Maternal protein restriction leads to pancreatic failure ......islets derived from LP fetuses and in islets from adult 3 month-old LP progeny. miR-375 regulates the proliferation and

1

Maternal protein restriction leads to pancreatic failure in

offspring: role of misexpressed microRNA-375

Short title: miR-375 and fetal programming of type 2 diabetes

Olivier Dumortier1,2,3

, Charlotte Hinault1,2,3,4

, Nadine Gautier1,2,3

,

Stéphanie Patouraux5, Virginie Casamento

1,2,3, Emmanuel Van

Obberghen1,2,3,4

1 INSERM, U1081, Institute for Research on Cancer and Aging of Nice (IRCAN), « Aging and Diabetes» team

06107 Nice, France

2 CNRS, UMR7284, IRCAN, Nice, France

3 University of Nice-Sophia Antipolis, Nice, France

4 Clinical Chemistry Laboratory, University Hospital, Nice, France

5 Central Pathology Laboratory, University Hospital, Nice, France

Corresponding author: Emmanuel Van Obberghen, IRCAN - CNRS UMR7284 / INSERM

U1081, 28 avenue Valombrose - 06107 NICE Cedex 2 – France, Tel. : +33 (0)4 93 37 77 85

- Fax : +33 (0)4 93 37 76 76 – mail: [email protected]>

Page 1 of 51 Diabetes

Diabetes Publish Ahead of Print, published online May 16, 2014

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Abstract

The intrauterine environment of the fetus is a preeminent actor in long-term health. Indeed,

mounting evidence shows that maternal malnutrition increases the risk of type 2 diabetes

(T2D) in progeny. Although the consequences of disturbed prenatal environment on pancreas

development are known, the underlying mechanisms are poorly defined. In rats, gestational

protein restriction alters the endocrine pancreas development and favors T2D appearance later

in life. Here we evaluate the potential role of perturbed microRNA (miRNA) expression in the

decreased β-cell mass and insulin secretion characterizing progeny of pregnant dams fed a

low protein (LP) diet. miRNA profiling shows increased expression of several miRNAs in

pancreas of LP fetuses including miR-375. Its expression remains augmented in neoformed

islets derived from LP fetuses and in islets from adult 3 month-old LP progeny. miR-375

regulates the proliferation and insulin secretion of dissociated islet cells, contributing to the

reduced β-cell mass and function of LP progeny. Remarkably, miR-375 normalization in LP

derived islet cells restores β-cell proliferation and insulin secretion. Our findings suggest the

existence of a developmental memory in islets that registers intrauterine protein restriction.

Hence, pancreatic failure after in utero malnutrition could result from transgenerational

transmission of microRNA misexpression in β-cells.

Page 2 of 51Diabetes

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Introduction

Over the last years the occurrence of type 2 diabetes (T2D) has increased at a frightening

pace. While sedentary lifestyle and overfeeding undoubtedly contribute to this world-wide

epidemic, the intrauterine environment of the fetus is an additional influential actor in long-

term health. Indeed, several epidemiological studies on human populations have highlighted a

direct correlation between intrauterine growth-retardation (IUGR) and the appearance of

insulin resistance and T2D in adult life (1; 2). Such observations have led to the concept of

developmental origins of adult disease, which proposes that crucial programming of human

disorders originates in early life (3; 4).

To unravel the mechanisms underlying the programming of adult diseases, several animal

models of IUGR have been generated (5). Despite differences in the nature, timing, and

duration of the intrauterine insult, most of the animal IUGR models produce comparable

outcomes. One of the most extensively studied models employs maternal protein restriction in

rats. Importantly, this situation exhibits phenotypic similarities to the human IUGR, which is

coupled to an age-dependent deterioration of glucose tolerance.

In the T2D pathogenesis, insulin resistance associated to β-cell failure leads to chronic

hyperglycemia defining diabetes. In IUGR animals and humans, functional disruption in

multiple tissues including muscle, adipose tissue, liver, and pancreas occurs at adulthood.

However, the endocrine pancreas appears to be the most severely affected at early

developmental stages, suggesting that in the context of intrauterine modifications T2D could

originate from developmental defects in this organ.

Obviously, any disturbance in the environment of the endocrine cells within a given

developmental time window may predispose to T2D, even in the next generation (6).


4

Although the consequences of an unfavorable intrauterine environment on fetal development

have been documented, the molecular processes by which they occur are only starting to

emerge (7).

An intriguing feature of the developmental origin of adult disease concerns the

generational transmission of the health disorder. Such programming has been associated to

changes in DNA methylation and/or histone modifications affecting gene expression

contributing to this cellular memory (8; 9). Furthermore, nutrient-dependent modulation of

microRNAs (miRNAs) may also trigger disease susceptibility and metabolic complications in

offspring. Indeed, misexpressed miRNAs participate in the programming of adipose tissue in

growth-retarded rats, causing lipotoxicity and insulin resistance, and hence they increase the

susceptibility to metabolic disease at adulthood (10).

The mammalian genome codes for several hundreds of miRNAs that regulate gene

expression through modulation of their target mRNAs. Most of these single-stranded 20-22

nucleotide-long RNAs interact with specific sequences in the 3’untranslated mRNA region.

By doing so, miRNAs induce mRNA degradation and/or translation inhibition (11). The

biological importance of miRNAs is demonstrated by the diverse and profound phenotypic

sequelae upon changes in their expression. These alterations are associated to perturbed

development and pathological situations. Indeed, miRNAs appear to be major regulators of

gene expression in many biological programs including organ development and metabolism

(12-17). Computational predictions of miRNA targets estimate that a single miRNA can

impact on a gamut of different mRNAs, suggesting that a large proportion of the

transcriptome is subjected to miRNA modulation. Using a rat model of maternal malnutrition

we address here the hypothesis that perturbation of the programmed expression of key


5

miRNAs in the endocrine pancreas of progeny contributes to the effects of early nutriture on

β-cell mass establishment, and consequently, on long-term organismal health.

Materials and methods

Animals and diets

All procedures are in accordance with the Guidelines for the care and use of laboratory

animals of the French National Institute of Health and Medical Research. Nulliparous female

Wistar rats weighing 200–250g (Janvier, Le Genest Saint Isle, France) were mated overnight

with male Wistar rats. The pregnant females were individually housed with free water access.

Dams were fed, ad libitum, during gestation and lactation, with the Control (20%, w/w

protein, C group) or isocaloric Low Protein diet (8%, w/w protein, LP group) (Hope Farm,

Woerden, the Netherlands) (18). After weaning, the progeny of both groups were fed ad

libitum on standard chow. A minimum of 3 litters per group were analyzed in each

experiment.

Islet collection

Neoformed fetal rat islets were obtained as described (19). Briefly, pancreases of 21 day-old

fetuses were removed aseptically, minced and digested with collagenase (Sigma, St Louis,

MO, USA). The digested pancreases were incubated for 7 days at 37°C in a humidified

atmosphere. Islets of 3 month-old rats were obtained as described (20). Following density-

gradient centrifugation using histopaque-1077 (Sigma), islets were washed and hand-picked

under a stereomicroscope. Finally, they were either dissociated for transfection experiments

(see further) or cultured in RMPI 1640 medium (Gibco, Grand Island, NY, USA)

supplemented with FBS (10%, v/v) and PenStrep (1%, v/v).


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Culture and transfection of dissociated islet cells

Before transfection, rat islets were dissociated by trypsinization (0.5mg/mL; Gibco) and

seeded in 60 mm diameter Petri dishes containing 5ml RMPI 1640 medium (Gibco, Grand

Island, NY, USA) containing FBS (10%, v/v) and PenStrep (1%, v/v). Cells were incubated

for 16h at 37°C to allow full recovery. Then, dissociated islet cells were seeded at a density of

25 × 103 cells/cm

2 on 35 mm well dishes coated with 804G-ECM (gift from Halban PA;

Geneva, Switzerland).

48h after plating, using Lipofectamine 2000 (Invitrogen), dissociated islet cells were

transfected with 100nM of double-stranded RNA oligonucleotides corresponding to mature

miR-375 or microRNA hairpin inhibitor to block endogenous miR-375 (Thermo Scientific,

Gometz le Châtel, France). 72h after transfection cells were analyzed.

Immunohistochemical and morphometrical measurements

Pancreases from fetuses and from 3-month-old rats were fixed in 3.7% (w/v) formalin,

dehydrated and embedded in paraffin. Sections (5µm) were collected, dewaxed, hydrated in

ethanol, and an antigen retrieval method was used. Tissues were permeabilized and incubated

for 30 min with a blocking solution containing BSA (3%, w/v) before overnight incubation

with primary antibodies (see supplemental information). Next samples were incubated for 1 h

at room temperature with secondary antibodies (see supplemental information).

All measurements were performed blindly to avoid influences from testers' expectations. At

F21 a minimum of 3 sections per animal (every 150 µm), a total of 6 animals from 3 different

litters were analyzed per group. On 3-month-old animals, a minimum of 6 sections per animal

(every 150 µm), a total of 6 animals were analyzed per group.


7

Insulin-positive and glucagon-positive areas were measured for the determination of β- and α-

cell fractions. These were measured as the ratio of the insulin-positive and glucagon-positive

cell area over the total tissue area on the entire section.

Size-frequency distribution of islets was measured with ImageJ software. We considered as an

islet a cluster of at least 3 insulin positive cells (21). For their size distribution, islets were

arbitrarily classified as small (<100µm), medium (100µm < diameter <200µm) or large

(>200µm). The islet number in each class was expressed as the percentage of total islets per

group. Diameter of individual β-cells was calculated with Image J software assuming that the

cells are spheres. For β-cell proliferation determination, pancreatic sections were stained with

antibodies to phosphorylated histone H3 and to insulin, and appropriate secondary antibodies.

RNA extraction and RT-qPCR

RNA from fetal pancreas and islet cells was isolated using TRIzol reagent (Invitrogen). 1 µg

total RNA was reverse-transcribed into cDNAs and analyzed using SYBR Green (ABI

PRISM 7000 Sequence Detector System). The cDNA amount used in each reaction was

normalized to the housekeeping gene cyclophilin A. The primers and qPCR assay conditions

are available upon request.

miRNA expression profiling (miRNome analysis)

miRNA qPCR array analysis was performed using the rno-miRNome miRNA profiling kit

(Systems Biosciences, Mountain View CA, USA ). RNA was extracted from individual C and

LP fetal pancreas. Then, RNA from a minimum of 4 pancreases per litter was pooled for the

array. 3 litters per group were analyzed. Per litter, 1µg of pooled RNA was reverse-

transcribed into first-strand cDNA using the QuantiMir RT Kit (System Biosciences,

Mountain View, CA). Profiling of mature miRNAs was performed on 3 litters per group by


8

qPCR in a 384 well-plate format including 380 miRNA-specific primers and using SYBR

green. Expression levels were normalized using U6 since its melting curve was more

satisfactory compared to that obtained for the 2 other reference genes.

Heatmap was performed using the open-source analysis software with the multi experiment

viewer (www.tm4.org). miR-375 expression was confirmed with the miRCURY LNA

Universal RT MicroRNA PCR kit (Exiqon, Vedbaek, Denmark) according to manufacturer’s

instructions.

Analysis of total cell extracts and western blotting

Fetal pancreas or dissociated islet cells were processed for protein isolation as in (22). For

western blotting, 10-50 µg of total proteins were separated by electrophoresis and transferred

to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA).

Immunoreactive proteins were revealed by enhanced chemiluminescence (Millipore,

Billerica, MA, USA). Antibody to PDK-1 was from Cell Signaling Technology (Danvers,

Massachusetts, USA) and to β actin from Sigma. Western blots were quantified by

densitometry with Image Quant software.

Insulin secretion assay

All experiments were performed with a modified Krebs-Ringer solution containing 2.8 mM

glucose (23). Dissociated islet cells or batches of 20 free-floating size-matched islets were

incubated at 37°C in 1 mL of Krebs-Ringer medium containing glucose at 2.8 mM or 20 mM.

After 120 min, the incubation medium was removed for insulin measurement. Islets or

dissociated islet cells were collected and homogenized by sonification to extract insulin. To

eliminate variations due to differences in individual islet batches, insulin secretion during


9

incubation was expressed as a percentage of the islet insulin content at the incubation start,

which is referred to as fractional insulin release.

Oral Glucose Tolerance Tests (OGTT)

After an overnight fast, C and LP progeny were given an oral glucose bolus (2g). Blood was

collected from the tail vein at 0, 15, 30, 60, 90 and 120 min. Glycemia was measured using

the One Touch glucometer (Lifescan Inc., Milpitas, CA, USA).

Statistical analysis

Results are shown as means ± SEM. n represents the number of litters analyzed. Student’s t-

test was used to compare 2 conditions (paired or unpaired) and two-way ANOVA test

followed by post-hoc Fisher test when more than 2 conditions were analyzed. A p value of

<0.05 was considered significant.

Results

Expression of miRNAs in LP pancreas of progeny at day 21 of gestation

To investigate whether miRNA expression in the fetal pancreas is altered by maternal LP diet,

we compared the miRNA expression profile obtained from normal fetal pancreas and

pancreas from LP fetuses at day 21 of gestation. We employed the qPCR-based miRNome

miRNA profiler to screen 380 mature rat miRNAs, in pancreas of 3 different litters of male C

and LP rats (Fig 1A). Of the 360 detectable (95%) miRNAs, 47 miRNAs are differentially

expressed between C and LP (Fig 1B). Remarkably, 43 are upregulated (28 with p<0.05 and


10

15 with p<0.09) in LP, while only 4 are downregulated (p<0.05). Interestingly, we found that

the expression of miR-375, highly expressed in endocrine pancreas and involved in β-cell

proliferation and function, is increased in fetal LP pancreas at day 21 of conception (1.4 ±

0.08 in LP compared with 1 ± 0.028 in C; see supplemental data 1).

PDK-1 protein is downregulated in fetal pancreatic islets of LP progeny

PDK-1, one of the miR-375 targets (24), is a key component of the insulin and growth factor

signaling pathway, downstream of PI3K in the PKB cascade. As miR-375 is upregulated in

the pancreas of LP fetuses, we measured PDK-1 protein levels in the pancreas of our

experimental groups. By western blotting, no difference was observed in total LP pancreas

compared with controls (Fig 2A), while qPCR analysis revealed a moderately decreased level

of PDK-1 mRNA (Fig 2B). However, immunohistochemical analyses showed that PDK-1

immunoreactivity is specifically reduced in the endocrine compartment of the pancreas (Fig

2C).

β-cell mass is reduced in LP progeny at fetal day 21 due to a reduction in β-cell

proliferation and size

PDK-1 is a key regulator of β-cell proliferation and growth of developing pancreatic cells

(25). To investigate whether the PDK-1 protein reduction observed in LP islets impacts

pancreas development, immunohistochemical and morphometric analyses were performed.

While in the LP progeny the number of islets measured as insulin positive aggregates per

pancreas area is the same as in the control progeny, the islets are smaller than in the control

progeny (Table 1). This diminished islet size in LP pancreas can be accounted by a 50%


11

decrease in β-cell fraction area, while the α-cell fraction was preserved (Table 1). In fact, the

maternal LP diet significantly increased the proportion of small islets, and concomitantly

reduced the proportion of large islets compared to the control progeny (Fig 3A).

To gain more insight into the mechanism underlying reduced β-cell growth, the proliferative

capacity of these cells was investigated. Co-immunostaining of pancreas sections with

antibodies to insulin and to phosphorylated histone H3 (pHH3) (Fig 3B) or to Ki-67

(supplemental Fig. 2A) showed a decreased mitotic index of β-cells in LP pancreas.

Quantification revealed that β-cell proliferation is reduced by approximately 50% in LP

compared to C fetuses. In contrast, apoptosis assessed by the TUNEL method (not shown)

and differentiation measured by β-cell markers seem to be intact (supplemental Fig. 2B). In

conclusion, protein restriction during pregnancy reduces in the progeny β-cell mass due to

decreased β-cell expansion.

In addition to its role in β-cell proliferation, PDK-1 has been found to exert a major impact on

β-cell size (26). To evaluate whether this is occurring in our experimental conditions we took

advantage of the fact that β-cells are the only islet cells that express GLUT-2 at the cell

membrane. Morphometric measurements indicate that fetal LP β-cells display a modest size

reduction (Fig 3C). Thus, LP diet during pregnancy diminishes the β-cell diameter of the

fetuses by approximately 10 percent. However, when extrapolated to the islet volume, this

decrease represents a reduction of approximately 25 percent. Note that the reduction in β-cell

size was confirmed by E-cadherin/insulin co-immunostaining (supplemental Fig. 2C).

Reduced proliferation and increased miR-375 expression in islets derived from LP

fetuses are maintained after 7 days in culture


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Animal studies have clearly demonstrated that poor nutrition during gestation irreversibly

leads to reduced cell numbers in tissues such as the pancreas (27). Here we evaluated whether

the decreased cell proliferation observed in vivo persists in LP neoformed islets in culture.

Indeed, our measurements revealed that the size of islets neoformed from LP fetal pancreas is

reduced compared to those from C pancreas (Fig 4A). Further, insulin content per islet is

diminished in LP neoformed islets (Fig 4B), while relative insulin mRNA expression is

preserved (Fig 4C). Together these data suggest that cell proliferation is decreased in LP

neoformed islets. Due to the scant quantity of available material, we were unable to accurately

measure protein levels of critical cell cycle actors, and hence we used qPCR to examine their

mRNA expression (Fig 4D). While cyclin D2, which accumulates during the G1 phase, was

increased in LP derived neoformed islets, cyclin A2, which is necessary for progression into

mitosis, was reduced. In addition, the expression of an important inhibitor of G1 cyclin/cdk

complexes, p57, is increased in LP neoformed islets (Fig 4D). These data are compatible with

an impaired β-cell proliferation of LP neoformed islets. To determine whether miR-375 might

be involved in perturbing the regulation of β-cell replication in LP neoformed islets, we

examined its expression. Notably, we found that miR-375 expression is significantly

increased in LP neoformed islets compared to controls (Fig 4E). Taken together our data

strongly suggest that β-cell alterations induced by maternal protein restriction are not the

result of direct regulation by the maternal milieu. Rather they represent changes transmitted to

next cell generations.

Forced expression of miR-375 in primary islet cells impairs cell proliferation and insulin

secretion


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To investigate whether miR-375 interferes with cell proliferation and/or insulin secretion, we

mimicked the changes in miR-375 expression observed in LP progeny by transfecting

dissociated primary rat islet cells with mature miR-375. 72h after transfection, cell

proliferation and insulin secretion were measured. Similar to our data in the INS-1E cell line

(supplemental Fig 3), miR-375 expression in dissociated primary islet cells reduces PDK-1

protein levels (Fig 5A-B), and inhibits cell proliferation by approximately 40% (Fig 5C).

Moreover, increased miR-375 expression decreases glucose-induced insulin secretion in

cultured primary islet cells (Fig 5D) without affecting insulin content (data not shown),

confirming observations by Poy and colleagues in the insulinoma MIN6 cell-line (28; 29).

Increased miR-375 expression and pancreatic deterioration persist in LP progeny at 3

months of age

As increased miR-375 expression and reduced proliferation are maintained in neoformed LP

islets, we evaluated whether this was also the case in adult 3 month-old male offspring.

Importantly, we found that miR-375 expression is increased in isolated islets from 3 month-

old LP animals (Fig 6A). Accordingly, mRNA levels of PDK-1 and MTPN, another miR-375

target (28), are diminished (Fig 6B). As expected from the decrease in PDK-1, β-cell mass

and proliferation are decreased (Fig 6C-D). Consistent with MTPN and PDK-1 reduction, the

β-cell functioning of 3 month-old LP animals is deteriorated, as glucose-stimulated insulin

secretion is severely blunted (Fig 6E). Given the diminished β-cell mass and insulin secretion

in LP progeny, we studied glucose homeostasis in 3 month-old offspring. As shown in Fig

6F, LP offspring are glucose-intolerant after an oral glucose challenge. However, insulin

injection did not reveal hormone resistance in LP compared to control progeny at 3 months of

age (data not shown), indicating that the glucose intolerance is due to insufficient insulin

secretion.


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Repressed miR-375 expression in LP-derived islet cells rescues cell proliferation and

insulin secretion

To address whether miR-375 mediates the pancreatic alterations in LP progeny, we used a

loss of function approach with anti-miR-375 oligonucleotides to reverse the elevated miR-375

expression in islet cells derived from 3 month-old LP rats. miR-375 is upregulated in LP

derived islet cells (supplemental Fig. 4), and glucose-induced insulin secretion and cell

proliferation are reduced (Fig 7A-B). Of significance, anti-miR-375 driven normalization of

miR-375 levels (supplemental Fig 4) in dissociated LP-derived islet cells improved glucose-

induced insulin secretion (Fig 7A) without affecting insulin content (data not shown) and cell

proliferation (Fig 7B). The latter is accompanied by a return to normal levels of PDK-1

protein level (Fig 7B). Taken together, we show that restoration of normal miR-375 levels in

LP islets tends to correct key deviations of the β-cell phenotype induced by maternal protein

deficiency.

Discussion

The biological importance of miRNAs has been placed in the limelight following the

demonstration of diverse and profound phenotypic alterations upon changes in their

expression. These modifications are associated to perturbed development and pathological

situations (13). Our present data demonstrate that an unfavorable environment during fetal

development results in changes in programmed miRNA expression in the endocrine pancreas

of the progeny. Amongst the miRNAs with aberrant expression, miR-375 which regulates the

proliferation and the size of β-cells, contributes, at least in part, to the reduction in fetal β-cell


15

mass observed at birth. Our results in adult 3 month-old animals reveal a sustained increase in

miR-375 expression continuously affecting β-cell physiology and function. We show that

early environmental factors, such as maternal nutrition, durably influence pancreatic

expression of miRNAs, including miR-375, which will impact β-cell physiology and control

long-term health.

MiR-375 is a highly conserved miRNA which was cloned from a mouse insulinoma β-cell

line (28). miR-375 is expressed in β-cells as well as in non-β-cells of the pancreas (30-32),

and in the pituary gland (33). In human and mouse islets, miR-375 appears to be the miRNA

with the most robust expression (30).

Using mice lacking miR-375 Poy et al. reported that miR-375 deletion influences not only β-

cell mass but also α-cell mass by regulating a cluster of genes controlling cellular growth and

proliferation (32). Indeed, in normal situations, mice lacking miR-375 exhibit an increased α-

cell number, while the miR-375 loss has little influence on β-cell mass. However, when the

metabolic demand increases, genetic miR-375 deletion in mice counteracts the normally

occurring β-cell hyperplasia (32). At first glance this result appears at variance with our

observations. However, several studies have reported that miR-375 acts not as a positive

regulator of cell proliferation, but rather as a negative modulator (24; 34-36). At least two

scenarios could account for this apparent divergence. One possible explanation relates to

animal species differences. Tattikota and colleagues (37) reported in mice models that miR-

375 effect on β-cell proliferation was mostly attributed to its target Cadm1 (cell adhesion

molecule 1). As shown in supplemental figure 5, Cadm1 expression is localized in all human

and rat islet cells, while its expression is particularly perceptible at the periphery of mouse

islets corresponding to α-cells. This could explain the robust influence of miR-375 deletion on

α-cell mass under normal physiological conditions (37). When the metabolic demand


16

augments (e.g. ob/ob mice), it is possible that the reduced β-cell mass results from α-cell

dysfunction. Hence the nature of miR-375 action on β-cell proliferation could be different in

mice compared to humans and rats.

Another scenario relates to recent views that miRNAs are responsive to cellular and

extracellular stress and are used by cells to adjust to changes in their environment (38). A

pioneering example in Drosophila revealed that miR-7 loss had no detectable impact on

photoreceptor development under uniform laboratory conditions, but miR-7 becomes

necessary under conditions of temperature fluctuation (39). It is thus conceivable that, under

intensified stress conditions due to increased metabolic pressure, miR-375 can negatively

influence cell proliferation, while under physiological circumstances it fosters cell

proliferation.

In addition to its role in β-cell proliferation, miR-375 is a regulator of chief β-cell functions.

Indeed, forced miR-375 expression in insulinoma cells leads to reduced glucose-stimulated

insulin secretion without interfering with glucose action on ATP production and intracellular

calcium levels. More precisely, miR-375 interacts with a series of gene products, including

MTPN mRNA, governing insulin granule fusion with the plasma membrane, and thereby

inhibits exocytosis (28; 29). In fetal neoformed LP islets, we fail to observe a difference in

insulin secretion compared to C (not shown). This is probably due to the variable ability of

neoformed pancreatic islets to respond to glucose. Indeed, in fetal islets the acquisition of

stimulus / secretion coupling of insulin in response to glucose occurs after birth (39). While

some reports view neonatal islets as developmentally immature (40), others explain the weak

glucose responsiveness by a high activity at low glucose but a lower one at high glucose (41).

In contrast to neoformed pancreatic islets, islets isolated from 3 month-old LP progeny, with

increased miR-375 expression, show a lower fractional insulin release in response to glucose.


17

At the same time MTPN and PDK-1 mRNA are decreased. Importantly, normalization of

miR-375 levels restores insulin secretion in islet cells of 3 month-old LP progeny.

One of the most intriguing and important aspects of disease programming concerns the

transmission of the disorder (42). Such phenomenon appears to occur in our LP animals

where increased miR-375 expression observed in fetal islets persists in neoformed islets.

Indeed, a similarly augmented level of miR-375 prevailed in LP neoformed islets after 7 days

of culture under the same conditions as control islets. Furthermore, in agreement with

previous publications, cell proliferation seems to be impaired in LP neoformed islets (43-45).

Indeed, LP neoformed islets were smaller than neoformed islets from control progeny and a

select set of genes involved in cell cycle regulation has altered expression, suggesting a

reduction in cell proliferation similar to the in vivo situation. Most notably, 3 month-old LP

progeny display persistently increased miR-375 expression, reduced β-cell proliferation and

decreased glucose-induced insulin secretion. As a whole our data indicate that the elevated

miR-375 level is unlikely to result from direct regulatory signals from the maternal milieu, but

rather reflects the occurrence of a developmental memory of the islet cells that recall the

intrauterine protein malnutrition. This induces long-lasting damaging effects on the pancreas

resulting in reduced β-cell proliferation and function.

Our results on miR-375 misexpression in LP endocrine pancreas are supported by the

existence of epigenetic marks adjacent to the miR-375 coding region. Indeed, the miR-375

gene is epigenetically regulated by DNA methylation consistent with the presence of 2 large

CpG-rich regions (46). Moreover, the miR-375 promoter region contains several consensus

binding sites for transcription factors, which have been implicated in the development and

function of islets such as HNF-6 and INSM-1 (47). PDX-1 was also found to interact with the


18

upstream region of the miR-375 gene (48). Most of these transcription factors are themselves

regulated by epigenetic mechanisms (49).

In conclusion, we propose that under deleterious in utero conditions, the altered control

exerted by miRNAs on gene regulation increases the postnatal risk of β-cell failure and hence

of T2D. This augmented risk could reflect a reduced ability of the β-cells to contend with

stress due to a deficiency in adaptive β-cell mass and function. While biological systems are

characterized by robustness against environmental changes to ensure proper development and

health, failure in the protective mechanisms set the stage for exacerbated disease risk. A

groundbreaking concept in this context is that miRNAs are instrumental in generating

biological robustness (39). Dovetailing with this view, our data suggest that the level of miR-

375 is key for the establishment of an adequate β-cell mass and function, which is necessary

for the homeostatic health preservation of the organism. Taking into account the fact that

miR-375 has been identified as a diabetes-related circulating miRNA (50) and that miR-375

misexpression is observable before and after birth in growth-retarded animals, miR-375

appears as a promising biomarker of β-cell failure.

Acknowledgments

Our research was supported by Inserm, Université de Nice Sophia Antipolis, Conseil

Régional PACA and Conseil Général des Alpes-Maritimes, by the European Foundation for

the Study of Diabetes (EFSD/Lilly; European Diabetes Research Programme; 2011), by

Aviesan/AstraZeneca , « Diabetes and the vessel wall injury » program and by the Agence

Nationale de la Recherche (ANR blanc n°RPV12004AAA). The authors thank Pr Phillipe

Halban, University of Geneva for providing the 804G matrix.


19

Author contributions: O.D. researched data, contributed to the manuscript and discussion.

C.H. researched data, contributed to the manuscript and discussion. N.G. and V.C researched

data. E.V.O. contributed to manuscript and discussion. E.V.O. is the guarantor of this work

and, as such, had full access to all the data and takes full responsibility for the integrity of data

and the accuracy of data analysis.

Conflict of Interests

The authors declare no conflicts of interests.

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23

Figure legends

Fig. 1. miRNA expression in fetal rat pancreas is altered by maternal LP diet. A. Heatmap

showing the quantification of 380 miRNAs expressed in the fetal pancreas of C and LP

progeny at day 21 of gestation. Each column represents one litter (pool of 4 fetuses). miRNA

qPCR array analysis was performed to examine mature miRNA expression in C and LP fetal

pancreas. B. The 47 most differentially expressed miRNAs in LP pancreas compared to C

pancreas.

Fig. 2. PDK-1 protein level is reduced in fetal LP endocrine pancreas at day 21. A. Western

blot analysis. Fetal pancreas from C and LP fetuses were harvested and protein extracts were

analyzed with antibody to PDK-1 or to β actin. Quantification was performed using Image

Quant software. Values are means ± SEM, n = 6. B. PDK-1 mRNA measurement. Fetal

pancreases were collected for RNA extraction. RNA extracts were reverse-transcribed and

analyzed by qPCR. Gene expression was normalized to the cyclophilin A transcript level.

Values are means ± SEM, n = 6. C. Detection of PDK-1 protein on sections of fetal pancreas.

Scale bar: 100µm. Immunostaining intensity was evaluated using Image J software on a

minimum of 90 islets per group. Graphs show means ± SEM, n = 6 fetal pancreases with each

3 slides, ** p<0.01, LP vs C.

Fig. 3. β-cell mass is diminished in fetal LP progeny at day 21 due to a reduction in β-cell

size and proliferation. Immunocytochemistry was performed on pancreatic sections. A. Size-

frequency distribution of pancreatic islets of 21 day-old fetuses. The profile diameter of the

islets was measured using the ImageJ software. The islet diameter was calculated assuming

that islets are spheres. Clusters of at least 3 insulin positive cells were considered as islets.


24

Values are means ± SEM, n=6, **p < 0.01, LP vs C. B. Left: pHH3 positive cells (green)

were counted on pancreatic sections together with insulin (red) and dapi (blue). Scale bar: 50

µm. Right: Percentage of proliferating β-cells over total β-cells. Values are means ± SEM, n =

6, ** p<0.01, LP vs C. C. β-cell size measurement. Left: Pancreatic sections were stained

with antibodies to GLUT-2 (red) to determine the size of individual β-cells. Scale bar: 10 µm.

Right: The β-cell diameter was calculated with Image J software assuming that β-cells are

spheres. Values are means ± SEM from at least 200 β-cells from each of 6 fetuses from LP

and C dams. **p < 0.01, LP vs C.

Fig. 4. Maternal LP diet induces alterations in cell cycle regulators in fetal neoformed LP

islets. After 7 days in culture, fetal C and LP neoformed islets were studied for their size,

insulin content, insulin mRNA and expression of genes involved in cell growth. A. Size of

fetal neoformed islets after 7 days in culture. The islet diameter was measured using the

ImageJ software and was calculated assuming that islets are spheres. Values are means of 100

observations pooled from 3 independent cultures. Values are means ± SEM, n = 3,

***p < 0.001, LP vs C. B. Insulin content of fetal neoformed islets after 7 days culture. After

the culture period, fetal islets were placed in 1 ml acid–ethanol to extract insulin. Insulin

content was determined by ELISA. Values are means ± SEM, n = 4, **p < 0.01, LP vs C. C.

Insulin mRNA measurement. RNA extracts were reverse-transcribed and analyzed by qPCR.

Insulin gene expression was normalized to the cyclophilin A transcript level. qPCR was

performed on RNA extracted from 4 independent cultures. Values are means ± SEM, n = 4.

The same procedure was used for panel D and E. D. Expression of cell cycle regulators in LP

neoformed islets after 7 days in culture. Values are means ± SEM, n = 4, *p < 0.05, LP vs C;

**p < 0.01, LP vs C. E. miR-375 expression in neoformed islets after 7 days in culture.


25

Expression of mature miR-375 precursor was normalized to the U6 transcript level. Values

are means ± SEM, n = 4, **p < 0.01, LP vs C.

Fig. 5. Forced miR-375 expression in dissociated primary rat islet cells impairs cell

proliferation and insulin secretion. 48h after plating, dissociated primary rat islet cells were

transfected with 100nM of double-stranded RNA oligonucleotides corresponding to the

mature miR-375 sequence or with 100nM of a scrambled control miR (CTL). 72h after

transfection, cells were fixed for 10 min with methanol for proliferation assessment, or

incubated for 60 min in modified Krebs-Ringer buffer for insulin secretion experiments. A.

Analysis of the miR-375 target, PDK-1. 72h after transfection, cells were harvested for

protein extraction. Protein extracts were analyzed by western blot with antibody to PDK-1 or

to actin. B. Relative quantification of PDK-1 protein. Data represent 3 independent

transfections. Values are means ± SEM, n = 3, *p < 0.05, miR-375 vs CTL. C. Cell

proliferation measurement. Proliferation index was evaluated as the ratio of pHH3 positive

cells to total cell number from 4 independent transfections. Values are means ± SEM, n = 4,

*p < 0.05, miR-375 vs CTL. D. Insulin secretion assay. For secretion experiments, dissociated

cells were starved in modified Krebs-Ringer buffer containing 2.8mM glucose for 60 min and

thereafter were stimulated or not with 20mM glucose for 2h. Values are means ± SEM, n = 4,

**p < 0.01, miR-375 vs CTL at 20mM glucose, ANOVA.

Fig. 6. Pancreatic deteriorations persist in adult 3 month-old male LP progeny. A. miR-375

expression. Islets were collected from 3 month-old C and LP progeny for RNA extraction.

Expression of mature miR-375 was normalized to the U6 transcript level. B. Expression of

miR-375 target genes. C. β-cell area. D. β-cell proliferation. pHH3 positive cells were

counted on pancreatic sections together with insulin. The percentage of proliferating β-cells


26

over total β-cells is shown. For panels A to D values are means ± SEM, n = 4, *p < 0.05, LP

vs C; **p < 0.01, LP vs C. E. Insulin secretion assay. Batches of 20 free floating islets were

starved for 2h in modified Krebs-Ringer buffer containing 2.8 mM glucose and were

incubated thereafter for 2h either with 2.8 mM glucose or 20 mM glucose. For the LP and C

progeny islets of similar size were used. Values are means ± SEM, n = 3, **p < 0.01, LP

20mM glucose vs C 20mM glucose, ANOVA. F. OGTT study at 3 months of age in C rat

(open circle) and LP (black circle). OGTTs were performed after a 16h overnight fast.

Glucose (2g) was administered orally. Glycemia was measured at different times. n = 5 for

each group, ***p<0.001, LP vs C, repeated measures ANOVA.

Fig. 7. Normalization of miR-375 in islet cells derived from 3 month-old LP progeny rescues

insulin secretion and cell proliferation. Islets from 3 month-old C and LP progeny were

isolated, dissociated by trypsinization and seeded at a density of 25 × 103 cells/cm

2. 48h after

plating, dissociated islet cells were transfected with 100 nM miR-375 hairpin-inhibitor to

block endogenous miR-375 (Anti-375) or with control hairpin inhibitor (Anti-CTL). 72h after

transfection, cells were used for measurement of insulin secretion (A) or of cell proliferation

(B). A. Insulin secretion assay. For secretion experiments, dissociated C and LP islet cells

were starved in modified Krebs-Ringer buffer containing 2.8 mM glucose for 60 min and

thereafter stimulated or not with 20 mM glucose for 2h. Values are means ± SEM, n = 4,

***p < 0.001, Anti-375 vs Anti-CTL from C derived islet cells at 20mM glucose, LP vs C

derived islet cells in CTL condition at 20mM glucose, **p < 0.01, Anti-375 vs Anti-CTL from

LP derived islet cells at 20 mM glucose, ANOVA. B. Cell proliferation. Proliferation index

was evaluated as the ratio of pHH3 positive cells to total cell number from 4 independent

transfections. Lower insert: analysis of the miR-375 target, PDK-1. 72h after transfection,

cells were harvested for protein extraction. Protein extracts were analyzed by western blot


27

with antibody to PDK-1. Values are means ± SEM, n = 4, *p < 0.05, LP vs C in Anti-CTL

condition, Anti-CTL vs Anti-375 in LP derived islet cells,**p < 0.01, Anti-375 vs Anti-CTL

in C derived islet cells, ANOVA.


28

Table 1: Morphometric parameters of β- and α- cells in C and LP fetuses at day 21

C LP

β-cell fraction (%) 3.06 ± 0.11 1.76 ± 0.09 ***

α-cell fraction (%) 0.69 ± 0.07 0.73 ± 0.05

Insulin positive aggregates per

pancreas area (number/cm2)

2,999 ± 223 3,154 ± 268

Islet size (µm2) 2,062 ± 112 1,388 ± 138 **

Immunocytochemistry for insulin and glucagon was performed on pancreatic sections. The β-

and α- cell fraction (%) were measured as the ratio of the insulin-positive and glucagon-

positive cell area over the total tissue area on the entire section. The number of insulin-stained

pancreatic islets in each image was manually counted using Adobe Photoshop 7.0 computer

software. Average islet density (number of islets per unit area of pancreatic tissue) was

calculated. Values are means ± SEM, n = 6 from three different dams, **p < 0.01, LP vs C;

***p < 0.001, LP vs C.


72x104mm (300 x 300 DPI)


72x104mm (300 x 300 DPI)


72x104mm (300 x 300 DPI)


72x104mm (600 x 600 DPI)


72x104mm (600 x 600 DPI)


72x104mm (600 x 600 DPI)


72x104mm (300 x 300 DPI)


1

Supplemental materials and methods

Primary Antibody used for Immunohistochemistry

Primary antibodies used for immunohistochemistry were mouse anti-insulin (1/1,000; Sigma),

mouse anti-phosphorylated histone H3 (Abcam), mouse anti-glucagon (1/1,000; Sigma),

mouse anti-Glut-2 (1/100; Santa Cruz) and rabbit anti-PDK-1 (Novus, Littleton, CO, U.S.A.).

Secondary antibodies (1/100) were FITC-labeled anti-rabbit or anti-mouse (Jackson Immuno-

Research Laboratories, West Grove, PA, USA), Texas Red-labeled anti-rabbit or anti-mouse

(Jackson Immuno-Research Laboratories).


2

Supplemental figure legends

Supplementary Figure 1. miRNA expression in C and LP fetal pancreas. 2-ddCt and fold

change for C and LP pancreas with the p-values (T test).

Supplementary Figure 2. β-cell differentiation is not impacted by LP diet. A. Percentage of

proliferating β-cells over total β-cells. Ki67 positive cells were counted on pancreatic sections

together with insulin positive cells. Values are means ± SEM, n = 6, ** p<0.01, LP vs C. B.

Detection of PDX-1-, insulin-, and Glut-2-positive cells. Scale bar 10 µm. C. β-cell size

measurement. Pancreatic sections were stained with antibodies to E-cadherin together with

insulin to determine the size of individual β-cells. The β-cells diameter was calculated with

Image J software assuming that β-cells are spheres. Scale bar: 100 µm. Values are means ±

SEM from at least 250 β-cells from each of 6 fetuses from LP and C dams. **p < 0.01, LP vs

C.

Supplementary Figure 3. Forced miR-375 expression in INS-1E cells reduces insulin

secretion and proliferation. INS-1E cells were seeded in 6-well plates (8 × 105/well) and

transfected with scramble miR (CTL) or miR-375. 48h later, cells were fixed for 1h with

formalin (4%, v/v) for proliferation, or incubated in modified Krebs-Ringer buffer for insulin

secretion measurement. A. Analysis of miR-375 target, PDK-1. INS-1E cells were seeded in

6-well plates (8 × 105/well) and transfected with scramble miR (CTL) or miR-375. 48h later,

cells were harvested for protein extraction. Protein extracts were analyzed by western blot

using antibody to PDK-1 or to actin. B. Relative quantification of PDK-1 protein. Data

represent 3 independent transfections. Values are means ± SEM, n = 3, **p < 0.01, miR-375

vs CTL. C. Cell proliferation. Proliferation index was evaluated as the ratio of pHH3 positive


3

cells to total cell number from 3 independent transfections. **p < 0.01, miR-375 vs CTL. D.

Insulin secretion. For secretion experiments, INS-1E cells were starved in modified Krebs-

Ringer buffer containing 2.8mM glucose for 2h and thereafter were stimulated or not with 20

mM glucose for 2h. Values are means ± SEM, n = 3, **p < 0.01, miR-375 vs CTL at 20mM

glucose, ANOVA.

Supplementary Figure 4. Normalization of miR-375 in dissociated LP-derived islet cells.

Islets from 3 month-old C and LP progeny were isolated, dissociated and seeded at a density

of 25 × 103 cells/cm

2. 48h after plating, dissociated islet cells were transfected with 100 nM

miR-375 hairpin-inhibitor to block endogenous miR-375 (Anti-375), or with control hairpin

inhibitor (Anti-CTL). 72h after transfection, RNA was isolated using TRIzol reagent and

mature miRNA expression was assessed with the miRCURY LNA Universal RT MicroRNA

PCR kit according to manufacturer’s instructions. miRNA expression was normalized to the

level of U6. Values are means ± SEM, n = 3, *p < 0.05, LP vs C in Anti-CTL condition,

**p<0.01, Anti-375 vs Anti-CTL in LP derived islet cells, ANOVA.

Supplementary Figure 5. CADM1 (cell adhesion molecule 1) expression in adult rat

pancreas compared with human and mouse adult pancreas. Tissues samples were dehydrated

and embedded in paraffin. Slides were then incubated overnight with primary antibody to

CADM1. Secondary antibody was FITC-labeled anti-rabbit IgG. Total nuclei were stained

with dapi. Scale bar 50 or 100 µm.


1

2-ddCt Pancreas F21 Males

Well MicroRNA C LP Fold T test

A1 rno-let-7a 4,5 3,5 0,8 0,72

A2 rno-let-7b 1,1 1,3 1,2 0,59

A3 rno-let-7b* 1,0 1,7 1,6 0,56

A4 rno-let-7c 1,1 1,0 0,9 0,77

A5 rno-let-7d 1,0 0,7 0,7 0,03

A6 rno-let-7d* 1,9 4,5 2,4 0,31

A7 rno-let-7e 1,1 0,9 0,8 0,56

A8 rno-let-7e* 1,4 2,0 1,4 0,61

A9 rno-let-7f 1,1 0,6 0,5 0,22

A10 rno-let-7i 1,0 0,9 0,8 0,38

A11 rno-let-7i* 1,1 1,3 1,2 0,58

A12 rno-miR-1 1,0 0,3 0,3 0,01

A13 rno-miR-1* 2,1 2,7 1,3 0,77

A14 rno-miR-7a 1,0 1,1 1,1 0,65

A15 rno-miR-7a* 1,3 1,9 1,5 0,57

A16 rno-miR-7b 1,1 1,1 1,0 0,90

A17 rno-miR-9 1,3 1,8 1,4 0,64

A18 rno-miR-9* 1,4 2,2 1,6 0,58

A19 rno-miR-10a-3p 3,9 8,1 2,1 0,19

A20 rno-miR-10a-5p 1,0 1,1 1,1 0,76

A21 rno-miR-10b 1,1 1,0 0,9 0,65

A22 rno-miR-15b 1,1 1,2 1,1 0,73

A23 rno-miR-16 1,0 0,7 0,7 0,04

A24 rno-miR-17-3p 0,9 1,5 1,6 0,09

B1 rno-miR-17-5p 1,0 1,4 1,4 0,19

B2 rno-miR-18a 1,0 1,3 1,3 0,08

B3 rno-miR-19a 1,1 1,8 1,7 0,09

B4 rno-miR-19b 1,1 2,0 1,7 0,09

B5 rno-miR-20a 1,0 0,9 0,9 0,42

B6 rno-miR-20a* 1,0 1,4 1,4 0,17

B7 rno-miR-20b-3p 1,0 1,4 1,4 0,44

B8 rno-miR-20b-5p 1,2 1,8 1,5 0,42

B9 rno-miR-21 1,0 1,1 1,1 0,77

B10 rno-miR-21* 1,3 2,6 2,0 0,43

B11 rno-miR-22 1,0 1,0 1,0 0,99

B12 rno-miR-22* 1,1 3,9 3,4 0,35

B13 rno-miR-23a 1,0 1,4 1,4 0,07

B14 rno-miR-23a* 1,0 2,4 2,3 0,16

B15 rno-miR-23b 1,0 1,4 1,4 0,08

B16 rno-miR-24 1,0 1,0 1,0 0,83


2

B17 rno-miR-24-1* 1,1 1,2 1,1 0,83

B18 rno-miR-24-2* 1,0 1,7 1,7 0,12

B19 rno-miR-25 1,0 1,6 1,6 0,05

B20 rno-miR-25* 1,1 1,4 1,3 0,33

B21 rno-miR-26a 1,0 1,4 1,4 0,00

B22 rno-miR-26b 1,3 1,0 0,8 0,57

B23 rno-miR-26b* 1,4 0,8 0,5 0,52

B24 rno-miR-27a 1,1 1,5 1,3 0,70

C1 rno-miR-27a* 1,0 1,2 1,1 0,63

C2 rno-miR-27b 1,1 2,1 1,9 0,12

C3 rno-miR-28 1,0 1,4 1,3 0,31

C4 rno-miR-28* 1,0 1,9 1,8 0,02

C5 rno-miR-29a 1,0 1,0 1,0 0,96

C6 rno-miR-29a* 1,0 1,7 1,6 0,56

C7 rno-miR-29b 1,1 2,0 1,9 0,11

C8 rno-miR-29b-1* Undetermined Undetermined Undetermined Undetermined

C9 rno-miR-29b-2* 1,0 1,3 1,3 0,74

C10 rno-miR-29c 1,0 1,6 1,5 0,05

C11 rno-miR-29c* 1,1 2,4 2,2 0,18

C12 rno-miR-30a 1,0 1,4 1,4 0,06

C13 rno-miR-30a* 1,0 1,8 1,7 0,01

C14 rno-miR-30b-3p 1,1 2,7 2,5 0,02

C15 rno-miR-30b-5p 1,0 1,2 1,2 0,11

C16 rno-miR-30c 1,0 1,1 1,1 0,34

C17 rno-miR-30c-1* 1,4 3,0 2,1 0,28

C18 rno-miR-30c-2* 1,3 2,7 2,0 0,46

C19 rno-miR-30d 1,0 1,4 1,3 0,05

C20 rno-miR-30d* 1,1 2,6 2,5 0,01

C21 rno-miR-30e 1,0 1,3 1,3 0,17

C22 rno-miR-30e* 1,0 1,5 1,5 0,12

C23 rno-miR-31 1,3 0,8 0,6 0,52

C24 rno-miR-32 1,4 2,0 1,5 0,58

D1 rno-miR-33 0,9 2,3 2,5 0,07

D2 rno-miR-34a 1,2 1,9 1,6 0,32

D3 rno-miR-34b 0,9 1,6 1,7 0,04

D4 rno-miR-34c 1,1 1,5 1,4 0,29

D5 rno-miR-34c* 1,1 1,2 1,1 0,75

D6 rno-miR-92a 1,0 1,4 1,4 0,46

D7 rno-miR-92b 1,2 1,9 1,6 0,42

D8 rno-miR-93 1,0 1,1 1,1 0,32

D9 rno-miR-96 1,0 1,0 0,9 0,73

D10 rno-miR-98 1,0 1,0 1,0 0,94

D11 rno-miR-99a 1,0 1,1 1,1 0,64

D12 rno-miR-99a* 1,5 2,1 1,4 0,69

D13 rno-miR-99b 1,0 1,3 1,3 0,06


3

D14 rno-miR-99b* 1,1 0,6 0,5 0,27

D15 rno-miR-100 1,0 1,1 1,1 0,62

D16 rno-miR-101a 1,4 1,4 1,0 1,00

D17 rno-miR-101a* 1,1 1,6 1,5 0,23

D18 rno-miR-101b 1,0 1,4 1,3 0,14

D19 rno-miR-103 1,0 1,2 1,2 0,03

D20 rno-miR-106b 1,0 1,3 1,2 0,56

D21 rno-miR-106b* 1,0 1,2 1,2 0,17

D22 rno-miR-107 1,0 1,4 1,4 0,03

D23 rno-miR-122 1,1 1,3 1,1 0,70

D24 rno-miR-124 1,0 1,0 1,0 0,79

E1 rno-miR-124* 1,2 1,0 0,9 0,85

E2 rno-miR-125a-3p 1,1 1,6 1,5 0,38

E3 rno-miR-125a-

5p 1,0 1,3 1,3 0,27

E4 rno-miR-125b-3p 1,0 1,2 1,2 0,42

E5 rno-miR-125b-5p 1,0 1,1 1,1 0,51

E6 rno-miR-125b* 1,0 1,2 1,2 0,48

E7 rno-miR-126 1,0 1,2 1,2 0,45

E8 rno-miR-126* 1,0 1,2 1,2 0,26

E9 rno-miR-127 1,0 1,1 1,0 0,66

E10 rno-miR-128 1,1 2,3 2,1 0,29

E11 rno-miR-129 1,6 1,5 0,9 0,93

E12 rno-miR-129* 1,1 1,7 1,6 0,42

E13 rno-miR-130a 1,1 0,9 0,8 0,49

E14 rno-miR-130b 1,1 0,9 0,9 0,62

E15 rno-miR-132 1,1 1,6 1,5 0,46

E16 rno-miR-133a 1,0 1,2 1,1 0,66

E17 rno-miR-133b 1,1 1,6 1,5 0,31

E18 rno-miR-134 0,9 1,3 1,4 0,08

E19 rno-miR-135a 2,0 1,8 0,9 0,91

E20 rno-miR-135a* 1,9 1,2 0,7 0,71

E21 rno-miR-135b 1,4 0,7 0,5 0,45

E22 rno-miR-136 1,2 1,6 1,3 0,64

E23 rno-miR-136* 1,3 0,7 0,5 0,31

E24 rno-miR-137 1,1 1,0 0,9 0,84

F1 rno-miR-138 1,0 1,5 1,5 0,29

F2 rno-miR-138* 1,7 3,8 2,2 0,47

F3 rno-miR-139-3p 1,5 0,6 0,4 0,29

F4 rno-miR-139-5p 1,3 1,7 1,3 0,50

F5 rno-miR-140 1,0 1,1 1,1 0,77

F6 rno-miR-140* 1,0 1,5 1,5 0,01

F7 rno-miR-141 1,0 1,7 1,7 0,00

F8 rno-miR-142-3p 1,0 1,6 1,5 0,06


4

F9 rno-miR-142-5p Undetermined Undetermined Undetermined Undetermined

F10 rno-miR-143 1,1 1,1 1,0 0,94

F11 rno-miR-144 Undetermined Undetermined Undetermined Undetermined

F12 rno-miR-145 1,5 0,9 0,6 0,46

F13 rno-miR-146a 1,0 0,9 0,9 0,39

F14 rno-miR-146b 1,0 2,2 2,2 0,07

F15 rno-miR-147 1,1 2,1 1,9 0,16

F16 rno-miR-148b-3p 1,1 1,7 1,5 0,52

F17 rno-miR-148b-5p 1,2 1,3 1,1 0,80

F18 rno-miR-150 1,0 1,1 1,1 0,74

F19 rno-miR-151 1,0 1,0 1,0 0,95

F20 rno-miR-151* 1,0 1,7 1,7 0,03

F21 rno-miR-152 0,9 1,4 1,5 0,00

F22 rno-miR-153 1,1 1,2 1,1 0,80

F23 rno-miR-154 1,0 1,7 1,6 0,06

F24 rno-miR-181a 1,0 1,7 1,6 0,32

G1 rno-miR-181a* 1,1 1,4 1,3 0,41

G2 rno-miR-181b 1,1 1,6 1,5 0,59

G3 rno-miR-181c 1,1 7,7 6,8 0,31

G4 rno-miR-181d 1,0 1,2 1,2 0,46

G5 rno-miR-182 1,0 1,6 1,6 0,14

G6 rno-miR-183 1,1 1,4 1,3 0,41

G7 rno-miR-184 1,1 1,1 1,1 0,91

G8 rno-miR-185 1,0 1,2 1,2 0,39

G9 rno-miR-186 1,0 1,1 1,1 0,59

G10 rno-miR-187 1,1 1,5 1,3 0,75

G11 rno-miR-188 1,8 1,6 0,9 0,90

G12 rno-miR-190 Undetermined Undetermined Undetermined Undetermined

G13 rno-miR-190b Undetermined Undetermined Undetermined Undetermined

G14 rno-miR-191 1,0 7,3 7,0 0,29

G15 rno-miR-192 1,1 2,0 1,9 0,10

G16 rno-miR-193 1,1 1,5 1,3 0,56

G17 rno-miR-193* 1,3 2,7 2,1 0,47

G18 rno-miR-194 1,0 2,3 2,2 0,18

G19 rno-miR-195 1,1 2,1 1,9 0,41

G20 rno-miR-196a 1,0 1,8 1,7 0,30

G21 rno-miR-196a* 1,7 3,4 2,0 0,53

G22 rno-miR-196b 1,0 1,5 1,4 0,62

G23 rno-miR-196c 3,2 6,0 1,8 0,65

G24 rno-miR-199a-3p 1,0 1,1 1,1 0,74

H1 rno-miR-199a-5p 1,0 1,3 1,3 0,28

H2 rno-miR-200a 1,0 0,9 0,9 0,80

H3 rno-miR-200b 1,2 2,2 1,9 0,30


5

H4 rno-miR-200c 1,0 1,9 1,8 0,02

H5 rno-miR-203 1,1 1,7 1,6 0,26

H6 rno-miR-204 1,1 1,5 1,4 0,51

H7 rno-miR-204* 1,7 4,6 2,8 0,42

H8 rno-miR-205 1,1 2,3 2,2 0,02

H9 rno-miR-206 1,1 1,7 1,6 0,43

H10 rno-miR-207 1,1 2,0 1,8 0,35

H11 rno-miR-208 1,1 1,5 1,4 0,63

H12 rno-miR-210 1,2 1,3 1,1 0,57

H13 rno-miR-211 1,1 1,9 1,8 0,34

H14 rno-miR-212 1,1 1,0 0,9 0,83

H15 rno-miR-214 1,0 1,3 1,3 0,17

H16 rno-miR-215 1,7 1,4 0,8 0,70

H17 rno-miR-216a 1,2 1,1 0,9 0,87

H18 rno-miR-217 1,1 1,4 1,3 0,56

H19 rno-miR-218 1,0 1,0 0,9 0,68

H20 rno-miR-218* 1,0 0,9 0,9 0,68

H21 rno-miR-219-1-

3p 1,1 1,7 1,5 0,54

H22 rno-miR-219-2-3p 1,2 1,6 1,3 0,81

H23 rno-miR-219-5p 1,1 1,9 1,6 0,43

H24 rno-miR-221 1,1 1,8 1,7 0,45

I1 rno-miR-222 1,3 1,3 0,9 0,84

I2 rno-miR-223 1,0 1,6 1,5 0,21

I3 rno-miR-224 1,1 1,2 1,1 0,75

I4 rno-miR-290 1,5 3,0 2,0 0,61

I5 rno-miR-291a-

3p 1,4 2,9 2,1 0,56

I6 rno-miR-291a-5p 1,2 1,5 1,2 0,71

I7 rno-miR-292-3p Undetermined Undetermined Undetermined Undetermined

I8 rno-miR-292-5p 0,6 1,1 2,0 0,43

I9 rno-miR-296 1,0 1,3 1,2 0,47

I10 rno-miR-296* 1,1 1,6 1,5 0,48

I11 rno-miR-297 1,2 1,2 1,0 0,93

I12 rno-miR-298 1,0 1,4 1,4 0,32

I13 rno-miR-299 1,0 1,3 1,3 0,31

I14 rno-miR-300-3p 1,0 1,2 1,2 0,45

I15 rno-miR-300-5p 1,1 2,1 1,8 0,41

I16 rno-miR-301a 1,0 1,6 1,5 0,22

I17 rno-miR-301b 1,0 1,6 1,5 0,32

I18 rno-miR-320 1,1 1,4 1,2 0,67

I19 rno-miR-322 0,9 1,6 1,8 0,05

I20 rno-miR-322* 1,1 1,2 1,2 0,08

I21 rno-miR-323 1,1 0,9 0,9 0,55

I22 rno-miR-323* 1,1 1,8 1,6 0,42


6

I23 rno-miR-324-3p 1,3 1,9 1,4 0,60

I24 rno-miR-324-5p 1,1 1,2 1,1 0,83

J1 rno-miR-325-3p 1,0 0,9 0,9 0,89

J2 rno-miR-325-5p 1,3 2,6 1,9 0,32

J3 rno-miR-326 1,2 1,5 1,3 0,60

J4 rno-miR-327 1,4 2,4 1,7 0,48

J5 rno-miR-328 1,1 2,7 2,5 0,25

J6 rno-miR-329 1,2 2,8 2,4 0,27

J7 rno-miR-330 1,0 1,3 1,3 0,48

J8 rno-miR-330* 1,3 2,3 1,8 0,57

J9 rno-miR-331 1,0 1,3 1,3 0,55

J10 rno-miR-335 1,5 2,5 1,7 0,23

J11 rno-miR-336 1,5 2,9 2,0 0,58

J12 rno-miR-337 1,2 1,3 1,1 0,90

J13 rno-miR-338 1,2 1,8 1,5 0,32

J14 rno-miR-338* 1,5 3,0 2,0 0,45

J15 rno-miR-339-3p 1,2 2,1 1,7 0,43

J16 rno-miR-339-5p 1,0 2,1 2,1 0,20

J17 rno-miR-340-3p 1,1 2,6 2,3 0,30

J18 rno-miR-340-5p 1,1 1,2 1,1 0,90

J19 rno-miR-341 1,0 1,1 1,1 0,75

J20 rno-miR-342-3p 1,1 1,8 1,7 0,22

J21 rno-miR-342-5p 0,8 2,7 3,3 0,05

J22 rno-miR-343 1,5 3,2 2,2 0,32

J23 rno-miR-344-3p Undetermined Undetermined Undetermined Undetermined

J24 rno-miR-344-5p 1,3 3,1 2,3 0,35

K1 rno-miR-345-3p 1,2 1,5 1,3 0,45

K2 rno-miR-345-5p 1,1 1,7 1,5 0,33

K3 rno-miR-346 1,1 1,7 1,5 0,27

K4 rno-miR-347 1,2 1,9 1,5 0,04

K5 rno-miR-349 1,5 2,1 1,4 0,73

K6 rno-miR-350 1,3 1,9 1,5 0,59

K7 rno-miR-351 1,1 1,4 1,2 0,50

K8 rno-miR-352 1,0 1,2 1,2 0,47

K9 rno-miR-361 1,3 1,7 1,3 0,16

K10 rno-miR-363 1,0 2,2 2,2 0,26

K11 rno-miR-363* 3,2 7,7 2,4 0,39

K12 rno-miR-365 1,3 1,2 1,0 0,93

K13 rno-miR-369-3p Undetermined Undetermined Undetermined Undetermined

K14 rno-miR-369-5p 1,0 1,3 1,3 0,03

K15 rno-miR-370 1,0 1,3 1,3 0,38

K16 rno-miR-374 1,0 1,4 1,4 0,34

K17 rno-miR-375 0,9 1,3 1,4 0,05

K18 rno-miR-376a 1,0 1,3 1,3 0,21

K19 rno-miR-376a* 1,1 1,3 1,2 0,67


7

K20 rno-miR-376b-3p 1,0 1,4 1,4 0,30

K21 rno-miR-376b-

5p 1,0 1,1 1,1 0,86

K22 rno-miR-376c 1,1 1,2 1,1 0,80

K23 rno-miR-377 1,0 1,6 1,6 0,01

K24 rno-miR-378 1,0 1,6 1,5 0,09

L1 rno-miR-378* 1,3 2,4 1,8 0,47

L2 rno-miR-379 1,0 1,5 1,5 0,10

L3 rno-miR-379* 1,1 1,3 1,2 0,44

L4 rno-miR-380 1,0 1,1 1,1 0,65

L5 rno-miR-381 1,0 1,3 1,3 0,12

L6 rno-miR-382 1,0 1,0 1,0 0,87

L7 rno-miR-382* 1,0 1,1 1,1 0,71

L8 rno-miR-383 1,1 1,5 1,4 0,30

L9 rno-miR-384-3p Undetermined Undetermined Undetermined Undetermined

L10 rno-miR-384-5p 1,0 1,1 1,1 0,56

L11 rno-miR-409-3p 1,1 1,5 1,3 0,29

L12 rno-miR-409-5p 1,0 1,2 1,2 0,28

L13 rno-miR-410 1,0 1,5 1,5 0,11

L14 rno-miR-411 1,0 1,4 1,4 0,02

L15 rno-miR-412 1,5 3,3 2,2 0,49

L16 rno-miR-421 1,1 3,8 3,4 0,30

L17 rno-miR-423 1,0 1,3 1,3 0,27

L18 rno-miR-425 1,0 1,6 1,6 0,27

L19 rno-miR-429 1,0 1,3 1,3 0,17

L20 rno-miR-431 1,0 1,1 1,1 0,62

L21 rno-miR-433 1,2 2,2 1,9 0,44

L22 rno-miR-434 1,0 1,1 1,1 0,69

L23 rno-miR-448 1,1 1,7 1,6 0,66

L24 rno-miR-449a 1,1 1,4 1,3 0,61

M1 rno-miR-450a 1,1 1,1 1,0 0,95

M2 rno-miR-451 1,0 1,2 1,1 0,65

M3 rno-miR-455 1,0 1,3 1,3 0,02

M4 rno-miR-463 1,4 1,3 0,9 0,86

M5 rno-miR-466b 1,5 2,0 1,4 0,72

M6 rno-miR-466c 1,1 1,1 1,0 0,93

M7 rno-miR-471 1,4 4,9 3,5 0,32

M8 rno-miR-483 1,1 1,5 1,4 0,49

M9 rno-miR-484 1,5 2,3 1,5 0,60

M10 rno-miR-485 1,0 1,4 1,3 0,09

M11 rno-miR-487b 1,1 1,0 1,0 0,93

M12 rno-miR-488 1,1 1,2 1,1 0,77

M13 rno-miR-489 1,1 2,4 2,1 0,03

M14 rno-miR-493 1,0 1,3 1,2 0,50

M15 rno-miR-494 1,0 1,0 0,9 0,78


8

M16 rno-miR-495 1,0 0,9 0,9 0,64

M17 rno-miR-497 1,0 1,0 1,0 0,95

M18 rno-miR-499 1,4 2,9 2,1 0,13

M19 rno-miR-500 1,1 1,2 1,1 0,64

M20 rno-miR-501 1,3 3,0 2,3 0,29

M21 rno-miR-503 1,1 1,0 1,0 0,94

M22 rno-miR-505 1,1 1,2 1,1 0,69

M23 rno-miR-532-3p 1,1 1,9 1,7 0,36

M24 rno-miR-532-5p 1,0 1,2 1,1 0,53

N1 rno-miR-539 1,3 4,3 3,4 0,34

N2 rno-miR-540 1,0 1,2 1,2 0,42

N3 rno-miR-541 1,0 1,2 1,2 0,31

N4 rno-miR-542-3p 1,2 1,1 0,9 0,86

N5 rno-miR-542-5p 1,0 1,4 1,4 0,18

N6 rno-miR-543 1,0 1,1 1,1 0,81

N7 rno-miR-543* 1,0 1,4 1,4 0,18

N8 rno-miR-551b 1,1 1,3 1,3 0,73

N9 rno-miR-598-3p 1,0 1,6 1,6 0,36

N10 rno-miR-598-5p 1,3 1,9 1,5 0,39

N11 rno-miR-652 1,0 1,0 1,0 0,75

N12 rno-miR-664 3,3 0,8 0,2 0,42

N13 rno-miR-671 1,1 2,2 2,0 0,25

N14 rno-miR-672 1,1 1,7 1,6 0,25

N15 rno-miR-673 1,0 1,3 1,3 0,34

N16 rno-miR-674-3p 1,4 1,3 1,0 0,95

N17 rno-miR-674-5p 1,2 0,9 0,7 0,61

N18 rno-miR-708 1,1 2,4 2,2 0,31

N19 rno-miR-708* 1,2 1,4 1,2 0,84

N20 rno-miR-742 2,3 4,0 1,7 0,69

N21 rno-miR-743a 1,6 3,6 2,2 0,50

N22 rno-miR-743b Undetermined Undetermined Undetermined Undetermined

N23 rno-miR-758 0,9 1,4 1,6 0,00

N24 rno-miR-760-3p 1,2 8,1 6,6 0,42

O1 rno-miR-760-5p 1,1 1,7 1,5 0,34

O2 rno-miR-770 1,0 1,5 1,4 0,39

O3 rno-miR-871 1,5 2,0 1,4 0,59

O4 rno-miR-872 1,0 1,4 1,3 0,27

O5 rno-miR-872* 1,1 1,1 1,0 0,96

O6 rno-miR-873 1,3 1,6 1,2 0,78

O7 rno-miR-874 1,1 1,6 1,4 0,39

O8 rno-miR-877 1,2 1,5 1,3 0,48

O9 rno-miR-878 1,2 1,4 1,1 0,87

O10 rno-miR-879 1,0 0,8 0,8 0,55

O11 rno-miR-880 Undetermined Undetermined Undetermined Undetermined

O12 rno-miR-881 3,1 15,8 5,2 0,32


9


O14 rno-miR-105 1,5 1,5 1,0 0,99

O15 rno-miR-1224 1,0 0,6 0,6 0,19

O16 rno-miR-201 1,8 0,9 0,5 0,45


O18 rno-miR-220 1,2 4,2 3,6 0,41

O19 rno-miR-293 1,2 1,6 1,3 0,51

O20 rno-miR-294 1,4 2,4 1,8 0,46

O21 rno-miR-295 1,1 1,5 1,3 0,42

O22 rno-miR-362 1,0 1,4 1,3 0,18


O24 rno-miR-490 1,1 1,0 0,9 0,75

P1 rno-miR-496 1,0 1,5 1,4 0,16

P2 rno-miR-504 1,2 2,1 1,8 0,41

P3 rno-miR-511 1,4 2,0 1,5 0,62

P4 rno-miR-513 1,1 2,7 2,5 0,01

P5 rno-miR-544 1,2 1,6 1,4 0,42

P6 rno-miR-547 1,2 2,9 2,5 0,22

P7 rno-miR-568 Undetermined Undetermined Undetermined Undetermined

P8 rno-miR-582 1,0 1,2 1,2 0,29

P9 rno-miR-592 1,0 1,4 1,3 0,69

P10 rno-miR-615 1,2 2,0 1,6 0,45

P11 rno-miR-628 1,5 1,7 1,1 0,89

P12 rno-miR-632 1,1 1,1 1,0 0,97

P13 rno-miR-653 1,9 4,1 2,1 0,37

P14 rno-miR-665 1,0 1,2 1,2 0,42

P15 rno-miR-666 1,0 0,6 0,6 0,05

P16 rno-miR-667 1,0 1,9 1,9 0,27

P17 rno-miR-668 1,1 1,3 1,2 0,85

P18 rno-miR-675 1,2 1,9 1,6 0,49

P19 rno-miR-678 1,3 1,8 1,3 0,72

P20 rno-miR-685 1,0 0,8 0,8 0,40


72x104mm (300 x 300 DPI)


72x104mm (300 x 300 DPI)


72x104mm (300 x 300 DPI)



Documents

Maternal protein restriction leads to pancreatic failure ......islets derived from LP fetuses and in islets from adult 3 month-old LP progeny. miR-375 regulates the proliferation and