Nesca2013 Article IdentificationOfParticularGrou PDF
Nesca2013 Article IdentificationOfParticularGrou PDF
Nesca2013 Article IdentificationOfParticularGrou PDF
DOI 10.1007/s00125-013-2993-y
ARTICLE
Received: 19 April 2013 / Accepted: 19 June 2013 / Published online: 11 July 2013
# Springer-Verlag Berlin Heidelberg 2013
in predisposed individuals this compensatory process fails, 8 weeks as described [11]. Male Wistar rats were purchased
resulting in beta cell dysfunction, eventually accompanied by from Charles River Laboratories (L’Arbresle, France). All
reduction of the beta cell mass and type 2 diabetes manifesta- animal procedures were performed in accordance with
tion [2]. A better knowledge of the molecular mechanisms National Institutes of Health (NIH) guidelines and were ap-
underlying beta cell adaptation and failure will be instrumental proved by the respective Australian, Canadian and Swiss
for designing new strategies to prevent or treat this disease. research councils and veterinary offices.
MicroRNAs (miRNAs) are small non-coding RNAs that
play central roles in a number of physiological and pathological Microarray profiling Total RNAwas isolated with the mirVana
processes [3]. Several studies have shown that miRNAs partic- RNA isolation kit (Ambion, Austin, TX, USA) from islets of
ipate in the control of beta cell differentiation, function and C57BL/KsJ db/db mice or control animals. Total RNA from islets
mass. These non-coding RNAs regulate insulin production by of C57BL/6 mice fed a normal diet or HFD was isolated with the
directly or indirectly affecting the expression of key transcrip- miRNeasy kit (Qiagen, Hombrechtikon, Switzerland). Global
tion factors and they contribute to fine-tuning of hormone miRNA expression profiling was carried out at the Genomic
release by modulating the levels of important components of Technologies Facility of the University of Lausanne using
the beta cell secretory machinery [4]. The expression of several miRNA gene microarrays (Agilent Technologies, Morges,
miRNAs is affected by prolonged exposure to elevated con- Switzerland). Microarrays included probes for mouse miRNAs
centrations of glucose, NEFA and proinflammatory cytokines listed on www.mirbase.org/ (release 14, 2009).
[4]. Moreover, alterations in the levels of many islet miRNAs
have been reported in different models of diabetes [5–9]. Isolation and culture of dissociated islet cells Pancreatic islets
However, the functional impact of these miRNA expression were isolated as described previously [12] by collagenase diges-
changes and their potential role in the development of diabetes tion followed by purification on a Histopaque (Sigma-Aldrich)
were, in most cases, not explored. density gradient. The islets were first cultured overnight in RPMI
In this study, we analysed the global variations in islet 1640 Glutamax medium (Invitrogen, Carlsbad, CA, USA)
miRNA expression in prediabetic and diabetic db/db mice supplemented with 10% (vol./vol.) FCS (Amimed,
[10] and in mice fed a high-fat diet (HFD) [11]. Differentially BioConcept, Allschwill, Switzerland), 50 U/ml penicillin,
expressed miRNAs in these models of obesity-associated dia- 50 μg/ml streptomycin, 1 mmol/l Na pyruvate and 250 μmol/l
betes were systematically investigated for their effects on rat HEPES, and then dissociated by incubation with trypsin (5 mg/ml
and human beta cell function and for their impact on cell at 37°C for 4–5 min). Human pancreatic islets were obtained
survival on chronic exposure to pro-apoptotic conditions. The from the Cell Isolation and Transplantation Center (University of
results indicate that specific changes in islet miRNA expression Geneva), through the ECIT ‘Islets for Research’ distribution
in prediabetic and diabetic states reflect the coexistence of programme sponsored by the JDRF. The use of human islets
adaptive processes elicited to compensate insulin resistance was approved by the Geneva institutional Ethics Committee.
and of pathological reactions promoting beta cell failure. The Dissociated human islet cells prepared using the procedure de-
balance between these opposing phenomena is likely to deter- scribed above were cultured in CMRL medium (Invitrogen)
mine progression from normoglycaemia to hyperglycaemic supplemented with 10% (vol./vol.) FCS, 100 U/ml penicillin,
states and the manifestation of diabetes. 100 μg/ml streptomycin, 2 mmol/l glutamine and 250 μmol/l
HEPES. Detailed information about the human islet preparations
used in this study is presented in electronic supplementary ma-
Methods terial (ESM) Table 1.
Materials TNFα and INFγ were obtained from R&D Systems MIN6B1 cell culture The murine insulin-secreting cell line
(Minneapolis, MN, USA). IL-1β, prolactin (PRL), exendin-4 MIN6B1 [13] was cultured at a density of 1.5×105 cells/cm2
and palmitate were purchased from Sigma-Aldrich (St Louis, in DMEM-Glutamax medium (Invitrogen) supplemented
MO, USA). with 15% (vol./vol.) FCS, 50 U/ml penicillin, 50 μg/ml
streptomycin, and 70 μmol/l β-mercaptoethanol.
Animals Prediabetic (6 weeks old) and diabetic (14–20 weeks
old) C57BL/KsJ db/db mice and age-matched C57BL/KsJ Transfection and modulation of miRNA levels MIN6B1 and
control animals were obtained from the Garvan Institute dissociated rat or human islet cells were transfected with
breeding colonies (Sydney, NSW, Australia) [10]. Five- Lipofectamine 2000 (Invitrogen) with RNA oligonucleotide
week-old male C57BL/6 mice were purchased from Charles duplexes (Eurogentec, Seraing, Belgium) corresponding to the
River Laboratories (Saint-Constant, QC, Canada) and fed a mature miRNA sequence (overexpression) or with single-
normal diet or HFD (Bio-Ser Diet number F3282, stranded miScript miRNA inhibitors (Qiagen, Hombrechtikon,
Frenchtown, NJ, USA; 60% [wt/wt] energy from fat) for Switzerland) that specifically block endogenous miRNAs [14].
Diabetologia (2013) 56:2203–2212 2205
A custom-designed small interfering (si)RNA duplex directed (Millipore, Zug, Switzerland) and then with anti-rabbit
against green fluorescent protein (sense 5′-GACGUAAACG Alexa-Fluor-488 and anti-mouse Alexa-Fluor-555 antibodies
GCCACAAGUUC-3′ and antisense 5′-ACUUGUGGCCGU (Invitrogen). At the end of the incubation, the cover slips were
UUACGU CGC-3′) and the miScript miRNA reference inhib- washed with PBS containing Hoechst 33342 (Invitrogen) and
itor (Qiagen) were used as negative controls for miRNA images of at least 1×103 cells per condition were collected
overexpression and downregulation, respectively. using a fluorescence microscope. PRL (500 ng/ml for 48 h)
was used as positive control.
Measurement of miRNA and mRNA expression Mature
miRNA expression was assessed with the miRCURY Protein extraction and western blotting Protein lysates (30–
LNATM Universal RT MicroRNA PCR kit (Exiqon, Vedbaek, 50 μg) from MIN6B1 cells prepared as described previously
Denmark). Measurement of the levels of putative target [9] were separated on polyacrylamide gels and transferred to
mRNAs was performed by conventional reverse transcription polyvinylidine fluoride membranes. The membranes were
(Promega, Dübendorf, Switzerland) followed by quantitative incubated overnight with antibodies against granuphilin [16]
RT-PCR (qRT-PCR; Biorad, Reinach, Switzerland) with (1:2,000); mammalian target of rapamycin (mTOR; 2972,
custom-designed primers (Microsynth, Balgach, Switzerland), 1:1,000 Cell Signaling, Danvers, MA, USA), met proto-
details of which are available on request. MiRNA expression oncogene (hepatocyte growth factor receptor) (cMET; Cell
was normalised to the level of U6 or miR-7 (an islet-specific Signaling, 3127, 1:1,000) and glycogen synthase kinase 3β
miRNA used as internal control) while mRNA expression was (GSK-3β; Cell Signaling 9315, 1:1,000). Antibodies against
normalised to 18S. α-tubulin (T9026, 1:10,000, Sigma-Aldrich) and actin (Clone
C4 MAB1501, 1:15,000, Millipore) were used to verify equal
Insulin secretion At 2 days after transfection, MIN6B1 or loading. After exposure to IRDye (Li-Cor Biosciences, Bad
dissociated rat islet cells were pre-incubated for 30 min at Homburg, Germany) or horseradish-peroxidase-coupled sec-
37°C in Krebs buffer (127 mmol/l NaCl, 4.7 mmol/l KCl, ondary antibodies for 1 h, the bands were visualised via the
1 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, Odyssey imaging system (Li-Cor Biosciences) and chemilumi-
5 mmol/l NaHCO3, 0.1% [wt/vol.] BSA, 25 mmol/l HEPES, nescence (GE Healthcare Europe, Glattbrugg, Switzerland), re-
pH 7.4) containing 2 mmol/l glucose. The pre-incubation me- spectively. Band intensity was quantified by ImageJ software.
dium was discarded and the cells incubated for 45 min in the
same buffer (basal conditions). After collecting the supernatant Statistical analysis Statistical differences were assessed using
fractions, the cells were incubated for 45 min in Krebs buffer a Student’s t test or, for multiple comparisons, one-way
containing 20 mmol/l glucose (stimulatory conditions). The ANOVA of the means, followed by a post-hoc Dunnett test
incubation medium was collected and total cellular insulin (SAS statistical package; SAS, Carry, NC, USA).
contents recovered in acidified ethanol (75% [vol./vol.] ethanol,
0.55% [vol./vol.] HCl). The amount of insulin in the samples
was determined using an insulin enzyme immunoassay kit
(SPI-Bio, Bertin Pharma, Montigny le Bretonneux, France). Prediabetic Diabetic HFD
db/db db/db
100
Cell death assessment Transfected MIN6B1, rat or human
dissociated islet cells were incubated with 1 μg/ml Hoechst
10
Fold change vs ctrl
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
miR-132
miR-203
miR-210
diabetes animal models. The *
miR184
4 1.0 1.0 1.0 *
expression level of the indicated * *
miRNAs was measured by qRT- 2 0.5 0.5 0.5
PCR in pancreatic islets of young 0 0.0 0.0 0.0
prediabetic (a–d) and diabetic (e–l)
trl
trl
trl
trl
b
/d
/d
/d
/d
C
C
C
db
db
db
yg
yg
yg
yg
mice and in mice fed a normal diet
or HFD (m–s). The results
correspond to the mean ± SD of e f g h
* *
miR-199a-5p
miR-199a-3p
12 12 12 12
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
three to four animals per group and *
miR-132
miR-21
trl
trl
trl
b
b
by unpaired Student’s t test). Ctrl,
/d
/d
/d
/d
C
C
db
db
db
db
control; ND, normal diet; yg,
young
i j k l
1.5 1.5 1.5 1.5
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
miR-203
miR-210
miR-383
miR-184
trl
trl
trl
b
b
/d
/d
/d
/d
C
C
db
db
db
db
m n o
4 * 2.0 2.0
miR-199a-5p
miR-199a-3p
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
miR-132
3 1.5 1.5
2 1.0 1.0
1 0.5 0.5
0 0.0 0.0
D
FD
D
FD
D
FD
N
N
H
H
p q r s
1.5 1.5 1.5 1.5
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
(fold vs ctrl)
miR-383
miR-210
miR-184
miR-203
D
FD
D
FD
D
FD
N
N
H
H
Diabetologia (2013) 56:2203–2212 2207
and miR-146a (ESM Table 6). The results obtained by micro- regulation of insulin biosynthesis and release. Most of the
array analysis were confirmed by qRT-PCR (Fig. 2a–l). Our studied miRNAs did not affect insulin content (Fig. 4a–c) or
microarray data also revealed a 2.2-fold increase in miR-21, insulin release in dissociated rat islet cells (Fig. 4d–f) and
which we have previously shown to inhibit insulin secretion MIN6B1 cells (ESM Fig. 2). However, overexpression of
[8], a 1.6-fold decrease in miR-26a, which controls insulin miR-132 resulted in improved glucose-stimulated insulin
biosynthesis [18], and an increase in miR-802 (sixfold), which release from dissociated rat islet cells (Fig. 4d). In contrast,
regulates Hnf1b expression [19] (ESM Tables 6). The role of upregulation of miR-199a-5p led to an insulin secretory
these miRNAs was not further investigated in this study. defect in MIN6B1 cells (ESM Fig. 2), but not in islet cells,
Islet miRNA expression was also analysed in HFD-fed where it only diminished the insulin content (Fig. 4a).
mice. For this purpose we selected the group of mice displaying We next investigated whether the miRNAs differentially
the strongest response to HFD. These animals were markedly expressed in type 2 diabetes models regulate beta cell ex-
obese, insulin resistant, hyperinsulinaemic and clearly pansion. In MIN6B1 cells, upregulation of miR-132 or
hyperglycaemic (ESM Table 4). HFD mice showed miRNA downregulation of miR-184, miR-203 and miR-383 led to
expression changes analogous to those observed in the islets of an increase in proliferation while modification of the levels
diabetic db/db mice, with the exception of miR-21, miR-34a, of other miRNAs had no significant effects (ESM Fig. 3).
miR-146a, miR-199a-5p and miR-199a-3p, which were not Proliferation of insulin-positive cells was also observed on
significantly modified (ESM Table 7 and Fig. 2m–s).
Overall, the data indicate that a subset of islet miRNAs is
similarly altered in two obesity-associated animal models of a b
300 150
miR-199a-5p
type 2 diabetes, suggesting a role of specific miRNAs in beta * * * *
(% of ctrl)
(% of ctrl)
cell failure and the development of hyperglycaemia. miR-132 200 100
100 50
miRNA expression is affected by glucolipotoxic conditions To 0 0
determine the possible causes of the changes in miRNA ex- 11 G 20 G 11 G 20 G
pression detected in the islets of db/db and HFD-fed mice, we
tested whether the levels of these non-coding RNAs are affect- c d
150 150
miR-199a-3p
(% of ctrl)
(% of ctrl)
miR-184
of glucose and NEFA. We found that prolonged incubation of 100 100 * * *
* *
rat islets (Fig. 3) under glucolipotoxic conditions mimicked the 50 50
modifications in miR-132, miR-184, miR-199a-3p, miR-203 0 0
and miR-383 expression observed in animal models. In con- 11 G 20 G 11 G 20 G
trast, under these glucolipotoxic conditions the levels of miR-
210 and miR-199a-5p were not affected (Fig. 3). e f
150 150
(% of ctrl)
(% of ctrl)
miR-203
miR-210
100 * * 100
Particular differentially expressed miRNAs influence beta cell * *
functions and survival Modifications of miRNA expression in 50 50
islets could reflect the activation of adaptive processes 0 0
counterbalancing the increased insulin needs caused by obesity 11 G 20 G 11 G 20 G
100 *
to compensatory beta cell mass expansion [9], whereas *
impacts on beta cell function [6, 8]. To assess the possible role 0
11 G 20 G
of other differentially expressed miRNAs in these phenomena,
we mimicked the changes observed in the animal models by Fig. 3 Effect of chronically elevated glucose and palmitate on the level
of islet miRNAs differentially expressed in type 2 diabetes animal
transfecting dissociated rat islet cells and MIN6B1 cells with models. Isolated rat islets were incubated at 11 or 20 mmol/l glucose
oligonucleotide duplexes corresponding to the mature miRNA with 0.5% BSA in the absence (black bars) or presence of 0.5 mmol/l
sequences or with anti-miRNA molecules that specifically inhibit palmitate for 48 h (grey bars) or 72 h (white bars). miRNA expression
miRNAs (ESM Fig. 1). The transfected cells were then analysed levels were measured by qRT-PCR, normalised by miR-7 and expressed
as percentage of control (11 mmol/l glucose with 0.5% BSA) (a–g).
for their functional properties. *
Significantly different from control condition, p≤0.05 by ANOVA anal-
We first assessed whether the miRNAs differentially ysis, Dunnett’s post-hoc test. 11 G, 11 mmol/l glucose; 20 G, 20 mmol/l
expressed in type 2 diabetes models are involved in the glucose; Ctrl, control
2208 Diabetologia (2013) 56:2203–2212
(ng insulin/well)
(ng insulin/well)
(ng insulin/well)
Insulin content
content and insulin secretion.
Insulin content
Insulin content
800 *
600 600
Dispersed rat islet cells were
600
transfected with oligonucleotides 400 400
400
leading to overexpression (a, d)
200 200 200
or downregulation (b, c, e, f) of
the indicated miRNAs. Insulin 0 0 0
content (a–c) and insulin
iR 3
m Ctrl
-1 32
5p
An iR trl
iR 4
10
An iR r l
83
m t
ti- -20
ti- -18
ti- t i - c
ti- t i - c
iR R-1
a-
-2
-3
secretion (d–f) in response to 2
99
An A n
An A n
i
m
m
(black bars) or 20 (white bars)
m
mmol/l glucose were measured
48 h post-transfection. Insulin
release is expressed as d e f
8 8 8
percentage of insulin content. *
Insulin release
Insulin release
Insulin release
*
Significantly different from 6 6 6
(% of IC)
(% of IC)
(% of IC)
control condition (control or
4 4 4
anti-control, as shown, incubated
at the same glucose 2 2 2
concentration), p≤0.05 by 0 0 0
ANOVA analysis, Dunnett’s
trl
32
m trl
83
5p
m trl
84
10
03
c
C
c
post-hoc test. Ctrl, control; IC,
-1
a-
-3
-2
An iR-2
An iR-1
An nti-
An nti-
iR
99
iR
iR
A
A
m
m
m
insulin content
-1
ti-
ti-
t i-
ti-
iR
m
(% of ins.-positive cells)
8 3 *
Ki67-positive cells
Ki67-positive cells
to our previous work with miR-338-3p [9], these findings *
6
suggest that modification of the levels of miR-132 and miR- 2
184 contributes to compensatory beta cell mass expansion 4
elicited in response to insulin resistance. 2
1
As an increase in beta cell apoptosis and a reduction in beta
cell mass are thought to play a role in the development of type 2 0 0
m trl
32
iR l
84
r
PR
t i - i-ct
C
-1
-1
A n A nt
iR
m
on beta cell survival. As previously observed for miR-34a and
miR-146a [6], upregulation of miR-199a-3p or reduction of
miR-203, miR-210 and miR-383 expression increased the c d
number of apoptotic MIN6B1 cells (ESM Fig. 4) as well as
(% of ins.-positive cells)
(% of ins.-positive cells)
3 3
Ki67-positive cells
Ki67-positive cells
dispersed rat islet cells (Fig. 6a, c, e). Similar results were
obtained using dissociated human islet cells (Fig. 6b, d, f). In 2 2
contrast, overexpression of miR-132 or silencing of miR-184
did not induce beta cell death, but rather protected dispersed rat 1 1
(Fig. 7a–d) and human (Fig. 7e–h) islet cells from apoptosis
when the cells were chronically exposed to elevated concen- 0 0
trations of NEFA or to proinflammatory cytokines. Analogous
m trl
03
iR l
83
r
ti- i-ct
t i - i-c
-2
-3
An Ant
An nt
iR
Impact of particular miRNA changes on candidate target Fig. 5 Effect of specific modifications in miRNA expression on beta
cell proliferation. Dispersed rat islet cells were transfected with oligo-
gene expression As described above, db/db mouse islets are
nucleotides leading to overexperession (a) or downregulation (b–d) of
characterised by a specific rise in the levels of miR-21, miR- the indicated miRNAs. Beta cell proliferation was assessed 72 h later by
34a, miR-146a, miR-199a-3p and -5p and a downregulation of staining the cells with anti-Ki67 and anti-insulin antibodies. PRL
miR-203, miR-210 and miR-383 that possibly result in beta (500 ng/ml for 48 h, grey bars) was used as positive control. The results
correspond to the mean ± SD of three to six independent experiments.
cell dysfunction and death. We previously found that miR-34a *
Significantly different from control condition (control or anti-control,
affects beta cell survival by directly targeting the anti-apoptotic as shown), p≤0.05 by ANOVA analysis, Dunnett’s post-hoc test. Ctrl,
protein B cell CLL/lymphoma 2 (BCL2) [6]. Combining control; ins., insulin
Diabetologia (2013) 56:2203–2212 2209
a b a b
Pycnotic nuclei
3 5
* *
Pycnotic nuclei
Pycnotic nuclei
*
(% of cells)
15 2.0
4
1.5 2
10 3
1.0
5 2
0.5 1
0 0.0 1
0 0
trl
32
5p
3p
ix
trl
5p
3p
ix
.m
.m
C
C
-1
a-
a-
a-
a-
trl
trl
4
iR
99
99
99
99
yt
yt
8
C
C
C
C
m
-1
-1
-1
-1
-1
-1
ti-
iR
iR
iR
iR
iR
iR
An
m
m
ti-
An
c d
10 (fold change vs ctrl) 2.5 c d
Pycnotic nuclei
Pycnotic nuclei
(% of cells)
Pycnotic nuclei
Pycnotic nuclei
6 1.5
4 1.0 2 2
2 0.5
0 0.0 1 1
t rl
84
10
83
rl
3
ct
i-c
-1
-2
-3
-1
-2
-3
ti-
0 0
t
iR
iR
iR
iR
iR
iR
An
An
i-m
i-m
m
ti-
ti-
ti-
ti-
trl
32
rl
84
t
t
An
An
An
An
An
An
ct
C
-1
-1
ti-
iR
iR
An
m
m
ti-
An
e f
(fold change vs ctrl)
10 * 2.5 *
Pycnotic nuclei
Pycnotic nuclei
* e f
(% of cells)
Pycnotic nuclei
4 1.0
2 0.5 2 2
0 0.0
rl
03
ix
rl
03
ix
1 1
ct
ct
.m
.m
-2
-2
ti-
ti-
iR
iR
yt
yt
An
An
C
C
m
m
ti-
ti-
0 0
An
An
trl
32
rl
84
ct
C
-1
ti-
iR
iR
An
survival. Dissociated rat (a, c, e) and human (b, d, f) islet cells were
m
m
ti-
An
transfected with the indicated miRNA mimics (a, b) or anti-miRNAs
(c–f). Cell death was assessed by scoring the cells displaying pycnotic
nuclei on Hoechst staining. Incubation for 24 h with a mix of g h
proinflammatory cytokines was used as a positive control for apoptosis
(grey bars). The results correspond to the mean ± SD of three to four
(fold change vs ctrl)
3 * 3
independent experiments. *Significantly different from control condi- *
Pycnotic nuclei
Pycnotic nuclei
32
rl
84
ct
C
-1
-1
ti-
iR
An
m
m
ti-
sion of mTOR and of the transcription factor cMET [21], two Fig. 7 Overexpression of miR-132 and inhibition of miR-184 protects
proteins known to play important roles in the control of beta beta cells against palmitate- or cytokine-induced apoptosis. Dissociated
cell mass and survival [22, 23]. We found that upregulation of rat (a–d) or human (e–h) islet cells were transfected with miR-132
mimic or with anti-miR-184. The cells were then incubated for 48 h
miR-199a-3p results in decreased expression of mTOR and with (white bars) or without (black bars) 0.5 mmol/l palmitate coupled
cMET also in MIN6B1 cells (ESM Fig. 6), possibly explaining to 0.5% BSA (a, b, e, f), or for 24 h with (grey bars) or without (black
the negative impact of this miRNA on beta cell survival. bars) a mix of proinflammatory cytokines (c, d, g, h). Apoptosis was
Increased expression of miR-132 displays beneficial ef- assessed at 48 h post-transfection by Hoechst staining of pycnotic
nuclei. The results correspond to the mean ± SD of three to four
fects on both beta cell mass and function. Computational independent experiments. *Significantly different from treated control
prediction algorithms (http://mirsystem.cgm.ntu.edu.tw/) in- condition (control or anti-control, as shown), p≤0.05 by ANOVA
dicate that granuphilin (also known as synaptotagmin-like 4 analysis, Dunnett’s post-hoc test. Ctrl, control
[SLP-4]), a granule-associated protein that negatively affects
insulin release [16], and GSK-3β, which negatively regulates
2210 Diabetologia (2013) 56:2203–2212
beta cell function and mass [24, 25], are potential miR-132 of type 2 diabetes [7]. Consistent with results obtained in
targets. Translational repression of these two genes could ob/ob mice [5], our microarray data did not reveal significant
explain, at least in part, the phenotypic traits of beta cells changes in the level of many miRNAs that play important
overexpressing miR-132. However, western blot analysis did roles in the control of beta cell functions, including miR-9,
not reveal any significant impact of miR-132 on the level of miR-24, miR-124a and miR-148 [18, 31–33]. Moreover, we
these proteins in MIN6B1 cells (ESM Fig. 6). MiRNAs often did not detect differences in the levels of miR-375, an islet-
have small impacts on the expression of single direct targets enriched miRNA that regulates insulin secretion and beta
[26]. However, cumulative effects can have major indirect cell proliferation and that is slightly upregulated (about 30%)
influences on gene expression and cellular activities. Thus, in ob/ob mice [34]. Thus, although appropriate expression of
instead of searching for direct targets, we measured the cellu- these miRNAs is required for ensuring optimal beta cell
lar level of a group of transcription factors known from the function, the development of type 2 diabetes appears not to
literature to improve survival and function of beta cells be associated with major changes in the level of these non-
[27–29]. We found that upregulation of miR-132 in rat islet coding RNAs. However, individuals expressing inappropri-
cells did not affect the mRNA levels of Foxm1 and Pdx1 but ate levels of these miRNAs may display defective beta cell
increased the level of Mafa (Fig. 8b). Downregulation of miR- functions [30] and may be more susceptible to type 2 diabe-
184 that induces overlapping phenotypic changes did not alter tes manifestation. Indeed, ob/ob mice lacking miR-375 de-
the expression level of these genes (not shown). velop diabetes [34].
The analysis of the functional impact of individual changes in
miRNA expression in isolated islet cells revealed that some of
Discussion them have beneficial effects on the activity of insulin-secreting
cells whereas others result in beta cell death. Upregulation of
We have identified two groups of miRNAs displaying dif- miR-132 and downregulation of miR-184 and miR-338-3p are
ferential expression in pancreatic islets isolated from two already observed in 6 week-old prediabetic obese db/db mice.
animal models characterised by obesity, insulin resistance These adaptive changes in miRNA expression that have a
and beta cell dysfunction: db/db mice and HFD-fed mice. positive impact on beta cell functions are conserved or even
The changes in expression of miR-21, miR-34a, miR-132, more pronounced in HFD-fed and 14–20-week-old diabetic
miR-146a, miR-184, miR-210 and miR-383 detected in this db/db mice. Indeed, when the level of these particular
study are consistent with those described by Zhao et al in the miRNAs was modulated in vitro, both tumoral and normal beta
islets of leptin-deficient ob/ob mice [5] and are in agreement cells displayed enhanced proliferation and resistance to pro-
with previous findings from our laboratory [6, 8]. Elevated apoptotic stimuli (present study and Jacovetti et al [9]).
miR-21 levels were also detected in islets of glucose- Moreover, a rise in the level of miR-132 improved the secretory
intolerant human donors [30]. Moreover, our microarray data response of the cells to glucose. These observations suggest that
confirm the upregulation of miR-802 in the islets of db/db adaptive expression of miR-132, miR-184 and miR-338-3p may
mice recently observed by Kornfeld et al [19]. Increased contribute to beta cell compensation processes.
expression of miR-132, miR-199a-5p and miR-199a-3p The increased miR-132 content and the decreased miR-184
have also been reported in the islets of GK rats, a lean model expression observed in db/db and HFD-fed mice were mim-
icked by incubation of dissociated rat islet cells in the presence
a b of chronically elevated concentrations of palmitate and glu-
* cose. This suggests that these miRNAs may be induced in
(fold change vs ctrl)
30 8
*
Gene expression
6
tions typically encountered in prediabetic and diabetic states.
miR-132
20
4 In neurons, the expression of miR-132 is triggered following
10
2 activation of the cAMP-dependent pathway and of the tran-
0 0
scription factor cAMP response element-binding protein
Mafa Foxm1 Pdx1 (CREB) [35–40]. Incubation of rat insulinoma INS-1 832/13
trl
32
C
-1
function and survival [28]. The expression of this transcription 34a, miR-146a, miR-199a-5p, miR-199a-3p, miR-203, miR-
factor is decreased by palmitate [42] and is strongly reduced in 210 and miR-383. A better understanding of the precise role
the islets of diabetic db/db mice [10, 43]. Moreover, nuclear of particular miRNAs involved in the natural history of
MAFA was recently reported to be diminished in the islets of the beta cell in diabetes may be harnessed to design novel
individuals affected by type 2 diabetes [44]. Our data suggest therapeutic strategies for diabetes prevention and treatment.
that the induction of miR-132 helps preserve the level of
MAFA during obesity-associated beta cell compensation. Acknowledgements We warmly thank Bryan Gonzalez (University
of Lausanne, Switzerland) for expert technical help.
Over the long term, the adaptive changes elicited by miR-132,
miR-184 and miR-338-3p may become insufficient to counter-
Funding The authors are supported by Grants from the Swiss National
balance insulin resistance; alterations in the levels of additional Science Foundation (31003A-127254) (to RR), the European Foundation
miRNAs with deleterious impacts on beta cells also add to the for the Study of Diabetes (to RR), the Canadian Institute of Health
effect. Indeed, the islets of HFD-fed and of diabetic db/db mice Research (to MP), the National Health and Medical Research Council of
Australia (to DRL) and Société Francophone du Diabète (SFD)-Servier (to
displayed changes in the levels of several other miRNAs, includ- CJ). MP is a recipient of a Canada research chair in diabetes and metab-
ing miR-21, miR-34a, miR-146a, miR-199a-5p, miR-199a-3p, olism. CG is supported by a fellowship from the Fonds de la Recherche en
miR-203, miR-210 and miR-383; variation in expression of these Santé du Québec (FRSQ), the SFD and the Canadian Diabetes Association.
miRNAs in vitro causes beta cell dysfunction and death (Lovis
et al [6], Roggli et al [8] and present study). We previously Duality of interest The authors declare that there is no duality of
interest associated with this manuscript.
showed that induction of miR-34a and miR-146a triggers beta
cell apoptosis and that miR-21 and miR-34a have a deleteri- Contribution statement VN and CG generated the data, wrote the
ous impact on insulin secretion [6, 8]. Experiments manuscript and approved its final version. CJ, VM and M-LP contributed
carried out in this study revealed an increase in apopto- to the acquisition of data, critically reviewed the manuscript and approved
sis after overexpression of miR-199a-3p or downregulation of its final version. DRL contributed to data acquisition and interpretation,
reviewed the manuscript and approved its final version. MP contributed
miR-203, miR-210 and miR-383 in dissociated rat and human to the interpretation of the data, critically reviewed the manuscript and
islet cells and in MIN6B1 cells. These phenotypic changes are approved its final version. RR conceived the experiments, interpreted the
not unique to beta cells as modifications in the level of some of data, reviewed the manuscript and approved its final version.
these miRNAs promote apoptosis in other cell systems [21,
45–47]. Overexpression of miR-199a-3p resulted in a reduction
of the levels of mTOR and cMET, two well-characterised References
targets of this miRNA [21, 48]. Disruption of the signalling
pathways involving these two proteins is detrimental for beta
1. Prentki M, Nolan CJ (2006) Islet beta cell failure in type 2 diabetes.
cells [23, 49]. Moreover, mTOR is an important regulator of J Clin Invest 116:1802–1812
autophagy, a process thought to contribute to type 2 diabetes 2. Schofield CJ, Sutherland C (2012) Disordered insulin secretion in the
onset [50]. Thus, the toxic effects of miR-199a-3p may be the development of insulin resistance and type 2 diabetes. Diabet Med
consequence of diminished expression of mTOR and cMET. 29:972–979
3. Sayed D, Abdellatif M (2011) MicroRNAs in development and
In conclusion, the present study is the first globally address- disease. Physiol Rev 91:827–887
ing the role of miRNAs in the aetiology of type 2 diabetes by 4. Guay C, Jacovetti C, Nesca V, Motterle A, Tugay K, Regazzi R
systematically investigating the impact on primary beta cell (2012) Emerging roles of non-coding RNAs in pancreatic beta-cell
function of miRNA changes observed in two animal models function and dysfunction. Diabetes Obes Metab 14(Suppl 3):12–21
5. Zhao E, Keller MP, Rabaglia ME et al (2009) Obesity and genetics
of obesity-associated diabetes. Our data demonstrate that obesity regulate microRNAs in islets, liver, and adipose of diabetic mice.
and insulin resistance are associated with modifications in two Mamm Genome 20:476–485
distinct groups of islet miRNAs that have opposing phenotypic 6. Lovis P, Roggli E, Laybutt DR et al (2008) Alterations in
effects on beta cells. Expression changes in miRNAs promoting microRNA expression contribute to fatty acid-induced pancreatic
beta-cell dysfunction. Diabetes 57:2728–2736
beta cell mass expansion and boosting glucose-induced insulin 7. Esguerra JL, Bolmeson C, Cilio CM, Eliasson L (2011) Differential
secretion already occur in normoglycaemic animals and proba- glucose-regulation of MicroRNAs in pancreatic islets of non-obese
bly belong to adaptive processes allowing beta cells to compen- type 2 diabetes model Goto–Kakizaki rat. PLoS One 6:e18613
sate for insulin resistance. If these mechanisms fail to compen- 8. Roggli E, Britan A, Gattesco S et al (2010) Involvement of
microRNAs in the cytotoxic effects exerted by proinflammatory
sate for the diminished insulin sensitivity, additional modifica- cytokines on pancreatic beta-cells. Diabetes 59:978–986
tions in miRNA expression may accumulate, causing beta cell 9. Jacovetti C, Abderrahmani A, Parnaud G et al (2012) MicroRNAs
failure and manifestation of type 2 diabetes. We propose that contribute to compensatory beta cell expansion during pregnancy
beta cell activities are tuned by a balance between the levels of and obesity. J Clin Invest 122:3541–3551
10. Chan JY, Luzuriaga J, Bensellam M, Biden TJ, Laybutt DR (2013)
particular miRNAs associated with enhanced function and Failure of the adaptive unfolded protein response in islets of obese
mass, such as miR-132, miR-184 and miR-338-3p, and mice is linked with abnormalities in beta-cell gene expression and
others having negative impacts, including miR-21, miR- progression to diabetes. Diabetes 62:1557–1568
2212 Diabetologia (2013) 56:2203–2212
11. Peyot ML, Pepin E, Lamontagne J et al (2010) Beta-cell failure in 31. Lovis P, Gattesco S, Regazzi R (2008) Regulation of the expression
diet-induced obese mice stratified according to body weight gain: of components of the exocytotic machinery of insulin-secreting
secretory dysfunction and altered islet lipid metabolism without cells by microRNAs. Biol Chem 389(3):305–312
steatosis or reduced beta-cell mass. Diabetes 59:2178–2187 32. Plaisance V, Abderrahmani A, Perret-Menoud V, Jacquemin P,
12. Gotoh M, Maki T, Satomi S et al (1987) Reproducible high yield of Lemaigre F, Regazzi R (2006) MicroRNA-9 controls the expres-
rat islets by stationary in vitro digestion following pancreatic ductal sion of Granuphilin/Slp4 and the secretory response of insulin-
or portal venous collagenase injection. Transplantation 43:725–730 producing cells. J Biol Chem 281:26932–26942
13. Lilla V, Webb G, Rickenbach K et al (2003) Differential gene 33. Baroukh N, Ravier MA, Loder MK et al (2007) MicroRNA-124a
expression in well-regulated and dysregulated pancreatic beta-cell regulates Foxa2 expression and intracellular signaling in pancreatic
(MIN6) sublines. Endocrinology 144:1368–1379 beta-cell lines. J Biol Chem 282:19575–19588
14. Roggli E, Gattesco S, Caille D et al (2012) Changes in microRNA 34. Poy MN, Hausser J, Trajkovski M et al (2009) miR-375 maintains
expression contribute to pancreatic beta-cell dysfunction in predi- normal pancreatic alpha- and beta-cell mass. Proc Natl Acad Sci U S
abetic NOD mice. Diabetes 61:1742–1751 A 106:5813–5818
15. Roche E, Buteau J, Aniento I, Reig JA, Soria B, Prentki M (1999) 35. Nudelman AS, DiRocco DP, Lambert TJ et al (2010) Neuronal
Palmitate and oleate induce the immediate-early response genes activity rapidly induces transcription of the CREB-regulated
c-fos and nur-77 in the pancreatic beta-cell line INS-1. Diabetes microRNA-132, in vivo. Hippocampus 20:492–498
48:2007–2014 36. Remenyi J, Hunter CJ, Cole C et al (2010) Regulation of the miR-
16. Coppola T, Frantz C, Perret-Menoud V, Gattesco S, Hirling H, Regazzi 212/132 locus by MSK1 and CREB in response to neurotrophins.
R (2002) Pancreatic beta-cell protein granuphilin binds Rab3 and Biochem J 428:281–291
Munc-18 and controls exocytosis. Mol Biol Cell 13:1906–1915 37. Pathania M, Torres-Reveron J, Yan L et al (2012) miR-132 en-
17. Kobayashi K, Forte TM, Taniguchi S, Ishida BY, Oka K, Chan L (2000) hances dendritic morphogenesis, spine density, synaptic integra-
The db/db mouse, a model for diabetic dyslipidemia: molecular charac- tion, and survival of newborn olfactory bulb neurons. PLoS One
terization and effects of Western diet feeding. Metabolism 49:22–31 7:e38174
18. Melkman-Zehavi T, Oren R, Kredo-Russo S et al (2011) miRNAs 38. Scott HL, Tamagnini F, Narduzzo KE et al (2012) MicroRNA-132
control insulin content in pancreatic beta-cells via downregulation regulates recognition memory and synaptic plasticity in the perirhinal
of transcriptional repressors. EMBO J 30:835–845 cortex. Eur J Neurosci 36:2941–2948
19. Kornfeld JW, Baitzel C, Konner AC et al (2013) Obesity-induced 39. Lin LF, Chiu SP, Wu MJ, Chen PY, Yen JH (2012) Luteolin induces
overexpression of miR-802 impairs glucose metabolism through microRNA-132 expression and modulates neurite outgrowth in
silencing of Hnf1b. Nature 494:111–115 PC12 cells. PLoS One 7:e43304
20. Lupi R, Del Prato S (2008) Beta-cell apoptosis in type 2 diabetes: 40. Numakawa T, Yamamoto N, Chiba S et al (2011) Growth factors
quantitative and functional consequences. Diabetes Metab 34(Suppl stimulate expression of neuronal and glial miR-132. Neurosci Lett
2):S56–S64 505:242–247
21. Fornari F, Milazzo M, Chieco P et al (2010) MiR-199a-3p regulates 41. Keller DM, Clark EA, Goodman RH (2012) Regulation of
mTOR and c-Met to influence the doxorubicin sensitivity of human microRNA-375 by cAMP in pancreatic beta-cells. Mol Endocrinol
hepatocarcinoma cells. Cancer Res 70:5184–5193 26:989–999
22. Xie J, Herbert TP (2012) The role of mammalian target of rapamycin 42. Hagman DK, Hays LB, Parazzoli SD, Poitout V (2005) Palmitate
(mTOR) in the regulation of pancreatic beta-cell mass: implications inhibits insulin gene expression by altering PDX-1 nuclear locali-
in the development of type-2 diabetes. Cell Mol Life Sci 69:1289– zation and reducing MafA expression in isolated rat islets of
1304 Langerhans. J Biol Chem 280:32413–32418
23. Mellado-Gil J, Rosa TC, Demirci C et al (2011) Disruption of 43. Matsuoka TA, Kaneto H, Miyatsuka T et al (2010) Regulation of
hepatocyte growth factor/c-Met signaling enhances pancreatic beta- MafA expression in pancreatic beta-cells in db/db mice with dia-
cell death and accelerates the onset of diabetes. Diabetes 60:525–536 betes. Diabetes 59:1709–1720
24. Liu Y, Tanabe K, Baronnier D et al (2010) Conditional ablation of 44. Butler AE, Robertson RP, Hernandez R, Matveyenko AV, Gurlo T,
Gsk-3beta in islet beta cells results in expanded mass and resistance Butler PC (2012) Beta cell nuclear musculoaponeurotic fibrosarco-
to fat feeding-induced diabetes in mice. Diabetologia 53:2600– ma oncogene family A (MafA) is deficient in type 2 diabetes.
2610 Diabetologia 55:2985–2988
25. Liu Z, Tanabe K, Bernal-Mizrachi E, Permutt MA (2008) Mice 45. Ru P, Steele R, Hsueh EC, Ray RB (2011) Anti-miR-203
with beta cell overexpression of glycogen synthase kinase-3beta upregulates SOCS3 expression in breast cancer cells and enhances
have reduced beta cell mass and proliferation. Diabetologia cisplatin chemosensitivity. Genes Cancer 2:720–727
51:623–631 46. Liu Y, Han Y, Zhang H et al (2012) Synthetic miRNA-mowers
26. Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian targeting miR-183-96-182 cluster or miR-210 inhibit growth and
microRNAs predominantly act to decrease target mRNA levels. migration and induce apoptosis in bladder cancer cells. PLoS One
Nature 466:835–840 7:e52280
27. Davis DB, Lavine JA, Suhonen JI et al (2010) FoxM1 is up-regulated 47. Li KK, Pang JC, Lau KM et al (2012) MiR-383 is downregulated in
by obesity and stimulates beta-cell proliferation. Mol Endocrinol medulloblastoma and targets peroxiredoxin 3 (PRDX3). Brain Pathol
24:1822–1834 23(4):413–425
28. Hang Y, Stein R (2011) MafA and MafB activity in pancreatic beta 48. Kim S, Lee UJ, Kim MN et al (2008) MicroRNA miR-199a*
cells. Trends Endocrinol Metab 22:364–373 regulates the MET proto-oncogene and the downstream extracellu-
29. Kulkarni RN, Jhala US, Winnay JN, Krajewski S, Montminy M, Kahn lar signal-regulated kinase 2 (ERK2). J Biol Chem 283:18158–
CR (2004) PDX-1 haploinsufficiency limits the compensatory islet 18166
hyperplasia that occurs in response to insulin resistance. J Clin Invest 49. Mori H, Inoki K, Opland D et al (2009) Critical roles for the TSC-
114:828–836 mTOR pathway in beta-cell function. Am J Physiol Endocrinol Metab
30. Bolmeson C, Esguerra JL, Salehi A, Speidel D, Eliasson L, Cilio 297:E1013–E1022
CM (2011) Differences in islet-enriched miRNAs in healthy and 50. Las G, Shirihai OS (2010) The role of autophagy in beta-cell
glucose intolerant human subjects. Biochem Biophys Res Commun lipotoxicity and type 2 diabetes. Diabetes Obes Metab 12(Suppl
404:16–22 2):15–19