced cells but was signifi- skeletal muscle cells (21,22). To determine whether Mon- cantly higher in cells with reduced MondoA expression doA regulates glucose uptake in human b-cells, glucose (Fig. 6F). uptake was measured in EndoC-bH1 cells. MondoA knockdown selectively suppressed TXNIP and ARRDC4 MondoA Regulates Glucose-Dependent Transcription in gene expression in EndoC-bH1 cells, whereas ChREBP si- Primary Human Islets lencing had no effect on these genes (Supplementary Fig. 4). Finally, we asked whether the glucose-dependent regula- Consistent with this observation, glucose uptake was tion of ARRDC4 and TXNIP is also MondoA dependent in diabetes.diabetesjournals.org Richards and Associates 469 primary human b-cells. For this purpose, we performed human b-cell physiology is crucial, as, despite many simi- siRNA-mediated knockdown of MondoA and ChREBP in larities, rodent and human b-cells differ on various specific human islets. ChREBP siRNA induced a nearly 80% de- points, including disparities in b-cell function (23,24). The crease in ChREBP RNA steady-state level, whereas MondoA limited number of studies using human b-cells mainly siRNA induced a nearly 70% decrease in MondoA RNA stems from the difficulty in accessing primary human islet steady-state levels without any impact on ChREBP mRNA preparations that derive from deceased donors. Available levels (Fig. 7A and B). Importantly, and consistent with human islets often come from transplantation rejection data obtained in EndoC-bH1 cells, ChREBP knockdown and therefore are not in an optimal state for further did not alter glucose-induced ARRDC4 and TXNIP upregu- ex vivo studies. Moreover, they contain b-cells in different lation, whereas such inductions were blunted after MondoA proportions from one preparation to another, giving rise knockdown (Fig. 7C and D). to data variability (25). The difficulty of generating functional human b-cell lines has also represented a major lim- DISCUSSION itation for decades (26). The present work mainly focuses Therehavebeenlimitedobservationsoftheeffectsofglucose on glucose-regulated gene expression in pancreatic b-cells in on the human b-cell transcriptome. Here, using EndoC-bH1 a human context, using both human islets from donors and cells and islets from organ donors, we demonstrated that the recently developed functional human b-cell line EndoC- the glucose-responsive transcription factor MondoA is ro- bH1 (17). In these two models, we observed a very limited bustly expressed in pancreatic b-cells. MondoA undergoes number of genes that were efficiently affected by short- nuclear translocation in high-glucose conditions where it term high-glucose treatment. This is in accordance with upregulates the expression of a limited number of genes a previous microarray analysis (16) reporting only 14 genes including TXNIP and ARRDC4, both of which are involved significantly upregulated in human islets upon 24-h expo- in glucose uptake inhibition. Therefore, we conclude that sure to high-glucose exposure. Of note, only five of these MondoA is the glucose-responsive transcription factor in genes were induced more than twofold, with TXNIP being human pancreatic b-cells. the most upregulated gene. In contrast, it was recently Studies have previously analyzed glucose-regulated gene reported that .2,000 genes are significantly induced by expression in rodent b-cells using either b-cell lines or islet glucose after a 12-h treatment in the rat b-cell line INS-1 preparations, whereas fewer data have been generated us- (15). Although improvements in the sensitivity of recent ing human b-cells (15). However, improving knowledge of transcriptomic 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)orMondoA(MLXIP) was knocked down in human islets using siRNA. Three days post-transfection, the cells were starved for 6 h in low-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. 470 MondoA and Human Pancreatic b-Cells Diabetes Volume 67, March 2018 number of detectable genes, a clear underlying difference as MondoA activity was inhibited by mannoheptulose, an between the two-species transcriptional response to glucose inhibitor of the GK enzyme, the hexokinase that catalyzes appears to exist. Among known glucose-related species dif- the first reaction in the glycolytic pathway in pancreatic ferences, glucose transport in rodent b-cells is mainly de- b-cells. pendent on GLUT2 (SLC2A2), a low-Km GLUT, whereas the Our experiments performed with either forskolin or the high-Km GLUT1 (SLC2A1) plays a major role in human GLP-1 receptor agonist Exendin-4 demonstrated that the b-cells (27); Moreover, the panel of voltage-gated ion chan- translocation of MondoA as well as its transcriptional nels differs between rodent and human b-cells (28), and activity were inhibited by cAMP signaling. Although this different set points for glucose-stimulated insulin secretion type of inhibitory effect has been observed for the have been reported in these two species (28). Regarding regulation of ChREBP activity in the liver (37), cAMP ef- glucose-induced cell cycle entry, glucose efficiently activates fects of MondoA translocation have not been previously alargesetofcellcycle–related genes that activate b-cell described. ChREBP is phosphorylated by PKA at Ser196 in proliferation in rodent b-cells (29), a process that is less response to glucagon, leading to a cytoplasmic localization efficient in the case of human b-cells (30). Altogether, these that involves the 14–3–3 protein (37). This specificPKA data suggest that human b-cells could be quantitatively less phosphorylation site has not been described in the MON- sensitive to glucose in terms of the regulation of gene ex- DOA sequence, and our results may suggest another PKA pression than rodent b-cells. phosphorylation site that needs to be characterized. Historically, it was first thought that in pancreatic b-cells Interestingly, previous data indicated that Exendin-4 the upstream stimulatory factors regulated glucose stimu- acts as an antiapoptotic agent on b-cells by decreasing lation of gene expression (31). ChREBP was next proposed txnip expression, though the authors did not elucidate as the b-cell carbohydrate-responsive transcription factor the mechanism by which Exendin-4 exerted this inhibitory based on a series of arguments. ChREBP was first reported effect (38). Our data would suggest that this antiapoptotic as expressed in pancreatic islets and in b-cell lines (32) and effect of Exendin-4 occurs through MondoA inhibition. Re- its overexpression in INS-1 cells using the Tetracycline-On cently, an inhibitor of MondoA has been reported to have system improved the glucose-dependent induction of its beneficial effects on insulin and glucose handling in high fat– target gene L-pyruvate kinase (LPK) (32). Moreover, over- fedmice(39).Basedonourfindings of MondoA actions in expression of a constitutively active mutant of ChREBP in b-cells, it would be interesting to explore whether its spe- INS-1 cells led to the induction of several ChREBP target cific inhibition here could be in part responsible for the genes, including TXNIP (33). Finally, glucose was shown to observed improved glucose handling. Of note, glucose, regulate gene expression in pancreatic b-cells through the which induces insulin secretion, also activates the expres- carbohydrate response element consensus sequence that sion of TXNIP and ARRDC4, both of which are involved in ChREBP binds (34). However, the direct evidence that glucose uptake inhibition. These new data suggest that glu- ChREBP acts as the primary glucose transcription factor cose entrance in human b-cells induces a transcriptional has not always been consistent. Indeed, although studies response that triggers a negative regulatory feedback loop have reported that chrebp knockdown in INS-1 cells alters on glucose uptake, which might contribute to the transitional glucose-stimulated txnip expression (34), others have found glucose signal in these cells. either no or limited effects in both INS-1 cells and rat islets Our data indicate that MondoA plays an essential role in 2 2 (35). Here, we demonstrate that islets from ChREBP / glucose-stimulated gene expression in human pancreatic animals have a similar upregulation of txnip compared b-cells. However, its functional role in b-cells is not yet with islets from wild-type littermates. Although this result elucidated. It has been demonstrated that its paralog does not preclude a role for ChREBP in pancreatic b-cells, it ChREBP is required for glucose-stimulated b-cell prolif- directly demonstrates that ChREBP is not necessary for eration (14). Indeed, the loss of ChREBP decreases glucose- glucose-stimulated txnip expression in mouse pancreatic is- induced BrdU incorporation in isolated human b-cells and let cells. Other functions for ChREBP in human islets may in b-cells isolated from ChREBP-deficient mice. Given the yet be discovered, which could relate to its described nuclear evidence for the involvement of MondoA in cancer cell pro- translocation and activation under periods of endoplasmic liferation through its transcriptional control of TXNIP and reticulum stress induced by thapsigargin (36). subsequent effects on glucose uptake (40), future studies Akeyfinding of the present work is that MondoA plays using MondoA-deficient mice will be useful to determine a major role in glucose-stimulated gene expression in whether MondoA is implicated in glucose-mediated b-cell human pancreatic b-cells. We observed that MondoA is proliferation in physiological conditions, but also after b-cell expressed in b-cells. Moreover, glucose induced MondoA injury. nuclear translocation and activated the expression of its Until now, studies on the regulation of gene expres- targetgenes,asisthecaseinmusclecells(10).Itwas sion by glucose in pancreatic b-cells has mainly focused on previously postulated, based on data from HEK293T ChREBP because of the many similarities between hepato- cells expressing different forms of hexokinase, that cytes and b-cells, including endodermal origins and meta- MondoA activity requires glucose metabolism. We dem- bolic functions. However, our study notably demonstrates onstrate here that this is also the case in human b-cells for the first time that its paralog, MondoA, is an essential diabetes.diabetesjournals.org Richards and Associates 471 glucose-responsive transcription factor in human pancreatic 12. Poungvarin N, Lee JK, Yechoor VK, et al. Carbohydrate response element- b-cells. binding protein (ChREBP) plays a pivotal role in beta cell glucotoxicity. Diabetologia 2012;55:1783–1796 13. da Silva Xavier G, Rutter GA, Diraison F, Andreolas C, Leclerc I. ChREBP Acknowledgments. The authors thank Dr. B.B. Kahn (Harvard Medical binding to fatty acid synthase and L-type pyruvate kinase genes is stimulated by 2 2 – School, Boston, MA) for sharing ChREBP / mice and Dr. D.E. Ayer (University glucose in pancreatic beta-cells. J Lipid Res 2006;47:2482 2491 of Utah, Salt Lake City, UT) for suggestions on MondoA antibodies. The authors 14. Metukuri MR, Zhang P, Basantani MK, et al. ChREBP mediates glucose- – also thank the transcriptomic platform from the Cochin Institute for performing stimulated pancreatic b-cell proliferation. Diabetes 2012;61:2004 2015 array hybridizations and N. Glaser (INSERM U1016) for help in further data 15. Schmidt SF, Madsen JG, Frafjord KO, et al. Integrative genomics outlines analyses. a biphasic glucose response and a ChREBP-RORg axis regulating proliferation in b – Funding. P.R. was supported by a postdoctoral grant from Agence Nationale cells. Cell Rep 2016;16:2359 2372 de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10- 16. Shalev A, Pise-Masison CA, Radonovich M, et al. Oligonucleotide microarray fi LABX-73). This study was funded by the Cochin Internal Program PIC (to L.R. and analysis of intact human pancreatic islets: identi cation of glucose-responsive S.G.). The R.S. laboratory is supported by Agence Nationale de la Recherche (ANR- genes and a highly regulated TGFbeta signaling pathway. Endocrinology 2002; – 10-LABX-73) and the Bettencourt Schueller Foundation. The C.P. and R.S. research 143:3695 3698 groups belong to the Département Hospitalo Universitaire (DHU). This project re- 17. Ravassard P, Hazhouz Y, Pechberty S, et al. A genetically engineered human ceived funding from the Innovative Medicines Initiative 2 Joint Undertaking under pancreatic b cell line exhibiting glucose-inducible insulin secretion. J Clin Invest – grant agreement number 115881 (RHAPSODY). This Joint Undertaking receives 2011;121:3589 3597 fi support from the European Union’s Horizon 2020 research and innovation pro- 18. Iizuka K, Bruick RK, Liang G, Horton JD, Uyeda K. De ciency of carbohydrate gramme and EFPIA. This work is supported by the Swiss State Secretariat for response element-binding protein (ChREBP) reduces lipogenesis as well as – Education Research and Innovation (SERI) under contract number 16.0097. glycolysis. Proc Natl Acad Sci U S A 2004;101:7281 7286 Duality of Interest. No potential conflicts of interest relevant to this article 19. Duvillié B, Attali M, Aiello V, Quemeneur E, Scharfmann R. Label-retaining cells were reported. in the rat pancreas: location and differentiation potential in vitro. Diabetes 2003; – Author Contributions. P.R. conceptualized the work, performed inves- 52:2035 2042 tigations, and wrote the manuscript. L.R. and S.G. conceptualized the work, 20. Filhoulaud G, Guilmeau S, Dentin R, Girard J, Postic C. Novel insights into – performed investigations, and reviewed and edited the manuscript. M.O. performed ChREBP regulation and function. Trends Endocrinol Metab 2013;24:257 268 investigations. P.M., M.B., and M.A. provided human islets. C.P. conceptualized the 21. Parikh H, Carlsson E, Chutkow WA, et al. TXNIP regulates peripheral glucose work and reviewed and edited the manuscript. R.S. conceptualized the work, wrote metabolism in humans. PLoS Med 2007;4:e158 the manuscript, and supervised the work. R.S. is the guarantor of this work and, as 22. Patwari P, Chutkow WA, Cummings K, et al. Thioredoxin-independent regu- such, had full access to all the data in the study and takes responsibility for the lation of metabolism by the alpha-arrestin proteins. J Biol Chem 2009;284: – integrity of the data and the accuracy of the data analysis. 24996 25003 23. Scharfmann R, Rachdi L, Ravassard P. 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