Exploring functional beta-cell heterogeneity in vivo using PSA-NCAM as a specific marker.

Abstract

The mass of pancreatic beta-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous beta-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of beta-cells and investigated their physiological relevance in increased insulin demand conditions in rats.
Two rat beta-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. beta(high) and beta(low)-cells. Insulin release, Ca(2+) movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, beta(high) and beta(low)-cell distribution and functionality were investigated in animal models with decreased or increased beta-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat.
We show that beta-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike beta(low)-cells, beta(high)-cells express functional beta-cell markers and are highly responsive to various insulin secretagogues. Whereas beta(low)-cells represent the main population in diabetic pancreas, an increase in beta(high)-cells is associated with gain of function that follows sustained glucose overload.
Our data show that a functional heterogeneity of beta-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in beta-cell defects in type 2 diabetes.

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Exploring Functional b-Cell Heterogeneity In Vivo Using
PSA-NCAM as a Specific Marker
Melis Karaca1*, Julien Castel1, Ce ´cile Tourrel-Cuzin1, Manuel Brun2, Anne Ge ´ant2, Mathilde Dubois3,
Sandra Catesson4, Marianne Rodriguez4, Serge Luquet1, Pierre Cattan5, Brian Lockhart4, Jochen Lang3,
Alain Ktorza2, Christophe Magnan1., Catherine Kargar2.
1Laboratoire de Physiopathologie de la Nutrition, Universite ´ Paris Diderot, CNRS UMR 7059, Paris, France, 2Division Diabe `te et Maladies Me ´taboliques, Institut de
Recherches Servier, Suresnes, France, 3Institut Europe ´en de Chimie et Biologie, Universite ´ de Bordeaux, CNRS UMR 5248, Pessac, France, 4Division Pharmacologie et
Physiopathologie Mole ´culaires, Institut de Recherches Servier, Suresnes, France, 5Service de chirurgie ge ´ne ´rale, digestive et endocrinienne and Unite ´ de The ´rapie
Cellulaire, Ho ˆpital Saint-Louis, Paris, France
Abstract
Background: The mass of pancreatic b-cells varies according to increases in insulin demand. It is hypothesized that
functionally heterogeneous b-cell subpopulations take part in this process. Here we characterized two functionally distinct
groups of b-cells and investigated their physiological relevance in increased insulin demand conditions in rats.
Methods: Two rat b-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. bhighand
blow-cells. Insulin release, Ca2+movements, ATP and cAMP contents in response to various secretagogues were analyzed.
Gene expression profiles and exocytosis machinery were also investigated. In a second part, bhighand blow-cell distribution
and functionality were investigated in animal models with decreased or increased b-cell function: the Zucker Diabetic Fatty
rat and the 48 h glucose-infused rat.
Results: We show that b-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike blow-cells, bhigh-cells express functional
b-cell markers and are highly responsive to various insulin secretagogues. Whereas blow-cells represent the main population in
diabetic pancreas, an increase in bhigh-cells is associated with gain of function that follows sustained glucose overload.
Conclusion: Our data show that a functional heterogeneity of b-cells, assessed by PSA-NCAM surface expression, exists in
vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in b-cell defects in type 2
diabetes.
Citation: Karaca M, Castel J, Tourrel-Cuzin C, Brun M, Ge ´ant A, et al. (2009) Exploring Functional b-Cell Heterogeneity In Vivo Using PSA-NCAM as a Specific
Marker. PLoS ONE 4(5): e5555. doi:10.1371/journal.pone.0005555
Editor: Kathrin Maedler, University of Bremen, Germany
Received February 25, 2009; Accepted April 15, 2009; Published May 18, 2009
Copyright: ? 2009 Karaca 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 supported by the Institut de Recherches Internationales Servier and the Centre National de la Recherche Scientifique. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Manuel Brun, Anne Geant, Sandra Catesson, Marianne Rodriguez, Brian Lockhart, Alain Ktorza and Catherine Kargar are employees of
Servier.
* E-mail: meliskaraca@gmail.com
. These authors contributed equally to this work
Introduction
Pancreatic b-cells synthesize and release insulin with a
remarkable degree of plasticity over time that allows adaptation
to the metabolic environment. This involves increased b-cell
function, i.e. an increase both in insulin production and secretion
as well as enlargement of the b-cell pool [1]. The functional b-cell
mass varies according to changes in insulin sensitivity. Thus,
increased insulin sensitivity displayed by master athletes is
associated with reduced insulin secretion [2]. On the contrary,
insulin resistance as part of pregnancy or obesity is compensated
via an increase in both b-cell number and responsiveness to
glucose. This mechanism allows the maintenance of euglycemia in
spite of decreased insulin sensitivity [3,4].
The concept of functional heterogeneity among b-cells proposes
that eachcelldiffersinitssensitivitytoglucose[5]andisrecruited ina
glucose-dependent manner into both biosynthetic [6,7,8] and
secretory active states in order to adapt insulin secretion to the
metabolic environment [5,8,9,10,11,12]. Therefore characterization
of such b-cell subpopulations with different metabolic sensitivities
would lead to the development of new therapeutic strategies.
The sialylated form of the Neural Cell Adhesion Molecule
(PSA-NCAM) is only expressed in structures undergoing func-
tional changes in the adult such as brain and pancreatic b-cells
[13,14,15]. In adult b-cells, the regulation of PSA-NCAM
abundance at the cell surface is controlled by cellular activity, i.e.
insulin exocytosis [15]. Interestingly, we have previously identified
PSA-NCAM as a marker of b-cell functionality. According to the
level of surface PSA-NCAM, b-cells were divided in two
subpopulations with different glucose responsiveness in rats [13].
Our present study aimed to characterize these two groups of b-
cells and to investigate the basis supporting their different insulin
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secretory capacities. Furthermore, we correlated the PSA-NCAM
labeling to the functional b-cell mass in animal models where it is
either decreased or increased: (i) the Zucker Diabetic Fatty (ZDF)
rat, a model of type 2 diabetes [16] and (ii) the 48 h glucose-
infused rat (HG/HI), a model in which a long-term imposed
hyperglycemia leads to pancreatic overactivity and to an
impressive increase of functional b-cell mass [17].
Taken together, our data (i) confirm that PSA-NCAM is a
prominent marker of functional b-cells, (ii) provide proof of
concept that a functional heterogeneity of b-cells exists in vivo and
(iii) explore mechanistic insights of heterogeneity. Moreover, they
support the idea that an alteration of pancreatic b-cell plasticity,
i.e. an inability to recruit fully functional b-cells, could contribute
to the impairment of insulin secretion in type 2 diabetes.
Materials and Methods
Animals
Experiments were carried out on 12 weeks old male Wistar rats
and on 12 weeks old male Zucker Diabetic Fatty rats (ZDF fa/fa)
and their age-matched lean littermates (ZDF fa/+). Animals were
purchased from Charles River laboratories. Rats were housed
under a 12 h light-dark cycle with free access to water. During the
acclimatization period, animals were fed ad libitum on standard diet
or on Purina 5008 chow (Charles River) for ZDF fa/fa rats. A
subset of Wistar rats were infused with 30% glucose during 48 h to
induce hyperglycemia and hyperinsulinemia (Wistar HG/HI) and
compared to 48 h 0.9% NaCl-infused Wistar rats (Wistar control)
as previously described [17]. In vivo insulin secretion in response to
glucose represented by the insulinogenic index (DI/DG) was
evaluated during oral glucose tolerance tests (OGTT). Insulin and
glucose responses during OGTT (3 g/kg) were calculated as the
incremental plasma insulin values integrated over a period of
30 min after the glucose gavage (DI) and the corresponding
increase in glucose concentration (DG). The insulinogenic index
represents the ratio of these two parameters. All procedures were
performed according to the French ethical rules for animal
experimentation.
Islet cell preparation
Rats were anesthetized with pentobarbital (Sanofi; 4 mg/100 g
body weight i.p.). Islets of Langerhans were isolated after
collagenase digestion of the pancreas as previously described
[18]. Islets were trypsinized with 0.1 mg/ml trypsin (1:250, Difco)
and digestion was stopped with cold Krebs-Ringer-Bicarbonate-
Hepes (KRBH) buffer-0.5% BSA (Interchim) and 5.5 mM glucose
[13]. Cell suspensions were used for immunocytochemistry or for
b-cell sorting.
Fluorescence-activated cell sorting of b-cells
Using a FACStarplus(Becton Dickinson), b-cells were distin-
guished from non-b cells and sorted based on their autofluores-
cence (FAD content) and cell size, resulting in a population with
95% (insulin-positive) b-cells, as previously described [19,20].
b-cells were analyzed for their surface PSA-NCAM expression
using the mouse anti-PSA-NCAM antibody (AbCys) and the anti-
mouse PE-conjugated secondary antibody (Invitrogren). The
geometric mean fluorescence for PSA-NCAM was determined in
control rats and used to arbitrarily separate between high PSA-
NCAM-labeled b-cell population (bhigh-cells) and low PSA-
NCAM-labeled b-cell population (blow-cells). In HG/HI and
ZDF fa/fa rat models, the geometric mean of respective controls
(ZDF fa/+ lean or NaCl-infused rats) was used to sort bhigh-cells
and blow-cells.
Data were analyzed using the attached FACStarplusanalysis
software (Becton Dickinson). Histograms are representative of
acquisitions performed on 7500 events for Wistar control and
HG/HI rats and 3500 for ZDF lean and fa/fa rats. Each
experiment was performed on 1–2.106cells.
Culture of sorted pancreatic b-cells
Sorted rat b-cells were resuspended in RPMI 1640 (MP
Biomedicals) supplemented with 5.5 mM glucose, 10% heat-
inactivated FCS, 100 mg/ml gentamycin, 2 mM L-glutamine and
10 mM Hepes and plated in miniculture dishes. For aggregates
formation, b-cells were incubated during 1 h in a rotary incubator
(30 cycles/min) at 37uC. Cells were then cultured for 18 h at 37uC
in 95% O2/5% CO2 and saturated humidity and washed in
KRBH buffer containing 5.5 mM glucose and 0.05% FAF-BSA
(fraction V, Roche) before use. The cell viability rate was assessed
by neutral red and was always between 90–95%.
Insulin content
Freshly sorted b-cells were homogenized in acid alcohol solution
(75% ethanol, 1.5% HCl 12N, 23.5% distilled water) and stored at
280uC until insulin determination.
In vitro analysis of insulin secretion
In vitro insulin release was assayed either by perifusion or under
static incubation on aggregated sorted b-cells (806103) after
overnight culture.
The perifusion of rat cells was performed using low (5.5 mM) or
stimulating (8.3 mM and/or 16.7 mM) glucose concentrations or
KCl (50 mM) in KRBH buffer-0.05% FAF-BSA, as described
previously [13]. The eluates were collected for insulin quantifica-
tion. Incremental insulin response (DI) to stimulating glucose
concentrations or KCl above basal release was obtained by
planimetry of perifusion profiles and was expressed as the difference
in insulin secretion rate relative to the mean hormonal output
recorded at the pre-stimulation period with 5.5 mM glucose.
For static incubation, rat cells were pre-incubated for 60 min at
37uC in silicone-coated glass tubes in a shaking water bath
containing KRBH buffer-0.05% FAF-BSA and 5.5 mM glucose.
Thereafter, the medium was replaced by KRBH-BSA containing
5.5 mM or 16.7 mM without or with 10 nM GLP-1, 5 mM db-
cAMP, 19 mM arginine or 10 mM leucine and b-cells were
further incubated for 30 min at 37uC. The supernatant was stored
at 220uC until assayed for insulin.
Intracellular free calcium measurements
Overnight-cultured aggregated sorted rat b-cells (806103) were
allowed to attach on polylysine-treated cover-glass then loaded for
1 h with 1.5 mM Fura-2/AM (Molecular Probes) at 37uC in
Krebs-Ringer-Bicarbonate (KRB) buffer containing (115 mM
NaCl, 5 mM KCl, 24 mM NaHCO3, 1 mM CaCl2, 1 mM
MgCl2, 5.5 mM glucose and) 0.05% FAF-BSA. Thereafter they
were transferred to a perifusion chamber placed on the stage of an
inverted fluorescent microscope (Nikon Diaphot) and were
perifused with 25 mM Hepes-buffered medium maintained at
37uC containing (125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl2,
1.2 mM MgCl2,) 0.1% FAF-BSA and supplemented with 5.5 mM
or 16.7 mM glucose. The 340 to 380 fluorescence ratios (F340/
F380) reflects the intracellular free calcium concentration [Ca2+]i
as previously described. The perifusion fluid was collected and
stored at 220uC until assayed for insulin. Incremental Ca2+
response to stimulating glucose (DR) was obtained in the same
manner as described above for insulin (DI).
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Insulin quantification
Insulin was measured using rat Ultra-Sensitive Insulin ELISA
kits (Mercodia).
Measurement of ATP content
ATP content wasmeasured under static incubation simultaneously
to insulin release on the same batches of b-cells (105), in the b-cell
pellets, using the ATP bioluminescence assay kit HSII (Roche).
Measurement of cAMP content
cAMP content was measured under static incubation simulta-
neously to insulin release on the same batches of b-cells, in the b-
cell pellets, by radioimmunoassay [21].
Quantitative real-time RT-PCR analysis
Total RNA extractions were carried out immediately after
sorting of rat b-cells with the RNeasy micro kit (Qiagen). RNA
quality was assessed using the 2100 Bioanalyser (Agilent) on the
basis of the RNA integrity number. Samples (1 mg) were then
subjected to reverse transcription using the high capacity cDNA
archive kit (Applied Biosystems). cDNAs (100 ng) were analyzed
by real-time quantitative RT-PCR using TaqMan Universal PCR
Master Mix (Applied Biosystems) on the 7900 HT Fast Real Time
PCR system with a TaqMan low density array (Applied
Biosystems). TaqMan gene expression assays (Applied Biosystems)
were used as a set of primers and TaqMan probe for amplification
of each gene of interest. Expression levels of target genes were
normalized to 18s RNA. The assay ID for each gene is given in
Table S1. Thermal cycle conditions were: 50uC 2 min, 94uC
10 min, followed by 40 cycles of 97uC 30 sec and 59.7u C 1 min.
Western blot analysis
Rat b-cells were suspended in PBS-1% Triton X-100 containing
a protease inhibitor cocktail (Sigma) and sonicated on ice. Proteins
(20 mg/lane) were resolved on 4–20% linear gradient (for PSA-
NCAM) and on 12% (for exocytosis proteins) SDS-PAGE gels and
transferred to PVDF membranes (Amersham). Treatment of
membranes, image acquisition and quantification was made as
previously described [22,23]. The following antibodies were
employed: anti-PSA-NCAM (AbCys), anti-cyclophilin (Upstate),
anti-syt9 (Beckton Dickinson), anti-Syntaxin1 (Sigma), anti SNAP-
25 (Sternberger Monoclonals), anti-VAMP-2 (Synaptic Systems),
HRP-linked anti-mouse (DAKO) and anti-rabbit antibodies
(Amersham).
Endoneuraminidase (EndoN) treatment of b-cells
Overnight-cultured aggregated sorted bhigh-cells (1.56105) were
treated 1 h with 0.7 U endoN (AbCys) at 37uC to cleave PSA and
subjected to western blot analysis for PSA-NCAM immediately
after EndoN digestion or 3 h after EndoN digestion. EndoN
treated bhigh-cells were tested for insulin secretion in response to
5.5 mM and 16.7 mM glucose in presence of EndoN to avoid
PSA-NCAM reexpression, during static incubation studies.
Immunolabeling on pancreas sections and isolated cells
Rat pancreata were fixed in 4% paraformaldehyde (PFA),
immersed in PBS 230% sucrose and frozen in liquid nitrogen. For
peroxydase labeling, successive tissue cryosections (7 mm thick-
ness), fixed in acetone, were treated with anti-PSA-NCAM
(AbCys) or anti-insulin (ICN) antibody overnight at 4uC. They
were then incubated 1 h with biotin-conjugated anti-mouse
(Vector) or with biotin-conjugated anti-Guinea pig (Jackson
ImmunoResearch) antibody followed by incubation with HRP-
labeled streptavidin (DAKO) and development using the Vectas-
tain VIP or DAB kit (Vector).
Multiple immunofluorescence labeling for PSA-NCAM and
islet hormones was performed on acetone-fixed rat pancreas
cryosections. After overnight incubation with a mix of anti-PSA-
NCAM (AbCys), anti-insulin (ICN), anti-glucagon (ICN), anti-
somatostatin (ICN) and anti-pancreatic polypeptide (ICN) primary
antibodies, sections were incubated 1 h with a mix of R-PE-
conjugated anti-mouse (Invitrogen), FITC-conjugated anti-Guinea
pig (Vector) or AMCA-conjugated anti-rabbit (Jackson Immu-
noResearch) secondary antibodies. This multiple labeling was also
performed on dissociated islet cells (506103) cultured on
polylysine-coated cover-glass as previously described [13]. Images
were analyzed with ImageJ (http://rsb.info.nih.gov/ij/).
Confocal microscopy and quantification of actin
filaments
Sorted rat b-cells (506103) were rinsed with PBS, fixed in 4%
PFA and permeabilized in PBS, 0.2% triton X-100. Cells were
washed, incubated with TRITC-phalloidin (Sigma) for 30 min,
washed in PBS, mounted on slides and examined with a laser
scanning confocal microscope (Leica SP2-AOBS). TRITC-phal-
loidin binds specifically to F-actin but not to actin monomers.
Images of TRITC-phalloidin stained b-cells were collected under
identical optical conditions and analyzed with ImageJ (http://rsb.
info.nih.gov/ij/). A contiguous series of 28 optical sections (1 mm
increments in the Z plane) was sufficient to capture most of the
actin staining in the whole b-cell.
Statistical methods
Data are reported as means6SEM. Statistical analyses were
assessed by one-way ANOVA for comparison among multiple
groups and by two-tailed Student’s t test for comparison between
two groups.
Results
Pancreatic b-cells are heterogeneous for PSA-NCAM
Staining of rat pancreas for PSA-NCAM and insulin showed
that PSA-NCAM was exclusively located in islets and was not
expressed in the exocrine tissue (Fig. 1A). Within islets, b-cells (in
green, Fig. 1B) expressed different levels of PSA-NCAM (in red,
Fig. 1B), i.e. they were heterogeneous for PSA-NCAM expression.
PSA-NCAM was not observed in the other islet cells (in blue,
Fig. 1B). Similarly, on dispersed islet cells, PSA-NCAM expression
was specific of b-cells. In this case too, PSA-NCAM heterogeneity
was obvious (Fig. 1C). This heterogeneity was further assessed by
FACS analyses on isolated b-cells. Total b-cells were gated based
on their FAD fluorescence and cell size (Fig. 1D), [19,20] then
analyzed for their PSA-NCAM surface expression. This latter was
a broad range continuous spectrum, characteristic of heterogeneity
(Fig. 1E). Using the geometric mean of fluorescence intensity
(481627 AU, black arrow in Fig. 1E), we arbitrarily divided the b-
cell population into two groups of b-cells expressing high levels
(bhigh-cells) and low levels (blow-cells) of surface PSA-NCAM
(Fig. 1E). Sorted bhigh-cells exhibited higher levels of total PSA-
NCAM (surface and intracellular) compared to blow-cells, as shown
by immunoblotting (Fig. 1F).
blow-cells are poorly responsive to glucose
We first investigated glucose-stimulated insulin secretion (GSIS)
in bhighand blowgroups of cells. In bhigh-cells, increasing the
glucose concentration from 5.5 mM to 8.3 mM and to 16.7 mM
in the perifusion medium induced a marked increase in insulin
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release (Fig. 2A). In blow-cells insulin secretion was significantly
lower than in bhigh-cells for all tested glucose concentrations
(Fig. 2A). In the rest of the study, 5.5 mM and 16.7 mM glucose
concentrations were used as low and high stimulating conditions.
This difference in GSIS between bhighand blow-cells did not result
from changes in insulin content (Fig. 2B) nor in cell size (Fig. 2C).
However FAD and SSC parameters were significantly higher in
bhigh-cells compared to blow-cells (Fig. 2D–E).
We next wondered whether PSA-NCAM itself was responsible for
the difference of GSIS between the two groups of cells. To this end,
Figure 1. Pancreatic b-cells are heterogeneous for PSA-NCAM. (A) Immunochemical analyses on successive frozen rat pancreas sections
stained for insulin (brown) and PSA-NCAM (purple). ex: exocrine tissue ; en: endocrine tissue. (B) Frozen rat pancreas. In islets, PSA-NCAM (red)
colocalizes only with insulin (green) and not with other islet hormones (blue). Scale bar: 30 mm. (C) Dissociated islet cells. PSA-NCAM (red) colocalizes
only with insulin (green) and not with other islet hormones (blue). Scale bar: 10 mm. (D) Representative dot plot analysis of dissociated islet cells
examined for their FAD content and cell size (FSC) in order to gate total b-cells (black frame). (E) Representative histogram of PSA-NCAM surface
labeling of total b-cells. Vertical arrows indicate the geometric mean (481627 for labeled b-cells). This value was used to arbitrarily separate and sort
highly (bhigh) and poorly (blow) PSA-NCAM labeled b-cells (n=7). (F) Immunoblot analysis for total PSA-NCAM expression. Cyclophilin was used as
loading control.
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Figure 2. blow-cells are poorly responsive to glucose. (A) Insulin release in response to 5.5 mM, 8.3 mM and 16.7 mM glucose in bhighand blow
cells determined by perifusion experiments (n=2). Quantification of insulin response to stimulating glucose is represented by DI. (B) Insulin content
(n=3). (C–E) Representative histogram of size (FSC) (C), FAD (autofluorescence) (D) and granularity (SSC) (E) of sorted bhighand blow-cells (n=5–7).
(F) Immunoblot analysis for PSA-NCAM expression after endoneuraminidase N (endoN) treatment (0.7 U/ml) of bhigh-cells. Cyclophilin was used as
loading control. (G) Insulin release in response to 5.5 mM and 16.7 mM glucose in endoN-treated bhigh-cells (0.7 U/ml) assessed by static incubation
experiments (n=2). n represents the number of independent cell preparations from 6 pooled rats each. Data are means6SEM. *, p,0.05; **, p,0.01;
***, p,0.005.
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sorted bhigh-cells were digested with endoneuraminase N (endoN) to
remove PSA, as shown by immunoblotting (Fig. 2F). GSIS was
similar in endoN-treated and non-treated bhigh-cells (Fig. 2G).
Therefore the difference in GSIS between bhighand blow-cells can
not be due to the sole difference of PSA-NCAM expression.
blow-cells exhibit altered Ca2+and ATP signaling in
response to glucose
In order to understand the differences between bhighand blow-cells,
we investigated the intracellular messengers implicated in insulin
secretion. Simultaneously to GSIS studies, the movements of
intracellular calcium ([Ca2+]i) were evaluated. In bhigh-cells,
16.7 mMglucose triggered a typical [Ca2+]ielevation that correlated,
as expected, with the stimulation of insulin secretion whereas only a
minor calcium response was observed in blow-cells (Fig. 3A).
Increased ATP levels being required for GSIS, ATP content
were measured in both groups of cells. Increasing glucose
concentration from 5.5 mM to 16.7 mM induced a GSIS in
bhigh-cells but did not further increase the ATP levels, as
previously described by others [24], (Fig. 3B). However ATP
levels were higher in bhigh-cells compared to blow-cells at low and
high glucose concentrations (Fig. 3B).
Thus, lower intracellular calcium movements and ATP levels
contribute to impaired GSIS in blow-cells.
blow-cells have an altered cAMP signaling
To investigate whether GSIS could be induced in blow-cells in the
presence of a potentiator, we tested Glucagon Like Peptide-1 (GLP-
1). At low glucose concentration, insulin secretion was similar in
both groups and wasnotaffectedbytheaddition ofGLP-1(Fig. 4A).
Insulin release in response to 16.7 mM glucose was potentiated by
GLP-1 in bhigh-cells whereas the hormone was not efficient in blow-
cells (Fig. 4A). As the GLP-1 receptor activates adenylate cyclase, we
next investigated cAMP content as part of the signaling cascade.
Indeed, cAMP levels were increased in the presence of GLP-1 at 5.5
and 16.7 mM glucose in bhigh-cells as compared to glucose alone.
By contrast, cAMP content remained very low in response to
glucose and/or GLP-1 in blow-cells (Fig. 4B).
Addition of dibutyryl-cAMP (db-cAMP), a cell permeable
cAMP analog known to potentiate insulin secretion, significantly
increased insulin release in bhigh-cells in the presence of 16.7 mM
glucose (Fig. 4C). Although the addition of db-cAMP partially
restored GSIS in blow-cells, insulin secretion was still lower than in
bhigh-cells (Fig. 4C).
These observations indicate that blow-cells exhibit altered cAMP
accumulation in response to GLP-1 signaling, consistent with the
low ATP content.
bhighand blow-cells respond equally to depolarizing
agents but differently to metabolizable secretagogues
The difference in functional activity between both groups of
cells may result from a metabolic or mechanistic failure or both.
To investigate this point, bhighand blow-cells were treated with the
metabolizable secretagogue L-leucine or the depolarizing agents
L-arginine and KCl.
Addition of 10 mM leucine alone elicited the same response as
5.5 mM glucose alone in both b-cell groups (Fig. 5A). When
combined, the respective effects of 5.5 mM glucose and of leucine
on insulin secretion were additive in blow-cells and in bhigh-cells.
Whereas 16.7 mM glucose combined with leucine induced a
stronger insulin release in bhigh-cells, it failed to further stimulate
insulin secretion in blow-cells (Fig. 5A).
Figure 3. blow-cells exhibit altered Ca2+and ATP signaling in response to glucose. (A) Insulin release (top panel) and cytoplasmic calcium
concentration ([Ca2+]i) oscillations (bottom panel) in response to 5.5 mM and 16.7 mM glucose in bhighand blow-cells during perifusion experiments
(n=3–6). Quantification of insulin and calcium responses to stimulating glucose are represented by DI and DR respectively. (B) Insulin release (top
panel) and intracellular ATP ([ATP]) levels (bottom panel) in response to 5.5 mM and 16.7 mM glucose in bhighand blowcells assessed by static
incubation experiments (n=2). n represents the number of independent cell preparations from 6 pooled rats each. Data are means6SEM. ***,
p,0.005.
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Unlike to leucine, 19 mM arginine stimulated insulin release in
both b-cell populations and this response was significantly higher
as compared to 5.5 mM glucose alone or 10 mM leucine alone
(Fig. 5A). 5.5 mM glucose had no additive effect on arginine-
stimulated insulin release. 16.7 mM glucose combined with
arginine further stimulated insulin release in bhigh-cells but failed
in blow-cells (Fig. 5A). Addition of 50 mM KCl led to an overall
similar amount of insulin released in both groups (Fig. 5B).
These data suggest that metabolic alterations contribute to low
insulin secretion in blow-cells.
The exocytosis machinery of blow-cells is not favorable to
GSIS
Exocytosis involves changes in the actin cytoskeleton and
requires a number of conserved proteins. Confocal microscopy
revealed a fragmented distribution of F-actin beneath the plasma
membrane in 8765% of bhigh-cells (Fig. 6A) whereas 7867% of
blow-cells displayed a strong and continuous signal potentially
reflecting a ring beneath the plasma membrane that may hamper
exocytosis (Fig. 6A) [25]. Proteins, such as SYT9, STX1, SNAP25
and VAMP2, required for exocytosis and its regulation by calcium
were 3- to 5-fold less expressed in blow-cells as compared to bhigh-
cells as shown by immunoblotting (Fig. 6B).
These data indicate that the exocytosis machinery of blow-cells is
not favorable to GSIS.
bhighvs blow-cellsexhibit different geneexpressionprofiles
We next investigated by RT-qPCR the expression of genes
considered as highly representative of b-cell function, differentiation
and survival (Table S1). The mRNA levels of transcription factors
such as Neurod1, Pdx1, Pax6 and Nkx6.1 were 2.6 to 3.3 times more
important in bhigh-cells than in blow-cells. Meanwhile, blow-cells
expressed 2.6 to 3.6 higher levels of genes involved in early b-cell
differentiation such as Ngn3 and Tcf7l2. Regarding metabolism,
some enzymes normally absent in b-cells, such as Hk1or Ldha, were
highly expressed in blow-cells compared with bhigh-cells (12.1 to 16.7
times). At the same time several b-cell-related metabolic enzymes
such as Glut2, Gck, Pk, mtGPDH and Pcx were expressed to a lesser
extent in blow-cells (2.8 to 3.3times). In addition, the levels of several
pumps/ion channels (Kir6.2, Sur1, Cav1.2, Kv2.1 and SERCA2/3)
were 2 to 4 times higher in bhigh-cells compared with blow-cells.
Similar results were obtained with genes implicated in the cAMP
pathway (Gcgr, Glp1r, PKA, Rap1a and Rab3a). Expression of genes
involved in insulin synthesis, maturation and secretion (Ins itself,
Iapp, PC1/3, PC2, Chga, Cx36 and Slc30a8) or exocytosis (Munc13.1,
Stx1a, Snap25, Vamp2 and Rim2) was downregulated in blow-cells up
to 3.8 times compared with bhigh-cells.
This gene expression profile suggests that blow-cells are
composed of immature and/or non-functional cells in contrast
to fully functional bhigh-cells.
The change in bhighto blow-cell ratio correlates with the
change in b-cell function in animal models with
increased insulin demand
In order to evaluate the physiological relevance of PSA-NCAM
distribution, we measured the proportion of bhighand blow-cells
and their insulin secretory capacity in response to increased insulin
demand in two animal models. We first used the ZDF fa/fa rat, a
model for type 2 diabetes with impaired functional b-cell mass
[16]. In vivo insulin secretion in response to glucose is markedly
impaired in these animals at 12 week age, as reflected by a 70%
decrease of the insulinogenic index compared with ZDF lean
controls (Fig. 7A). The geometric mean of PSA-NCAM fluores-
cence of b-cells of ZDF fa/fa rats (red arrow, Fig. 7B) was strongly
left-shifted compared to that of ZDF fa/+ lean controls (blue
arrow, Fig. 7B), indicating a striking decrease of the expression of
PSA-NCAM on b-cells of ZDF fa/fa rats (Fig. 7B). The blow-cell
population was strongly predominant in ZDF fa/fa rats
(74.6%64.3% of total b-cells) in remarkable contrast to the age-
matched ZDF lean controls in which the blowpopulation only
represented 37.2%61.7% of total b-cells (Fig. 7B). In addition,
bhigh-cells from ZDF fa/fa rats were significantly less responsive to
glucose compared to the ZDF lean controls (Fig. 7C) whereas the
insulin secretion of blow-cells was unchanged (Fig. 7C).
Figure 4. blow-cells have an altered cAMP signaling. Effects of
5.5 mM and 16.7 mM glucose610 nM GLP-1 on insulin release (A) and
cAMP content (B) in bhighand blow-cells assessed by static incubation
experiments (n=3–5). (C) Effects of 5.5 mM and 16.7 mM gluco-
se65 mM db-cAMP on insulin secretion in bhighand blow-cells (n=3–5).
n represents the number of independent cell preparations from 6
pooled rats each. Data are means6SEM. *, p,0.05 and **, p,0.01.
doi:10.1371/journal.pone.0005555.g004
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Figure 5. bhighand blow-cells respond equally to depolarizing agents but differently to metabolizable secretagogues. (A) Effects of
5.5 mM and 16.7 mM glucose610 mM leucine (Leu) or 19 mM arginine (Arg) on insulin release in bhighand blow-cells (n=3–5). Data are means6SEM.
*, p,0.05 and **, p,0.01 for stimulating conditions (16.7 mM glucose6Arg or Leu) compared to basal conditions (5.5 mM glucose6Arg or Leu). ¤,
p,0.05 and ¤¤, p,0.01 between treated (glucose+Arg or Leu) and non-treated conditions (glucose alone) for same glucose concentrations. ££,
p,0.01 for Arg or Leu alone compared to 5.5 mM glucose alone. (B) Insulin secretory response to 5.5 mM glucose650 mM KCl in bhighand blow-cells
(n=3–5). Quantification of insulin response to KCl is represented by DI. Data are means6SEM. *, p,0.05 between bhighand blow-cells. n represents
the number of independent cell preparations from 6 pooled rats each.
doi:10.1371/journal.pone.0005555.g005
Figure 6. The exocytosis machinery of blow-cells is not favorable to GSIS. (A) Representative pictures of actin filament distribution in bhigh
and blow-cells. Scale bar: 10 mm (B) Representative immunoblots of exocytotic proteins in bhighand blow-cells (n=3). n represents the number of
independent cell preparations from at least 6 pooled rats each.
doi:10.1371/journal.pone.0005555.g006
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Figure 7. The change in bhighto blow-cell ratio correlates with the change in b-cell function in animal models with increased insulin
demand. (A) Insulinogenic index (DI/DG) after OGTT in ZDF fa/fa rats compared to ZDF lean controls (12 rats). (B) Representative FACS analyses of
dissociated islet cells in type 2 diabetic ZDF rat, a model of loss of b-cell function. The geometric mean of PSA-NCAM fluorescence is represented by a
blue arrow in control ZDF lean rats and by a red arrow in diabetic ZDF fa/fa rats. The distribution of bhighand blow-cells is determined by the
geometric mean of PSA-NCAM of the control ZDF lean rats (blue arrows) and expressed in percent of total b-cells (n=3–6). (C) Insulin release in
response to 5.5 mM and 16.7 mM glucose in bhighand blow-cells of ZDF rats during perifusion experiments (n=3–6). Quantification of insulin
response to stimulating glucose is represented by DI. (D) Insulinogenic index (DI/DG) after OGTT in HG/HI rats compared to saline-infused controls
(Wistar control). (E) Representative FACS analyses of dissociated islet cells in HG/HI rats and Wistar control rats. The geometric mean of PSA-NCAM
fluorescence is represented by a blue arrow in saline-infused Wistar control rats and by a red arrow in HG/HI rats. The distribution of bhighand blow-
cells is determined by the geometric mean of PSA-NCAM of the control Wistar rats (blue arrows) and expressed in percent of total b-cells (n=3–6). (F)
Insulin release in response to 5.5 mM and 16.7 mM glucose in bhighand blow-cells of control and HG/HI rats during perifusion experiments (n=3–6).
Quantification of insulin response to stimulating glucose is represented by DI. n represents the number of independent cell preparations from at least
6 rats pooled each. Data are means6SEM. *, p,0.05; ***, p,0.005.
doi:10.1371/journal.pone.0005555.g007
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The second model was the HG/HI rat in which the functional
b-cell mass is improved [17]. The insulinogenic index was 2-fold
increased compared with control saline-infused Wistar rats
(Fig. 7D). The geometric mean of PSA-NCAM fluorescence of
b-cells of HG/HI rats (red arrow, Fig. 7E) was strongly right-
shifted compared to that of saline-infused Wistar rats (blue arrow,
Fig. 7E), indicating a striking increase of the expression of PSA-
NCAM on b-cells of HG/HI rats (Fig. 7E). b-cells were
redistributed infavor of
73%63.0% of total b-cells in HG/HI rats as compared to bhigh-
cells of controls (55.3%61.7% of total b-cells) (Fig. 7E). bhighand
blow-cells from HG/HI rats were more responsive than bhighand
blow-cells of control rats (Fig. 7F).
These data show that the distribution of bhighand blow-cells
correlates with physiological or pathological states regarding
functional b-cell mass. Therefore surface expression of PSA-
NCAM reflects the ability of the pancreas to secrete insulin.
bhigh-cells,now representing
Discussion
The main outcome of our study is the demonstration of the
existence of the functional b-cell heterogeneity in vivo. Based on the
specificity of PSA-NCAM expression in pancreatic b-cells, we
correlated this marker with b-cell functional activity in rats and
explored the differences between both groups of cells.
b-cells are heterogeneous in their sensitivity to glucose and to
non-glucidic nutrient secretagogues [5,12]. Their metabolic redox
state differs when stimulated with glucose and it is correlated with
their capacity to secrete insulin [5,8]. Recently expression of E-
cadherin at the surface of b-cells was correlated with their insulin
secretory capacity, promoting E-cadherin as a functional marker of
b-cells [26]. However E-cadherin is expressed in both exocrine and
endocrinetissues [26] incontrast to PSA-NCAM whichis specific to
b-cells as shown here. In the present study, using the arbitrary
parameter of the geometric mean for PSA-NCAM fluorescence, we
discriminated fully functional bhigh-cells from poorly glucose
responsive blow-cells in rats, i.e. bhigh-cells were highly responsive
to glucose whereas blow-cellsshowed weaknessesin insulinsecretion.
The most striking difference between bhighand blow-cells is the
small amplitude of glucose-induced raises in [Ca2+]iand insulin
secretion in blow-cells, reflecting a ticking over glucose metabolism
in blow-cells. The effects of the metabolizable amino acid leucine
were similar to those of glucose which reinforces the hypothesis of
metabolic impairments in blow-cells. Glut2, Gck, Pk, mtGPDH and
Pcx are metabolic enzymes implicated in the delivery of
metabolites to mitochondria and thus the generation of metabolic
signals, such as ATP, required for an appropriate insulin secretory
response to glucose [27,28]. The expression of these genes as well
as the ATP levels were decreased in blow-cells while normally
suppressed metabolic genes (Ldha, Hk1) were upregulated. Such
alterations may be sufficient to interfere with glucose recognition
mechanism by regulating metabolic pathways diversionary to
normal b-cell metabolism [29]. blow-cells also showed deficiencies
in cAMP-dependent pathways as evidenced by the use of GLP-1.
Although the expression of the GLP-1 receptor and ATP content
were diminished in blow-cells, the differential effects of db-cAMP
also imply differences downstream of cAMP generation. Indeed
expression of genes of the cAMP pathway such as Gcgr, Glp1r, PKA
and Rap1a were decreased in blow-cells. Finally blow-cells expressed
high levels of markers of progenitor cells Ngn3 and Tcf7l2 [30]
while islet-associated transcription factors such as Neurod1, Pdx1,
Nkx6.1, Pax6 were downregulated. None of these transcription
factors was completely shut off in blow-cells but modest reductions
have wide repercussions [31,32] including insulin secretion.
bhighand blow-cells also differ from a mechanistic point of view.
First they showed different cellular complexity, implying differences
in intracellular components. bhigh-cells exhibited a fragmented
distribution of F-actin. Conversely blow-cells displayed a strong and
continuous signal as a ring beneath the plasma membrane. This
physical barrier modulates the access of insulin vesicles to the
plasma membrane hence hampering exocytosis [25,33]. In addition
several proteins required in distal steps of exocytosis and its calcium
regulation [34], such as SYT9, STX1, SNAP25 and VAMP2, were
considerably downregulated in blow-cells as well as key pumps and
ion channels such as Kir6.2, Sur1 and Cav1.2, mediators of insulin
secretion. The similar amount of released insulin in response to the
depolarizing agents arginine and KCl may result from the fact that
dynamics of insulin granules differs between K+stimulation and
glucose stimulation[35].Indeedmost ofthe granules responsible for
fusion events induced by K+stimulation consist of already docked
granules to the plasma membrane whereas fusion in glucose
stimulation implies newly recruited granules [35]. Collectively our
data suggest that bhighand blow-cells may differ in their capacity to
recruit new insulin granules to the cell membrane and that blow-cells
are not able to properly respond to glucose in contrast to fully
functional bhigh-cells. Therefore, in normal animals, PSA-NCAM is
a specific marker to detect glucose responsive b-cells.
The physiological relevance of such functional heterogeneity is
underscored by our findings in two animal models in which insulin
demand is increased. In the HG/HI rat, a 48 h glucose infusion
results in an increased functional b-cell mass [17]. In this model,
insulin release in response to glucose of both bhighand blowgroups
of cells was strongly enhanced compared to cells from saline-
infused control rats. In the meantime the proportion of bhigh-cells
rose to 75% of total b-cells compared to 55% in control rats. This
expansion of fully functional bhigh-cells and of their capacity to
release insulin easily explains the functional improvement in HG/
HI rats [17]. It could be hypothesized that blow-cells may be a pool
of cells recruited in presence of hyperglycemia as a component of
endocrine pancreas plasticity. In ZDF fa/fa rats, a genetic model
of type 2 diabetes with decreased functional b-cell mass [16],
insulin release of bhigh-cells from ZDF fa/fa rats was strongly
diminished compared to bhigh-cells from control ZDF lean rats.
Moreover the distribution of bhighand blow-cells was completely
inversed in ZDF fa/fa rats, poorly functional blow-cells becoming
the predominant population (75% of total b-cells versus 37% in
ZDF lean rats). These data perfectly fits with the failure of b-cell
function in diabetic rats, i.e. impaired control of glucose
homeostasis and onset of permanent hyperglycemia. Furthermore
they suggest that an alteration of pancreatic b-cell plasticity, i.e.
inability to recruit fully functional b-cells, is a key component in
the development of type 2 diabetes [36]. Several reasons may
account for the increased number of blow-cells in ZDF fa/fa rats.
ZDF rats have a decreased b-cell mass due to massive apoptosis
[37]. So the glucolipotoxic environment may induce the death of
highly glucose responsive bhigh-cells. Otherwise, it may induce a
loss of their activity favoring the loss of PSA-NCAM mobilization
to the cell surface [15] which increases the number of blow-cells.
The follow-up of diabetic patients and anti-diabetic strategies
requires reliable methods to assess the b-cell mass with non-invasive
imagery procedures. Such approaches rely on the specific labeling of
b-cells using enzymes, cell receptors or surface structures that are
expressed on b-cells. Several b-cell structures, including GLP-1
receptors, sulfonylurea receptors, vesicular amin transporter 2
(VMAT2) or gangliosides have been used as labeling targets.
However none of them has yet provided an accurate estimation of
b-cell mass to justify a routine application in humans [38]. Because
PSA-NCAM is a surface marker specific of functional b-cells at least
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Page 11

in rat,itcan be proposed as a potential tag to investigate functional b-
cell mass by imagery approaches in vivo.
Here we characterized two groups of b-cells with different
functional and gene expression profiles. Taken together the data
suggest that blow-cells are poorly glucose responsive in contrast to
highly responsive bhigh-cells. Moreover, the proportion of bhighand
blow-cellsvariedaccordingtotheinsulindemand andwascorrelated
with the progression of diabetes. PSA-NCAM constitutes defini-
tively a powerful functional b-cell marker which may provide
further insight in b-cell plasticity and lead to the identification of
new targets in order to improve the functional status of b-cells with
low secretory capacities such as in advanced type 2 diabetes.
Supporting Information
Table S1
profiles. Gene expression profiles of bhighand blow-cells (n=3). n
bhighvs blow-cells exhibit different gene expression
represents the number of independent cell preparations from at
least 12 pooled rats each. Date are means6SEM. *, p,0.05, **,
p,0.01 and ***, p,0.005.
Found at: doi:10.1371/journal.pone.0005555.s001 (0.04 MB
DOC)
Acknowledgments
We are very grateful to Drs Y. Emre, F. Criscuolo, H. Oudart for helpful
discussion and comments. We thank N. Kassis, B. Durel and D. Bailbe for
expert technical assistance.
Author Contributions
Conceived and designed the experiments: MK CTC AK CM CK.
Performed the experiments: MK JC CTC MB AG MD SC MR PC BL.
Analyzed the data: MK CTC CM CK. Wrote the paper: MK SL JL CM
CK.
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