Sirt1 and mir-9 expression is regulated during
glucose-stimulated insulin secretion in pancreatic b-islets
Deepti Ramachandran*, Upasana Roy*, Swati Garg, Sanchari Ghosh, Sulabha Pathak and
Ullas Kolthur-Seetharam
Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India
Introduction
MicroRNAs (mirs) regulate protein expression due to
their ability to target the 3¢UTRs of mRNAs [1].
Although, in the recent past, there have been numer-
ous studies reporting mir targets and their physiologi-
cal implications, we still do not understand fully the
mechanisms that regulate their expression. This is cru-
cial as they are now known to play diverse roles and
are being considered as potential therapeutic targets.
Mirs have also been found to be important modulators
of changes in metabolic response, including endocrine
functions [2]. Several mirs involved in the control of
pancreatic development and insulin secretion have
been discovered recently [3,4]. Mir-375 was one of the
first mirs to be identified as a key factor affecting insu-
lin secretion by inhibiting glucose-stimulated insulin
secretion (GSIS) [4]. Another mir that has been impli-
cated in the control of insulin secretion is mir-9 [5].
Plaisance et al. [5] indicated a possible role for mir-9
in insulin secretion by showing that mir-9 targets
Onecut-2 (OC-2) mRNA and down-regulates its
expression in insulin-secreting cells. This decrease in
OC-2 consequently leads to an increase in the levels of
its target gene, granuphilin. Granuphilin has been well
characterized as a key player in insulin secretion and is
known to negatively regulate insulin exocytosis [6].

and regulates Sirt1 expression in insulin-secreting cells. This targeting is
relevant in pancreatic b-islets, where we show a reduction in Sirt1 protein
levels when mir-9 expression is high during glucose-dependent insulin secre-
tion. This functional interplay between insulin secretion, mir-9 and Sirt1
expression could be relevant in diabetes. It also highlights the crosstalk
between an NAD-dependent protein deacetylase and microRNA in pancre-
atic b-cells.
Abbreviations
ARBP, acidic ribosomal binding protein; GSIS, glucose-stimulated insulin secretion; LNA, locked nucleic acid; mir, microRNA; OC-2,
Onecut-2; pS, pSuper vector; pS9, pSuper mir-9 vector.
FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1167
relevant, particularly under conditions in which insulin
secretion is regulated in vivo.
The Sir2 family of proteins (sirtuins) are NAD-
dependent protein deacetylases that have been impli-
cated in several physiological processes [7]. Mamma-
lian Sirt1, one of the most well-studied members of the
family, is a nuclear protein. It is known to deacetylate
histones, transcription factors and co-regulators in an
NAD-dependent manner [8–10]. Interestingly, levels of
Sirt1 protein are known to fluctuate in tissues such as
the liver, white adipose tissue, brown adipose tissue
and muscle under different metabolic conditions (such
as calorie restriction and starvation) [11,12]. In addi-
tion to changes in its activity, as a result of fluctua-
tions in NAD levels [13–15], the modulation of Sirt1
protein levels is also important for its functions [11].
In the pancreatic islets, insulin secretion is linked to
glucose availability and is controlled by several factors,
including changes in ADP ⁄ ATP, mitochondrial mem-

with mir-9 levels. In conclusion, our results indicate
that, in insulin-secreting cells, Sirt1 protein levels are
altered in response to changes in glucose availability,
and this is brought about by mir-9.
Results
Glucose-dependent changes in mir-9 expression
affect its levels in pancreatic b-islets
Mir-9 has been implicated in insulin secretion and has
been proposed to be regulated by glucose levels [18].
We therefore wished to determine whether mir-9
expression was regulated in vivo in pancreatic b-islets.
To this end, we isolated pancreatic b-islets from mice
that had been starved for 24 h and administered glu-
cose, intraperitoneally, to stimulate insulin secretion
(GSIS). Following glucose injections, sera and b-islets
were isolated at different time intervals. We assayed
for serum insulin levels in these mice (Fig. 1A). Mir-9
levels during GSIS were quantified from total RNA
isolated from b-islets. It was very interesting to see
that mir-9 levels showed no change at 30 min, but
increased significantly by 60 min post-glucose injection
(Fig. 1B). Importantly, this increase in mir-9 coincided
with the time point at which insulin levels started to
decline (Fig. 1A, B). Further, we also observed that
mir-9 levels remained high until 4 h post-glucose injec-
tion, when insulin secretion is expected to be low or
decreasing (Fig. 1A). To our knowledge, this is the
first report to clearly map the kinetics of mir-9 induc-
tion in vivo in b-islets during GSIS.
Given the implications of mir-9 function in the pan-

these loci, including the identification of transcription
factors.
Sequence analyses of mir-9 upstream regions indi-
cated that there were CpG islands at these loci in
humans and mice (Fig. S1). Indeed, mir-9 expression is
known to be altered by hypermethylation in cancers
[19,20]. Our results showed that pri- ⁄ pre-mir-9-3
expression was very low and barely detectable
(Fig. 1C). The analysis of CpG methylation at the mir-
9-3 locus using methylation-sensitive enzymes, followed
by PCR, showed that a low level of pri- ⁄ pre-mir-9-3 in
the islets was probably not caused by hypermethyla-
tion of this locus (Doc. S2 and Fig. S2).
Mir-9 negatively regulates Sirt1 protein
Mir-9 has been shown to target OC-2 in INS-1E cells
and to regulate the exocytosis of over-expressed human
growth hormone in these cells [5]. To further elucidate
the role of mir-9 in insulin secretion, we looked for
possible mir-9 targets using the online prediction tools
Pictar and Targetscan. We found Sirt1 mRNA to be
one of the candidate mRNAs for mir-9 targeting,
among several others, based on seed complementarity
and evolutionary conservation (Fig. 2A, B).
Taking into consideration the importance of both
Sirt1 and mir-9 in insulin secretion, we wished to
determine whether this targeting was true. In order to
assess this, Sirt1 3¢UTR from mouse cDNA was
cloned into pmir-Report plasmid that encodes a firefly
luciferase (Fig. S3). The pre-mir-9 sequence cloned
into pSuper vector (pS9) was used for the over-expres-

transcripts pri ⁄ pre-mir-9-1, pri ⁄ pre-mir-9-2 and pri ⁄ pre-mir-9-3. ARBP
was used as the normalization control. (A–C) One-way ANOVA was
used for statistical analysis (n =3; *P < 0.05, **P < 0.01). In (B),
* and # indicate significance with respect to the 0- and 30-min time
points, respectively. In (C), # and * indicate significance with respect
to the 0-min time point for 9-1 and 9-2, respectively.
D. Ramachandran et al. Mir-9-dependent regulation of Sirt1 in b-cells
FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1169
Sirt1 mRNA and post-transcriptionally regulate its
expression.
Sirt1 is regulated in vivo in response to GSIS
Sirt1 is known to affect GSIS in b-islets [17]. However,
it is unclear whether its expression is regulated in vivo.
We found that mir-9 levels were regulated in response
to GSIS in vivo and that it targeted Sirt1 and down-
regulated its expression in cells in vitro. Hence, we
wished to determine whether Sirt1 levels were regu-
lated in vivo in pancreatic b-islets. If mir-9 targets Sirt1
in the b-islets, we would expect to see a decrease in
Sirt1 levels under conditions in which mir-9 levels are
high. Therefore, Sirt1 protein was assayed and, inter-
estingly, we found that Sirt1 levels were modulated in
pancreatic b-islets during GSIS (Fig. 3A). We observed
that there was a significant decrease in Sirt1 expression
240 min post-glucose injection. Importantly, the reduc-
tion in Sirt1 protein correlated well with increased
mir-9 levels in the b-islets when serum insulin secretion
Fig. 2. Mir-9 targets the 3¢UTR of Sirt1 and down-regulates its
expression. (A) Alignment of mature mir-9 with the target sequence
on the 3¢UTR of mouse Sirt1 (using Target Scan). (B) Conservation

amount of Sirt1 protein is regulated (post-transcrip-
tionally) during GSIS in insulin-secreting b-islets
in vivo.
Sirt1 is down-regulated in insulin-secreting cells
in a mir-9-dependent manner
Although we have described the targeting in NIH3T3
cells, we wished to ascertain whether the post-tran-
scriptional regulation of Sirt1 expression in b-cells was
brought about by mir-9. We therefore used insulin-
secreting b-TC-6 cells to address this. b-TC-6 cells
were transfected with pre-mir-control ⁄ -9 and pS ⁄ pS9.
From Fig. 4A, B, it can be seen that Sirt1 protein is
reduced in cells transfected with pre-mir-9 and pS9,
respectively. Increased expression of mir-9 in pS9
transfected cells is shown in Fig. 4C. These findings
clearly demonstrate that mir-9 is indeed able to target
Sirt1 in b-TC-6 cells. In order to confirm this, b-TC-6
cells were transfected with control and anti-mir-9
locked nucleic acid (LNA). To mimic a declining insu-
lin secretion phase (Fig. 4D), the cells were subjected
to 8 h of glucose withdrawal, 16 h post-transfection.
The reduction in mir-9 after LNA transfection was
quantified by RT-qPCR (Fig. 4E). It was very interest-
ing to see that Sirt1 protein levels were significantly
higher in cells that had been transfected with anti-mir-
9 LNA (Fig. 4F). This result is consistent with reduced
Sirt1 expression in the pancreatic b-islets in vivo at
240 min post-glucose injection (when insulin secretion
is decreasing) (Fig. 3A, B). Importantly, this provides
mechanistic insight into the regulation of Sirt1 protein

withdrawal. Insulin levels in cell culture
supernatants were measured. (E, F) b-TC-6
cells were transfected with 100 n
M LNA
anti-mir-control and anti-mir-9 for 16 h and
subjected to glucose withdrawal for 8 h as
detailed in Experimental procedures. (C, E)
Mir-9 expression was quantified by
RT-qPCR normalized to U6 levels. (C–E)
Student’s t-test was used for statistical anal-
ysis (n =3;*P < 0.05, **P < 0.01). (A, B, F)
Western blot analysis for Sirt1 protein with
b-actin as the loading control.
D. Ramachandran et al. Mir-9-dependent regulation of Sirt1 in b-cells
FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS 1171
process. On the basis of our finding of the temporal
regulation of its expression and the earlier report on
exocytosis [5], we implicate mir-9 as a crucial factor in
the control of insulin secretion in response to glucose
stimulation in vivo.
Furthermore, we have also identified mir-9 to be a
key factor in the modulation of Sirt1 expression
in vivo. Recently, Saunders et al. [22] have shown that
mir-9 targets Sirt1 in mouse embryonic stem cells.
However, our results show that mir-9 targeting of Sirt1
is physiologically significant in insulin-secreting cells
(Figs 3 and 4).
Earlier studies have linked Sirt1 to GSIS. In Sirt1
transgenic (BESTO) mice, Sirt1-mediated control of
insulin secretion was found to be a result of the differ-

Given the known functions of Sirt1 in regulating
insulin secretion, our study adds a new facet by show-
ing, that Sirt1 protein levels are altered in insulin-secret-
ing cells during GSIS. To conclude, we have discovered
a functional interplay between glucose-dependent insu-
lin secretion, mir-9 levels and Sirt1 protein in b-cells. In
addition, we provide some evidence for the dynamic
(and differential) nature of mir-9 expression in pancre-
atic b-islets. Further insights into these mechanisms
may help in the understanding and tackling of diseases
such as diabetes.
Experimental procedures
Animal experiments
Adult Swiss male mice were maintained at the Tata Insti-
tute of Fundamental Research animal facility in accordance
with the institute’s animal ethics regulations. These mice
were used for GSIS. Briefly, the mice were starved over-
night and injected with glucose (3 gÆkg
)1
body weight)
intraperitoneally. Serum and tissue samples were collected
at 0, 15, 30, 60 and 240 min post-glucose injection. Three
animals per group were used and the experiment was
repeated at least twice.
Cell culture
NIH3T3 and HEK293T cells were grown in DMEM
(Sigma, St. Louis, MO, USA cat. no. D7777) supplemented
with 10% newborn calf serum (Gibco, USA cat. no. 16010-
159) and 10% fetal bovine serum (Gibco cat. no. 16000),
respectively. b-TC-6 cells were grown in DMEM supple-

Real-time PCR
qPCRs were performed in triplicate using SYBR green
(Qiagen, USA cat. no. 204056) according to the manufac-
turer’s instructions. qPCR for mir was performed as
described in ref. [26]. Briefly, short-mir-9 and MP-
fwd ⁄ MP-rev primers (Table S1) were used at concentra-
tions of 4 and 100 nm, respectively. U6 and ARBP were
used for the normalization of mir and mRNA expression,
respectively.
Pancreatic b-islet isolation
b-Islets were harvested by perfusing the pancreas as
described by Szot et al. [27] (Doc. S1).
Luciferase and b-galactosidase assays
HEK293T cells were transfected with pmir-Report-Sirt1
3¢UTR (wild-type or mutant), b-galactosidase vector and
pSuper or pSuper-mir-9 (pS ⁄ pS9) in 24-well plates. Cells
were harvested 24 h later and luciferase assay was carried
out using the Stratagene Luciferase Assay kit (Agilent
Technologies, Santa Clara, CA, USA cat. no. 219020)
according to the manufacturer’s instructions. Luciferase
activities were normalized to the b-galactosidase activity in
each case.
Western blots
Equal amounts of protein (estimated using the BCA kit,
Sigma-Aldrich, USA) were run on SDS ⁄ PAGE and trans-
ferred to poly(vinylidene difluoride) membranes (Roche,
Basel, Schweiz cat. no. 3 010 040 ⁄ ThermoFisher cat. no.
88518). Anti-Sirt1 (Millipore-Upsatate, MA, USA cat. no.
07-131) and anti-b-actin (Sigma cat. no. A1978) antibodies
were used for immunoblotting. Horseradish peroxidase-

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Doc. S4. Sirt1 3¢UTR wild-type and mutant luciferase
construct.
Table S1. Primer sequences.
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Mir-9-dependent regulation of Sirt1 in b-cells D. Ramachandran et al.
1174 FEBS Journal 278 (2011) 1167–1174 ª 2011 The Authors Journal compilation ª 2011 FEBS