The mitochondrial carrier uncoupling protein (UCP) 2 belongs to the family of the UCPs. Despite its name, it is now accepted that UCP2 is rather a metabolite transporter than a UCP. UCP2 can regulate oxidative stress and/or energetic metabolism. In rodents, UCP2 is involved in the control of α- and β-cell mass as well as insulin and glucagon secretion. Our aim was to determine whether the effects of UCP2 observed on β-cell mass have an embryonic origin. Thus, we used Ucp2 knockout mice. We found an increased size of the pancreas in Ucp2−/− fetuses at embryonic day 16.5, associated with a higher number of α- and β-cells. This phenotype was caused by an increase of PDX1+ progenitor cells. Perinatally, an increase in the proliferation of endocrine cells also participates in their expansion. Next, we analyzed the oxidative stress in the pancreata. We quantified an increased nuclear translocation of nuclear factor erythroid 2–related factor 2 (NRF2) in the mutant, suggesting an increased production of reactive oxygen species (ROS). Phosphorylation of AKT, an 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.
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 (UCP) 2 belongs to the family of UCPs (2). Despite the well-accepted role of UCP1 as a proton transporter and a UCP 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 reactive oxygen species (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 knockout 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 β-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.
Research Design and Methods
Experiments were in agreement with the French animal care committee guidelines. Ucp2−/− mice (C57Bl6/J background) were previously described (14). N-acetyl-l-cysteine (NAC) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) treatment was initiated at embryonic day 9.5 (E9.5) until E13.5, and at E12.5 until E19.5.
Tissues were fixed in 10% formalin and processed for immunohistochemistry, as described previously (15). The following 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), and rabbit anti–phospho Akt (Ser 473) (1:25) (nos. 9272 and 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, U.K.), and Alexa Fluor anti-rabbit antibody (1:400; Biogenex, Fremont, CA). For NGN3, revelation was performed using the vectastain ABC kit (Vector Laboratories, Peterborough, U.K.). Fluorescent image acquisition was performed using the Zeiss AxioObserver Z1 inverted fluorescence microscope coupled with the Zeiss Axiocam MRm (Zeiss, Marly-le-Roi, France).
Determination of Cellular ATP Levels
Detection of ATP levels was assessed using a luminescence-based assay kit (Roche, Meylan, France).
RNA Extraction and PCR
Procedures are described in Hoarau et al. (16). The oligonucleotide sequences for RT-PCR are available on request.
Western Blot Analysis
For Western blotting analysis, cells were lysed in Laemmli. Proteins (20 µg) were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride 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), mouse anti–β-actin (Sigma-Aldrich), mouse anti–α-tubulin (Sigma-Aldrich), rabbit anti-Akt, and rabbit anti–phospho Akt (Ser 473) (nos. 9272 and 9271; Cell Signaling). Immunoreactive bands were visualized with the SuperSignal System (Pierce, Fisher Scientific, Illkirch, France).
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 previously (17,18). In brief, cell suspensions were stained in Hanks’ balanced salt solution (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), and anti-CD133 APC (clone 3152C11). For each antibody, optimal dilution was determined by titration. Cells were incubated for 15–30 min 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 (1:4,000; Sigma-Aldrich). Stained cells were analyzed and sorted with FACS Aria III (BD Biosciences). Data were analyzed in FlowJo (Ashland, OR) software.
Neonatal islets from wild-type (WT) and Ucp2−/− mice were harvested as described previously (19). Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase (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 secretion was quantified as described previously (20) using an ultrasensitive mouse insulin ELISA kit (Crystal Chem, Zaandam, the Netherlands).
To quantify the absolute surface of PDX1-, insulin-, glucagon-, and amylase-expressing and Hoechst-stained cells, 5-µm-thick sections of each pancreas were digitized at E13.5 and E16.5. At E19.5 and postnatal day 2, one of the five slides of the total pancreas was digitized (17). On every image, the surface of immunostaining was quantified by ImageJ (National Institutes of Health, Bethesda, MD). 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 t test. To measure proliferation of early progenitors, we counted the frequency of Ki67+ nuclei among 1,000 PDX1+ cells. At least three rudiments per condition were analyzed. Statistical significance was determined using Student t test.
Increased Pancreas Growth in the Ucp2 Knockout 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 twofold compared with controls (Figs. 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 (Figs. 1 and 2). Using an antibody directed against 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+ 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 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 nonsignificant decrease of ATP per cell was quantified at E16.5 (6.93 × 10−4 pmol in Ucp2−/− vs. 9.83 × 10−4 pmol 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. In the 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 whereas 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 β-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 twofold 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 ratio of phospho-AKT to total AKT, confirmed by Western blot at E16.5 (Supplementary Figs. 9 and 10). Thus, these data suggest that the activation of the ROS-AKT signaling pathway is involved in the growth of Ucp2−/− mouse pancreas.
NAC Treatment Reverses the Pancreatic Phenotype of the Ucp2−/− Fetuses
To further analyze the implication of ROS, we treated pregnant mice with the antioxidant NAC 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 and β-cell and α-cell masses were increased in untreated Ucp2−/− fetuses, compared with controls (Fig. 4). This effect was abolished when Ucp2−/− fetuses received NAC treatment (Fig. 4). Interestingly, α- and β-cell proliferation was increased in E19.5 Ucp2−/− pancreata versus controls (Supplementary Figs. 12 and 13). This effect was abrogated when an NAC treatment was administrated. Thus, the knockout of Ucp2 controls the proliferation of endocrine cells in an ROS-dependent manner. It leads to a nonsignificant 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.
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 negatively control 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. (23) 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. On the contrary, using similar experiments, Lee et al. (12) 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. 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 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 current 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, whereas here in vivo, an increased proliferation of the progenitor cells mainly occurs in Ucp2−/− pancreata prior to differentiation. We hypothesize that this difference may 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 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 on 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 Le Belle et al. (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.
Our study demonstrates that UCP2 deficiency enhances the growth of the pancreas during embryogenesis and the 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.
Acknowledgments. The authors thank Latif Rachdi (INSERM, U1016, Institut Cochin; CNRS, UMR8104; Université Paris Descartes, Sorbonne Paris Cité) for helping to isolate the mouse neonatal islets. The authors thank Diane Girard (INSERM, U1016, Institut Cochin; CNRS, UMR8104; Université Paris Descartes, Sorbonne Paris Cité) for the English editing of the manuscript. The research leading to these results received support from Société Francophone du Diabéte–Boehringer Ingelheim-Lilly.
Duality of Interest. No other potential conflicts of interest relevant to this article were reported.
Author Contributions. B.B., S.B.F., E.A., and C.B. designed and performed experiments 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 wrote the 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 of the data and the accuracy of the data analysis.