Mitochondrial Protein UCP2 Controls PancreasDevelopmentBenjamin Broche,1,2,3 Selma Ben Fradj,1,2,3 Esther Aguilar,1,2,3 Tiphaine Sancerni,1,2,3,4 Matthieu Bénard,1,2,3

Fatna Makaci,1,2,3 Claire Berthault,1,2,3 Raphaël Scharfmann,1,2,3 Marie-Clotilde Alves-Guerra,1,2,3 andBertrand Duvillié1,2,3

Diabetes 2018;67:78–84 | https://doi.org/10.2337/db17-0118

The mitochondrial carrier uncoupling protein (UCP) 2 be-longs to the family of the UCPs. Despite its name, it is nowaccepted that UCP2 is rather a metabolite transporterthan a UCP. UCP2 can regulate oxidative stress and/orenergetic metabolism. In rodents, UCP2 is involved in thecontrol of a- and b-cell mass as well as insulin and glu-cagon secretion. Our aim was to determine whether theeffects of UCP2 observed on b-cell mass have an embry-onic origin. Thus, we used Ucp2 knockout mice. We foundan increased size of the pancreas in Ucp22/2 fetuses atembryonic day 16.5, associated with a higher number ofa- and b-cells. This phenotype was caused by an increaseof PDX1+ progenitor cells. Perinatally, an increase in theproliferation of endocrine cells also participates in theirexpansion. Next, we analyzed the oxidative stress in thepancreata. We quantified an increased nuclear transloca-tion of nuclear factor erythroid 2–related factor 2 (NRF2) inthe mutant, suggesting an increased production of reac-tive oxygen species (ROS). Phosphorylation of AKT, anROS target, was also activated in the Ucp22/2 pancreata.Finally, administration of the antioxidantN-acetyl-L-cysteineto Ucp22/2 pregnant mice alleviated the effect of knock-ing out UCP2 on pancreas development. Together, thesedata demonstrate that UCP2 controls pancreas develop-ment through the ROS-AKT signaling pathway.

During the last decade, the impact of mitochondrial dys-function in pancreatic islet development and diabetes hasbeen widely studied (1). However the underlying mecha-nisms involving the mitochondria are still not well under-stood. The mitochondrial uncoupling protein (UCP) 2

belongs to the family of UCPs (2). Despite the well-acceptedrole of UCP1 as a proton transporter and a UCP in the brownadipose tissue, it was shown that UCP2 is a metabolite trans-porter with no or little mitochondrial uncoupling activity (3).UCP2 is expressed in the spleen, lungs, stomach, adiposetissue, and pancreas (4,5). Moreover, several studies in-dicate that UCP2 is a repressor of reactive oxygen species(ROS) production in different cell types (6,7). In addition,UCP2 can regulate the balance between glycolysis and oxi-dative phosphorylation in murine embryonic fibroblasts (7)and in different types of cancer cells (8,9). Recently, Ucp2mutations were discovered in humans and were associatedwith congenital hyperinsulinism (10). In mice, the absenceof UCP2 also leads to increased insulin secretion (11), sup-porting the observation in humans. The knockout of UCP2induces an increase in the number of endocrine cells, andthis phenotype is amplified by a high-fat diet (12,13).

The aim of our study was to determine whether theb-cell hyperplasia observed in adult Ucp22/2 mice has anembryonic origin. For this, we used Ucp22/2 mouse em-bryos at different stages and we analyzed the developmentof the pancreas.

RESEARCH DESIGN AND METHODS

AnimalsExperiments were in agreement with the French animalcare committee guidelines. Ucp22/2 mice (C57Bl6/J back-ground) were previously described (14). N-acetyl-L-cysteine(NAC) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) treat-ment was initiated at embryonic day 9.5 (E9.5) until E13.5,and at E12.5 until E19.5.

1INSERM, U1016, Institut Cochin, Paris, France2CNRS, UMR8104, Paris, France3Université Paris Descartes, Sorbonne Paris Cité, Paris, France4Université Paris Diderot, Sorbonne Paris Cité, Paris, France

Corresponding authors: Bertrand Duvillié, bertrand.duvillie@curie.fr, and Marie-Clotilde Alves-Guerra, clotilde.alves-guerra@inserm.fr.

Received 27 January 2017 and accepted 23 October 2017.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0118/-/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.

78 Diabetes Volume 67, January 2018

ISLETSTUDIES

ImmunohistochemistryTissues were fixed in 10% formalin and processed forimmunohistochemistry, as described previously (15). Thefollowing antibodies were used: mouse anti-insulin(1:2,000; Sigma-Aldrich), rabbit anti-glucagon (1:1,000;Euromedex, Souffelweyerrsheim, France), rabbit anti-PDX1(1:1,000), mouse anti-Ki67 (1:50; BD Pharmingen, Le Pont-de-Claix, France), rabbit anti-amylase (1:300; Sigma-Aldrich),rabbit anti–neurogenin 3 (anti-NGN3; 1:1,000), rabbit anti–nuclear factor erythroid 2–related factor 2 (anti-NRF2;1:1,000; GeneTex, Irvine, CA), rabbit anti-Akt (1:200), andrabbit anti–phospho Akt (Ser 473) (1:25) (nos. 9272 and9271; Cell Signaling, Saint-Quentin, France). The fluores-cent secondary antibodies were fluorescein isothiocyanateanti-rabbit and Texas Red anti-mouse antibodies (1:200;Jackson ImmunoResearch, Suffolk, U.K.), and Alexa Fluoranti-rabbit antibody (1:400; Biogenex, Fremont, CA). ForNGN3, revelation was performed using the vectastainABC kit (Vector Laboratories, Peterborough, U.K.). Fluo-rescent image acquisition was performed using the ZeissAxioObserver Z1 inverted fluorescence microscope coupledwith the Zeiss Axiocam MRm (Zeiss, Marly-le-Roi, France).

Determination of Cellular ATP LevelsDetection of ATP levels was assessed using a luminescence-based assay kit (Roche, Meylan, France).

RNA Extraction and PCRProcedures are described in Hoarau et al. (16). The oligo-nucleotide sequences for RT-PCR are available on request.

Western Blot AnalysisFor Western blotting analysis, cells were lysed in Laemmli.Proteins (20 mg) were separated by SDS-PAGE and electro-phoretically transferred onto polyvinylidene fluoride mem-brane (Bio-Rad, Marnes-la-Coquette, France). After blockingwith milk, membranes were probed with mouse anti–phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204; CellSignaling), mouse anti–b-actin (Sigma-Aldrich), mouseanti–a-tubulin (Sigma-Aldrich), rabbit anti-Akt, and rabbitanti–phospho Akt (Ser 473) (nos. 9272 and 9271; Cell Sig-naling). Immunoreactive bands were visualized with theSuperSignal System (Pierce, Fisher Scientific, Illkirch, France).

Protein OxidationProtein oxidation of total pancreas homogenates wasmeasured by assaying the amount of carbonyl groups onproteins using the OxyBlot kit (Protein Oxidation DetectionKit; Millipore, Molsheim, France).

Cell Suspension and Cell SortingThe procedures were described previously (17,18). In brief,cell suspensions were stained in Hanks’ balanced salt solu-tion (HBSS) without calcium/magnesium supplementedwith 20% FCS with the following anti-mouse antibodiespurchased from BD Biosciences (Le Pont-de-Claix, France):anti-CD45 PercpCy5.5 (clone 30F11), anti-CD31 PercpCy5.5(clone MEC13.3), anti-TER119 PercpCy5.5 (clone TER119),anti-EpCam BV421 (clone G8.8), anti-CD49f PE (clone GoH3),

and anti-CD133 APC (clone 3152C11). For each antibody,optimal dilution was determined by titration. Cells were in-cubated for 15–30 min at 4°C in the dark, washed, andsuspended in HBSS without calcium/magnesium supple-mented with 20% FCS, and dead cells were excluded withpropidium iodide (1:4,000; Sigma-Aldrich). Stained cells wereanalyzed and sorted with FACS Aria III (BD Biosciences).Data were analyzed in FlowJo (Ashland, OR) software.

Islet IsolationNeonatal islets from wild-type (WT) and Ucp22/2 micewere harvested as described previously (19). Freshly dis-sected whole pancreata were digested with 0.5 mg/mLcollagenase (Sigma-Aldrich) dissolved in HBSS at 37°C.Tubes were tapped regularly to aid tissue dispersal. Next,lysates were washed with HBSS containing 10% FBS. Then,islets were handpicked under a dissecting stereoscope(Leica, Nanterre, France).

Insulin SecretionInsulin secretion was quantified as described previously (20)using an ultrasensitive mouse insulin ELISA kit (CrystalChem, Zaandam, the Netherlands).

QuantificationTo quantify the absolute surface of PDX1-, insulin-,glucagon-, and amylase-expressing and Hoechst-stainedcells, 5-mm-thick sections of each pancreas were digitized atE13.5 and E16.5. At E19.5 and postnatal day 2, one of thefive slides of the total pancreas was digitized (17). On everyimage, the surface of immunostaining was quantified byImageJ (National Institutes of Health, Bethesda, MD). AtE16.5, the total number of immunopositive cells for NGN3was counted on all sections of a complete pancreas. Statisti-cal significance was determined by Student t test. To mea-sure proliferation of early progenitors, we counted thefrequency of Ki67+ nuclei among 1,000 PDX1+ cells. Atleast three rudiments per condition were analyzed. Statis-tical significance was determined using Student t test.

RESULTS

Increased Pancreas Growth in the Ucp2 Knockout MiceFirst, the expression pattern of UCP2 was analyzed. E12.5pancreatic epithelial and mesenchymal cells were separatedby FACS (17). Ucp2 expression was enriched in the epithe-lial fraction containing the progenitors (Supplementary Fig.1A). At E16.5, we separated mesenchymal, acinar, NGN3+,and endocrine cells (18). Ucp2 expression was found pref-erentially in endocrine cells and in a lesser extent in othercell types (Supplementary Fig. 1B). To investigate its role,Ucp2+/2 mice were intercrossed. The weight, islet insulinsecretion, and glycemia of the homozygous neonates wereall similar to the controls (Supplementary Fig. 2). Duringthe embryonic and fetal periods, the overall external mor-phology of Ucp22/2 animals was normal (SupplementaryFig. 3). As shown by the Hoechst staining, the size of theUcp22/2 pancreas at E16.5, E19.5, and PN2 was increasedby nearly twofold compared with controls (Figs. 1, 2, and 4).

diabetes.diabetesjournals.org Broche and Associates 79

This difference was not observed at E13.5 (Fig. 3C). More-over, the absolute surfaces of insulin, glucagon, and amylasewere also increased in the Ucp22/2 pups and fetuses (Figs.1 and 2). Using an antibody directed against NGN3, weshowed that the number of endocrine precursors increasedproportionally to the pancreas size at E16.5 in the mutants(Supplementary Fig. 4). To investigate the mechanism re-sponsible for the increased growth of the Ucp22/2 pancreata,we quantified progenitor proliferation using anti-PDX1 andanti-Ki67 antibodies. At E13.5, we found an increased pro-liferation of PDX1+ progenitor cells (Fig. 3A and B), but notat E12.5 (Supplementary Fig. 5). Together, these data dem-onstrate that Ucp2 deletion induces an overgrowth of thepancreas due to an increased proliferation of progenitor cells.

UCP2 Controls Oxidative Stress and AKT Signalingin the Ucp22/2 Fetal PancreasTwo main mechanisms have been described to explain thebiological effects of UCP2. First, UCP2 can modulate theenergetic metabolism by controlling the balance betweenglycolysis and oxidative phosphorylation (8). To examine theenergetic status of Ucp22/2 pancreas, we quantified theATP content in Ucp22/2 and WT pancreata. No differencewas found at the pancreatic level (Supplementary Fig. 6).

However, a nonsignificant decrease of ATP per cell wasquantified at E16.5 (6.93 3 1024 pmol in Ucp22/2 vs.9.833 1024 pmol in WT, P = 0.25). The second hypothesisis that UCP2 is involved in the regulation of the productionof ROS (5,6). To examine this possibility, we performedimmunofluorescence experiments to visualize the nucleartranslocation of the ROS-sensitive factor NRF2. In the ab-sence of oxidative stress, NRF2 is associated with the pro-tein Keap1, which promotes the degradation of NRF2 bythe ubiquitin proteasome pathway. Also, oxidants can mod-ify the cysteine residues of Keap1, leading to nuclear trans-location of NRF2. Interestingly, we observed NRF2 only atthe periphery of the nuclei at E13.5 in the WT pancreatawhereas nuclear translocation of NRF2 was observed in themutants (Supplementary Fig. 7). At E16.5, nuclear trans-location was found both in WT and Ucp22/2 pancreata, inan area containing b-cells, and this event was increased inthe mutants (Supplementary Fig. 7). These results suggestthat ROS are involved in endocrine development. We alsoquantified protein oxidation levels using the OxyBlot assay(Supplementary Fig. 7). We found a nearly twofold increaseof the protein oxidation levels in the mutant pancreata.Together, these results indicate that oxidative stress is

Figure 1—UCP2 deficiency increases pancreas development. WT and Ucp22/2 pancreata were analyzed at postnatal day 2. The white dottedlines demarcate the limits of the pancreas. A: Nuclei were stained with Hoechst 33342 (blue). Islets were detected using anti-insulin (red) (B) andanti-glucagon (green) (C) antibodies. Exocrine cells were detected using anti-amylase antibody (green) (D). The absolute surface areas occupiedby Hoechst-positive cells, insulin-positive cells, glucagon-positive cells, and amylase-positive cells were quantified. Each point represents themean 6 SEM of n $ 3 individual pancreata. *P , 0.05. Scale bar = 50 mm.

80 UCP2 and b-Cell Development Diabetes Volume 67, January 2018

higher in the Ucp22/2 pancreata. To further investigatesignaling pathways involved in the pancreatic phenotypeof Ucp22/2 fetuses, we first analyzed the ERK1/2 pathway.No difference was found between mutants and controls(Supplementary Fig. 8). Second, using immunofluorescence,we analyzed the AKT signaling pathway, sensitive to ROSlevels (21). The total AKT level was slightly increased in themutants at E16.5 but not at E13.5. At both stages in themutants, we found an increased ratio of phospho-AKT tototal AKT, confirmed by Western blot at E16.5 (Supplemen-tary Figs. 9 and 10). Thus, these data suggest that theactivation of the ROS-AKT signaling pathway is involvedin the growth of Ucp22/2 mouse pancreas.

NAC Treatment Reverses the Pancreatic Phenotypeof the Ucp22/2 FetusesTo further analyze the implication of ROS, we treatedpregnant mice with the antioxidant NAC between E12.5and E19.5. In Ucp22/2 pancreata, the number of NRF2+

cells decreased when treated with NAC, validating its anti-oxidant effect (Supplementary Fig. 11). Pancreatic weightand b-cell and a-cell masses were increased in untreatedUcp22/2 fetuses, compared with controls (Fig. 4). This

effect was abolished when Ucp22/2 fetuses receivedNAC treatment (Fig. 4). Interestingly, a- and b-cell prolif-eration was increased in E19.5 Ucp22/2 pancreata versuscontrols (Supplementary Figs. 12 and 13). This effect wasabrogated when an NAC treatment was administrated.Thus, the knockout of Ucp2 controls the proliferation ofendocrine cells in an ROS-dependent manner. It leads to anonsignificant increased fraction of endocrine cells at PN2(Supplementary Fig. 14) but not at E19.5. Finally, we treatedUcp22/2 and control mice with NAC from E9.5 to E13.5.Such treatment reduced progenitor proliferation inducedby the deletion of Ucp2 (Fig. 3B). Altogether, these datademonstrate that increased oxidative stress caused by thelack of UCP2 is responsible for the increased fetal pancreatagrowth.

DISCUSSION

Our main finding is that UCP2 is a negative regulator ofpancreas development. Indeed, the absence of UCP2 in-duces an increase in cell proliferation and a larger pancreas.Moreover, this effect is induced by oxidative signals, throughthe activation of the AKT pathway.

Figure 2—Pancreas growth is enhanced in the Ucp22/2 E16.5 fetuses. WT and Ucp22/2 pancreata were analyzed at E16.5. The white dottedlines demarcate the limits of the pancreas. A: Photographs of the WT and Ucp22/2 pancreata. Scale bar = 200 mm. B: Nuclei were stained withHoechst 33342 (blue). Endocrine development was investigated using anti-insulin (red) (C) and anti-glucagon (green) (D) antibodies. The absolutesurface areas occupied by Hoechst-positive cells, insulin-positive cells, and glucagon-positive cells were quantified. Each point represents themean 6 SEM of n $ 3 individual pancreata. *P , 0.05. Scale bar = 50 mm.

diabetes.diabetesjournals.org Broche and Associates 81

The Roles of UCP2 in Physiological and PathologicalProcessesHere we show that the deletion of Ucp2 increases progen-itor and endocrine cell proliferation, two cell types thatnormally express Ucp2 (Supplementary Fig. 1). This sug-gests a potential autocrine effect of UCP2, but we do notexclude other paracrine effects. For example, the mesen-chyme that expresses lower levels of Ucp2 also controlsprogenitor proliferation (15). Moreover, several recent stud-ies indicate that UCP2 plays a crucial role in the developmentof several cell types. Indeed, during human stem cell differ-entiation, UCP2 expression decreases, suggesting its role as a

repressor of stem cell differentiation (22). Moreover, in mu-rine embryonic fibroblasts, UCP2 was shown to negativelycontrol their proliferation (7). Finally, the roles of UCP2were investigated in different pathologies. In cancer celllines expressing low levels of UCP2, its overexpression de-creases cell proliferation through metabolic changes and inconsequence represses the malignant phenotype. Moreover,in diabetes, the involvement of UCP2 is still controversial(23). Indeed, Emre et al. (23) treated WT and Ucp22/2 micewith low doses of streptozotocin to generate an experimentalmodel of diabetes. They found that autoimmune diabeteswas accelerated in Ucp22/2 mice, with the presence of an

Figure 3—The proliferation of the progenitor cells is increased in theUcp22/2 E13.5 pancreata. A: The proliferation of the PDX1+ progenitor cellswas analyzed using anti-PDX1 (green) and anti-Ki67 (red) antibodies. B: Proliferation percentage was also quantified. Each point represents themean6 SEM of n$ 3 individual pancreata. *P, 0.05. Scale bar = 50 mm. For higher magnification, scale bar = 10 mm.C: E13.5 pancreata wereanalyzed using anti-PDX1 antibodies (green). Nuclei were stained with Hoechst 3342 (blue). The white dotted lines demarcate the limits of thepancreas. The absolute areas of Hoechst-positive cells and PDX1-positive cells were quantified. Each point represents the mean6 SEM of n $ 3individual pancreata. Scale bar = 50 mm.

82 UCP2 and b-Cell Development Diabetes Volume 67, January 2018

increased lymphocytic infiltration. On the contrary, usingsimilar experiments, Lee et al. (12) found that treatment ofWT and Ucp22/2 mice with low doses of streptozotocinresulted in hyperglycemia that was much less severe inUcp22/2 mice than controls. The difference between thesetwo studies was suggested to be connected to the geneticbackground of the mice. Moreover, in humans, another re-cent illustration is that variants of the Ucp2 gene are asso-ciated with diabetes and diabetic retinopathy in a Chinesepopulation (24). The exact mechanism responsible for di-abetes in these patients still needs to be elucidated.

UCP2 and Oxidative StressPreviously, we used a culture model to analyze the effects ofROS on endocrine pancreas development (16). Embryonicpancreata were cultured at the air/medium interface, and

different doses of hydrogen peroxide were added to themedium. We found that ROS stimulate endocrine differen-tiation by increasing the expression of NGN3. Moreover,this effect was ERK dependent. Despite similarities with thecurrent study, some of these ROS effects are different fromthe in vivo Ucp22/2 model. Indeed, ROS-induced endocrinedevelopment in vitro is mainly due to an increased differ-entiation, whereas here in vivo, an increased proliferationof the progenitor cells mainly occurs in Ucp22/2 pancreataprior to differentiation. We hypothesize that this differencemay be associated with the ROS levels in these two models.Moreover, in other cell types, such as embryonic stem cells,induced pluripotent stem cells, adipocytes, and neural pro-genitors, oxidative stress was shown to stimulate eithercell proliferation or cell differentiation, or both (16). Thus,

Figure 4—The antioxidant NAC normalizes the phenotype of the Ucp22/2 embryonic pancreata. Pregnant WT and Ucp22/2 mice were treatedwith 10 mmol/L NAC (drinking water) from E12.5 to E19.5 days postcoitum. Fetal pancreata were analyzed at E19.5. The white dotted linesdemarcate the limits of the pancreas. A: Nuclei were detected using Hoechst staining (blue) and a- and b-cells were detected with anti-insulin(red) and anti-glucagon (green) antibodies. Scale bar = 50 mm. B: The pancreas weights, the a-cell mass, and the b-cell mass were thencalculated. Each point represents the mean 6 SEM of three individual pancreata. *P , 0.05.

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these observations indicate that the effects of ROS are highlydependent on the cellular context. Moreover, downstream ofROS production, we found an activation of AKT in the Ucp22/2

pancreata. This link between ROS and AKT is similar to Le Belleet al. (21), which established that proliferative neural stemcells have high endogenous ROS levels that regulate both self-renewal and neurogenesis in a PI3K/AKT-dependent manner.

ConclusionsOur study demonstrates that UCP2 deficiency enhances thegrowth of the pancreas during embryogenesis and theperinatal period. This effect is mediated by an activationof the ROS-AKT signaling pathway. These mechanisms areimportant to better understand congenital hyperinsulinismobserved in children.

Acknowledgments. The authors thank Latif Rachdi (INSERM, U1016, InstitutCochin; CNRS, UMR8104; Université Paris Descartes, Sorbonne Paris Cité) for helpingto isolate the mouse neonatal islets. The authors thank Diane Girard (INSERM,U1016, Institut Cochin; CNRS, UMR8104; Université Paris Descartes, Sorbonne ParisCité) for the English editing of the manuscript. The research leading to these resultsreceived support from Société Francophone du Diabéte–Boehringer Ingelheim-Lilly.Duality of Interest. No other potential conflicts of interest relevant to thisarticle were reported.Author Contributions. B.B., S.B.F., E.A., and C.B. designed and performedexperiments and analyzed the data. T.S., M.B., and F.M. performed experiments.R.S. contributed to discussion and wrote the manuscript. M.-C.A.-G. and B.D.designed research experiments, performed experiments, analyzed data, and wrotethe manuscript. B.D. and M.-C.A.-G. are the guarantors of this work and, as such,had full access to all the data in the study and take responsibility for the integrity ofthe data and the accuracy of the data analysis.

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