Expression of the NH(2)-terminal fragment of RasGAP in pancreatic beta-cells increases their resistance to stresses and protects mice from diabetes.
ABSTRACT Our laboratory has previously established in vitro that a caspase-generated RasGAP NH(2)-terminal moiety, called fragment N, potently protects cells, including insulinomas, from apoptotic stress. We aimed to determine whether fragment N can increase the resistance of pancreatic beta-cells in a physiological setting.
A mouse line, called rat insulin promoter (RIP)-N, was generated that bears a transgene containing the rat insulin promoter followed by the cDNA-encoding fragment N. The histology, functionality, and resistance to stress of RIP-N islets were then assessed.
Pancreatic beta-cells of RIP-N mice express fragment N, activate Akt, and block nuclear factor kappaB activity without affecting islet cell proliferation or the morphology and cellular composition of islets. Intraperitoneal glucose tolerance tests revealed that RIP-N mice control their glycemia similarly as wild-type mice throughout their lifespan. Moreover, islets isolated from RIP-N mice showed normal glucose-induced insulin secretory capacities. They, however, displayed increased resistance to apoptosis induced by a series of stresses including inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N mice were also protected from multiple low-dose streptozotocin-induced diabetes, and this was associated with reduced in vivo beta-cell apoptosis.
Fragment N efficiently increases the overall resistance of beta-cells to noxious stimuli without interfering with the physiological functions of the cells. Fragment N and the pathway it regulates represent, therefore, a potential target for the development of antidiabetes tools.
[show abstract] [hide abstract]
ABSTRACT: beta-Cell apoptosis is a key event contributing to the pathogenesis of type 1 diabetes mellitus. In addition to apoptosis being the main mechanism by which beta cells are destroyed, beta-cell apoptosis has been implicated in the initiation of type 1 diabetes mellitus through antigen cross-presentation mechanisms that lead to beta-cell-specific T-cell activation. Caspase-3 is the major effector caspase involved in apoptotic pathways. Despite evidence supporting the importance of beta-cell apoptosis in the pathogenesis of type 1 diabetes, the specific role of caspase-3 in this process is unknown. Here, we show that Caspase-3 knockout (Casp3(-/-) mice were protected from developing diabetes in a multiple-low-dose streptozotocin autoimmune diabetes model. Lymphocyte infiltration of the pancreatic islets was completely absent in Casp3(-/-) mice. To determine the role of caspase-3-dependent apoptosis in disease initiation, a defined antigen-T-cell receptor transgenic system, RIP-GP/P14 double-transgenic mice with Casp3 null mutation, was examined. beta-cell antigen-specific T-cell activation and proliferation were observed only in the pancreatic draining lymph node of RIP-GP/P14/Casp3(+/-) mice, but not in mice lacking caspase-3. Together, our findings demonstrate that caspase-3-mediated beta-cell apoptosis is a requisite step for T-cell priming, a key initiating event in type 1 diabetes.Molecular and Cellular Biology 06/2005; 25(9):3620-9. · 5.53 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Type 2 diabetes mellitus manifests itself in individuals who lose the ability to produce sufficient amounts of insulin to maintain normoglycaemia in the face of insulin resistance. The ability to secrete adequate amounts of insulin depends on beta-cell function and mass. Chronic hyperglycaemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and playing an essential role in the regulation of beta-cell turnover. This paper will address the effect of chronically elevated glucose levels on beta-cell turnover and function. In previous studies we have shown that elevated glucose concentrations induce apoptosis in human beta-cells due to an interaction between constitutively expressed Fas ligand and upregulated Fas. Human beta-cells produce interleukin (IL)-1beta in response to high glucose concentrations, independently of an immune-mediated process. This was antagonized by the IL-1 receptor antagonist (IL-1Ra), a naturally occurring anti-inflammatory cytokine also found in the beta-cell. Therefore the balance of IL-1beta and IL-1Ra may play a crucial role in the pathogenesis of diabetes. Inhibition of glucotoxicity represents a promising therapeutic stratagem in diabetes therapy to preserve functional beta-cell mass.Hormone Research 01/2004; 62 Suppl 3:67-73. · 2.48 Impact Factor
Article: Decreased beta-cell mass in diabetes: significance, mechanisms and therapeutic implications.[show abstract] [hide abstract]
ABSTRACT: Increasing evidence indicates that decreased functional beta-cell mass is the hallmark of both Type 1 and Type 2 diabetes. This underlies the absolute or relative insulin insufficiency in both conditions. In this For Debate, we consider the possible mechanisms responsible for beta-cell death and impaired function and their relative contribution to insulin insufficiency in diabetes. Beta-cell apoptosis and impaired proliferation consequent to hyperglycaemia is one pathway that could be operating in all forms of diabetes. Autoimmunity and other routes to beta-cell death are also considered. Recognition of decreased functional beta-cell mass and its overlapping multifactorial aetiology in diabetic states, leads us to propose a unifying classification of diabetes.Diabetologia 04/2004; 47(3):581-9. · 6.81 Impact Factor
Expression of the NH2-Terminal Fragment of RasGAP in
Pancreatic ?-Cells Increases Their Resistance to Stresses
and Protects Mice From Diabetes
Jiang-Yan Yang,1,2Jo ¨el Walicki,1,2Evrim Jaccard,1,2Gilles Dubuis,1,2Natasa Bulat,1,2
Jean-Pierre Hornung,2Bernard Thorens,1,3and Christian Widmann1,2
OBJECTIVE—Our laboratory has previously established in
vitro that a caspase-generated RasGAP NH2-terminal moiety,
called fragment N, potently protects cells, including insulinomas,
from apoptotic stress. We aimed to determine whether fragment
N can increase the resistance of pancreatic ?-cells in a physio-
RESEARCH DESIGN AND METHODS—A mouse line, called
rat insulin promoter (RIP)-N, was generated that bears a trans-
gene containing the rat insulin promoter followed by the cDNA-
encoding fragment N. The histology, functionality, and resistance
to stress of RIP-N islets were then assessed.
RESULTS—Pancreatic ?-cells of RIP-N mice express fragment
N, activate Akt, and block nuclear factor ?B activity without
affecting islet cell proliferation or the morphology and cellular
composition of islets. Intraperitoneal glucose tolerance tests
revealed that RIP-N mice control their glycemia similarly as
wild-type mice throughout their lifespan. Moreover, islets iso-
lated from RIP-N mice showed normal glucose-induced insulin
secretory capacities. They, however, displayed increased resis-
tance to apoptosis induced by a series of stresses including
inflammatory cytokines, fatty acids, and hyperglycemia. RIP-N
mice were also protected from multiple low-dose streptozotocin-
induced diabetes, and this was associated with reduced in vivo
CONCLUSIONS—Fragment N efficiently increases the overall
resistance of ?-cells to noxious stimuli without interfering with
the physiological functions of the cells. Fragment N and the
pathway it regulates represent, therefore, a potential target for
the development of antidiabetes tools. Diabetes 58:2596–2606,
is therefore of critical importance to delay or prevent the
development of the disease.
limination of pancreatic ?-cells by apoptosis is a
culminating event leading to type 1 diabetes (1)
and possibly type 2 diabetes (2,3). The develop-
ment of tools favoring ?-cell survival in patients
Apoptosis is induced when a family of proteases called
the caspases is activated (4,5). These enzymes cleave a
subset of cellular proteins, inducing the characteristic
biochemical and morphological features of apoptosis. Pan-
creatic islet cells undergo apoptosis in response to many
stimuli (6), including anoxia (7), nutrient deprivation (8),
hyperglycemia (9), and inflammatory cytokines (10).
Counteracting the proapoptotic effects of caspases would
therefore be advantageous to render islet cells more
resistant to a series of noxious stimuli.
Many proapoptotic signaling pathways have been char-
acterized in ?-cells. These include the Fas death receptor
pathway, the endoplasmic reticulum stress response, and
the activation of the nuclear factor (NF)?B transcription
factor (6,11). The detrimental effect of sustained NF?B
activity observed in ?-cells contrasts with the prosurvival
effect of NF?B activation in many other cell types (7,8). An
elegant in vivo support for the notion that NF?B can be
deleterious in ?-cells comes from the demonstration that
transgenic mice expressing specifically in ?-cells a degra-
dation-resistant NF?B inhibitor are protected from diabe-
togenic agents (12).
On the other hand, antiapoptotic pathways can be
induced in ?-cells to allow for survival in stress conditions.
Akt is a kinase that inhibits apoptosis in many cell types by
regulating a vast variety of pro- and antiapoptotic mole-
cules (13,14). Expression of a constitutively active form of
Akt in ?-cells in mice protected them from experimentally
induced diabetes (15,16). In at least one of the models, this
was accompanied by disturbed ?-cell and islet morphol-
ogy, islet hyperplasia, and, paradoxically, a very significant
increase in the basal ?-cell apoptotic rate (15). The in-
creased rate of proliferation was therefore compensating
for the loss of cells through apoptosis. These data indicate
that expression of an active form of Akt1 in ?-cells
generates two opposing forces: an increase in basal apo-
ptosis and a stimulation of proliferation/growth. The latter
effect eventually promotes the development of insulino-
mas (17). The potential beneficial effects of Akt activity in
?-cells are therefore mitigated by a predisposition toward
malignancy and by an increased susceptibility to cell death
that is most likely mediated by the concomitant activation
of NF?B (6). Thus, unless Akt is prevented from stimulat-
ing NF?B (and hence apoptosis) and from inducing ex-
cessive cell proliferation, it remains unclear whether
expression of an active form of Akt is advantageous for the
long-term survival and functionality of ?-cells.
RasGAP, a regulator of Ras and Rho, is a caspase-3
substrate bearing two cleavage sites. RasGAP is cleaved in
a stepwise manner as caspase activity increases in cells. At
low caspase-3 activity, RasGAP is cleaved only once,
generating an NH2-terminal fragment, called fragment N,
Switzerland; the2Department of Cell Biology and Morphology, University of
Lausanne, Lausanne, Switzerland; and the3Center for Integrative Genomics,
Genopode Building, University of Lausanne, Lausanne, Switzerland.
Corresponding author: Christian Widmann, email@example.com.
Received 22 January 2009 and accepted 2 August 2009. Published ahead of
print at http://diabetes.diabetesjournals.org on 20 August 2009. DOI:
J.-Y.Y., J.W., and E.J. contributed equally to this article.
© 2009 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Department of Physiology, University of Lausanne, Lausanne,
2596 DIABETES, VOL. 58, NOVEMBER 2009diabetes.diabetesjournals.org
that induces a potent antiapoptotic response (18,19). At
higher caspase activity, fragment N is further processed
into two additional fragments, called fragments N1 and N2,
that no longer protect cells (18,20). It is possible, however,
to prevent cleavage of fragment N by replacing, in the
second caspase cleavage site, the aspartate residue at
position 157 with an alanine (18). Fragment N induces cell
survival by activating the Ras-PI3K-Akt pathway (19).
Importantly, not only does fragment N not require NF?B
activity for its antiapoptotic properties, it inhibits the
ability of Akt to activate NF?B (19). This indicates that
different ways of activating Akt (i.e., via expression of an
active mutant of Akt or via expression of fragment N) does
not lead to the same cellular responses. We have recently
demonstrated that expression of fragment N in ?-cells in
vitro leads to the stimulation of Akt-dependent protective
signals while blocking the ability of Akt to activate the
proapoptotic NF?B pathway (21). To determine whether
fragment N would display its protective functions in an in
vivo setting, a transgenic mouse was generated that ex-
presses an uncleavable form of fragment N under the
control of the rat insulin promoter to restrict its expres-
sion in pancreatic ?-cells. This mouse model displayed an
increased resistance to experimentally induced diabetes,
and its ?-cells were less susceptible to apoptosis induced
by a variety of death stimuli.
RESEARCH DESIGN AND METHODS
The supplemental methods for cell culture, chemicals and antibodies, trans-
gene detection by PCR, quantitative PCR, mouse islet isolation and dissocia-
tion, preparationof tissue sections
quantitation, Western blot analysis, Southern blot, nuclear protein extract
preparation, and electromobility shift assay (EMSA) are found in the online
appendix (available at http://diabetes.diabetesjournals.org/cgi/content/full/
Apoptosis assay. Apoptosis ex vivo was assessed by scoring the number of
cells with pycnotic nuclei after Hoechst 33342 staining (20). Apoptosis in vivo
was assessed by a terminal transferase dUTP nick-end labeling (TUNEL)
assay (DeadEnd Fluorometric TUNEL system, catalog no. G3250; Promega,
Basel, Switzerland) on islet paraffin sections as per the manufacturer’s
Animal experimentation. All procedures on mice were performed accord-
ing to the Swiss legislation for animal experimentation. Unless noted other-
wise, the animals were used at an age of 8–12 weeks.
Transgenic lines. The transgenic construct (RIP-HA-N[D157A].xf3) bears
fragment N of RasGAP under the control of the rat insulin promoter (RIP). It
was obtained by ligation of a blunt-ended BamHI/SalI 1.4-kb fragment from
plasmid HA-N(D157A).bs (22) with a blunt-ended XbaI/HindIII 4-kb fragment
from RIP-vMos.xf3 plasmid. The correctness and functionality of the plasmid
were controlled by sequencing and transfection into insulinoma cell lines.
Finally, a BamHI 2.8-kb fragment from RIP-N.xf3 was microinjected into
FVB/N oocytes at the transgenic animal facility of the University of Lausanne.
Four independent RIP-N–expressing founders were obtained. Founders 1 and
2 were used in the experiments described here.
Blood glucose level measurements and intraperitoneal glucose toler-
ance test. Blood glucose content of mice under feeding or fasting (16 h)
conditions was determined with an Accu-Check Compact Plus glucometer
(Roche Diagnostics). For the intraperitoneal glucose tolerance tests (IPGTTs),
fasted (16 h) animals were injected intraperitoneally with 2 mg glucose per
kilogram body weight. Blood glucose levels were determined from a blood
drop taken after a short incision of the tail tip at increasing time intervals
(?30, 0, 15, 30, 60, 90, 120, and 150 min) following glucose injection.
Streptozotocin-induced diabetes. Type 1–like diabetes was induced by
multiple low-dose streptozotocin injections. Briefly, 4-h–fasted female RIP-N
mice were injected intraperitoneally with 50 mg streptozotocin per kg of mice.
This procedure was repeated every day for a total period of 5 days.
Streptozotocin was prepared and diluted in citrate buffer (pH 4.5) (sodium
citrate 25 mmol/l, citric acid 23 mmol/l) just before injection. Control mice
were injected with the citrate buffer alone. Blood glucose levels were assessed
In vitro insulin secretion measurement. Islets were isolated from mice
pancreas as described in the supplemental RESEARCH DESIGN AND METHODS
and immunochemistry, insulin
section in the online appendix. The islets (200 per 100-mm dish in 10 ml
culture medium) were incubated overnight at 37°C, 5% CO2. The next day, the
islets were hand-picked and cultured in Krebs-Ringer bicarbonate HEPES
buffer (KRBH)-BSA (120 mmol/l NaCl, 4 mmol/l KH2PO4, 20 mmol/l HEPES, 1
mmol/l MgCl2, 1 mmol/l CaCl2, 5 mmol/l NAHCO3, pH 7.4, with 0.5% BSA) at
37°C and 5% CO2. The following day, well preserved and good-quality islets
were again hand-picked and placed into 12-well plates (10 islets/well) in 1 ml
KRBH-BSA containing 2.8 mmol/l glucose for 1 h. The islets were then
transferred to new wells containing 2.8 or 20 mmol/l glucose with or without
10 nmol/l exendin-4 (catalog no. H-8370; Bachem) in 1 ml KRBH-BSA and
incubated for 2 additional hours. The supernatant and islets were collected
into separate tubes and placed on ice. The islets were lysed in 500 ?l
acid/ethanol (75% ethanol/1.5% concentrated HCl) and sonicated 15 sec (using
a W-375 cell disruptor from Kontron equipped with a 3-mm tip). Insulin in the
supernatant and extracted islets was measured using an radioimmunoassay
kit (catalog no. RI-13K; Linco).
Statistical analysis. Unless stated otherwise, the statistical analyses were
done with Microsoft Office Excel 2003 SP1 using the two-tailed unpaired
Student t test. Significance is indicated by an asterisk when P ? 0.05/n, where
P is the probability derived from the t test analysis and n is the number of
comparisons done (Bonferroni correction). All the other statistical analyses
were performed with the SAS/STAT software (version 9.1.3; SAS Institute,
Generation of a transgenic mouse expressing frag-
ment N in pancreatic ?-cells. A transgenic vector was
constructed (see RESEARCH DESIGN AND METHODS) so as to
encode an HA-tagged form of fragment N bearing the
D157A mutation (preventing it from being cleaved by
caspases) under the control of the RIP and regulatory
sequences of the simian virus 40 (SV40) gene (Fig. 1A).
The construct was injected into FVB/N oocytes, and
transgene-positive mice were identified by Southern blot-
ting (Fig. 1B). In total, four founder mice were obtained.
The results presented here all include data from founder 1
(labeled mouse 5 in Fig. 1B). When indicated, some
experiments were also performed with mice derived from
founder 2 (labeled mouse 28 in supplemental Fig. S1). By
comparison, with the endogenous insulin promoters, it
was estimated that founders 1 and 2 bore 12–15 and 1
copies of the transgene in their genome, respectively (Fig.
1B and supplemental Fig. S1).
To determine the expression pattern of fragment N in
the transgenic line, lysates from pancreatic islets, liver,
brain, and spleen were analyzed by Western blotting using
antibodies specific for the HA tag or for the NH2-terminal
part of RasGAP. Figure 1C shows that fragment N was, as
expected, only expressed in islet cells. Immunofluores-
cence analysis of both founders revealed that fragment N
was restricted to the endocrine part of the pancreas (Fig.
1D and supplemental Fig. S2A and B) and that the vast
majority of fragment N–expressing cells corresponded to
?-cells (i.e., insulin-containing cells) (Fig. 1E).
Regulation of Akt and NF?B by fragment N in RIP-N
?-cells. In various cell types, fragment N, when ectopi-
cally expressed or when generated in response to mild
stress, activates Akt (19,21,23). As adaptive mechanisms
can take place in vivo, it was important to determine
whether fragment N could induce a chronic Akt activity in
islet cells in mice. Islets isolated from control and RIP-N
mice were therefore analyzed for the presence of activated
Akt. As shown in Fig. 2A, there was a significant approx-
imately threefold increase in Akt activity in islet cells from
RIP-N compared with control islets. This indicates that
fragment N can stimulate Akt on a long-term basis when
expressed in vivo.
A potential important property of fragment N in the
context of ?-cell protection is its ability to block NF-?B
J.-Y. YANG AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 58, NOVEMBER 20092597
: Mice #
729 30 33 34 35 36 38 392 3456
Fragment N HARIPprom
EcoRIEcoRI EcoRIEcoRIEcoRI EcoRI EcoRIEcoRIEcoRI
FIG. 1. Expression and function of fragment N in RIP-N mice. A:
Schematic representation of the RIP-N transgene together with the
strategy for its detection by Southern blot. An HA-tagged form of
fragment N (amino acids 1–455 of RasGAP) followed by an SV40-
derived poly-A sequence was placed under the control of the RIP.
Band A corresponds to the transgene-specific EcoRI Southern blot
fragment. B1 and B2 are examples of EcoRI Southern blot frag-
ments derived from random insertions of the transgene into the
host’s genome. B: Identification of RIP-N transgenic mice. The
progeny of the injected pseudo-pregnant mice were genotyped by
Southern blot (see RESEARCH DESIGN AND METHODS for details). Band A
(2.8 kb) is specific for the transgene. Founder 1 (mouse 5) was
able to transmit the transgene to the F1 generation. C: Tissue expression of fragment N. Lysates from the indicated tissues were analyzed
for the presence of fragment N by Western blot using anti-HA and anti-RasGAP antibodies. D: Expression of fragment N in the pancreas.
The presence of fragment N was assessed by immunofluorescense analysis of paraformaldehyde-fixed cryosections using an antibody
recognizing the HA tag borne by fragment N. E: Colocalization of insulin and fragment N. The specific location of fragment N in pancreatic
?-cells was determined by immunofluorescence of paraformaldehyde-fixed cryo-sections from RIP-N mice using anti-insulin and anti-HA
antibodies. (A high-quality color digital representation of this figure is available in the online issue.)
FRAGMENT N PROTECTS ?-CELLS
2598DIABETES, VOL. 58, NOVEMBER 2009diabetes.diabetesjournals.org
activation. This property, however, had so far only been
evidenced in cultured immortalized cell lines (19,21). As
shown in Fig. 2B, binding of nuclear factors to NF?B
binding elements was markedly diminished in nuclear
extracts from RIP-N mouse–isolated islet cells stimulated
with cytokines compared with similarly treated islets
isolated from control mice. Moreover, cytokine-induced
expression of the transcript encoding inducible nitric
oxide (iNOS) synthase, which participates in ?-cells apo-
ptosis (24) and the gene of which is an NF?B target (25),
also appeared to be impaired in islets cells isolated from
RIP mice compared with wild-type islets (Fig. 2C).
These results indicate that fragment N regulates Akt and
NF?B in ?-cells in vivo in a manner similar to what has
been described using cultured cell lines. As cytokines can
induce apoptosis of ?-cells via NF?B–mediated NO pro-
duction (24), these results also suggest that the ability of
fragment N to protect ?-cells might rely, at least in part, on
its capacity to target the NF?B–iNOS axis.
No detection of fragment N in the brain of RIP-N
mice. It was reported in transgenic models done using
RIP-Cre mice that the RIP promoter can also be active in
the brain (more specifically in the hypothalamus) (26,27).
Immunohistochemical analysis, however, did not reveal
the presence of fragment N in hypothalamic sections from
adult RIP-N mice (supplemental Fig. S3). This indicates
that the RIP-N transgene is not expressed in adult mouse
brain or, if it is expressed, at levels that are much lower
than those detected in the endocrine pancreas and that are
under the sensitivity limit of our assay.
Fragment N expression does not affect islet morphol-
ogy and cellularity. Expression of fragment N in insuli-
nomas and islet cells leads to Akt activation (Fig. 1F) (21).
Since Akt signaling has the potential to stimulate cell
survival and proliferation (28), and since transgenic mice
expressing a constitutively active form of Akt (myr-Akt)
show an increase in both ?-cell size and total islet mass
(15), the presence of fragment N in islets might affect the
morphology and cellularity of the endocrine pancreas.
However, neither the proportion of ?- and ?-cells (Fig.
3A), nor the insulin content of the pancreas (Fig. 3B), were
affected by the presence of fragment N. Moreover, the size
of the islets did not appear to be different in RIP-N
transgenic mice compared with control mice (Fig. 3C).
Finally, the percentage of cells positive for the nuclear
protein Ki67 that is preferentially expressed in dividing
cells was similar in both types of mice (Fig. 3D). These
results indicate that fragment N does not favor ?-cell
proliferation in an in vivo setting and that it does not affect
the normal development of the endocrine pancreas. Con-
sistent with this notion is the observation that RIP-N mice
did not develop insulinomas over an 18-month period (as
assessed by a drop in glucose blood level and increased
mortality) (Fig. 8).
iNOS mRNA levels
(normalized to S18 levels;
% value at 6 hour)
FIG. 2. Fragment N activates Akt and inhibits NF?B in islet ? cells. A:
Lysates from islets isolated from the indicated mice were analyzed by
Western blot for the presence of fragment N using an HA-specific
antibody and for the activation of Akt using a phospho-specific anti-
Akt antibody (p-Akt). An Akt-specific antibody was used to assess
evenness in loading (total Akt). The numbers under the blots corre-
spond to the quantitation (arbitrary units) of the detected bands
(means ? SD of three independent determinations). The asterisk
indicates a statistically significant difference as assessed by a paired t
test analysis. B: Islets isolated from wild-type (?/?) and RIP-N mice
(?/RIP-N) were stimulated or not for 30 min with inflammatory
cytokines (1,000 units/ml tumor necrosis factor-?, 1,000 units/ml in-
terleukin-1?, and 50 units/ml interferon-?). The ability of nuclear
proteins to interact with an NF?B-binding element-bearing radioactive
probe was then monitored by EMSA as described in RESEARCH DESIGN AND
METHODS. The locations of p65-p50 and p50-p50 complexes are indi-
cated. The asterisk denotes a nonspecific band. This experiment was
repeated once with similar results. C: Islets isolated from wild-type
(?/?) and RIP-N mice (?/RIP-N) were stimulated or not for the
indicated periods of time with 1,000 units/ml of interleukin-1?. The
expression of iNOS mRNA was then measured by quantitative real-time
PCR, normalized as described in RESEARCH DESIGN AND METHODS and
expressed as percent of the 6-h values. The results correspond to the
means ? SE of three independent experiments performed in triplicate.
The asterisk indicates a significant difference as determined by a
nonparametrical Wilcoxon’s signed-rank test.
J.-Y. YANG AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 58, NOVEMBER 20092599
Islets from RIP-N transgenic mice display increased
resistance to basal- and stress-induced apoptosis. In
wild-type mice, the basal apoptotic rate in islets is very low
(? 0.5%; Fig. 6B) or undetectable (15). In contrast, islets
from transgenic mice expressing a constitutively active
form of Akt show a marked increase in ?-cell apoptosis
(15). Despite the ability of fragment N to activate Akt (Fig.
2A), there was no associated increase in the basal apoptotic
rate in islets from RIP-N mice compared with the wild-type
controls, either in vitro or in vivo (Fig. 4A and first two bars
of Fig. 6B). Moreover, islets isolated from RIP-N mice were
more resistant than those isolated from control mice when
subjected to a variety of stress stimuli, including inflamma-
tory cytokines, the free fatty acid palmitate, and high glucose
concentrations (Fig. 4). These results demonstrate that frag-
ment N efficiently protects pancreatic ?-cells against various
noxious conditions and stimuli, including some that are
associated with the development of type 1 and type 2
diabetes (e.g., inflammatory cytokines and free fatty acids).
Fragment N does not adversely affect ?-cell functions
in vivo. Transgenic mice expressing a nondegradable
form of I?B? under the control of Pdx1 promoter, which
% total islet cells
(ng insulin per mg pancreas)
p = 0.87
p = 0.41
p = 0.73
Number of cells per islet section
p = 0.18
FIG. 3. Fragment N expression does not affect islet morphology and cellularity. A: The identification of ?- and ?-cells was determined by
immunohistochemistry of paraffin-embedded pancreas sections with antibodies directed against glucagon (dark brown staining) and insulin
(purple-red staining). The graph depicts the proportion of ?- and ?-cells in islets derived from the analysis of an average of 25 islets per 9- to
12-week-old wild-type and ?/RIP-N mice. Data from individual mice are shown (the numbers in the sex symbols indicate which founder the animals
are derived from) as well as the means ? SD values (indicated by the ? symbol in the gray area). B: Freshly isolated pancreata were homogenized
and extracted with acid ethanol. Insulin concentration in the supernatant was determined by enzyme-linked immunosorbent assay. The results
correspond to the means ? SD of nine (wild-type) and six (RIP-N) pancreata. C: The graph depicts the number of cells-per-islet section counted
on hematoxylin-eosin–stained paraffin-embedded pancreas sections. The results are presented as in A and were derived from the analysis of 9-
to 12-week-old wild-type and ?/RIP-N animals (12 mice per genotype; an average of 90 islets per mouse were analyzed). D: The percentage of
proliferating cells was determined by scoring Ki67-positive cells on paraffin-embedded pancreas sections (the arrow points to a Ki67-positive
cell). The bar graph depicts the percentage of proliferating cells in islets (means ? SD) derived from the analysis of at least 20 histological slices
obtained from nine mice per genotype. (A high-quality color digital representation of this figure is available in the online issue.)
FRAGMENT N PROTECTS ?-CELLS
2600DIABETES, VOL. 58, NOVEMBER 2009 diabetes.diabetesjournals.org