Essential Role of the Small GTPase Ran in Postnatal
Pancreatic Islet Development
Fang Xia1,2, Takehiko Dohi1, Nina M. Martin1, Christopher M. Raskett1,2, Qin Liu3, Dario C. Altieri1*
1Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, Pennsylvania, United States of America, 2Department of Cancer Biology,
University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 3Center for Computational and Systems Biology, The Wistar Institute,
Philadelphia, Pennsylvania, United States of America
The small GTPase Ran orchestrates pleiotropic cellular responses of nucleo-cytoplasmic shuttling, mitosis and subcellular
trafficking, but whether deregulation of these pathways contributes to disease pathogenesis has remained elusive. Here, we
generated transgenic mice expressing wild type (WT) Ran, loss-of-function Ran T24N mutant or constitutively active Ran
G19V mutant in pancreatic islet b cells under the control of the rat insulin promoter. Embryonic pancreas and islet
development, including emergence of insulin+b cells, was indistinguishable in control or transgenic mice. However, by one
month after birth, transgenic mice expressing any of the three Ran variants exhibited overt diabetes, with hyperglycemia,
reduced insulin production, and nearly complete loss of islet number and islet mass, in vivo. Deregulated Ran signaling in
transgenic mice, adenoviral over-expression of WT or mutant Ran in isolated islets, or short hairpin RNA (shRNA) silencing of
endogenous Ran in model insulinoma INS-1 cells, all resulted in decreased expression of the pancreatic and duodenal
homeobox transcription factor, PDX-1, and reduced b cell proliferation, in vivo. These data demonstrate that a finely-tuned
balance of Ran GTPase signaling is essential for postnatal pancreatic islet development and glucose homeostasis, in vivo.
Citation: Xia F, Dohi T, Martin NM, Raskett CM, Liu Q, et al. (2011) Essential Role of the Small GTPase Ran in Postnatal Pancreatic Islet Development. PLoS
ONE 6(11): e27879. doi:10.1371/journal.pone.0027879
Editor: Giorgio Sesti, Universita Magna-Graecia di Catanzaro, Italy
Received June 22, 2011; Accepted October 27, 2011; Published November 17, 2011
Copyright: ? 2011 Xia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health grants CA78810, CA140043, CA118005 and HL54131. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
As a member of the Ras family of small GTPases , the Ran
protein orchestrates a multitude of cellular responses, including
nucleo-cytoplasmic shuttling , various aspects of mitosis , and
other cytoplasmic transport mechanisms in specialized cell types
. These functions require regulated subcellular compartmen-
talization of Ran , spatial control of its guanine nucleotide
cycling , and a finely-tuned balance involving plethora of Ran
regulatory molecules that monitor the guanine-nucleotide state
Ran signaling is highly evolutionary conserved, and is thought
to be essential for cellular homeostasis . However, except for
transformed cells, where Ran is frequently over-expressed ,
controls the distribution , and/or stability [9,10] of various
cancer genes, and correlates with unfavorable outcome [11,12], a
mechanistic link between deregulated Ran signaling and disease
pathogenesis has not been determined.
In this study, we generated transgenic mice that express wild
type (WT) Ran, the Ran loss-of-function mutant T24N, or the
Ran gain-of-function mutant G19V  in insulin-producing
pancreatic islet b cells. Unexpectedly, we found that deregulated
Ran signaling under these conditions dramatically impairs
postnatal, but not embryonic islet development, triggering
hypoinsulinemia, reduced b cell proliferation and overt diabetes,
Materials and Methods
Plasmid construction and generation of transgenic mice
All experiments involving animals were approved by an Institu-
tional Animal Care and Use Committee. A full-length human Ran
WT cDNA or cDNA encoding the Ran mutant T24N or G19V was
fused to an HA tag at the 59end, and cloned intoBamHI and SpeI sites
downstream of the Rat Insulin Promoter (RIP)  in pBluescript II
KS, containing SV40 polyadenylation sequences at the 39 end. Each
RIP-HA-Ran construct (WT, T24N or G19V) was confirmed by
Valencia, CA), and microinjected (5 ng/ml) into C57Bl/6 embryos
that were implanted into syngeneicrecipient pseudopregnant females,
as described . Littermates were screened by PCR of tail genomic
CTCGAGGGCTGCAGGAATTCGATA-39; reverse 59-GCCT-
TCACTTTCCTGTCCTTAATA-39) or RIP-Ran (forward 59-
TGGACTA TAAAGCTAGTGGGGATT-39; reverse, (59-GCT-
GTGTCCCATACATTGAACTTA-39) sequences. PCR reactions
(35 cycles) were carried out at 95uC for 1 min, 56uC for 1 min and
72uC for 1 min plus a 10 min extension at 72uC. Colonies from
independent transgene-positive founder mice or control littermates
were established, and bred with C57Bl/6 mice. Two independent
colonies per each condition were analyzed for blood glucose levels
with comparable results. Plasmid adenoviral (pAd) constructs
encoding GFP-Ran-WT, GFP-Ran-T24N or GFP-Ran-G19V were
generated using the pAdEasy system, as described previously .
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Cell culture, antibodies, and Western blotting
The rat insulinoma cell line INS-1 was the kind gift of R.S.
Sherwin (Yale University School of Medicine, New Haven, CT),
and was maintained in culture as described . The following
antibodies to Ran (Novus Biologicals, Cell Signaling, Santa Cruz),
HA (Santa Cruz, Roche Applied Science), insulin (Invitrogen),
glucagon (Dako), somatostatin (Dako), Ki-67 (Dako), PDX-1
(Upstate Biotechnology), or b-actin (Sigma-Aldrich), were used.
Restriction enzymes were purchased from New England BioLabs.
Pancreas or liver tissues isolated from non-transgenic (non-TG) or
Ran transgenic mice were washed in PBS (pH 7.2), suspended in 4
to 5 volumes of cold lysis buffer containing 50 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5%
Nonidet P-40, plus protease inhibitors (Roche Applied Science).
After 30-min incubation at 4uC, tissue extracts were cleared by
Figure 1. Characterization of Ran transgenic mice. A, Tail DNA
was extracted from transgenic mouse lines expressing Ran-WT, Ran
T24N or Ran G19V in pancreatic b cells under RIP control, and PCR
products were amplified using RIP-HA (top) or RIP-Ran (bottom) primers.
(+) or (2) refers to Ran-positive or –negative genotype, or positive or
negative control primers for the amplification reaction. M, molecular
weight markers. B, Pancreas tissues extracted from the various mouse
cohorts genotyped for the presence (+) or absence (2) of Ran
transgenes were analyzed by Western blotting. 3T3, extracts from
NIH3T3 cells transiently transfected with HA-Ran cDNA used as control.
a-HA, antibody to HA. C: Pancreas or liver tissues were isolated from
PCR-confirmed Ran transgenic mice, and analyzed by Western blotting.
b-actin was used as a loading control.
Figure 2. Transgenic expression of Ran impairs glucose metabolism. A and B, The indicated PCR-confirmed transgenic mice were analyzed
for blood glucose content at 2 mo of age under ad libitum feeding (A) or fasting (B) conditions. Non-TG, non-transgenic mice. Glucose concentrations
(mg/dl) in each group in A (number of mice in parentheses), and statistical analyses (unpaired t test) are as follows: Non-TG (n=24), 12562; Ran-WT
(n=26), 189.4617.8, p=0.013; Ran-G19V (n=24), 153.766.5, p=0.003; Ran-T24N (n=21), 172.4621.1, p=0.02. Statistical data re-analysis of the
groups in A using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p,0.0001; Ran-G19V, 0.017; Ran-T24N,
p=0.029. C, The indicated non-TG or Ran transgenic mice were analyzed for blood insulin concentrations. Insulin levels (ng/ml) in each group
(number of mice in parentheses), and statistical analyses (unpaired t test) are as follows: Non-TG (n=8), 1.2660.1; Ran-WT (n=11), 0.760.16,
p=0.019; Ran-G19V (n=5), 0.6160.03, p=0.0008; Ran-T24N (n=7), 0.4960.14, p=0.0009. Statistical data re-analysis using ANOVA and post-hoc
multiple tests with Bonferroni procedure was as follows: Ran-WT, p=0.023; Ran-G19V, p=0.031; Ran-T24N, p=0.004. One outlier mouse in the Ran-
G19V group with aberrantly high insulin level (2.35 ng/ml) was excluded from the analysis. For panels A–C, each point corresponds to an individual
mouse. D, Islets (20/well) isolated from non-TG or Ran-WT transgenic mice were incubated with 5 mM D-glucose, and analyzed for insulin release in
the supernatant at the indicated time intervals. Mean6SD of replicates. E, Islets (20/well) isolated from non-TG (black) or Ran-WT (purple), Ran-G19V
(grey) or Ran-T24N (blue) transgenic mice were incubated with 16.7 mM glucose, and analyzed for insulin release in the supernatant at the indicated
time intervals. Mean6SD of replicates.
Figure 3. Gender analysis of the diabetic phenotype in Ran
transgenic mice. The indicated non-TG or Ran transgenic mice were
analyzed for gender differences in blood glucose levels at 2 mo of age.
Each point corresponds to an individual mouse.
Ran Regulation of Pancreatic b Cells
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centrifugation at 14,000 rpm for 20 min at 4uC, and analyzed by
Western blotting. Alternatively, INS-1 cells were transduced with a
lentivirus expressing control pLKO or Ran-directed short hairpin
RNA (shRNA) (Open Biosystems), selected with 1 mg/ml puro-
mycin, and analyzed by Western blotting. Protein content was
determined using a BCA-200 protein assay kit (Pierce).
Pancreatic islet isolation
Pancreatic islets were harvested from control or Ran transgenic
mice by collagenase P (1 mg/ml) (Sigma-Aldrich) perfusion, as
described . After filtration through a 100 mm cell strainer,
islets were hand-picked under a dissecting microscope. Islets
isolated from non-TG mice were transduced in vitro with pAd-
GFP or pAd-GFP-Ran-WT, pAd-GFP-Ran-G19V or pAd-GFP-
Ran-T24N, as described previously , and analyzed after 48 h
for GFP expression by fluorescence microscopy or endogenous
PDX-1 levels, by Western blotting.
Islet glucose stimulation, in vitro
Islets isolated from non-TG or transgenic mice expressing Ran-
WT, Ran-G19V or Ran-T24N (7 mice/group) at 2–5 mo of age
were plated onto six-well plates (20 islets/well), and cultured in
2 ml DMEM medium for 24 h. Cells were suspended in 1 ml
OPTI-MEM for 4 h, separately incubated with 5 mM or
16.7 mM D-glucose, and 200 ml aliquots of the supernatants were
collected after 0.5, 1, and 2 h for determination of insulin levels, as
described . Similar experiments were carried out with parental
or shRNA-transduced stable INS-1 clones.
Glucose and insulin secretion
Non-TG or Ran transgenic mice were maintained with feeding
ad libitum, or, alternatively, under fasting conditions for 16 h. At
the end of the fasting period, thirty ml of blood was collected from
the tail vein, and analyzed for glucose or insulin content using an
ELISA assay kit (Crystal Chem).
Pancreas tissues from non-TG or Ran transgenic mice were
fixed in 10% neutral-buffered formalin, dehydrated through
graded ethanol passages, and embedded in paraffin wax. Five
mm-thick tissue sections were cut, put on high-adhesive slides, and
stained with hematoxylin and eosin (H&E). Immunohistochemical
staining was performed as described , using primary
antibodies to insulin (1:500) or PDX-1 (1:200). Quantification of
tissue staining was carried out by morphometry, as described .
For islet cell proliferation or apoptosis, pancreas sections were
incubated with an antibody to Ki-67 (1:100), or analysed for
terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL, Roche), respectively, as described , and
positively stained islet cells were quantified. Images were collected
on an Olympus microscope with on-line charge-coupled device
camera. Islet surface area normalized to mm2or islet number was
quantified from insulin-labeled, serial pancreas sections by
morphometry, as described .
Data were analyzed by two-sided unpaired t-tests using a
GraphPad software package (Prism 4.0) for Windows. In some
Figure 4. Defective islet development in Ran transgenic mice.
A, Pancreas tissues from non-TG, Ran-WT, Ran-G19V or Ran-T24N
transgenic mice were analyzed by H&E or immunohistochemical
staining with an antibody to insulin. B, Pancreas sections from non-
TG, asymptomatic Ran-WT or diabetic Ran-WT transgenic mice were
analyzed for islet number or islet surface area by morphometry of
insulin-stained areas. Islet number, non-TG (n=2), 29.561.5; Ran-WT
(n=3), 2361.73, n.s., not significant; Ran-WT diabetic (n=3), 9.3360.88,
**, p=0.001; islet surface area, non-TG (n=2), 0.5160.02; Ran-WT
(n=3), 0.3560.021, *, p=0.017; Ran-WT diabetic (n=3), 0.05760.004,
**, p,0.0001. C, Pancreas tissues from Ran-WT, Ran-G19V or Ran-T24N
transgenic mice were analyzed by immunohistochemical staining with
antibodies to somatostatin or glucagon.
Ran Regulation of Pancreatic b Cells
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experiments, ANOVA and post-hoc multiple tests with Bonferroni
procedure or Mann-Whitney tests were used for data analysis. A p
value of 0.05 was considered as statistically significant.
Characterization of Ran-b cell transgenic mice
We generated transgenic mice that express HA-tagged Ran
WT, a Ran T24N mutant that is unable to hydrolyze GTP, or a
Ran G19V mutant with constitutively active GTPase function 
in insulin-producing pancreatic b cells . PCR products
corresponding to Ran-WT, Ran-T24N or Ran-G19V construct
were amplified from tail DNA using transgene-specific primers for
RIP-HA (Fig. 1A, top), or RIP-Ran (Fig. 1A, bottom) sequences.
Pancreas extracts from PCR-confirmed transgenic mice reacted
with an antibody to HA, whereas PCR-negative samples were
unreactive (Fig. 1B). In addition, HA-reactive material was
demonstrated in pancreas, but not liver extracts of PCR-confirmed
Ran transgenic mice (Fig. 1C), confirming tissue-specific expres-
sion of the various cDNA constructs. Endogenous Ran levels in
pancreas of control littermates were negligible, by Western
blotting of isolated islet tissues (see below).
Deregulated Ran signaling in b cells impairs glucose
metabolism and islet maintenance
Ran transgenic mice did not present overt developmental
defects, were born at expected rates and were fertile. However, by
1–2 mo of age, subsets of transgenic mice expressing Ran-WT
(57%), Ran-G19V (58%) or Ran-T24N (42%) exhibited blood
glucose levels .150 mg/dl under ad-libitum feeding (Fig. 2A), or
fasting (Fig. 2B) conditions. In contrast, non-transgenic mice had
blood glucose levels of 1256.3.2 mg/dl, which was considered
within the normal range. Hyperglycemia in Ran transgenic mice
was associated with reduced blood insulin levels, compared to non-
transgenic littermates (Fig. 2C). In addition, pancreatic islets
isolated from non-transgenic mice responded to glucose stimula-
tion with a transient increase in insulin release, peaking at 1 h, and
returning to baseline 2 h after challenge (Fig. 2D). In contrast,
islets from representative asymptomatic Ran-WT transgenic mice
(Fig. 1C) had constitutively higher basal insulin levels, potentially
reflecting a compensatory response to decreasing postnatal b cell
mass (see below), which were not further modulated by stimulation
with low glucose concentrations (5 mM) (Fig. 2D). Conversely,
islets isolated from all three transgenic mouse lines expressing Ran-
WT, Ran-G19V or Ran-T24 responded to stimulation with
higher glucose concentrations (16.7 nM) with an increase in
insulin release that peaked 1 h after challenge (Fig. 2E). Most
diabetic Ran transgenic mice died of hyperglycemia by 4–6 mo of
age and none was alive by 8 mo. Different from other mouse
models of diabetes [16,17], no gender-specific differences were
observed in the development of hyperglycemia in Ran transgenic
mice (Fig. 3).
Pancreas tissue isolated from non-TG mice contained morpho-
logically developed endocrine islets that stained intensely positive
for insulin (Fig. 4A). In contrast, by 2 mo of age, symptomatic
transgenic mice expressing any of the three Ran variants, WT,
G19V or T24N in pancreatic b cells exhibited largely undetectable
insulin staining, in situ (Fig. 4A). Consistent with these data,
overtly diabetic Ran-WT transgenic mice at 2 mo of age exhibited
nearly complete loss of pancreatic islet number and islet mass,
compared to control littermates (Fig. 4B). In contrast, asymptom-
atic Ran-WT transgenic mice (Fig. 2A) of comparable age had
intermediate defects in postnatal islet development, with partial
reduction in islet number and islet mass, compared to the control
Figure 5. Postnatal defect in islet development in Ran
transgenic mice. A, E18 non-TG or Ran-WT embryos were analyzed
by H&E or immunohistochemical staining with an antibody to insulin.
Arrows, insulin-stained islets. B, Islet number (left), or islet surface area
(right), was quantified in E18 non-TG or Ran-WT transgenic embryos by
morphometry of insulin-stained sections. The differences among
groups are not statistically significant. C, Littermates of Ran-WT diabetic
mice crossed with normal mice were analyzed for changes in blood
glucose levels at the indicated time intervals. E, embryonic; P, postnatal.
Glucose levels in PCR-confirmed non-TG (open circles) or Ran-WT
transgenic (closed circles) mice are shown. Each point corresponds to an
individual mouse. Glucose concentrations (mg/dl) in each group
(number of mice in parentheses), and statistical analyses versus E18
values are as follows: E10 (n=15), 108.764.93; E18 (n=15), 120.762.7;
P30 Ran-WT (n=7), 196.3617.8, p,0.0001; P30 non-TG (n=4), 13465.4;
P45 Ran-WT (n=7), 187.1613.5, p,0.0001; P45 non-TG (n=4),
134.863.4; P60 Ran-WT (n=7), 193.1615.5, p,0.0001; P60 non-TG
(n=4), 132.5613.9. Statistical data re-analysis using Mann-Whitney test
was as follows: P30 Ran-WT, p=0.013; P45 Ran-WT, p=0.0006; P60 Ran-
Ran Regulation of Pancreatic b Cells
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group (Fig. 4B). Conversely, production of glucagon by pancreatic
a cells, or somatostatin by d cells was unaffected in all three
transgenic mouse lines expressing Ran-WT, Ran-G19V or Ran-
T24N (Fig. 4C).
Postnatal defect of pancreatic islet maintenance in Ran
PCR-confirmed non-TG E18 embryos contained morpholog-
ically discrete endocrine islets, which stained positive for insulin,
by immunohistochemistry (Fig. 5A). Ran-WT E18 embryos also
revealed the presence of insulin-stained islets (Fig. 5A), quantita-
tively indistinguishable for number and surface area from those of
non-TG embryos (Fig. 5B). Metabolically, embryos collected at
E10 or E18 had normal blood glucose levels (Fig. 5C). However,
by postnatal d 30 (P30), a subset of mice (63%) developed
hyperglycemia, which was maintained at analyses on P45 and P60
(Fig. 5C). Six out of seven of these diabetic mice were confirmed
positive for the Ran-WT transgene by PCR, whereas none of the
non-transgenic mice showed hyperglycemia (Fig. 5C).
Time-course analysis of islet cell proliferation revealed no
significant differences in the number of Ki-67+islet cells between
transgenic and non-transgenic specimens at P14 or P21 (Fig. 6A,
B). In contrast, islet cell proliferation was significantly reduced in
Ran-WT transgenic mice by P50 (Fig. 6A, B). Conversely, no
differences in islet cell apoptosis were observed between transgenic
and non-transgenic mice, by TUNEL staining of pancreas sections
at comparable age (Fig. 6C).
Deregulated Ran signaling affects PDX-1 expression
In addition to the loss of insulin staining (Fig. 4A), pancreas
tissue from overtly diabetic transgenic mice expressing Ran-WT,
Ran-G19V or Ran-T24N at 2 mo of age exhibited significantly
decreased expression of the pancreatic and duodenal homeobox
transcription factor, PDX-1 , compared to non-transgenic
mice or asymptomatic Ran-WT transgenic animals, by quantita-
tive morphometryof immunohistochemically-stained
(Fig. 7A, B). Similar results were obtained by Western blotting
analysis of PDX-1 expression in pancreas tissues isolated from
diabetic Ran-WT transgenic mice, compared to control, non-TG
littermates (Fig. 7C). We next used two independent approaches to
examine the dependence of PDX-1 expression on Ran signaling.
First, we transduced pancreatic islets isolated from non-TG mice
with GFP-encoding adenoviral constructs (pAd) expressing HA-
tagged Ran-WT, Ran-G19V or Ran-T24N. In these experiments,
adenoviral transduction of isolated islets was associated with GFP
reactivity, by fluorescence microscopy (Fig. 7D, left), and
expression of HA-tagged recombinant Ran proteins, by Western
blotting (Fig. 7D, right). In addition, adenoviral over-expression of
all three Ran isoforms in isolated islets resulted in reduced levels of
endogenous PDX-1, compared to pAd-GFP transduction, by
Western blotting (Fig. 7D, right), thus mirroring the phenotype of
Ran transgenic mice (Fig. 7B, C). Second, we targeted Ran by
gene silencing approaches in model insulinoma INS-1 cells. Due to
their transformed phenotype, these cells express elevated levels of
endogenous Ran, in agreement with previous observations . In
these experiments, INS-1 cells stably transfected with lentivirus
expressing Ran-directed shRNA exhibited nearly complete loss of
endogenous Ran levels, which was associated with loss of PDX-1
expression, by Western blotting (Fig. 7E). In control experiments,
INS-1 clones stably transduced with a control non-targeting
lentivirus (pLKO) had endogenous levels of Ran or PDX-1
comparable to those of parental cells (Fig. 7E). In addition, stable
shRNA silencing of Ran in four independent INS-1 clones was
associated with reduced insulin production, compared to parental
INS-1 cells (Fig. 7F).
In this study, we have shown that transgenic expression of
functional variants of the Ran GTPase [6,19] in pancreatic b cells
induces catastrophic defects of postnatal islet development,
resulting in hypoinsulinemia, and an overt diabetic phenotype.
Although a subset of Ran transgenic mice were asymptomatic
(,40%) at the time of diabetic onset (2 mo), these animals also
revealed reduced islet number and islet mass, compensatory
Figure 6. Defective islet cell proliferation in Ran transgenic mice. A, Pancreas section from non-TG or Ran-WT transgenic mice were
harvested at the indicated postnatal (P) age and analyzed for Ki-67 reactivity, by immunohistochemistry. Sections from normal mouse spleen were
used as control. B, The number of Ki-67+cells/islet surface area (0.02 mm2) was quantified by morphometry. **, p=0.0081. C, Pancreas section from
non-TG or Ran-WT transgenic mice (P50) were analyzed for TUNEL reactivity and the numbers of positive cells was quantified. Mean6SD.
Ran Regulation of Pancreatic b Cells
PLoS ONE | www.plosone.org5 November 2011 | Volume 6 | Issue 11 | e27879
hyperinsulinemia, and impaired insulin response to glucose
stimulation of isolated islets, in vitro. Mechanistically, deregulated
Ran signaling in islet b cells was associated with decreased
expression of the transcriptional regulator of islet development,
PDX-1 , and impaired postnatal islet cell proliferation, but not
apoptosis, in vivo.
GTP-binding proteins, or G proteins, function as important
effectors of glucose homeostasis , orchestrating cytoskeletal
remodeling , and vesicle fusion  for insulin secretion, as
well as docking of insulin secretory granules at the plasma
membrane [23,24]. Genetic models support this view, as deletion
of the Rab3A , or Rab27a  G protein in mice results in
hyperglycemia, hypoinsulinemia and glucose intolerance, whereas
transgenic expression of a dominant negative Rac1 mutant in b
cells impairs islet morphogenesis, potentially by interfering with
migration of pancreatic b cells away from the ductal epithelium
Conversely, a role of the Ran G protein [6,19] in glucose
homeostasis has not been previously described. Here, transgenic
expression of three functionally different Ran variants in b cells
caused a comparable phenotype of overt diabetes, suggesting that
a finely-tuned balance of Ran GTPase signaling [6,19] is required
for b cell maintenance, in vivo. Mechanistically, this pathway was
associated with decreased expression of the transcriptional
regulator of islet development, PDX-1 . A large body of
literature points to an essential role of PDX-1 in pancreas and islet
formation in mice [28,29], and humans , orchestrating the
differentiation and expansion of precursor cells within the
pancreatic buds (E8.5–9.5), formation of acinar cells and islets
(E12.5), and completion of secondary transition (E14.5–E15.5)
[17,31]. There is also evidence that persistent PDX-1 activity is
required to maintain a functional b cell mass in the adult pancreas
[32,33], where its expression becomes restricted to b, but not
acinar cells . Accordingly, post-developmental haploinsuffi-
ciency of PDX-1 causes an overtly diabetic phenotype in mice
[35,36], mechanistically associated with increased b cell apoptosis
[35,36], loss of downstream PDX-1 target genes implicated in
glucose-stimulated insulin transcription [37,38], and impaired b
cell proliferation/regeneration . Together, these data are
consistent with a model in which loss of PDX-1 expression in Ran
transgenic mice triggers developmental defects of postnatal islet
formation, associated with decreased islet cell proliferation (i.e. b
cell neogenesis), in situ . Conversely, deregulated Ran
signaling did not affect islet formation in E10 or E18 transgenic
embryos, suggesting that PDX-1 activity at this developmental
stages  does not depend on Ran GTPase function.
Additional work is required to conclusively elucidate how Ran
GTPase activity controls PDX-1 expression and/or function in
mature b cells [32,33]. This pathway may not involve modulation
of PDX-1 nucleo-cytoplasmic shuttling , as shRNA silencing
of Ran in INS-1 cells comparably depleted PDX-1 in cytosolic and
nuclear compartments, by Western blotting of isolated subcellular
fractions (our unpublished observations). Instead, data presented
here suggest that Ran may help maintaining PDX-1 protein
stability in postnatal b cells, similar to its function on other
downstream Ran target proteins, including survivin , or TPX-
2/Aurora A . It is also plausible that other Ran target proteins
contribute to postnatal islet development, a possibility suggested by
the partial decrease in PDX-1 expression in Ran transgenic mice,
as opposed to the nearly complete loss of insulin production under
these conditions, in vivo.
In summary, we have identified the small GTPase, Ran as an
essential effector of postnatal islet development. The requirements
of this pathway differ from previously known examples of G
Figure 7. Deregulated PDX-1 expression in Ran-targeted cells.
A, Pancreas sections from non-TG, Ran-WT, Ran-G19V or Ran-T24N
transgenic mice were stained with an antibody to insulin or PDX-1, by
immunohistochemistry. B, The number of insulin- or PDX-1-stained cells
was quantified by morphometry in the indicated surface area. Insulin+
cells, non-TG (n=2), 28.260.4; Ran-WT (asymptomatic, n=3), 29.861.2;
Ran-WT (diabetic, n=3), 6.260.6, ***, p,0.0001; PDX-1+cells, Non-TG
(n=2), 34.264.1; Ran-WT (asymptomatic, n=3), 37.562.3; Ran-WT
(diabetic, n=3), 22.861.8, **, p=0.007. C, Islets from 2 mo-old non-TG
or a Ran-WT transgenic mouse were analyzed by Western blotting. D,
Pancreatic islets isolated from non-TG mice were transduced ex vivo
with control pAd-GFP or pAd-GFP-Ran-WT, pAd-GFP-Ran-G19V or pAd-
GFP-T24N and analyzed after 48 h by fluorescence microscopy for GFP
expression (left), or Western blotting (right). *, non-specific. E, INS-1 cells
were left untreated (INS-1) or transduced with control lentivirus (pLKO)
or lentivirus encoding Ran-directed shRNA (Ran, 74V1), and analyzed by
Western blotting. F, Parental INS-1 cells or four independent clones of
INS-1 cells stably transduced with Ran-directed shRNA (77V-2, 74V-1,
75V-1, 75V-2) were analyzed for changes in insulin release in the
supernatant. Representative experiment out of at least two indepen-
Ran Regulation of Pancreatic b Cells
PLoS ONE | www.plosone.org6 November 2011 | Volume 6 | Issue 11 | e27879
protein regulation of b cell morphogenesis , or insulin
secretion , and involve a temporal control of postnatal
PDX-1 expression , and mature b cell proliferation. Future
studies will elucidate the functional partners of Ran GTPase
signaling in postnatal islet development, and PDX-1-dependent
Conceived and designed the experiments: DCA FX. Performed the
experiments: FX TD NMM CMR. Analyzed the data: DCA FX TD
NMM QL. Contributed reagents/materials/analysis tools: TD. Wrote the
paper: DCA FX.
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PLoS ONE | www.plosone.org7 November 2011 | Volume 6 | Issue 11 | e27879