Mitochondrial Protein UCP2 Controls Pancreas Development · 27/10/2017 · Our aim was to...

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Mitochondrial protein UCP2 controls pancreas development. Benjamin Broche

1, 2, 3, Selma Ben Fradj

1, 2, 3, Esther Aguilar

1, 2, 3, Tiphaine Sancerni

1, 2, 3, 4,

Matthieu Bénard1, 2, 3

, Fatna Makaci1, 2, 3

, Claire Berthault 1, 2, 3

, Raphaël Scharfmann1, 2, 3

,

Marie-Clotilde Alves-Guerra1, 2, 3 *

and Bertrand Duvillié1, 2, 3 *

.

1 Inserm, U1016, Institut Cochin, Paris, France.

2 CNRS, UMR8104, Paris, France.

3 Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

4 Université Paris Diderot, Sorbonne Paris Cité, Paris, France.

*Corresponding authors:

Bertrand Duvillié

Institut Cochin, Inserm U1016

Groupe Hospitalier Cochin-Port-Royal

Bâtiment Cassini

123 bd du Port-Royal

75014 Paris

e-mail: [email protected]

Phone: (33) 1 69 86 30 41

and

Marie-Clotilde Alves-Guerra

Institut Cochin, Inserm U1016

Bâtiment Faculté

24 rue du Faubourg Saint Jacques

75014 Paris

e-mail: [email protected]

Phone: (33) 1 53 73 27 06

Running Title: UCP2 and beta-cell development

Keywords: UCP2, Reactive Oxygen Species, development, pancreas, beta-cell

Word count: 2332

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Diabetes Publish Ahead of Print, published online October 27, 2017

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Abbreviations

AKT AKT serine/threonine kinase 1

ATP adenosine 5’-triphosphate

DNPH dinitrophenylhydrazine

ERK1/2 extracellular signal-regulated kinase 1

FACS Fluorescent activated cell sorting

KO knock-out

NAC N-acetyl-L-cysteine

NGN3 neurogenin3

NRF2 nuclear factor, erythroid 2-related factor 2 (Nfe2l2)

PDX1 pancreatic and duodenal homeobox 1

Ki67 antigen Ki-67

ROS reactive oxygen species

SDS sodium dodecyl sulfate

UCP2 uncoupling protein 2

WT wild type

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Abstract

The mitochondrial carrier UCP2 belongs to the family of the uncoupling proteins. Despite its

name, it is now accepted that UCP2 is rather a metabolite transporter than an uncoupling

protein. UCP2 can regulate oxidative stress and/or energetic metabolism. In rodents, UCP2 is

involved in the control of alpha- and beta-cell mass as well as insulin- and glucagon-secretion.

Our aim was to determine whether the effects of UCP2 observed on beta-cell mass have an

embryonic origin. Thus, we used Ucp2 knock-out mice. We found an increased size of the

pancreas in Ucp2-/-

fetuses at E16.5, associated with a higher number of alpha- and beta-cells.

This phenotype was caused by an increase of PDX1+ progenitor-cells. Perinatally, an increase

in the proliferation of endocrine cells also participates to their expansion. Next, we analyzed

the oxidative stress in the pancreata. We quantified an increased nuclear translocation of

NRF2 in the mutant suggesting an increased production of ROS. Phosphorylation of AKT, a

ROS-target, was also activated in the Ucp2-/-

pancreata. Finally, administration of the

antioxidant N-acetyl-L-cysteine to Ucp2-/-

pregnant mice alleviated the effect of knocking-out

UCP2 on pancreas development. Together, these data demonstrate that UCP2 controls

pancreas development through the ROS-AKT signaling pathway.

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Introduction

During the last decade, the impact of mitochondrial dysfunction in pancreatic islet

development and diabetes has been widely studied (1). However the underlying mechanisms

involving the mitochondria are still not well understood. The mitochondrial uncoupling

protein 2 belongs to the family of Uncoupling Proteins (UCPs) (2). Despite the well-accepted

role of UCP1 as a proton transporter and an uncoupling protein in the brown adipose tissue, it

was shown that UCP2 is a metabolite transporter with no or little mitochondrial uncoupling

activity (3). UCP2 is expressed in the spleen, lungs, stomach, adipose tissue and pancreas

(4,5). Moreover, several studies indicate that UCP2 is a repressor of ROS production in

different cell types (6,7). In addition, UCP2 can regulate the balance between glycolysis and

oxidative phosphorylation in murine embryonic fibroblasts (7) and in different types of cancer

cells (8,9). Recently, Ucp2 mutations were discovered in humans and were associated with

congenital hyperinsulinism (10). In mice, the absence of UCP2 also leads to increased insulin

secretion (11) supporting the observation in humans. The knock-out of UCP2 induces an

increase in the number of endocrine cells, and this phenotype is amplified by a high fat diet

(12,13).

The aim of our study was to determine whether the beta-cell hyperplasia observed in adult

Ucp2-/-

mice has an embryonic origin. For this, we used Ucp2-/-

mouse embryos at different

stages and we analyzed the development of the pancreas.

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Research Design and Methods

Animals.

Experiments were in agreement with the French animal care committee guidelines. Ucp2-/-

mice (C57Bl6/J background) were previously described (14). N-acetyl-L-cysteine (Sigma-

Aldrich, Saint-Quentin Fallavier, France) treatment was initiated at E9.5 until E13.5, and at

E12.5 until E19.5.

Immunohistochemistry.

Tissues were fixed in 10% formalin and processed for immunohistochemistry, as described

previously (15). The following antibodies were used: mouse anti-insulin (1 : 2000; Sigma-

Aldrich, Saint-Quentin Fallavier, France), rabbit anti-glucagon (1 : 1000; Euromedex,

Souffelweyerrsheim, France), rabbit anti-PDX1 (1 : 1000), mouse anti-Ki67 (1 : 50, BD,

Pharmingen, Le Pont-De Claix, France), rabbit anti-amylase (1 : 300, Sigma-Aldrich), rabbit

anti-NGN3 (1 : 1000), rabbit anti-NRF2 (1 : 1000, GeneTex, Irvine, CA, USA), rabbit anti-

Akt (1 : 200) and rabbit anti-Phospho Akt (Ser 473) (1: 25) (#9272, #9271 Cell signaling,

Saint Quentin, France). The fluorescent secondary antibodies were fluorescein isothiocyanate

anti-rabbit and Texas Red anti-mouse antibodies (1 : 200, Jackson Immunoresearch, Suffolk,

UK), and Alexa-fluor anti-rabbit antibody (1 : 400, Biogenex, Fremont, CA, USA). For

NGN3, revelation was performed using the vectastain ABC kit (Vector LAB, Peterborough,

UK). Fluorescent image acquisition was performed using an inverted fluorescence microscope

Zeiss AxioObserver Z1 coupled with MRm Axiocam Zeiss (Zeiss, Marly le Roi, France).

Determination of cellular ATP levels

Detection of ATP levels was assessed using a luminescence-based assay kit (Roche, France).

RNA extraction and PCR

Procedures are described in (16). The oligonucleotide sequences for RT-PCR are available on

request.

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Western blot analysis

For western blotting analysis, cells were lysed in Laemmli. Proteins (20 µg) were separated

by SDS-PAGE and electrophoretically transferred onto PVDF membrane (Bio-Rad, Marnes-

la-Coquette, France). After blocking with milk, membranes were probed with mouse anti-

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, Cell signaling, Saint Quentin, France),

mouse anti-β−actin (Sigma-Aldrich), mouse anti-α−tubulin (Sigma-Aldrich), rabbit anti-Akt

and rabbit anti-Phospho Akt (Ser 473) (#9272, #9271 Cell signaling, Saint Quentin, France).

Immunoreactive bands were visualized with the SuperSignal System (Pierce, Fisher

Scientific, Illkirch, France).

Protein oxidation

Protein oxidation of total pancreas homogenates was measured by assaying the amount of

carbonyl groups on proteins using the OxyBlot kit (Protein Oxidation Detection kit, Millipore,

Molsheim, France).

Cell suspension and cell sorting

The procedures were described in (17,18). Briefly, cells suspensions were stained in HBSS

without Calcium/Magnesium supplemented with 20% FCS with the following anti-mouse

antibodies purchased 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),

anti-CD133 APC (clone 3152C11). For each antibody, optimal dilution was determined by

titration. Cells were incubated for 15 to 30 minutes at 4°C in the dark, washed and suspended

in HBSS without Calcium/Magnesium supplemented with 20% FCS, and dead cells were

excluded with Propidium Iodide (1 : 4000, Sigma Aldrich). Stained cells were analyzed and

sorted with FACS Aria III (BD Biosciences). Data was analyzed in FlowJo (Ashland, Oregon,

USA) software.

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Islet isolation

Neonatal islets from WT and Ucp2-/-

mice were harvested as described in (19). Freshly

dissected whole pancreases were digested with 0.5 mg/ml collagenase (Sigma-Aldrich)

dissolved in HBSS at 37°C. Tubes were tapped regularly to aid tissue dispersial. Next, lyzates

were washed with HBSS containing 10% fetal bovine serum FBS. Then, islets were

handpicked up under dissecting stereoscope (Leica, Nanterre, France).

Insulin secretion

Insulin secretion was quantified as described in (20) using ultra-sensitive mouse insulin Elisa

kit (Zaadam, Netherlands).

Quantification

To quantify the absolute surface of PDX1-, insulin-, glucagon-, amylase-expressing, and

Hoechst-stained cells, all 5 µm thick sections of each pancreas were digitized at E13.5 and

E16.5. At E19.5 and PN2, one of the 5 slides of the total pancreas was digitized (17). On

every image, the surface of immuno-staining was quantified by ImageJ (NIH, Washington,

WA, USA). At E16.5, the total number of immunopositive cells for NGN3 was counted on all

sections of a complete pancreas. Statistical significance was determined by Student's t-testing.

To measure proliferation of early progenitors, we counted the frequency of Ki67+ nuclei

among 1000 PDX1+ cells. At least three rudiments per condition were analyzed. Statistical

significance was determined using Student's t-test.

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Results

Increased pancreas growth in the Ucp2 knock-out mice.

First, the expression pattern of UCP2 was analyzed. E12.5 pancreatic epithelial and

mesenchymal cells were separated by FACS (17). Ucp2 expression was enriched in the

epithelial fraction containing the progenitors (Supplementary Fig. 1A). At E16.5, we

separated mesenchymal-, acinar-, NGN3+- and endocrine-cells (18). Ucp2 expression was

found preferentially in endocrine-cells, and in a lesser extent in other cell types

(Supplementary Fig. 1B). To investigate its role, Ucp2+/-

mice were intercrossed. The weight,

islet insulin secretion and glycemia of the homozygous neonates were all similar to the

controls (Supplementary Fig. 2). During the embryonic and fetal periods, the overall external

morphology of Ucp2-/-

animals was normal (Supplementary Fig. 3). As shown by the

Hoechst-staining, the size of the Ucp2-/-

pancreas at E16.5, E19.5 and PN2 was increased by

nearly 2-folds compared to controls (Fig. 1, 2 and 4). This difference was not observed at

E13.5 (Fig. 3C). Moreover, the absolute surfaces of insulin, glucagon and amylase were also

increased in the Ucp2-/-

pups and fetuses (Fig. 1 and 2). Using an antibody directed against

Neurogenin 3 (NGN3), we showed that the number of endocrine precursors increased

proportionally to the pancreas size at E16.5 in the mutants (Supplementary Fig. 4). To

investigate the mechanism responsible for the increased growth of the Ucp2-/-

pancreata, we

quantified progenitor proliferation using anti-PDX1 and anti-Ki67 antibodies. At E13.5, we

found an increased proliferation of PDX1-positive progenitor cells (Fig. 3A and B), but not at

E12.5 (Supplementary Fig. 5). Together, these data demonstrate that Ucp2 deletion induces an

overgrowth of the pancreas due to an increased proliferation of progenitor cells.

UCP2 controls oxidative stress and AKT signaling in the Ucp2-/- fetal pancreas.

Two main mechanisms have been described to explain the biological effects of UCP2. First,

UCP2 can modulate the energetic metabolism by controlling the balance between glycolysis

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and oxidative phosphorylation (8). To examine the energetic status of Ucp2-/-

pancreas, we

quantified the ATP content in Ucp2-/-

and WT pancreata. No difference was found at the

pancreatic level (Supplementary Fig. 6). However, a non-significant decrease of ATP per cell

was quantified at E16.5 (6.93.10-4

pmoles in Ucp2-/-

vs 9.83.10-4

pmoles in WT, P=0.25). The

second hypothesis is that UCP2 is involved in the regulation of the production of ROS (5,6).

To examine this possibility, we performed immunofluorescence experiments to visualize the

nuclear translocation of the ROS-sensitive factor NRF2 (nuclear factor erythroid 2-related

factor 2). In absence of oxidative stress, NRF2 is associated with the protein Keap1, which

promotes the degradation of NRF2 by the ubiquitin proteasome pathway. Also, oxidants can

modify the cysteine residues of Keap1, leading to nuclear translocation of NRF2.

Interestingly, we observed NRF2 only at the periphery of the nuclei at E13.5 in the WT

pancreata while nuclear translocation of NRF2 was observed in the mutants (Supplementary

Fig. 7). At E16.5, nuclear translocation was found both in WT and Ucp2-/-

pancreata, in an

area containing beta-cells and this event was increased in the mutants (Supplementary Fig. 7).

These results suggest that ROS are involved in endocrine development. We also quantified

protein oxidation levels using the Oxyblot assay (Supplementary Fig. 7). We found a nearly

2-fold increase of the protein oxidation levels in the mutant pancreata. Together, these results

indicate that oxidative stress is higher in the Ucp2-/-

pancreata. To further investigate signaling

pathways involved in the pancreatic phenotype of Ucp2-/-

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 ROS levels (21). The total-AKT level was slightly increased in the mutants at

E16.5 but not at E13.5. At both stages in the mutants, we found an increased phospho-

AKT/total-AKT ratio, confirmed by western blot at E16.5 (Supplementary Fig. 9-10). Thus,

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these data suggest that the activation of the ROS-AKT signaling pathway is involved in the

growth of Ucp2-/-

mice pancreas.

N-acetyl-L-cysteine treatment reverses the pancreatic phenotype of the Ucp2-/- fetuses.

To further analyze the implication of ROS, we treated pregnant mice with the antioxidant N-

acetyl-L-cysteine between E12.5 and E19.5. In Ucp2-/-

pancreata, the number of NRF2+ cells

decreased when treated with NAC, validating its antioxidant effect (Supplementary Fig. 11).

Pancreatic weight, beta-cell, and alpha-cell masses were increased in non-treated Ucp2-/-

fetuses, compared to controls (Fig. 4). This effect was abolished when Ucp2-/-

fetuses received

NAC treatment (Fig. 4). Interestingly, alpha- and beta-cell proliferation was increased in

E19.5 Ucp2-/-

pancreata versus controls (Supplementary Fig. 12 and 13). This effect was

abrogated when a NAC-treatment was administrated. Thus, the knock-out of Ucp2 controls

the proliferation of endocrine cells in a ROS-dependent manner. It leads to a non-significant

increased fraction of endocrine cells at PN2 (Supplementary Fig. 14) but not at E19.5. Finally,

we treated Ucp2-/-

and control mice with NAC from E9.5 to E13.5. Such treatment reduced

progenitor proliferation induced by the deletion of Ucp2 (Fig. 3B). Altogether, these data

demonstrate that increased oxidative stress caused by the lack of UCP2 is responsible for the

increased fetal pancreata growth.

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Discussion

Our main finding is that UCP2 is a negative regulator of pancreas development. Indeed, the

absence of UCP2 induces an increase in cell-proliferation and a larger pancreas. Moreover,

this effect is induced by oxidative signals, through the activation of the AKT pathway.

The roles of UCP2 in physiological and pathological processes.

Here we show that the deletion of Ucp2 increases progenitor and endocrine-cell proliferation,

two cell types that normally express Ucp2 (Supplementary Fig. 1). This suggests a potential

autocrine effect of UCP2 but we do not exclude other paracrine effects. For example, the

mesenchyme that expresses lower levels of Ucp2 also controls progenitor proliferation (15).

Moreover, several recent studies indicate that UCP2 plays a crucial role in the development of

several cell types. Indeed, during human stem cell differentiation, UCP2 expression decreases

suggesting its role as a repressor of stem cell differentiation (22). Moreover, in murine

embryonic fibroblasts, UCP2 was shown to control negatively their proliferation (7). Finally,

the roles of UCP2 were investigated in different pathologies. In cancer cell lines expressing

low levels of UCP2, its overexpression decreases cell proliferation through metabolic changes

and in consequence represses the malignant phenotype. Moreover, in diabetes, the

involvement of UCP2 is still controversial (23). Indeed, Emre et al. treated WT and Ucp2-/-

mice with low doses of streptozotocin to generate an experimental model of diabetes. They

found that autoimmune diabetes was accelerated in Ucp2-/-

mice, with the presence of an

increased lymphocytic infiltration (23). On the contrary, using similar experiments, Lee et al.

found that treatment of WT and Ucp2-/-

mice with low doses of streptozotocin resulted in

hyperglycemia that was much less severe in Ucp2-/-

mice than controls (12). The difference

between these two studies was suggested to be connected to the genetic background of the

mice. Moreover, in humans, another recent illustration is that variants of the Ucp2 gene are

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associated with diabetes and diabetic retinopathy in a chinese population (24). The exact

mechanism responsible for diabetes in these patients still needs to be elucidated.

UCP2 and oxidative stress

Previously, we used a culture model to analyze the effects of ROS on endocrine pancreas

development (16). Embryonic pancreata were cultured at the air/medium interface, and

different doses of hydrogen peroxide were added to the medium. We found that ROS

stimulate endocrine differentiation by increasing the expression of NGN3. Moreover, this

effect was ERK-dependent. Despite similarities with the present study, some of these ROS

effects are different from the in vivo Ucp2-/-

model. Indeed, ROS-induced endocrine

development in vitro is mainly due to an increased differentiation, while here in vivo an

increased proliferation of the progenitor cells mainly occurres in Ucp2-/-

pancreata prior to

differentiation. We hypothesize that this difference may be associated to the ROS levels in

these two models. Moreover, in other cell types such as embryonic stem cells, induced

pluripotent stem cells, adipocytes and neural progenitors, oxidative stress was shown to

stimulate either cell-proliferation, or cell-differentiation, or both (16). Thus, these

observations indicate that the effects of ROS are highly dependent of the cellular context.

Moreover, downstream of ROS production, we found an activation of AKT in the Ucp2-/-

pancreata. This link between ROS and AKT is similar to (21), which established that

proliferative neural stem cells have high endogenous ROS levels that regulate both self-

renewal and neurogenesis in a PI3K/AKT-dependent manner.

In conclusion, our study demonstrates that UCP2 deficiency enhances the growth of the

pancreas during embryogenesis and perinatal period. This effect is mediated by an activation

of the ROS-AKT signaling pathway. These mechanisms are important to better understand

congenital hyperinsulinism observed in children.

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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements.

We thank Latif Rachdi (Inserm, U1016, Institut Cochin, CNRS, UMR8104, Université Paris

Descartes, Sorbonne Paris Cité, Paris, France) for helping to isolate the mouse neonatal islets.

We thank Diane Girard (Inserm, U1016, Institut Cochin, CNRS, UMR8104, Université Paris

Descartes, Sorbonne Paris Cité, Paris, France) for the english editing of the manuscript. The

research leading to these results has received support from Société Francophone du Diabète

(SFD-Boehringer Ingelheim-Lilly).

Bertrand Duvillié and Marie-Clotilde Alves-Guerra 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 of the

data and the accuracy of the data analysis.

Author contributions

B.B, S.BF, E.A, C.B designed and performed experiments and analyzed the data. T.S, M.B,

F.M, performed experiments. RS contributed to discussion and wrote the manuscript. B.D and

M-C.A-G designed research experiments, performed experiments, analyzed data, and wrote

the manuscript.

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Figure legends

Figure 1. UCP2 deficiency increases pancreas development.

WT and Ucp2-/-

pancreata were analyzed at postnatal day 2. The white dotted lines demarcate

the limits of the pancreas. (A) Nuclei were stained with Hoechst 33342 (blue). (B-C) Islets

were detected using anti-insulin (red, B) and anti-glucagon (green, C) antibodies. Exocrine

cells were detected using anti-amylase antibody (green, D). The absolute surfaces area

occupied by Hoechst-positive cells, insulin-positive cells, glucagon-positive cells and

amylase-positive cells were quantified. Each point represents the mean ± S.E.M of n≥3

individual pancreata. * P<0.05. Scale bar: 50 µm.

Figure 2. Pancreas growth is enhanced in the Ucp2-/- E16.5 fetuses.

WT and Ucp2-/-

pancreata were analyzed at E16.5. The white dotted lines demarcate the limits

of the pancreas. (A) Photographs of the WT and Ucp2-/-

pancreata. Scale bar: 200 µm. (B)

Nuclei were stained with Hoechst 33342 (blue). (C-D) Endocrine development was

investigated using anti-insulin (red, C) and anti-glucagon (green, D) antibodies. The absolute

surface area occupied by Hoechst-positive cells, insulin-positive cells, glucagon-positive cells

were quantified. Each point represents the mean ± S.E.M of n≥3 individual pancreata. *

P<0.05. Scale bar: 50 µm.

Figure 3. The proliferation of the progenitor cells is increased in the Ucp2-/- E13.5

pancreata.

(A) The proliferation of the PDX1+ progenitor cells was analyzed using anti-PDX1 (green)

and anti-Ki67 (red) antibodies. (B) Proliferation percentage was also quantified. Each point

represents the mean ± S.E.M of n≥3 individual pancreata. * P<0.05. Scale bar: 50 µm. For

higher magnification, bar: 10 µm. (C) E13.5 pancreata were analyzed using anti-PDX1

antibodies (green). Nuclei were stained with Hoechst 3342 (blue). The white dotted lines

demarcate the limits of the pancreas. The absolute areas of Hoechst-positive cells and PDX1-

positive cells were quantified. Each point represents the mean ± S.E.M of n≥3 individual

pancreata. NS: not significant.

Figure 4. The antioxidant N-acetyl-L-cysteine normalizes the phenotype of the Ucp2-/-

embryonic pancreata.

Pregnant WT and Ucp2-/-

mice were treated with 10 mM NAC (drinking water) from E12.5 to

E19.5 days post-coïtum. Fetal pancreata were analyzed at E19.5. The white dotted lines

demarcate the limits of the pancreas. (A) Nuclei were detected using Hoechst staining (blue)

and alpha- and beta-cells were detected with anti-insulin (red) and anti-glucagon (green)

antibodies. (B) The pancreas weights, the alpha-cell mass and the beta-cell mass were then

calculated. Each point represents the mean ± S.E.M of three individual pancreata. * P<0.05.

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Supplementary Fig. 1. FACS analysis of the expression of Ucp2.

(A) Analysis of Ucp2 expression at E12.5. (B) Analysis of Ucp2 expression at E16.5.

E12.5 and E16.5 fetal pancreata were dissociated and stained as described in

Supplementary Materials with anti-CD45, anti-CD31, anti-TER119, anti-EpCam,

anti-CD133 and anti-CD49f antibodies. CD45, CD31 and TER119 were used to

exclude hematopoietic cells, endothelial cells and erythrocytes, respectively. The

remaining CD45- CD31- TER119- fraction was subdivided into mesenchymal

(EpCam-) and epithelial (EpCam+) enriched fractions. At E16.5, the EpCam+

epithelial enriched fraction was further subdivided into 3 fractions using anti-CD133

and anti-CD49f antibodies: fraction I (CD133+ CD49f high) is enriched in acinar

cells, fraction II (CD133+ CD49f intermediate) is enriched in NGN3 positive cells,

fraction III (CD133- CD49f intermediate) is enriched in hormone positive cells. Dot

plots are representative of 3 independent stainings of 7 to 15 pooled fetal pancreata.

Quantitative RT-PCR gene expression analysis of Ucp2, Pdx1 and Nkx3.2 was done

in E12.5 pancreas CD45-CD31-TER119- EpCam- and EpCam+ fractions.

Quantitative RT-PCR gene expression analysis of Ucp2, Nkx3.2, Ins, Amy2, Ngn3

was done in E16.5 pancreas (CD45-CD31-TER119-) EpCam- and EpCam+ (I, II and

III) fractions. Histograms display relative quantity of expression normalized to Ppia.

Data is a pool of 3 independent experiments. * P<0.05.

Supplementary Fig. 2. Physiological parameters of the WT and Ucp2-/- neonates.

(A) WT and Ucp2-/- neonates were weighed at birth. Graph represents the mean±

S.E.M of n≥5 animals. (B) Glycemia was measured using a glucometer (in mg/dl).

Each point represents the mean of n≥5 animals ± S.E.M. (C) Pancreatic islets were

isolated one day after birth from Ucp2-/- and WT pancreata. Glucose stimulated

insulin secretion is represented. Data are expressed as average percentage of secreted

insulin. Results are representative of two independent experiments performed.

Supplementary Fig. 3. The external morphology of the WT and Ucp2-/- animals is

normal during embryonic and fetal development.

E13.5 and E16.5 WT and Ucp2-/- fetuses from the same litter were photographed

using a binocular microscope (Leica, France). Bar: 2000 µm.

Supplementary Fig. 4. The number of endocrine progenitor cells is increased in

the Ucp2-/- E16.5 pancreata.

Fetal pancreata were analyzed at E16.5. (A) NGN3 expression (in brown) was

detected by immunohistochemistry and the absolute number of NGN3-positive cells

was quantified. Arrows indicate positive nuclei. (B) The number of NGN3-positive

cells per surface of pancreas (mm2) is represented. Each point represents the mean±

S.E.M of three individual pancreata. * P<0.05. NS: not significant. Scale bar: 50 µm.

Supplementary Fig. 5. The proliferation of the progenitor cells is unaltered in the

Ucp2-/- E12.5 pancreata.

The proliferation of the PDX1+ progenitor cells was analyzed using anti-PDX1

(green) and anti-Ki67 (red) antibodies. Proliferation percentage was also quantified.

Each point represents the mean ± S.E.M of three individual pancreata. Scale bar: 50

µm.

Supplementary Fig. 6. The pancreatic ATP content is unaltered by Ucp2

deficiency.

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ATP was extracted from n=5 pancreatic rudiments from E13.5 and E16.5 Ucp2-/-

and

WT fetuses. Each point represents the mean ± S.E.M of five individual pancreata.

Supplementary Fig. 7. NRF2 translocation analysis.

(A) Photomicrographs showing immunohistochemical staining for NRF2 (green) and

insulin (red) on E13.5 pancreatic sections. NRF2 was found at the periphery of the

nuclei in WT pancreata while it was detected in the nuclei of the Ucp2-/- pancreata.

Arrows indicate the presence of NRF2 in the insulin-positive cells. (B)

Immunohistochemical staining for NRF2 (green) and insulin (red) on E16.5

pancreatic sections. Higher magnification shows the presence of NRF2 in the nuclei.

Arrows indicate the NRF2+ Insulin+ cells. The white dotted lines demarcate the

limits of the pancreas. (C) Quantification of the total surface of immunoreactive cells

for NRF2, and the surface of NRF2-positive cells per mm2

of pancreas surface. (D)

Quantification of protein oxidation in extracts from WT and Ucp2-/- pancreata. Each

point represents the mean ± S.E.M of five individual pancreata. * P<0.05. Scale bar:

50 µm. For higher magnification, bar: 10 µm.

Supplementary Fig. 8. The ERK1/2 pathway is not modified in the Ucp2-/-

pancreata.

(A) Protein extracts from WT and Ucp2-/- pancreata were analyzed by western blot to

quantify Phospho-ERK and Total ERK. (B) Ratio of Phospho-ERK/ERK was

quantified for each group of pancreata. Each point represents the mean ± S.E.M n=5.

Supplementary Fig. 9. Phosphorylation of AKT is activated in the Ucp2-/- E13.5

pancreata. (A) Phospho-AKT and Total AKT were analyzed by immunofluorescence

on WT and Ucp2-/- consecutive sections. Higher magnification shows increased

staining of P-AKT in the Ucp2-/-

pancreata versus WT. The white dotted lines

demarcate the limits of the pancreas. (B) The signals were quantified using ImageJ

software and the P-AKT percentage with regards to Total AKT was calculated. Each

point represents the mean ± S.E.M n=3 pancreata. Scale bar: 50 µm. For higher

magnification, scale bar: 10 µm. * P<0.05.

Supplementary Fig. 10. Phosphorylation of AKT is activated in the Ucp2-/- E16.5

pancreata. (A) Phospho-AKT and Total AKT were analyzed by immunofluorescence

on WT and Ucp2-/- consecutive sections. The signals were quantified using ImageJ

software and the P-AKT percentage with regards to Total AKT was calculated.

Higher magnification shows increased staining of P-AKT in the Ucp2-/-

pancreata

versus WT. Each point represents the mean ± S.E.M n=3 pancreata. * P<0.05. Scale

bar: 50 µm. For higher magnification, bar: 10 µm. (B) Protein extracts from WT and

Ucp2-/-

E16.5 pancreata were analyzed by western blot to quantify P-AKT and Total

AKT. Densitometry was performed and P-AKT was normalized to ß-actin and

quantified for each group of tissue. Each point represents the mean ± S.E.M of n=4. *

P<0.05. Ratio P-AKT/ Total AKT was also determined. Each point represents the

mean ± S.E.M of n=4. ** P<0.01.

Supplementary Fig. 11. NAC treatment decreases expression of nuclear NRF2

protein in the Ucp2-/- beta-cells.

Pregnant WT and Ucp2-/-

mice were treated with 10 mM NAC (drinking water) from

E12.5 to E19.5 days post-coïtum. Fetal pancreata were analyzed at E19.5. NRF2 was

detected by immunofluorescence (green) and beta-cells were detected with anti-

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insulin (red). The NRF2+ Ins+ cell percentage of the total number of insulin-positive

cells is shown. Each point represents the mean ± S.E.M of three individual pancreata.

* P<0.05. Scale bar: 10 µm.

Supplementary Fig. 12. The proliferation of beta-cells is increased in the Ucp2-/-

E19.5 pancreata and depends on oxidative stress.

The proliferation of the beta-cells was analyzed using anti-insulin (green) and anti-

Ki67 (red) antibodies. When treated with NAC from E12.5 to E19.5, the proliferation

of beta-cells decreased in Ucp2-/- but not in WT pancreata. Each point represents the

mean ± S.E.M of three individual data pools. *** P<0.001. Scale bar: 50 µm.

Supplementary Fig. 13. The proliferation of alpha-cells is increased in the Ucp2-/-

E19.5 pancreata and decreased after NAC treatment.

The proliferation of the alpha-cells was analyzed using anti-glucagon (green) and

anti-Ki67 (red) antibodies. When treated with NAC from E12.5 to E19.5, the

proliferation of alpha-cells decreased in Ucp2-/- but not in WT pancreata. Each point

represents the mean ± S.E.M of five individual pancreata. *** P<0.001. Scale bar: 50

µm.

Supplementary Fig. 14. Fractions of alpha and beta cells in Ucp2-/- and wild type

pancreata at different developmental stages.

(A) The insulin staining percentage of the total pancreatic surface was quantified at

E16.5. Each point represents the mean ± S.E.M of five individual pancreata. (B) The

glucagon staining percentage of the total pancreatic surface was quantified. Each

point represents the mean ± S.E.M of five individual pancreata. (C-D) Percentage of

beta- and alpha-cell mass at E19.5 in Ucp2-/-

and WT pancreata treated or not with

NAC. (C) The beta-cell mass percentage of the total pancreatic mass was quantified.

Each point represents the mean ± S.E.M of five individual pancreata. (D) The alpha-

cell mass percentage of the total pancreatic mass was quantified. Each point

represents the mean ± S.E.M of five individual pancreata. (E-F) beta- (E) and alpha-

cell (F) masses normalized to pancreas weight at PN2. Each point represents the mean

± S.E.M of four individual pancreata. (F-G) beta- (F) and alpha-cell (G) masses

normalized to body weight at PN2. Each point represents the mean ± S.E.M of four

individual pancreata.

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Mitochondrial Protein UCP2 Controls Pancreas Development · 27/10/2017 · Our aim was to...

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