Olivier Dumortier,1,2,3 Charlotte Hinault,1,2,3,4 Nadine Gautier,1,2,3 Stéphanie Patouraux,5

Virginie Casamento,1,2,3 and Emmanuel Van Obberghen1,2,3,4

Maternal Protein RestrictionLeads to Pancreatic Failure inOffspring: Role of MisexpressedMicroRNA-375Diabetes 2014;63:3416–3427 | DOI: 10.2337/db13-1431

The intrauterine environment of the fetus is a preeminentactor in long-term health. Indeed, mounting evidenceshows that maternal malnutrition increases the risk oftype 2 diabetes (T2D) in progeny. Although the conse-quences of a disturbed prenatal environment on thedevelopment of the pancreas are known, the underlyingmechanisms are poorly defined. In rats, restriction ofprotein during gestation alters the development of theendocrine pancreas and favors the occurrence of T2Dlater in life. Here we evaluate the potential role ofperturbed microRNA (miRNA) expression in the de-creased b-cell mass and insulin secretion characterizingprogeny of pregnant dams fed a low-protein (LP) diet.miRNA profiling shows increased expression of severalmiRNAs, including miR-375, in the pancreas of fetuses ofmothers fed an LP diet. The expression of miR-375remains augmented in neoformed islets derived fromfetuses and in islets from adult (3-month-old) progenyof mothers fed an LP diet. miR-375 regulates the prolif-eration and insulin secretion of dissociated islet cells,contributing to the reduced b-cell mass and function ofprogeny of mothers fed an LP diet. Remarkably, miR-375normalization in LP-derived islet cells restores b-cell pro-liferation and insulin secretion. Our findings suggest theexistence of a developmental memory in islets that regis-ters intrauterine protein restriction. Hence, pancreaticfailure after in utero malnutrition could result from trans-generational transmission of miRNA misexpression inb-cells.

Over the past 10 years the occurrence of type 2 diabetes(T2D) has increased at a frightening pace. While sedentarylifestyle and overfeeding undoubtedly contribute to thisworldwide epidemic, the intrauterine environment of thefetus is an additional influential actor in long-term health.Indeed, several epidemiological studies of human popula-tions highlighted a direct correlation between intrauterinegrowth retardation (IUGR) and the appearance of insulinresistance and T2D in adult life (1,2). Such observationshave led to the concept of developmental origins of adultdisease, which proposes that crucial programming of hu-man disorders originates in early life (3,4).

To unravel the mechanisms underlying the programmingof adult diseases, several animal models of IUGR have beengenerated (5). Despite differences in the nature, timing, andduration of the intrauterine insult, most of the animalIUGR models produce comparable outcomes. One of themost extensively studied models uses maternal protein re-striction in rats. Importantly, this situation exhibits pheno-typic similarities to the human IUGR, which is coupled toan age-dependent deterioration of glucose tolerance.

In the pathogenesis of T2D, insulin resistance associ-ated with b-cell failure leads to chronic hyperglycemia,defining diabetes. In IUGR in animals and humans, func-tional disruption in multiple tissues including muscle, ad-ipose tissue, liver, and pancreas occurs during adulthood.However, the endocrine pancreas seems to be affectedmost severely at early developmental stages, suggesting

1INSERM, U1081, Institute for Research on Cancer and Aging of Nice (IRCAN),Aging and Diabetes Team, Nice, France2CNRS, UMR7284, IRCAN, Nice, France3University of Nice Sophia Antipolis, Nice, France4Clinical Chemistry Laboratory, University Hospital, Nice, France5Central Pathology Laboratory, University Hospital, Nice, France

Corresponding author: Emmanuel Van Obberghen, vanobbeg@unice.fr.

Received 17 September 2013 and accepted 7 May 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1431/-/DC1.

© 2014 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, andthe work is not altered.

3416 Diabetes Volume 63, October 2014

ISLETSTUDIES

that, in the context of intrauterine modifications, T2Dcould originate from developmental defects in this organ.

Obviously, any disturbance in the environment of theendocrine cells within a given developmental time windowmay predispose to T2D, even in the next generation (6).Although the consequences of unfavorable intrauterineenvironment on fetal development have been documented,the molecular processes by which they occur are only start-ing to emerge (7).

An intriguing feature of the developmental origin ofadult disease concerns the generational transmission ofthe health disorder. Such programming has been associatedto changes in DNA methylation and/or histone modifica-tions affecting gene expression and contributing to thiscellular memory (8,9). Furthermore, nutrient-dependentmodulation of microRNAs (miRNAs) may also trigger dis-ease susceptibility and metabolic complications in off-spring. Indeed, misexpressed miRNAs participate in theprogramming of adipose tissue in rats with retardedgrowth, causing lipotoxicity and insulin resistance, andhence they increase the susceptibility to metabolic diseaseduring adulthood (10).

The mammalian genome codes for several hundreds ofmiRNAs that regulate gene expression through modula-tion of their target mRNAs. Most of these single-stranded20– to 22-nucleotide-long RNAs interact with specificsequences in the 39 untranslated region of the mRNA.By doing so, miRNAs induce mRNA degradation and/ortranslation inhibition (11). The biological importance ofmiRNAs is demonstrated by the diverse and profoundphenotypic sequelae upon changes in their expression.These alterations are associated with perturbed develop-ment and pathological situations. Indeed, miRNAs seemto be major regulators of gene expression in many biolog-ical programs, including organ development and metab-olism (12–17). Computational predictions of miRNA targetsestimate that a single miRNA can affect a gamut of differ-ent mRNAs, suggesting that a large proportion of the tran-scriptome is subjected to miRNA modulation. Using a ratmodel of maternal malnutrition, we address here the hy-pothesis that perturbation of the programmed expressionof key miRNAs in the endocrine pancreas of progeny con-tributes to the effects of early nutriture on the establish-ment of b-cell mass and, consequently, on the long-termhealth of the organism.

RESEARCH DESIGN AND METHODS

Animals and DietsAll procedures were performed in accordance with theguidelines for the care and use of laboratory animals ofthe French National Institute of Health and MedicalResearch. Nulliparous female Wistar rats weighing 200–250 g (Janvier, Le Genest-Saint-Isle, France) were matedovernight with male Wistar rats. The pregnant femaleswere individually housed with free access to water. Damswere fed ad libitum during gestation and lactation witha control (20% w/w protein) or isocaloric low-protein (LP)

diet (8% w/w protein; LP group) (Hope Farm, Woerden, theNetherlands) (18). After weaning, the progeny of bothgroups were fed standard chow ad libitum. A minimum of3 litters per group were analyzed in each experiment.

Islet CollectionNeoformed fetal rat islets were obtained as describedpreviously (19). Briefly, pancreases of 21-day-old fetuseswere removed aseptically, minced, and digested with collage-nase (Sigma-Aldrich, St. Louis, MO). The digested pancreaseswere incubated for 7 days at 37°C in a humidified atmo-sphere. Islets of 3-month-old rats were obtained as describedelsewhere (20). Following density-gradient centrifugationusing a histopaque-1077 (Sigma-Aldrich), islets were washedand hand-picked under a stereomicroscope. Finally, theywere either dissociated for transfection experiments (seeCULTURE AND TRANSFECTION OF DISSOCIATED ISLET CELLS) or cul-tured in RPMI 1640 Medium (Gibco, Grand Island, NY)supplemented with FBS (10% v/v) and Pen Strep (1% v/v).

Culture and Transfection of Dissociated Islet CellsBefore transfection, rat islets were dissociated bytrypsinization (0.5 mg/mL; Gibco) and seeded in 60 mm–diameter Petri dishes containing 5 mL RPMI 1640 medium(Gibco) containing FBS (10% v/v) and Pen Strep (1% v/v).Cells were incubated for 16 h at 37°C to allow full recovery.Then, dissociated islet cells were seeded at a density of25 3 103 cells/cm2 on 35-mm well dishes coated with804G-ECM (gift from Phillipe Halban, Geneva, Switzerland).

Using Lipofectamine 2000 (Invitrogen), 48 h after platingdissociated islet cells were transfected with 100 nmol/L ofdouble-stranded RNA oligonucleotides corresponding tomature miR-375 or miRNA hairpin inhibitor to blockendogenous miR-375 (Thermo Scientific, Gometz-le-Châtel,France). Cells were analyzed 72 h after transfection.

Immunohistochemical and MorphometricMeasurementsPancreases from fetuses and from 3-month-old rats werefixed in 3.7% (w/v) formalin, dehydrated, and embeddedin paraffin. Sections (5 mm) were collected, dewaxed, andhydrated in ethanol, and an antigen retrieval method wasused. Tissues were permeabilized and incubated for 30min with a blocking solution containing BSA (3% w/v)before overnight incubation with primary antibodies(see Supplementary Data). Next samples were incubatedfor 1 h at room temperature with secondary antibodies(see Supplementary Data).

All measurements were performed blindly to avoidinfluences from testers’ expectations. At fetal day 21, a min-imum of 3 sections (every 150 mm) from each of 6 animalsfrom 3 different litters per group were analyzed. A mini-mum of 6 sections (every 150 mm) from each of 6 animalsper group of 3-month-old rats were analyzed.

Insulin-positive and glucagon-positive areas were mea-sured to determine b- and a-cell fractions, respectively.These were measured as the ratio of the insulin-positiveand glucagon-positive cell area over the total tissue area ofthe entire section.

diabetes.diabetesjournals.org Dumortier and Associates 3417

Frequency distribution of various islet sizes wasmeasured with ImageJ software. We considered as anislet a cluster of at least three insulin-positive cells (21).For their size distribution, islets were arbitrarily classifiedas small (,100 mm), medium (100 mm, diameter, 200mm), or large (.200mm). The number of islets in eachclass was expressed as the percentage of total islets pergroup. Assuming that the cells are spheres, the diameterof individual b-cells was calculated with ImageJ software.To determine b-cell proliferation, pancreatic sectionswere stained with antibodies to phosphorylated histoneH3 and to insulin and appropriate secondary antibodies.

RNA Extraction and Quantitative RT-PCRRNA from fetal pancreas and islet cells was isolated usingTRIzol reagent (Invitrogen). Total RNA (1 mg) was reverse-transcribed into complementary DNAs (cDNAs) and an-alyzed using SYBR Green (ABI PRISM 7000 SequenceDetector System). The amount of cDNA used in eachreaction was normalized to the housekeeping genecyclophilin A. The primers and quantitative PCR assayconditions are available upon request.

miRNA Expression Profiling (miRNome Analysis)miRNA quantitative PCR array analysis was performedusing the rno-miRNome miRNA profiling kit (SystemBiosciences, Mountain View, CA). RNA was extractedfrom individual pancreases of fetuses in the control andLP groups. Then, RNA from a minimum of four pan-creases per litter was pooled for the array. Three littersper group were analyzed. Per litter, 1 mg of pooled RNAwas reverse transcribed into first-strand cDNA using theQuantiMir RT Kit (System Biosciences). Profiling of ma-ture miRNAs was performed on 3 litters per group byquantitative PCR in a 384-well plate format, including380 miRNA-specific primers and using SYBR Green. Ex-pression levels were normalized using U6 since its meltingcurve was more satisfactory compared with that obtainedfor the two other reference genes.

A heat map was created using open-source analysissoftware with a multiexperiment viewer (www.tm4.org). miR-375 expression was confirmed with themiRCURY LNA Universal RT MicroRNA PCR kit (Exiqon,Vedbaek, Denmark), according to the manufacturer’sinstructions.

Analysis of Total Cell Extracts and Western BlottingFetal pancreas or dissociated islet cells were processed forprotein isolation, as described by Dumortier et al. (22). ForWestern blotting, 10–50 mg of total proteins were sepa-rated by electrophoresis and transferred to polyvinylidenedifluoride membranes (Immobilon-P; Millipore, Bedford,MA). Immunoreactive proteins were revealed by enhancedchemiluminescence (Millipore, Billerica, MA). Antibody tophosphoinositide-dependent kinase-1 (PDK-1) was fromCell Signaling Technology (Danvers, MA) and antibody tob-actin was from Sigma-Aldrich. Western blots were quan-tified by densitometry using ImageQuant software.

Insulin Secretion AssayAll experiments were performed with a modified Krebs-Ringer solution containing 2.8 mmol/L glucose (23). Disso-ciated islet cells or batches of 20 free-floating, size-matchedislets were incubated at 37°C in 1 mL of Krebs-Ringermedium containing glucose at a concentration of 2.8 or20 mmol/L. After 120 min, the incubation medium wasremoved so insulin could be measured. Islets or dissociatedislet cells were collected and homogenized by sonificationto extract insulin. To eliminate variations due to differ-ences in individual islet batches, insulin secretion duringincubation was expressed as a percentage of the islet in-sulin content at the start of incubation, which is referredto as fractional insulin release.

Oral Glucose Tolerance TestsAfter an overnight fast, progeny of the rats in the controland LP groups were given an oral glucose bolus (2 g).Blood was collected from the tail vein at 0, 15, 30, 60, 90,and 120 min. Glycemia was measured using the OneTouchglucometer (LifeScan Inc., Milpitas, CA).

Statistical AnalysisResults are shown as means 6 SEM; n represents thenumber of litters analyzed. The Student t test was usedto compare two conditions (paired or unpaired), andtwo-way ANOVA followed by post hoc Fisher test wasused when more than two conditions were analyzed. AP value ,0.05 was considered significant.

RESULTS

Expression of miRNAs in Pancreas of Progeny in LPGroup at Day 21 of GestationTo investigate whether miRNA expression in the fetalpancreas is altered by a maternal LP diet, we comparedthe miRNA expression profile obtained from normal fetalpancreas and pancreas from fetuses in the LP group atday 21 of gestation. We used the quantitative PCR–basedmiRNome miRNA profiler to screen 380 mature rat miRNAsin the pancreas of 3 different litters of male rats in thecontrol and LP groups (Fig. 1A). Of the 360 detectable(95%) miRNAs, 47 are differentially expressed betweenthe control and LP groups (Fig. 1B). Remarkably, 43 areupregulated (28 with P , 0.05 and 15 with P , 0.09) inthe LP group, while only 4 are downregulated (P , 0.05).Interestingly, we found that the expression of miR-375,which is highly expressed in endocrine pancreas and in-volved in b-cell proliferation and function, is increased atday 21 of gestation in the pancreas of fetuses in the LPgroup (1.4 6 0.08 arbitrary units in LP group comparedwith 1 6 0.028 arbitrary units in control group; see Sup-plementary Fig. 1).

PDK-1 Protein Is Downregulated in Pancreatic Islets ofProgeny in the LP GroupPDK-1, one of the targets of miR-375 (24), is a key com-ponent of the insulin and growth factor signaling path-way, downstream of phosphoinositide 3-kinase in theprotein kinase B cascade. Because miR-375 is upregulated

3418 miR-375 and Fetal Programming of T2D Diabetes Volume 63, October 2014

in the pancreas of fetuses in the LP group, we measuredPDK-1 protein levels in the pancreases of our experimen-tal groups. Using Western blotting, we observed no dif-ference in total PDK-1 in pancreases of the LP groupcompared with controls (Fig. 2A), whereas quantitativePCR analysis revealed a moderately decreased level ofPDK-1 mRNA (Fig. 2B). However, immunohistochemicalanalyses showed that PDK-1 immunoreactivity is specifi-cally reduced in the endocrine compartment of the pan-creas (Fig. 2C).

b-Cell Mass Is Reduced in Progeny in the LP Group atFetal Day 21 Because of a Reduction in b-CellProliferation and SizePDK-1 is a key regulator of b-cell proliferation and growthin the developing pancreas (25). To investigate whetherthe reduction of PDK-1 protein observed in islets of ani-mals in the LP group affects pancreatic development,immunohistochemical and morphometric analyses wereperformed. While in the progeny in the LP group thenumber of islets measured as insulin-positive aggregatesper pancreas area is the same as in the control progeny,the islets are smaller than in the control group progeny(Table 1). This diminished islet size in pancreas of animalsin the LP group can be accounted for by a 50% decrease inb-cell fraction area, whereas the a-cell fraction was pre-served (Table 1). In fact, the maternal LP diet significantly

increased the proportion of small islets and concomitantlyreduced the proportion of large islets compared with thecontrol progeny (Fig. 3A).

To gain more insight into the mechanism underlyingreduced b-cell growth, the proliferative capacity of thesecells was investigated. Co-immunostaining of pancreassections with antibodies to insulin and phosphorylatedhistone H3 (Fig. 3B) or antibodies to Ki-67 (Supplemen-tary Fig. 2A) showed a decreased mitotic index of b-cellsin pancreases from animals in the LP group. Quantifica-tion revealed that b-cell proliferation is reduced by ap-proximately 50% in fetuses in the LP compared withcontrol groups. In contrast, apoptosis assessed using theTUNEL method (data not shown) and differentiationmeasured by b-cell markers seem to be intact (Supple-mentary Fig. 2B). In conclusion, protein restriction duringpregnancy reduces in the progeny b-cell mass because ofdecreased b-cell expansion.

In addition to its role in b-cell proliferation, PDK-1 hasbeen found to exert a major effect on b-cell size (26). Toevaluate whether this was occurring in our experimentalconditions, we took advantage of the fact that b-cells arethe only islet cells that express glucose transporter-2at the cell membrane. Morphometric measurements in-dicate that fetal b-cells in the LP group display a modestreduction in size (Fig. 3C). Thus, an LP diet during preg-nancy diminishes the b-cell diameter of the fetuses by

Figure 1—miRNA expression in fetal rat pancreas is altered by a maternal LP diet. A: Heat map showing the quantification of 380 miRNAsexpressed in the fetal pancreases of progeny from the control (C) and LP group at day 21 of gestation. Each column represents one litter (apool of four fetuses). miRNA quantitative PCR analysis was performed to examine mature miRNA expression in the pancreases of fetusesfrom the control and LP groups. B: The 47 most differentially expressed miRNAs in pancreas from LP animals compared with controlanimals.

diabetes.diabetesjournals.org Dumortier and Associates 3419

approximately 10%. When extrapolated to the islet vol-ume, however, this decrease represents a reduction ofapproximately 25%. Note that the reduction in b-cell sizewas confirmed by E-cadherin/insulin co-immunostaining(Supplementary Fig. 2C).

Reduced Proliferation and Increased miR-375Expression in Islets Derived From Fetuses in the LPGroup Are Maintained After 7 Days in CultureAnimal studies clearly demonstrated that poor nutritionduring gestation irreversibly leads to reduced numbers ofcells in tissues such as the pancreas (27). Here we evalu-ated whether the decreased cell proliferation observed invivo persists in LP neoformed islets in culture. Indeed,our measurements revealed that the size of islets neo-formed from fetal pancreas from the LP group is reducedcompared with those from control pancreas (Fig. 4A). Fur-ther, insulin content per islet is diminished in neoformed

islets in the LP group (Fig. 4B), whereas relative insulinmRNA expression is preserved (Fig. 4C). Together thesedata suggest that cell proliferation is decreased in LPneoformed islets. Because of the scant quantity of avail-able material, we were unable to accurately measure lev-els of critical proteins in the cell cycle, and hence we usedquantitative PCR to examine their mRNA expression(Fig. 4D). While cyclin D2, which accumulates duringthe G1 phase, was increased in neoformed islets derivedfrom animals in the LP group, cyclin A2, which is neces-sary for progression into mitosis, was reduced. In addi-tion, the expression of an important inhibitor of G1cyclin/cdk complexes, p57, is increased in neoformedislets from the LP group (Fig. 4D). These data are com-patible with an impaired b-cell proliferation of neoformedislets from the LP group. To determine whether miR-375might be involved in perturbing the regulation of b-cellreplication in neoformed islets from the LP group, weexamined its expression. Notably, we found that miR-375 expression is significantly increased in neoformedislets from the LP group compared with controls (Fig.4E). Taken together, our data strongly suggest thatb-cell alterations induced by maternal protein restrictionare not the result of direct regulation by the maternalmilieu. Rather, they represent changes transmitted tothe next generation of cells.

Forced Expression of miR-375 in Primary Islet CellsImpairs Cell Proliferation and Insulin SecretionTo investigate whether miR-375 interferes with cellproliferation and/or insulin secretion, we mimicked thechanges in miR-375 expression observed in LP groupprogeny by transfecting dissociated primary rat islet cellswith mature miR-375. Cell proliferation and insulinsecretion were measured 72 h after transfection. Similarto our data from the INS-1E cell line (Supplementary Fig.3), miR-375 expression in dissociated primary islet cellsreduces levels of the PDK-1 protein (Fig. 5A and B) andinhibits cell proliferation by approximately 40% (Fig. 5C).

Figure 2—PDK-1 protein level is reduced in fetal endocrine pan-creas from the LP group at day 21. A: Western blot analysis. Pan-creas from control (C) and LP group fetuses were harvested andprotein extracts were analyzed with antibody to PDK-1 or to b-actin.Samples were quantified using ImageQuant software. Values aremeans 6 SEM (n = 6). B: PDK-1 mRNA measurement. Fetal pan-creases were collected for RNA extraction. RNA extracts werereverse transcribed and analyzed by quantitative PCR. Gene ex-pression was normalized to the cyclophilin A (CyA) transcript level.Values are means 6 SEM (n = 6). C: Detection of PDK-1 protein onsections of fetal pancreas (circled). Scale bar = 100 mm. Immunostain-ing intensity of a minimum of 90 islets per group was evaluated usingImageJ software. Graphs show means 6 SEM (n = 6 fetal pancreaseswith 3 slides each). **P < 0.01, LP vs. control group.

Table 1—Morphometric parameters of b- and a-cells infetuses of control and LP dams at day 21

Control fetus LP fetus

b-Cell fraction (%) 3.06 6 0.11 1.76 6 0.09*

a-Cell fraction (%) 0.69 6 0.07 0.73 6 0.05

Insulin-positive aggregatesper pancreas area (n/cm2) 2,999 6 223 3,154 6 268

Islet size (mm2) 2,062 6 112 1,388 6 138**

Immunocytochemistry for insulin and glucagon was performedon pancreatic sections. The b- and a-cell fractions (%) were mea-sured as the ratio of the insulin-positive and glucagon-positivecell area and the total tissue area of the entire section. Thenumber of insulin-stained pancreatic islets in each image wasmanually counted using Adobe Photoshop 7.0 computer soft-ware. Average islet density (number of islets per unit area ofpancreatic tissue) was calculated. Values are means 6 SEM(n = 6 from three different dams). *P , 0.001, LP vs. controlfetus; **P , 0.01, LP vs. control fetus.

3420 miR-375 and Fetal Programming of T2D Diabetes Volume 63, October 2014

Moreover, increased miR-375 expression decreases glucose-induced insulin secretion in cultured primary islet cells(Fig. 5D) without affecting insulin content (data notshown), confirming observations from the insulinomaMIN6 cell-line made by Poy and colleagues (28,29).

Increased miR-375 Expression and PancreaticDeterioration Persist in LP Group Progeny at 3 Monthsof AgeSince increased miR-375 expression and reduced pro-liferation are maintained in neoformed islets from the LPgroup, we evaluated whether this was also the case inadult 3-month-old male offspring. Importantly, we foundthat miR-375 expression is increased in isolated islets from3-month-old animals in the LP group (Fig. 6A). Accord-ingly, mRNA levels of PDK-1 and myotrophin (MTPN),another miR-375 target (28), are diminished (Fig. 6B). Asexpected from the decrease in PDK-1, b-cell mass and pro-liferation are decreased (Fig. 6C and D). Consistent with

MTPN and PDK-1 reduction, the b-cell functioning is dete-riorated in 3-month-old animals in the LP group, as glucose-stimulated insulin secretion is severely blunted (Fig. 6E).Given the diminished b-cell mass and insulin secretion inLP group progeny, we studied glucose homeostasis in 3-month-old offspring. As shown in Fig. 6F, offspring fromthe LP group are glucose intolerant after an oral glucosechallenge. However, insulin injection did not reveal hormoneresistance in LP compared with control progeny at 3 monthsof age (data not shown), indicating that the glucose intoler-ance is due to insufficient insulin secretion.

Repressed miR-375 Expression in Islet Cells DerivedFrom LP Animals Rescues Cell Proliferation and InsulinSecretionTo address whether miR-375 mediates the pancreaticalterations in LP group progeny, we used a loss-of-function approach with anti-miR-375 oligonucleotides toreverse the elevated expression of miR-375 in islet cells

Figure 3—b-Cell mass is diminished at day 21 in fetal progeny in the LP group because of a reduction in b-cell size and proliferation.Immunocytochemistry was performed on pancreatic sections. A: A frequency distribution of the size of pancreatic islets of 21-day-oldfetuses. Assuming that islets are spheres, the profile diameter of the islets was measured using ImageJ software. Clusters of at least threeinsulin-positive cells were considered as islets. Values are means 6 SEM (n = 6). **P < 0.01, LP vs. control (C) group. B: Phosphorylatedhistone H3 (pHH3)-positive cells (green) were counted on pancreatic sections together with insulin (red) and DAPI (blue) (left). Scale bar =50 mm. The graph (right) shows the percentage of proliferating b-cells over total b-cells. Values are means6 SEM (n = 6). **P< 0.01, LP vs.control group. C: Measurements of b-cell size. Left: Pancreatic sections were stained with antibodies to glucose transporter-2 (red) todetermine the size of individual b-cells. Scale bar = 10 mm. Right: Assuming that b-cells are spheres, the b-cell diameter was calculatedusing ImageJ software. Values are means 6 SEM from at least 200 b-cells from each of 6 fetuses from dams in the LP and control groups.**P < 0.01, LP vs. control group.

diabetes.diabetesjournals.org Dumortier and Associates 3421

derived from 3-month-old rats in the LP group. miR-375is upregulated in islet cells derived from LP animals(Supplementary Fig. 4), and glucose-induced insulin secre-tion and cell proliferation are reduced (Fig. 7A and B). Ofsignificance, anti-miR-375-driven normalization of miR-375 levels (Supplementary Fig. 4) in dissociated islet cellsderived from LP animals improved glucose-induced insu-lin secretion (Fig. 7A) without affecting insulin content(data not shown) and cell proliferation (Fig. 7B). The lat-ter is accompanied by a return to normal levels of PDK-1protein (Fig. 7B). Taken together, these data show thatrestoration of normal miR-375 levels in islets from LPanimals tends to correct key deviations of the b-cell phe-notype induced by maternal protein deficiency.

DISCUSSION

The biological importance of miRNAs has been placed inthe limelight following the demonstration of diverse and

profound phenotypic alterations upon changes in theirexpression. These modifications are associated with per-turbed development and pathological situations (13). Thedata presented here demonstrate that an unfavorable envi-ronment during fetal development results in changes inprogrammed miRNA expression in the endocrine pancreasof the progeny. Among the miRNAs with aberrant expres-sion, miR-375, which regulates the proliferation and thesize of b-cells, contributes, at least in part, to the reductionin fetal b-cell mass observed at birth. Our results from adult3-month-old animals reveal a sustained increase in miR-375expression continuously affecting b-cell physiology andfunction. We show that early environmental factors, suchas maternal nutrition, durably influence pancreatic expres-sion of miRNAs, including miR-375, which affects b-cellphysiology and controls long-term health.

miR-375 is a highly conserved miRNA that was clonedfrom a mouse insulinoma b-cell line (28). miR-375 is

Figure 4—A maternal LP diet induces alterations in cell cycle regulators in fetal neoformed islets. After 7 days in culture, the size, insulincontent, insulin mRNA, and expression of genes involved in cell growth in fetal neoformed islets in the control (C) and LP groups werestudied. A: Size of fetal neoformed islets after 7 days in culture. The islet diameter was measured using ImageJ software and was calculatedassuming that islets are spheres. Values (means 6 SEM) are calculated from 100 observations pooled from 3 independent cultures (n = 3).***P < 0.001, LP vs. control. B: Insulin content of fetal neoformed islets after 7 days in culture. After the culture period, fetal islets wereplaced in 1 mL acid–ethanol to extract insulin. Insulin content was determined by ELISA. Values are means 6 SEM (n = 4). **P < 0.01, LPvs. control. C: Insulin mRNA measurement. RNA extracts were reverse transcribed and analyzed by quantitative PCR. Insulin geneexpression was normalized to the cyclophilin A (CyA) transcript level. Quantitative PCR was performed on RNA extracted from fourindependent cultures. Values are means 6 SEM (n = 4). The same procedure was used for the data presented in panels D and E. D:Expression of cell cycle regulators in neoformed islets in the LP group after 7 days in culture. Values are means 6 SEM (n = 4). *P < 0.05,LP vs. control; **P < 0.01, LP vs. control. E: miR-375 expression in neoformed islets after 7 days in culture. Expression of mature miR-375precursor was normalized to the U6 transcript level. Values are means 6 SEM (n = 4). **P < 0.01, LP vs. control.

3422 miR-375 and Fetal Programming of T2D Diabetes Volume 63, October 2014

expressed in b-cells as well as in non–b-cells of the pan-creas (30–32) and in the pituary gland (33). In human andmouse islets, miR-375 seems to be the miRNA with themost robust expression (30).

Using mice lacking miR-375 Poy et al. (32) reportedthat miR-375 deletion influences not only b-cell mass butalso a-cell mass by regulating a cluster of genes control-ling cellular growth and proliferation. Indeed, in normalsituations, mice lacking miR-375 exhibit an increasednumber of a-cells, whereas the loss of miR-375 has littleinfluence on b-cell mass. When the metabolic demandincreases, however, genetic miR-375 deletion in mice coun-teracts the normally occurring b-cell hyperplasia (32). Atfirst glance this result seems to vary from our observations.However, several studies have reported that miR-375 acts

not as a positive regulator of cell proliferation but rather asa negative modulator (24,34–36). At least two scenarioscould account for this apparent divergence. One possibleexplanation relates to differences among animal species.Using mice models, Tattikota and colleagues (37) reportedthat the effect of miR-375 on b-cell proliferation wasmostly attributed to its target Cadm1 (cell adhesion mole-cule 1). As shown in Supplementary Fig. 5, Cadm1 expres-sion is localized in all human and rat islet cells, while itsexpression is particularly perceptible at the periphery ofmouse islets corresponding to a-cells. This could explainthe robust influence of miR-375 deletion on a-cell massunder normal physiological conditions (37). When the met-abolic demand augments (e.g., ob/ob mice), it is possiblethat the reduced b-cell mass results from a-cell dysfunc-tion. Hence the nature of miR-375 action on b-cell pro-liferation could be different in mice compared with humansand rats.

Another scenario relates to recent views that miRNAsare responsive to cellular and extracellular stress and areused by cells to adjust to changes in their environment(38). A pioneering example in Drosophila revealed thatmiR-7 loss had no detectable effect on photoreceptor de-velopment under uniform laboratory conditions, but miR-7becomes necessary under conditions of temperature fluc-tuation (39). It is thus conceivable that, under intensifiedstress conditions caused by increased metabolic pressure,miR-375 can negatively influence cell proliferation,whereas under physiological circumstances it fosterscell proliferation.

In addition to its role in b-cell proliferation, miR-375is a regulator of chief b-cell functions. Indeed, forced miR-375 expression in insulinoma cells leads to reduced glucose-stimulated insulin secretion without interfering with theeffect of glucose action on ATP production and intracel-lular calcium concentrations. To be more precise, miR-375interacts with a series of gene products, including MTPNmRNA, governing insulin granule fusion with the plasmamembrane, and thereby inhibits exocytosis (28,29). Inneoformed islets from LP group fetuses, we failed to ob-serve a difference in insulin secretion compared with thecontrol group (data not shown). This is probably becauseof the variable ability of neoformed pancreatic islets torespond to glucose. Indeed, in fetal islets the acquisitionof stimulus/secretion coupling of insulin in response toglucose occurs after birth. While some reports view neo-natal islets as developmentally immature (40), others ex-plain the weak responsiveness to glucose by a high activityat low glucose concentrations but a lower one at highglucose concentrations (41). In contrast to neoformed pan-creatic islets, islets isolated from 3-month-old progeny inthe LP group, with increased miR-375 expression, showa lower fractional insulin release in response to glucose.At the same time MTPN and PDK-1 mRNA are decreased.Importantly, normalization of miR-375 levels restores in-sulin secretion in islet cells of 3-month-old progeny in theLP group.

Figure 5—Forced miR-375 expression in dissociated primary rat isletcells impairs cell proliferation and insulin secretion. Dissociated pri-mary rat islet cells were transfected 48 h after plating with 100 nmol/Lof double-stranded RNA oligonucleotides corresponding to the ma-ture miR-375 sequence or with 100 nmol/L of a scrambled controlmiR (CTL). After transfection (72 h), cells were fixed with methanol for10 min to assess proliferation or incubated for 60 min in modifiedKrebs-Ringer buffer for insulin secretion experiments. A: Analysis ofthe miR-375 target PDK-1. Cells were harvested for protein extrac-tion 72 h after transfection. Protein extracts were analyzed by West-ern blot with antibody to PDK-1 or to actin. B: Relative quantificationof PDK-1 protein. Data represent three independent transfections.Values are means 6 SEM (n = 3). *P < 0.05, miR-375 vs. CTL.C: Cell proliferation measurement. Proliferation index was evaluatedas the ratio of phosphorylated histone H3–positive cells to total cellnumber from four independent transfections. Values are means 6SEM (n = 4). *P < 0.05, miR-375 vs. CTL. D: Insulin secretion assay.For secretion experiments, dissociated cells were starved in modifiedKrebs-Ringer buffer containing 2.8 mmol/L glucose (G) for 60 minand thereafter were stimulated or not with 20 mmol/L glucose for 2 h.Values are means 6 SEM (n = 4). **P < 0.01, miR-375 vs. CTL at20 mmol/L glucose, ANOVA.

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One of the most intriguing and important aspects ofdisease programming concerns the transmission of thedisorder (42). Such phenomenon seems to occur in theanimals in our LP group, where increased miR-375 expres-sion observed in fetal islets persists in neoformed islets.Indeed, a similarly augmented level of miR-375 prevailedin neoformed islets from LP animals after 7 days of

culture under the same conditions as control islets. Fur-thermore, in agreement with previous publications, cellproliferation seems to be impaired in neoformed isletsfrom LP animals (43–45). Indeed, neoformed islets fromprogeny in the LP group were smaller than neoformedislets from control progeny, and a select set of genes in-volved in cell cycle regulation has altered expression,

Figure 6—Pancreatic deteriorations persist in adult 3-month-old male progeny in the LP group. A: miR-375 expression. Islets were collectedfrom 3-month-old progeny in the control (C) and LP groups for RNA extraction. Expression of mature miR-375 was normalized to the U6transcript level. B: Expression of miR-375 target genes. C: b-Cell area. D: b-Cell proliferation. Phosphorylated histone H3–positive cells werecounted on pancreatic sections, together with insulin. The percentage of proliferating b-cells over total b-cells is shown. Values in panels A toD are means 6 SEM (n = 4). *P < 0.05, LP vs. control; **P < 0.01, LP vs. control. E: Insulin secretion assay. Batches of 20 free-floating isletswere starved for 2 h in modified Krebs-Ringer buffer containing 2.8 mmol/L glucose (G) and were incubated thereafter for 2 h either with 2.8 or20 mmol/L glucose. For the LP and control progeny, islets of similar size were used. Values are means6 SEM (n = 3). **P< 0.01, LP 20 mmol/Lglucose vs. control 20 mmol/L glucose, ANOVA. F: Oral glucose tolerance tests (OGTTs) at 3 months of age in rats from the control (opencircle) and LP groups (black circle) (n = 5 for each group). OGTTs were performed after a 16-h overnight fast. Glucose (2 g) was administeredorally. Glycemia was measured at different times.***P < 0.001, LP vs. control, repeated measures ANOVA. CyA, cyclophilin A.

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suggesting a reduction in cell proliferation similar to thein vivo situation. Most notably, 3-month-old progeny in theLP group display persistently increased miR-375 expression,reduced b-cell proliferation, and decreased glucose-induced

insulin secretion. As a whole our data indicate that theelevated miR-375 level is unlikely to result from directregulatory signals from the maternal milieu; rather, theyreflect the occurrence of a developmental memory in theislet cells that recalls the intrauterine protein malnutri-tion. This induces long-lasting damaging effects on thepancreas, resulting in reduced b-cell proliferation andfunction.

Our results of miR-375 misexpression in endocrinepancreas from the LP animals are supported by theexistence of epigenetic marks adjacent to the miR-375coding region. Indeed, the miR-375 gene is epigeneticallyregulated by DNA methylation, consistent with thepresence of two large CpG-rich regions (46). Moreover,the miR-375 promoter region contains several consensusbinding sites for transcription factors; these have beenimplicated in the development and function of islets suchas hepatocyte nuclear factor-6 and insulinoma-associated 1(47). Pancreatic and duodenal homeobox-1 also was foundto interact with the upstream region of the miR-375 gene(48). Most of these transcription factors are themselvesregulated by epigenetic mechanisms (49).

In conclusion, we propose that under deleterious inutero conditions, the altered control exerted by miRNAson gene regulation increases the postnatal risk of b-cellfailure and hence of T2D. This augmented risk could re-flect a reduced ability of b-cells to contend with stressbecause of a deficiency in adaptive b-cell mass and func-tion. While biological systems are characterized by robust-ness against environmental changes to ensure properdevelopment and health, failure of protective mechanismssets the stage for exacerbated disease risk. A groundbreak-ing concept in this context is that miRNAs are instrumen-tal in generating biological robustness (39). Dovetailingwith this view, our data suggest that the level of miR-375 is key to establishing adequate b-cell mass and func-tion, which are necessary for preserving the homeostatichealth of the organism. Taking into account the fact thatmiR-375 has been identified as a diabetes-related circulat-ing miRNA (50) and that miR-375 misexpression is ob-servable before and after birth in animals with retardedgrowth, miR-375 seems to be a promising biomarker ofb-cell failure.

Acknowledgments. The authors thank Phillipe Halban, University of Ge-neva, for providing the 804G matrix.Funding. Our research was supported by INSERM, Université Nice SophiaAntipolis, Conseil Régional PACA and Conseil Général des Alpes-Maritimes; bythe European Foundation for the Study of Diabetes (EFSD/Lilly, European Di-abetes Research Programme 2011); and by the Agence Nationale de laRecherche (grant no. RPV12004AAA).Duality of Interest. Our research was also supported by Aviesan/AstraZeneca’s“Diabetes and the Vessel Wall Injury” program. No other potential conflicts ofinterest relevant to this article were reported.Author Contributions. O.D. and C.H. researched data, wrote the man-uscript, and contributed to discussion. N.G. and V.C. researched data. S.P.researched data on human tissue shown in supplementary figures. E.V.O. wrote

Figure 7—Normalization of miR-375 in islet cells derived from3-month-old progeny from the LP group rescues insulin secretionand cell proliferation. Islets from 3-month-old progeny in both thecontrol and LP groups were isolated, dissociated by trypsinization,and seeded at a density of 25 3 103 cells/cm2. Dissociated isletcells were transfected with 100 nmol/L miR-375 hairpin inhibitor 48 hafter plating to block endogenous miR-375 (anti-375) or with con-trol hairpin inhibitor (anti-CTL). Cells were used to measure insulinsecretion (A) or cell proliferation (B) 72 h after transfection. A:Insulin secretion assay. For secretion experiments, dissociatedcontrol and LP islet cells were starved in modified Krebs-Ringerbuffer containing 2.8 mmol/L glucose for 60 min and thereafterstimulated or not with 20 mmol/L glucose for 2 h. Values aremeans 6 SEM (n = 4). ***P < 0.001, anti-375 vs. anti-CTL fromcontrol group (C)–derived islet cells at 20 mmol/L glucose, LP- vs.control-derived islet cells in CTL condition at 20 mmol/L glucose.**P < 0.01, anti-375 vs. anti-CTL from LP group–derived islet cellsat 20 mmol/L glucose, ANOVA. B: Cell proliferation. Proliferationindex was evaluated as the ratio of phosphorylated histone H3–positive cells to total cell number from four independent transfec-tions. Bottom: Analysis of the miR-375 target PDK-1. Cells wereharvested for protein extraction 72 h after transfection. Proteinextracts were analyzed by Western blot with antibody to PDK-1.Values are means 6 SEM (n = 4). *P < 0.05, LP vs. control in anti-CTL condition, anti-CTL vs. anti-375 in LP group–derived islet cells.**P < 0.01, anti-375 vs. anti-CTL in control group–derived isletcells, ANOVA.

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the manuscript and contributed to discussion. E.V.O. is the guarantor of this workand, as such, had full access to all the data and takes full responsibility for theintegrity of data and the accuracy of data analysis.

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