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From the Pacific Northwest Research Institute and Department of
Pharmacology, University of Washington, Seattle, Washington 98122
Received for publication, August 27, 2002, and in revised form, October 3, 2002
Free fatty acids (FFA) have been reported to
reduce pancreatic In insulin-resistant states such as obesity, there is a
compensation of increased pancreatic The correct balance between the level of apoptosis and cell
proliferation is a crucial factor in maintaining an appropriate mass of
fully functional In In this study, we have determined that FFA can induce apoptosis in the
pancreatic Materials--
The oleic acid (OA) and OA methyl ester were
purchased from Alltech (State College, PA). Hoechst 33342 (HO) and
propidium iodide (PI) were purchased from Sigma, and the annexin
V-fluorescein isothiocyanate (FITC) kit was form Molecular Probes
(Eugene, OR). The p53wt and mutant vectors were purchased from BD
Biosciences Clontech (Palo Alto, CA). Total-FKHR
(for the detection of rat FoxO1) was from Upstate Biotechnology, Inc.
(Lake Placid, NY), and phospho-Ser256 FKHR antibody (for
the detection of rat phospho-FoxO1) was from Cell Signaling (Beverly,
MA). The phospho-GSK3 Cell Culture--
The glucose-sensitive pancreatic Construction of Adenoviruses--
The pUSEamp expression vectors
containing Myc-His-tagged mouse Akt1 (wild type), myr-Akt1 (activated),
and Akt1-K179M (kinase-dead) from Upstate Biotechnology, Inc. were used
for construction of the PKB adenoviruses, which have been described
previously (14). The constitutively active form has c-Src-derived
residues fused to PKB, and the consequent myristoylation directly
targets PKB to the plasma membrane leading to its constitutive
activation (19). The kinase-dead form has a point mutation, K179M,
removing the ATP-binding site, resulting in loss of kinase activity
(20). The p53 wild type and p53 mutant cDNAs (from BD Biosciences
Clontech) express the wild type or a dominant
negative p53 (p53mt135) tumor suppressor protein, respectively. The
dominant negative mutant differs form the wild type p53 by a G to A
conversion at nucleotide 1017, which causes a conformational change
that not only prevents interaction with p53 DNA binding sites, but may
also bind to and inhibit wild type p53. The p53 cDNAs were digested
out of the vector pCMV-p53 or pCMV-p53mt135 with the restriction
enzymes HindIII and EcoRI, then inserted between
the HindIII and EcoRI sites of pBluescript,
providing the necessary restriction sites for insertion into
pACCMV.pLpA between KpnI and XbaI. The p53 adenoviruses were generated and purified as previously described (14,
21, 22). As control a recombinant adenovirus expressing green
fluorescent protein (GFP) was used and generated as previously described (21).
Adenovirus (AdV) Infection--
Initially, the appropriate titer
for each recombinant AdV was determined by the addition of various
dilutions of each adenovirus to INS-1 cells subcultured in six-well
plates (9.5 cm2) to 60% confluence (~2 × 106 cells), giving a multiplicity of infection (m.o.i.)
ranging from 50 to 2500 based on 0.5-2.0 × 106
plaque-forming units/ml as measured by A260. For
the OA/BSA-treated experiments, INS-1 cells were subcultured on 10-cm
plates to ~60% confluence and then infected with the indicated dose
of adenovirus. For all infections, the viral stock was replaced with
complete medium after 2 h and the cells were incubated at 37 °C
in 5% CO2 for ~16 h. The AdV-infected cells were then
used experimentally as indicated.
FFA Treatment--
INS-1 cells were subcultured on 10-cm plates
to ~60% confluence with or without recombinant AdV infection (see
"Adenovirus (AdV) Infection") as indicated for each experiment. The
cells were then incubated in the modified INS-1 cell RPMI 1640 medium at 5 or 15 mM glucose ± 10 ng/ml IGF-1 with 0.5%
(w/v) BSA alone or 0.4 mM OA complexed to 0.5% (w/v) BSA
for 16 to 18 h. Preparation of the 0.4 mM OA, 0.5%
BSA complex solution was carried out as described previously (5).
Briefly, a 100 mM OA stock solution was prepared in 0.1 mM NaOH by heating at 70 °C. A 10% FFA-free BSA
solution was prepared in H2O and maintained at 55 °C in
an adjacent water bath. The appropriate amount of 100 mM OA
stock solution was added to the BSA and incubated for another 30 min at
55 °C. The OA/BSA complex stock solution was then cooled to 25 °C, filter sterilized, and stored at Apoptosis Assay--
Apoptotic measurements were carried out
using a fluorometric method and counting cell numbers under a
fluorescent microscope as described previously (23). This method
involves the use of DNA-binding dyes HO and PI. The HO compound is
known to cross the plasma membrane of all cells, whether they are
damaged or not, causing a blue fluorescence of their nuclei. The polar
PI only penetrates cells with damaged membranes and leads to nuclear fluorescence. The percentage of apoptotic cells were counted by viewing
cells under an inverted fluorescence microscope. After treating cells
for 18 h with various treatments (see "OA/BSA Treatment"), the
cells were incubated with 20 µg/ml HO and 10 µg/ml PI at 37 °C,
5% CO2 for 15 min. The medium was then removed by
aspiration, and the cells were washed once with PBS and then fixed by
incubation with 4% formaldehyde for 15 min at room temperature. The
formaldehyde was removed by aspiration, mounting fluid added and the
number of apoptotic cells counted under an inverted fluorescent
microscope. A minimum of 500 cells were counted for each plate under
randomized conditions. In a few instances, the levels of apoptosis and
necrosis were also assessed using an annexin V-FITC staining kit (24) and complementary findings to that using the HO/PI method were found.
In these studies, the incidence of necrosis only accounted for <1% of
dead cells.
Immunoblot Analyses--
Following the 18-h incubation at 5 or
15 mM glucose with or without IGF-1 ± OA/BSA, the
cells/media were centrifuged at 1200 rpm for 5 min to pool floating and
attached cells. The INS-1 cells were then lysed in ice-cold cell lysis
buffer consisting of 50 mM HEPES (pH 7.5), 1% (v/v)
Nonidet P-40, 2 mM activated sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 4 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
1 µg/ml leupeptin, and 1 µg/ml aprotinin, then sonicated (25 watts;
10 s, on ice), and particulate material removed by centrifugation
(10,000 × g; 10 min; 4 °C). The supernatants were
collected and stored at Other Procedures--
Where appropriate, data are presented as a
mean ± S.E. Statistically significant differences between groups
were analyzed using Student's t test, where
p FFA-induced Apoptosis in the Pancreatic FFA Reduced PKB Phosphorylation Activation in Pancreatic Adenoviral Mediated Expression of Constitutively Active PKB
Prevented FFA-induced INS-1 Cell Apoptosis--
Having ascertained
that FFA can decrease the activation of PKB in association with
FFA-induced
In the AdV-GFP-infected control INS-1 cells in the absence of FFA, the
incidence of apoptosis was low, ranging from 1.0 to 1.7% of the INS-1
cell population irrespective of the glucose/IGF-1 conditions (Fig. 3).
In contrast, in the presence of FFA, the level of INS-1 cell apoptosis
in AdV-GFP-infected control cells incubated at a basal 5 mM
glucose was significantly increased 23-fold to 39.6 ± 5.9%
(p
In AdV-PKB-KD-infected INS-1 cells, there was negligible difference in
FFA-induced apoptosis compared with that in AdV-GFP-infected control
INS cells, with comparable rates of apoptosis at the various incubation
conditions of 5 or 15 mM glucose ± IGF-1 (Fig. 3). However, in AdV-PKB-WT-infected INS-1 cells, FFA-induced apoptosis was
generally reduced compared with that in AdV-GFP-infected control cells.
At 5 mM glucose + FFA, the incidence of apoptosis in
AdV-PKB-WT-infected INS-1 cells was 21.1 ± 2.3%
(n = 7), significantly decreased compared with
FFA-treated AdV-GFP-infected INS-1 cells (p Inhibition of FFA-induced
For INS-1 cells infected with the various AdV-PKB constructs, the
"infected PKB" could be distinguished from the endogenous PKB on
the immunoblot analyses as a result of the addition of a Myc-His tag
that increased the apparent Mr (Fig.
4A) as previously observed (14). In FFA-treated
Adv-GFP-infected control cells as well as AdV-PKB-WT-, AdV-PKB-CA-, and
AdV-PKB-KD-infected INS-1 cells, the 15 mM glucose- and
IGF-1-induced Ser473 and Thr308 phosphorylation
of the endogenous PKB was inhibited compared with Adv-GFP-infected
control cells not incubated with FFA (Fig. 4A), as
demonstrated elsewhere (Fig. 2). Nonetheless, despite the presence of
FFA, in AdV-PKB-WT-infected INS-1 cells there was an IGF-1, and 15 mM glucose, stimulation of both Ser473 and
Thr308 phosphorylation of the "infected PKB-WT" (Fig.
4A) that correlated with the modest decrease in FFA-induced
apoptosis in AdV-PKB-WT-infected INS-1 cells over the 18-h incubation
period (Fig. 3). As predicted, the PKB Ser473 and
Thr308 phosphorylation activation state of the "infected
PKB-CA" in AdV-PKB-CA-infected INS-1 cells was markedly increased to
an equivalent extent regardless of the glucose/IGF-1 incubation
conditions (Fig. 4A), consistent with the constitutive
activation of this PKB variant and correlative with the prevention of
FFA-induced apoptosis in AdV-PKB-CA-infected INS-1 cells (Fig. 3). As
previously observed (14), glucose/IGF-1-induced PKB Thr308
phosphorylation of the "infected PKB-KD" in AdV-PKB-KD-infected INS-1 cells was equivalent to that of the "infected PKB-WT" in AdV-PKB-WT-infected INS-1 cells, but that of PKB Ser473
phosphorylation was very much reduced (Fig. 4A). This is
consistent with the idea that Ser473 phosphorylation is an
autophosphorylation by PKB and that infected PKB-KD lacks PKB activity
(14, 16). Moreover, this observation correlated with a lack of
protection from FFA-induced apoptosis in AdV-PKB-KD-infected INS-1
cells (Fig. 3). The total levels of endogenous PKB and infected PKB did
not appreciably change in these experiments, underlining the specific
effects observed on PKB phosphorylation (Fig. 4A).
The final stages of apoptosis are executed by the family of caspases,
including caspase-9 and the effector caspase-3 (26). Consistent with
the low incidence of apoptosis in AdV-GFP-infected control cells not
incubated with FFA (Fig. 3), negligible activation of caspase-9 or -3 was detected (Fig. 4B). In contrast, in FFA-treated AdV-GFP-infected INS-1 cells, significant activated caspase-9 and -3 could be detected at 5 mM glucose that was correlatively decreased upon addition of IGF-1 and/or increasing to a 15 mM glucose concentration (Fig. 4B). A similar
pattern of FFA-induced caspase-9 and -3 activation was observed in
AdV-PKB-WT- and AdV-PKB-KD-infected INS-1 cells (Fig. 4B).
However, in AdV-PKB-CA-infected INS-1 cells, negligible activated
caspase-9 or -3 was observed irrespective of the glucose/IGF-1
incubation conditions (Fig. 4B). Hence, the pattern of
caspase-9 and -3 activation observed in these studies (Fig.
4B) correlated with the degree of FFA-induced apoptosis (Fig. 3) and inversely correlated with the extent of PKB
phosphorylation activation (Fig. 4A). These measurements of
caspase-9 and -3 activation corroborate well with the other
determinations of apoptosis using the HO/PI staining technique (Figs. 1
and 3), and annexin V-FITC analysis. This further suggested that most
of the FFA-induced FFA-induced Inhibition of Glucose/IGF-1-stimulated GSK3 and FoxO1
Phosphorylation in INS-1 Cells Is Rescued by Expression of
Constitutively Active PKB--
The effect of FFA on the PKB-mediated
phosphorylation inhibition of two known pro-apoptotic proteins,
GSK3
The phosphorylation state of FoxO1 was analyzed by immunoblotting with
a phosphospecific antibody that recognizes FoxO1 when phosphorylated at
Ser256. In AdV-GFP-infected INS-1 cells incubated in the
absence of FFA, phosphorylation of FoxO1 was increased above that at
basal 5 mM glucose by addition of IGF-1 or raising the
glucose concentration to 15 mM (Fig.
5A). The combination of 15 mM glucose and IGF-1 further increased FoxO1
phosphorylation in these AdV-GFP-infected control cells (Fig.
5A). In FFA-treated AdV-GFP-infected INS-1 cells, this
glucose/IGF-1-induced FoxO1 phosphorylation was clearly decreased (Fig.
5A). Likewise, in FFA-treated AdV-PKB-KD-infected INS-1
cells, the glucose/IGF-1-induced FoxO1 phosphorylation was similarly
decreased, as it was in FFA-treated AdV-PKB-WT-infected INS-1 cells,
albeit to a lesser extent, particularly at 15 mM glucose + IGF-1 (Fig. 5A). In contrast, in FFA-treated
AdV-PKB-CA-infected cells, the levels of FoxO1 phosphorylation were
comparable with those for untreated AdV-GFP-infected control cells,
especially under stimulated conditions at 15 mM
glucose ± IGF-1 (Fig. 5A). Immunoblot analysis of the
total FoxO1 indicated equivalent FoxO1 levels under all the conditions
(Fig. 5A). It should be noted that several FoxO1 bands were
observed on the immunoblot analysis, likely reflective of the
phosphorylation state of FoxO1 at multiple Ser/Thr residues (27), a
notion substantiated in resolving fewer FoxO1 bands after treating
INS-1 cell lysates with alkaline phosphatase prior to immunoblot
analysis (data not shown; Ref. 28).
Phosphorylation of GSK3 Effects of Adenoviral Mediated Expression of p53 Wild Type and a
p53 Variant on FFA-induced Apoptosis in INS-1 Cells--
One potential
mechanism through which PKB may promote
The effect of p53-WT and p53-MT expression on FFA-induced apoptosis was
examined. INS-1 cells were infected with 15 × 102
m.o.i. of AdV-p53-WT and AdV-p53-MT, as well as AdV-GFP and AdV-PKB-CA as negative and positive controls, respectively, and treated with or
without 0.4 mM OA plus 0.5% BSA at basal 5 mM
glucose for 18 h as indicated. In the absence of FFA, the
incidence of apoptosis in the INS-1 cell population was low and similar
(between 1.4 and 1.9%) in the AdV-GFP-, AdV-PKB-CA-, and
AdV-p53-MT-infected INS-1 cells, whereas, in AdV-p53-WT-infected cells,
there was a significant 4-fold increase in the incidence of apoptosis
to 6.1 ± 0.9% of the INS-1 cell population compared with the
AdV-GFP-infected control cells (p In this study we have shown that prolonged exposure to the FFA,
oleate, induced Increasing the glucose concentration from a basal 5 mM
glucose, and/or addition of IGF-1, tended to decrease the degree of FFA-induced Notwithstanding, insufficient PKB activation can induce Remarkably, the In this regard, we examined several downstream PKB targets to determine
whether their regulation, as influenced by PKB, could be associated
with the degree of FFA-induced In addition to GSK3 and FoxO1, MDM2, an ubiquitin-protein isopeptide
ligase, has also been identified as a substrate for PKB (17). MDM2 is
involved in the regulation of the tumor suppressor protein, p53, which
is a transcription factor known to induce apoptosis (30). Following
phosphorylation by PKB, MDM2 translocates to the nucleus, whereupon it
interacts with p53, suppressing the activity and promoting p53
degradation, which in turn further reflects the anti-apoptotic action
of PKB activation (17). We did not have the tools at hand to directly
examine MDM2 phosphorylation by PKB; however, we determined whether p53
might be involved in FFA-induced In conclusion, chronic exposure of FFA can promote We thank Cynthia Jacobs for assistance in
preparation of this manuscript.
*
This work was supported by National Institutes of Health
Grants DK 55269 and DK60266 and by a grant from the Juvenile Diabetes Research Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Current address: Klinik und Poliklinik Fuer Innere Medizin I,
Universität Regensburg, Franz-Josef-Strauss Allee 11, 93042 Regensburg, Germany.
¶
To whom correspondence should be addressed: Pacific Northwest
Research Inst., 720 Broadway, Seattle, WA 98122. Tel.: 206-860-6777; Fax: 206-726-1202; E-mail: cjr@pnri.org.
Published, JBC Papers in Press, October 21, 2002, DOI 10.1074/jbc.M208756200
2
C. Yu, Y. Chen, H. Zong, Y. Wang, R. Bergeron,
J. K. Kim, G. W. Cline, S. W. Cushman, G. J. Cooney, B. Atcheson, M. F. White, E. W. Kraegen, and G. I. Shulman, manuscript in preparation.
The abbreviations used are:
FFA, free fatty
acid;
IGF-1, insulin-like growth factor 1;
PI3K, phosphatidylinositol
3'-kinase;
ERK1/ERK2, extracellular signal-regulated protein kinases 1 and 2;
IRS-2, insulin receptor substrate 2;
PKB, protein kinase B (also
known as Akt);
PDK1, 3-phosphoinositide-dependent kinase;
Fox, forkhead box;
GSK3
Protein Kinase B/Akt Prevents Fatty Acid-induced Apoptosis in
Pancreatic
-Cells (INS-1)*
§,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell mitogenesis and to increase apoptosis. Here
we show that the FFA, oleic acid, increased apoptosis 16-fold in
the pancreatic -cell line, INS-1, over a 18-h period as assessed by
Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the
phosphorylation activation of protein kinase B (PKB) showed this was
reduced in the presence of FFA that correlated with the incidence of
apoptosis. At stimulatory 15 mM glucose and/or in the
added presence of insulin-like growth factor 1, FFA-induced -cell
apoptosis was lessened compared with that at a basal 5 mM
glucose. However, most strikingly, adenoviral mediated expression of a
constitutively active PKB, but not a "kinase-dead" PKB variant,
essentially prevented FFA-induced -cell apoptosis under all
glucose/insulin-like growth factor 1 conditions. Further analysis of
pro-apoptotic downstream targets of PKB, implicated a role for
PKB-mediated phosphorylation inhibition of glycogen synthase
kinase-3
/ and the forkhead transcription factor, FoxO1, in
protection of FFA-induced -cell apoptosis. In addition,
down-regulation of the pro-apoptotic tumor suppresser protein, p53, via
PKB-mediated phosphorylation of MDM2 might also play a role in
partially protecting -cells from FFA-induced apoptosis. Adenoviral
mediated expression of wild type p53 potentiated FFA-induced -cell
apoptosis, whereas expression of a dominant negative p53 partly
inhibited -cell apoptosis by ~50%. Hence, these data demonstrate
that PKB activation plays an important role in promoting pancreatic
-cell survival in part via inhibition of the pro-apoptotic proteins
glycogen synthase kinase-3/, FoxO1, and p53. This, in turn,
provides novel insight into the mechanisms involved in FFA-induced
-cell apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell mass and function so that insulin production is up-regulated and diabetes does not develop. However, with time and/or severity of the insulin resistance, pancreatic -cell dysfunction and an inadequate -cell mass
develop, so that it can no longer compensate for the peripheral insulin resistance and type 2 diabetes results (1-3). Although the
pathophysiological relevant factors that prevent expansion or lead to a
reduction in -cell mass have not been identified, studies have led
to the proposal that chronic exposure to high levels of long chain free fatty acids (FFA)1 are a
contributing factor (4). The FFA-induced reduction in -cell mass has
been attributed, in part, to a decrease in -cell proliferation (5)
and also decreased -cell survival (1, 6, 7).
-cells within the pancreatic islet (8). A number of
nutrients and growth factors can activate mitogenic signaling pathways
to increase -cell proliferation (3). For example, glucose can
independently induce -cell mitogenesis, and intriguingly growth
factors such as insulin-like growth factor-1 (IGF-1) and growth hormone
induce -cell proliferation in a glucose-dependent manner
(9, 10). Characterization of the pathways involved in this mitogenic
stimulation have shown that the phosphatidylinositol 3'-kinase (PI3K)
and mitogen-activated protein kinase (ERK-1/ERK-2) signaling pathways,
downstream of insulin receptor substrate-2 (IRS-2), play an important
role in -cell growth and survival (3, 11). However, little is known
about the signaling mechanisms that are involved in preventing
pancreatic -cell death. Several studies in other cell types have
implicated an important role for early upstream activation of protein
kinase B (PKB, also known as Akt) in signaling pathways for maintaining
cell survival (12). In this regard and relevant to pancreatic
-cells, it has recently been shown that -cell-specific expression
of a constitutively active form of PKB in transgenic mice markedly
increases -cell mass, mostly by preserving -cell survival and
increasing -cell size (13). In contrast, there appears to be less of
a role for PKB in promoting -cell proliferation (14).
-cells, PKB activation can be mediated by IGF-1-induced tyrosine
phosphorylation of IRS-2 leading to PI3K activation that generates
phosphatidylinositol 3,4,5-triphosphate (11). The increase in
phosphatidylinositol 3,4,5-triphosphate results in PKB translocation to
the plasma membrane via its pleckstrin homology domain (15), where the
constitutively active 3-phosphoinositide-dependent kinase-1
(PDK1) phosphorylates PKB at Thr-308 followed by another phosphorylation at Ser-473 thought to be by PKB autophosphorylation (16). PKB has a myriad of protein substrates that could influence apoptosis, including the family of forkhead box (Fox) transcription factors, glycogen synthase kinase-3 / (GSK3/), BAD, caspase 9 (12), and murine double minute 2 (MDM2) (17). There are three known
isoforms of forkhead factors, FoxO1, FoxO3a, and FoxO4 (also known as
FKHR, FKHR-L1, and AFX, respectively), which, upon phosphorylation by
PKB, appear to be retained in the cytoplasm preventing their
translocation into the nucleus, where they mediate transcription of
pro-apoptotic factors. GSK3 is also an important pro-apoptotic
signaling protein and the kinase activity of GSK3 is inhibited by PKB
phosphorylation. PKB phosphorylation has also been shown to inhibit the
activity of components of the apoptotic machinery including the Bcl2
family member, BAD, and the cysteine protease, caspase 9. A recent
finding has identified the ubiquitin ligase protein, MDM2, as a direct
target of PKB, and interestingly, when phosphorylated by PKB, MDM2
negatively regulates the tumor suppresser protein, p53, a known
pro-apoptotic transcription factor. Although several pro-apoptotic
substrates are directly inhibited by PKB-induced phosphorylation, it is
currently unclear to what extent PKB activation is protective of
-cell apoptosis and, if so, which of the PKB substrates might be
relevant in terms of promoting -cell survival.
-cell line, INS-1, in a dose-dependent manner. Moreover, FFA also induced an inhibition of IGF-1-induced PKB
phosphorylation activation complementary to previous observations of
FFA-induced inhibition of PKB activity (5). Intriguingly, adenoviral
mediated expression of constitutively active PKB completely protected
-cells from FFA-induced apoptosis. As such, we provide evidence that
PKB plays an important role in protecting pancreatic -cells against
a physiologically relevant factor, FFA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ (Ser21/9), total-PKB,
phospho-Ser473 PKB, phospho-Thr308 PKB, p53,
and the caspase-9 cleaved antibodies were from Cell Signaling (Beverly,
MA). Caspase-3 antibody was a gift from Dr. Nancy Thornberry (Merck
Research Laboratories, Rahway, NJ). The total and phospho-ERK1/ERK2
antibodies were obtained from Promega Corp. (Madison, WI) and total
GSK3 / antibody was from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-rabbit and anti-sheep IgG horseradish peroxidase
conjugates were from Jackson ImmunoResearch (West Grove, PA) and the
anti-mouse IgG horseradish peroxidase conjugate was from Upstate
Biotechnology, Inc. IGF-1 was purchased from Gro Pep Pty Ltd (Adelaide,
Australia). DNA purification kits and Superfect transfection reagents
were purchased from Qiagen (Valencia, CA). Restriction enzymes were
from New England Biolabs. The bicinchoninic acid (BCA) protein assay
kit was purchased from Pierce. The chemiluminescence reagent was from
NEN Life Sciences. Unless otherwise stated, all other reagents
were of analytical grade from either Sigma or Fisher Scientific.
-cell
line, INS-1 (18), was maintained in the complete medium RPMI 1640 (11.2 mM glucose) containing 10% (v/v) fetal calf serum, 50 µm
-mercaptoethanol, 10 mM HEPES, 2 mM
glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin,
100 µg/ml streptomycin and incubated at 37 °C, 5% CO2 as described (18).
20 °C until use.
80 °C pending immunoblot analysis. For
immunoblot analysis, cell lysates were first normalized for equivalent
total protein levels, as determined using the BCA protein assay kit.
Immunoblot analysis on cell lysates was then performed as previously
described (9, 14, 21).
0.05 was considered statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell, INS-1--
OA
was used as a "model FFA" in these studies, especially because OA
has been previously shown to inhibit glucose/IGF-1-induced -cell
mitogenesis independent of ceramide production (5), and induce
apoptosis, while having only a minor effect on cell necrosis in
-cells (7, 25). In dose-response experiments, it was examined what
concentration of OA would induce apoptosis in pancreatic -cells,
INS-1, over an 18-h period at a constant 5 mM glucose. The
concentration of BSA present was fixed at 0.5% (w/v), and the OA
concentration complexed was varied at 0.25, 0.3, 0.35, and 0.4 mM OA. The basal rate of apoptosis in the presence of 0.5%
BSA and absence of OA was 2.9 ± 0.1% of the total INS-1 cell
population undergoing apoptosis (n = 7; Fig.
1). At 0.25 mM OA and 0.5%
BSA (Fig. 1), the rate of apoptosis was unaltered compared with the
basal rate at 2.1 ± 0.2% (n = 3; Fig. 1).
However, at 0.3 mM OA and 0.5% BSA, the level of apoptosis
was increased to 5.6 ± 1.6% (n = 3), and at 0.35 mM OA and 0.5% BSA, it was significantly increased 4-fold
above the basal rate to 11.1 ± 3.6% (p 0.05;
n = 3) (Fig. 1). The highest rate of OA-induced apoptosis was observed at 0.4 mM OA and 0.5% BSA, which
was 16-fold (p 0.05) above the basal rate at
46.4 ± 6.4% (n = 8) of INS-1 cells being
apoptotic (Fig. 1). Interestingly, in INS-1 cells incubated with 0.4 mM methyl OA in the presence of 0.5% (w/v) BSA, the rate
of apoptosis was significantly lower (p 0.01) compared with those exposed to 0.4 mM OA and 0.5% BSA in
parallel experiments, and not significantly different from the basal
rate at 3.7 ± 1.5% (n = 3) apoptotic INS-1 cells
(Fig. 1). We found that there was negligible change in the occurrence
of INS-1 cell necrosis under these OA/BSA incubation conditions, as
judged by the HO/PI staining assay (Fig. 1), and also as assessed by
annexin V-FITC analysis (data not shown), in agreement with previous
studies (7). In a limited number of experiments, it was also found that
palmitate could also induce INS-1 cell apoptosis similarly to that by
oleate (data not shown), as previously observed (7).
View larger version (50K):
[in a new window]
Fig. 1.
FFA-induced apoptosis in the pancreatic
-cell, INS-1. INS-1 cells were cultured on
10-cm plates and incubated for 18 h with either 0.5% BSA
or 0.25, 0.3, 0.35, or 0.4 mM OA complexed to 0.5% BSA or
0.4 mM methyl OA plus 0.5% BSA. The percentage of
apoptosis was then assessed as described under "Apoptotic Assay."
Sample photographs of the HO- and PI-stained cells are shown for each
treatment. Arrows indicate HO-stained cells that have formed
apoptotic bodies. The results shown are representative of three
independent experiments.
-Cells,
INS-1--
INS-1 cells ± OA as a model FFA (0.4 mM OA + 0.5% BSA, or 0.5% BSA), were incubated for 18 h in the presence
of 5 or 15 mM glucose ± 10 ng/ml IGF-1 and then
phosphorylation activation of PKB assessed by immunoblot analysis using
PKB phosphospecific antibodies directed at phospho-Ser473
and phospho-Thr308. In the absence of FFA, PKB
phosphorylation was similarly stimulated in INS-1 cells by IGF-1, at
either 5 or 15 mM glucose (Fig.
2), indicative of the glucose-independent
aspect of IGF-1-induced PKB activation as previously reported (14).
However, a slight increase in PKB phosphorylation activation was also
observed in the absence of FFA in INS-1 cells chronically incubated for
18 h at a stimulatory 15 mM glucose compared with
basal 5 mM glucose (Fig. 2). However, in INS-1 cells
incubated with FFA, this modest 15 mM glucose-induced
increase in PKB phosphorylation was not apparent (Fig. 2). Moreover,
IGF-1-induced PKB phosphorylation at both 5 and 15 mM
glucose was significantly reduced in the presence of FFA (Fig. 2).
Immunoblot analysis of total PKB indicated equivalent levels of PKB
were present in INS-1 cells under the various incubation conditions
(Fig. 2). These observations of specific FFA-induced decrease in
IGF-1-induced PKB phosphorylation correlate with our previous
observations of FFA-induced decrease in PKB activity (5).
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Fig. 2.
FFA reduced PKB phosphorylation activation in
pancreatic -cells, INS-1. Cell lysates
were prepared from INS-1 cells treated with 5 or 15 mM
glucose ± 10 ng/ml IGF-1 with either control 0.5% BSA or 0.4 mM OA + 0.5% BSA for 18 h (see "Experimental
Procedures"). The cell lysates were subjected to immunoblot analysis
(as described under "Experimental Procedures") using
phospho-Ser473 PKB, phospho-Thr303 PKB, and
total PKB antibodies. The results shown are representative of three
independent experiments.
-cell apoptosis, we investigated the effect of
expressing a constitutively active form of PKB on INS-1 cell apoptosis.
INS-1 cells were infected with adenoviral vectors of Myc/His-tagged
PKB, consisting of a wild type (AdV-PKB-WT), a constitutively active
(AdV-PKB-CA), and a kinase-dead form (AdV-PKB-KD) as previously
described (14). A GFP-expressing adenovirus (AdV-GFP) was used as a
control (21). INS-1 cells were infected with equivalent levels
(0.5-2.0 × 106 plaque-forming units/ml) of
AdV-PKB-WT, AdV-PKB-CA, and AdV-PKB-KD or the control AdV-GFP (14), and
the rates of apoptosis were determined after 18 h treatment with 5 or 15 mM glucose ± 10 ng/ml IGF-1 + 0.4 mM OA
and 0.5% BSA, as a model FFA (Fig.
3).
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Fig. 3.
Adenoviral mediated expression of
constitutively active PKB prevented FFA-induced INS-1 cell
apoptosis. INS-1 cells were cultured on 10-cm plates and infected
with AdV-GFP, AdV-PKB-WT, AdV-PKB-CA, or AdV-PKB-KD at an m.o.i. of
10 × 102 (as described under "Experimental
Procedures"). The infected cells were treated with 5 or 15 mM glucose ± 10 ng/ml IGF-1 and 0.4 mM OA + 0.5% BSA for 18 h. As a control, AdV-GFP-infected cells were
treated under the same glucose and IGF-1 conditions, but with 0.5% BSA
only. The percentage of apoptotic cells was measured as described under
"Experimental Procedures." The results shown are representative of
four to eight independent experiments.
0.001; n = 7; Fig. 3). In the
additional presence of IGF-1 at 5 mM glucose, apoptosis in
AdV-GFP-infected control cells was significantly reduced to 20.2 ± 3.3% (p 0.05; n = 8) compared
with that at 5 mM glucose alone, but nonetheless significantly 20-fold higher than that in the absence of FFA under the
same conditions (p 0.01; Fig. 3). At 15 mM glucose in the absence of IGF-1 in FFA-treated
AdV-GFP-infected control -cells, the rate of apoptosis was further
decreased to 12.5 ± 2.1% (n = 4), but this was
significantly 8-fold higher than that in the absence of FFA under the
same conditions (p 0.05; Fig. 3). The addition of
IGF-1 at 15 mM glucose additionally reduced the degree of
INS-1 apoptosis to 5.4 ± 1.8% (n = 4), yet this
was 3-fold higher than that in the absence of FFA under the same
conditions (p 0.05; Fig. 3).
0.001),
yet still 12-fold (p 0.01) above the basal level of
apoptosis in AdV-GFP-infected control cells in the absence of FFA (Fig.
3). In the added presence of IGF-1 at 5 mM glucose, the
proportion of FFA-induced apoptotic cells was reduced to 13 ± 2.0% (n = 8), but this was 13-fold (p 0.05) above the basal level of apoptosis in AdV-GFP-infected control
cells not exposed to FFA (Fig. 3). At 15 mM glucose,
FFA-induced apoptosis in AdV-PKB-WT-infected cells was modestly
decreased by 4.9% to 7.6 ± 1.3% (n = 4)
compared with FFA-treated AdV-GFP-infected INS-1 cells, but remained
5-fold (p 0.05) above the basal level of apoptosis
in FFA-untreated AdV-GFP-infected control cells (Fig. 3). In
AdV-PKB-WT-infected INS-1 cells incubated in the added presence of
IGF-1 at 15 mM glucose, the apoptosis rate was cut to
2.6 ± 1.7% (n = 4) compared with FFA-treated
AdV-GFP-infected control cells, which was not significantly different
from the basal level of apoptosis of AdV-GFP-infected control cells not
incubated with FFA (Fig. 3). Notwithstanding, adenoviral mediated
transfer of a constitutively active PKB dramatically decreased
FFA-induced apoptosis in -cells. In AdV-PKB-CA-infected INS-1 cells
incubated at 5 mM glucose, the FFA-induced apoptosis was
markedly reduced to 5.4 ± 1.5% (n = 6), compared
with FFA-treated AdV-GFP-infected control cells (p 0.01), which was only 3-fold (p 0.001) above the
basal level of apoptosis in FFA-untreated AdV-GFP-infected control
cells (Fig. 3). In the added presence of IGF-1 at 5 mM
glucose, the incidence of FFA-induced apoptosis was significantly
reduced to 4.2 ± 0.9% (n = 7), compared with FFA-treated AdV-GFP-infected control cells (p 0.05),
only 4-fold (p 0.01) above the basal level of
apoptosis in the absence of FFA (Fig. 3). At 15 mM glucose,
FFA-induced apoptosis in AdV-PKB-CA-infected INS-1 cells was decreased
to 2.5 ± 0.2% (n = 4), compared with FFA-treated
AdV-GFP-infected control cells (p 0.05;
n = 4), and not significantly different from the
base-line incidence of apoptosis in FFA-untreated AdV-GFP-infected
control cells (Fig. 3). Likewise, in the added presence of IGF-1 at 15 mM glucose, FFA-induced apoptosis was not significantly
different in AdV-PKB-CA-infected INS-1 cells compared with the basal
rate of apoptosis in cells not exposed to FFA at 1.6 ± 0.7%
(n = 4), which was a significant decrease compared with
FFA-treated AdV-GFP-infected control cells (p 0.05)
(Fig. 3). As such, these data implicate an important role of PKB in
maintaining -cell survival.
-Cell Apoptosis by Glucose, IGF-1, and
PKB Inversely Correlated with Caspase-9 and -3 Activation--
The
caspase family of cysteine proteases play a key role in the execution
of apoptotic cell death (26). As such, we examined whether the degree
of caspase activation correlated with FFA-induced -cell apoptosis,
and protection from that by PKB activation. INS-1 cells infected with
the AdV-PKB constructs or control AdV-GFP were incubated with or
without 0.4 mM OA and 0.5% BSA at 5 or 15 mM
glucose ± IGF-1 for 18 h, as indicated (Fig.
4). The activation of the initiator
caspase-9 and the effector caspase-3 were measured by immunoblot
analysis with specific antibodies that only recognize the active
cleaved forms of these proteins in parallel with immunoblot analysis of
PKB phosphorylation activation using the PKB phosphospecific antibodies.
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Fig. 4.
Inhibition of FFA-induced
-cell apoptosis by glucose, IGF-1, and PKB
inversely correlated with caspase-9 and -3 activation. INS-1 cells
were cultured on 10-cm plates and infected with AdV-GFP, AdV-PKB-WT,
AdV-PKB-CA, or AdV-PKB-KD at an m.o.i. of 10 × 102
(as described under "Experimental Procedures"). The infected cells
were treated with 5 or 15 mM glucose + 10 ng/ml of IGF-1
and 0.4 mM OA + 0.5% BSA for 18 h. As a control,
AdV-GFP-infected cells were treated under the same glucose and IGF-1
conditions, but with 0.5% BSA only. Cell lysates were subjected to
immunoblot analysis (as described under "Experimental Procedures")
using phospho-Ser473 PKB, phospho-Thr303 PKB,
and total PKB antibodies (A) and activated caspase-9 and
caspase-3 antibodies (B). The results shown are
representative of three independent experiments.
-cell death observed was via an apoptotic, rather
than a necrotic, mechanism.
/ and FoxO1 (12), was also examined in INS-1 cells. INS-1
cells were infected with control AdV-GFP and the AdV-PKB variants, and
treated with or without 0.4 mM OA plus 0.5% BSA at 5 or 15 mM glucose ± IGF-1 for 18 h as indicated.
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Fig. 5.
FFA-induced inhibition of glucose/IGF-1
stimulated GSK3 and FoxO1 phosphorylation in INS-1 cells is rescued by
expression of constitutively active PKB. INS-1 cells were cultured
on 10-cm plates and infected with AdV-GFP, AdV-PKB-WT, AdV-PKB-CA, or
AdV-PKB-KD at an m.o.i. of 10 × 102 (as described
under "Experimental Procedures"). The infected cells were treated
with 5 or 15 mM glucose ± 10 ng/ml IGF-1 and 0.4 mM OA + 0.5% BSA for 18 h. As a control,
AdV-GFP-infected cells were treated under the same glucose and IGF-1
conditions, but with 0.5% BSA only. Cell lysates were subjected to
immunoblot analysis (as described under "Experimental Procedures")
using phospho-GSK3 / and total GSK3/ antibodies
(A) and phospho-FoxO1and total FoxO1 antibodies
(B). The results shown are representative of three
independent experiments.
/ by PKB occurs at residues
Ser9 and Ser20 on GSK3 and GSK3,
respectively (29). To assess the level of GSK3/ phosphorylation,
immunoblot analysis was performed using a phospho-GK3/
(Ser21/9) antibody, which only recognizes GSK3 when it
is phosphorylated at residue Ser21 or GSK3 when
phosphorylated at Ser9. In AdV-GFP-infected cells incubated
without FFA, GSK3/ phosphorylation inhibition was relatively low
at basal 5 mM glucose, but increased by either addition of
IGF-1 or increasing to a 15 mM glucose concentration for
the 18-h incubation period (Fig. 5B). In contrast, in
FFA-treated AdV-GFP-infected cells, glucose/IGF-1-stimulated
GSK3/ phosphorylation was decreased (Fig. 5B). A
similar reduction in the phosphorylation of GSK3/ in the presence
of FFA occurred in the AdV-PKB-KD-infected cells (Fig. 5B).
In AdV-PKB-WT-infected INS-1 cells, a glucose/IGF-1-induced GSK3/
phosphorylation was observed comparable with that in AdV-GFP-infected control cells incubated in the absence of FFA (Fig. 5B). A
marked increase of GSK3/ phosphorylation was observed in
AdV-PKB-CA-infected cells, which did not correlate with glucose/IGF-1
incubation conditions, but rather the constitutive activation of PKB in
these -cells (Fig. 5B). In this regard, the
phosphorylation state of GSK3/ observed was generally consistent
with the PKB phosphorylated activation state at residue PKB
Ser473 as previously found (Fig. 4A). Immunoblot
analysis indicated that total levels of GSK/ did not appreciably
vary (Fig. 5B).
-cell survival is by
inhibition of the tumor suppressor protein, p53 (which has been shown
to play a role in the induction of apoptosis; Ref. 30) via PKB-mediated
phosphorylation of MDM2 (17). To examine the role of p53 in FFA-induced
-cell apoptosis, we generated adenoviruses of wild type p53
(AdV-p53-WT) and mutant p53 (AdV-p53-MT). The p53-MT carries a G to A
mutation at nucleotide 1017 (Lys135 to Tyr), which not only
leads to a DNA-binding deficient conformational change in mutant p53,
but may also interact with and inhibit the wild type p53 (31, 32). The
titer of the adenoviruses was examined by infecting INS-1 cells with
0-25 × 102 m.o.i. and subsequent p53 immunoblot
analysis of AdV-p53-WT- and AdV-p53-MT-infected cells (Fig.
6A).
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Fig. 6.
Effects of adenoviral mediated expression of
p53 wild type and a p53 variant on FFA-induced apoptosis in INS-1
cells. INS-1 cells were infected with AdV-p53-WT and AdV-p53-MT
using an m.o.i. from 2 to 25 × 102 (as described
under "Experimental Procedures"), and an uninfected control was
included. After the 16-h incubation in complete medium, cell lysates
were subjected to immunoblot (IB) analysis as described
under "Experimental Procedures" using the total p53 antibody
(A). INS-1 cells were cultured on 10-cm plates and infected
with AdV-GFP, AdV-PKB-CA, AdV-p53-WT, or AdV-p53-MT at an m.o.i. of
10 × 102 (as described under "Experimental
Procedures"). The infected cells were treated with 5 mM
glucose and 0.5% BSA or 0.4 mM OA + 0.5% BSA for 18 h. The percentage of apoptotic cells was measured as described under
"Experimental Procedures." The results shown are representative of
four to six independent experiments.
0.05;
n = 4) (Fig. 6B). In the presence of FFA,
the incidence of apoptosis in AdV-GFP-infected INS-1 cells was
significantly increased to 40.8 ± 3.2% (p 0.01; n = 6), 29-fold higher compared with that in
AdV-GFP-infected control cells in the absence of FFA (Fig.
6B), similar to previous observations (Fig. 3). Likewise, as
previously observed (Fig. 3), FFA-induced apoptosis in
AdV-PKB-CA-infected cells was markedly reduced (p 0.05) to an incidence of 7.6 ± 1.7% (n = 4)
compared with FFA-induced apoptosis in AdV-GFP-infected INS-1 cells
(Fig. 6B). In AdV-p53-WT-infected cells, FFA-induced
apoptosis was further increased 35-fold to 50.0 ± 3.9% of the
INS-1 cell population compared with the AdV-GFP-infected control cells
in the absence of FFA (p 0.01; n = 4) (Fig. 6B). In contrast, FFA-induced apoptosis was reduced
in AdV-p53-MT-infected INS-1 cells to an incidence of 15.6 ± 3.5% (n = 4; Fig. 6B), significantly decreased in comparison to FFA-treated AdV-p53-WT-infected cells (p 0.005), and AdV-GFP-infected INS-1 cells
(p 0.02). However, FFA-induced apoptosis in
AdV-p53-MT-infected INS-1 cells was still a significant 11-fold higher
(p 0.05) compared with AdV-GFP-infected control
cells incubated in the absence of FFA (Fig. 6B).
Nonetheless, these results indicated that, at least in part,
FFA-induced -cell apoptosis might involve regulation of p53, perhaps
mediated downstream of PKB activation (17).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell apoptosis in INS-1 cells, as did palmitate (data not shown), similar to the FFA-induced apoptosis previously observed in islet -cells (7). It has been proposed that FFA-induced -cell apoptosis might occur, in part, via intracellular production of ceramide from palmitate (6). However, because, unlike palmitate, oleate is not a significant source for de novo synthesis of
ceramide (33, 34), the oleate-induced -cell apoptosis observed in this study was most likely ceramide-independent. Intriguingly, we also
found that methyl oleate did not induce -cell apoptosis. This
verifies that the oleate-induced -cell death we observed was
mediated via a programmed apoptotic mechanism, rather than a necrotic
one caused by a nonspecific detergent effect of the FFA. In addition,
because methylated FFA cannot undergo esterification to fatty acyl-CoA
(35), these data indicate that oleate-induced apoptosis was mediated by
a prerequisite for oleoyl-CoA formation, as previously indicated in
studies where inhibition of fatty-acyl CoA synthetase reduced
FFA-induced apoptosis (6).
-cell apoptosis (Fig. 3), suggesting a requirement for
activation of signaling pathways to promote -cell survival. In this
regard, we found that glucose/IGF-1-mediated phosphorylation activation
of the anti-apoptotic signaling protein, PKB, was reduced in the
presence of FFA, correlating with previous findings of FFA-induced
inhibition of PKB activity in -cells (5). This is consistent with a
number of reports in other cell types, which have shown that FFA and
ceramide inhibit insulin-induced PKB activation (36, 37). Currently,
the mechanism of FFA-induced inhibition of PKB activation in -cells
is unclear; however, several possibilities have been proposed in other
cell types. For example, inhibition of PKB by FFA and/or ceramide does
not appear to be caused by the down-regulation of signaling components
upstream of PKB (5), such as PI3K or PDK1, but instead is thought to
prevent PKB translocation to the plasma membrane (38, 39).
Alternatively, it has been proposed that PKB dephosphorylation may be
increased by FFA-induced activation of certain protein phosphatases,
such as protein phosphatase 2A, in turn reducing PKB activity (40, 41).
There are also reports that suggest there can be a FFA-induced
activation of certain PKC isoforms that increase Ser/Thr
phosphorylation of IRS-1/2, dampening downstream IRS signal
transduction, including decreased PI3K/PKB activation
(42-44).2 For the moment, it
remains to be shown whether any of these potential mechanisms might be
pertinent to FFA-induced inhibition of PKB in -cells.
-cell
apoptosis, especially in the light of our findings in this study, which
show activation of PKB plays a key role in promoting pancreatic
-cell survival. Adenoviral mediated gene transfer of a
constitutively active PKB variant into -cells almost completely prevented FFA-induced apoptosis especially in the presence of IGF-1.
These findings are complementary to that of transgenic expression of a
constitutively active PKB- (also known as Akt-1) specifically in
mouse pancreatic -cells (myr-Akt1 mice), where there was increased
-cell mass attributable to increased -cell survival and increased
size of -cells, but not up-regulation of -cell proliferation
where the actual number of -cells per islet was decreased (13). We
have previously shown that increased expression of constitutively
active or wild type PKB in -cells has little effect on
glucose/IGF-1-induced -cell proliferation (14), and because in this
study we have used the INS-1 cell line, PKB-mediated changes in
-cell neogenesis are irrelevant. As such, our findings emphasize the
importance of PKB activation in promoting -cell survival.
-cell specific myr-Akt1 mice are resistant to low
dose streptozotocin-induced diabetes (13). Here we show that a marked
increase in PKB activity in -cells is protective against a
physiologically relevant mediator of -cell death, FFA. In the
pathogenesis of obesity-linked type 2 diabetes, chronic exposure to FFA
has been proposed to be a key factor in promoting reduced -cell
mass, so that peripheral insulin resistance can no longer be
compensated for and, together with -cell dysfunction, the disease is
acquired (3, 4). Although we believe our findings to be informative,
for the moment it is premature to consider that PKB protection of the
-cell from FFA-induced apoptosis has therapeutic possibilities in
protecting -cell mass delaying the onset of obesity-linked type 2 diabetes (13, 45). We recognize the limitation of this study in using
the INS-1 cell line, and it will be important, first of all, to examine
whether these observations are reflected in the in vivo
setting in animal models of obesity-linked type 2 diabetes. Moreover,
because PKB has a plethora of protein substrates, it will also be
important to establish which are the appropriate substrates downstream
of PKB involved in promoting -cell survival.
-cell apoptosis. Phosphorylation of
two known pro-apoptotic PKB targets, GSK3/ and FoxO1, leads to
their inactivation (12). Here we found that increased phosphorylation
state of GSK3/ and FoxO1 correlated well with increased PKB
phosphorylation activation (especially in cells expressing the
constitutively active PKB), and this in turn inversely correlated with
the extent of FFA-induced apoptosis. Hence, PKB-mediated
phosphorylation inactivation of GSK3/ and FoxO1 likely promotes
-cell survival. Inhibition of GSK3/ has indeed previously been
implicated to play a role in the protection of cells from apoptosis
(12); however, little is known in regard to GSK3 activity in -cells,
and it will be important to elucidate the pro-apoptotic protein
phosphorylation substrates of GSK3 in future studies. FoxO1 is one of a
family of forkhead transcription factors, suggested to be involved in
the expression of a number of pro-apoptotic genes, as well as the
transcriptional activation of other genes (27). PKB phosphorylation of
FoxO1 prevents its translocation to the nucleus, and it is retained in
the cytoplasm (via interaction with 14.3.3 proteins), thus decreasing
FoxO1 transcriptional activity in driving expression of pro-apoptotic factors such as p27, a cyclin inhibitor, and Fas ligand (27). However,
as with GSK3 substrates, the potential pro-apoptotic genes regulated by
FoxO1 in the -cells need to be better determined.
-cell apoptosis. It was found that
adenoviral mediated increase in wild type p53 expression increased the
incidence of -cell apoptosis in the presence or absence of FFA,
suggesting it plays a role, at least in part, in the general mechanism
of -cell apoptosis. In contrast, adenoviral mediated expression of a negative variant of p53, which is deficient in DNA binding (31,
32), gave a partial protection of FFA-induced apoptosis. These data
implicate a contributing role for p53 in the mechanism of FFA-induced
-cell apoptosis, perhaps because of reduced p53 down-regulation as a
consequence of FFA-induced inhibition of PKB activation. However,
because protection from FFA-induced -cell apoptosis in
AdV-p53-MT-infected cells was only partial, and to a lesser extent than
that of expressing constitutively active PKB, clearly phosphorylation
inhibition of other factors downstream of PKB, including GSK3/
and FoxO1, will play contributing roles in promoting -cell survival.
-cell apoptosis,
at least in part by dampening PKB activation. This may have
implications for the development of an inadequate -cell mass that no
longer compensates for peripheral insulin resistance in the
pathogenesis of obesity-linked type 2 diabetes (3, 4). However, it will
be important to first substantiate that our findings in this study can
be replicated in an in vivo setting. Nonetheless, it is
interesting to note that FFA has been implicated to interfere with
insulin signal transduction in muscle, including downstream inhibition
of PI3K and PKB, which contribute to an insulin-resistant state (46).
As such, there might well be some intriguing parallels between
FFA-induced inhibition of IRS-mediated signal transduction pathways
that causes insulin resistance, as well as promoting apoptosis of
pancreatic -cells.
ACKNOWLEDGEMENT
FOOTNOTES
These authors made an equivalent contribution to this study and
share first authorship.
ABBREVIATIONS
/, glycogen synthase kinase-3 and ;
BAD, Bcl-2/Bcl-XL-antagonist causing cell death;
MDM2, murine double minute 2;
FoxO1, forkhead rhabdomyosarcoma transcription
factor (also known as FKHR);
OA, oleic acid or
cis-9-octadecenoic acid;
HO, Hoechst 33342;
PI, propidium
iodide;
AdV, adenovirus;
m.o.i., multiplicity of infection;
FITC, fluorescein isothiocyanate;
BSA, bovine serum albumin;
GFP, green
fluorescent protein.
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RESULTS
DISCUSSION
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