| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Pacific Northwest Research Institute, and Department of Pharmacology, University of Washington, Seattle, Washington 98122; and Research Division, Joslin Diabetes Center (M.G.M.), Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Christopher J. Rhodes, Ph.D., Pacific Northwest Research Institute, 720 Broadway, Seattle, Washington 98122. E-mail: cjr{at}pnri.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C12) inhibited 15
mM glucose-induced [3H]thymidine
incorporation (±10 nM IGF-I) by 95% or more within
24 h above 0.2 mM FFA complexed to 1% BSA
(K0.5 for palmitate/1% BSA = 65–85 µM
for 24 h; t0.5 for 0.2 mM palmitate/1%
BSA = 
6 h). FFA-mediated inhibition of glucose/IGF-I-induced
ß-cell DNA synthesis was reversible, and FFA oxidation did not appear
to be required, nor did FFA interfere with glucose metabolism in INS-1
cells. An examination of mitogenic signal transduction pathways in
INS-1 cells revealed that glucose/IGF-I induction of early signaling
elements in SH2-containing protein (Shc)- and insulin receptor
substrate-1/2-mediated pathways leading to downstream mitogen-activated
protein kinase and phosphoinositol 3'-kinase activation, were
unaffected by FFA. However, glucose-/IGF-I-induced activation of
protein kinase B (PKB) was significantly inhibited, and protein
kinase C
was chronically activated by FFA. It is possible that
FFA-mediated inhibition of ß-cell mitogenesis contributes to the
reduction of ß-cell mass and the subsequent failure to compensate for
peripheral insulin resistance in vivo that is key to the
pathogenesis of obesity-linked diabetes. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In states of peripheral insulin resistance, such as obesity, an increased demand for insulin can be compensated for by an increase in ß-cell mass and relatively increased insulin production (6, 7). However, when pancreatic ß-cell mass does not increase, there is a failure to compensate for the insulin resistance, and obesity-linked type II diabetes ensues (8). Thus, control of ß-cell growth is a key factor contributing to the pathogenesis of type II diabetes. IRS-mediated signal transduction is important for glucose- and glucose-dependent growth factor-induced ß-cell DNA synthesis, especially that via IRS-2 and PI3'K (3, 9). Moreover, it has been demonstrated that IRS-mediated mitogenic signal transduction pathways in the ß-cell could play an important role in the pathogenesis of type II diabetes (10, 11). In the IRS-1 knockout mouse there is peripheral insulin resistance that is compensated for by an increase in ß-cell mass, and there is no diabetes (10). However, in the IRS-2 knockout mouse the ß-cell mass is markedly reduced, and in the face of additional peripheral insulin resistance, type II diabetes ensues (10, 11). Although there are likely genetic susceptibilities involved in the pathogenesis of type II diabetes (12), it appears that alterations in the IRS-2 primary gene structure do not contribute (13). However, this does not rule out that adverse biochemical affects in IRS-2 function could lead to a diabetic state. As such, an unresolved question is what are the pathophysiologically relevant factors that prevent an increase and/or decrease ß-cell mass in type II diabetes so that there is no longer compensation for peripheral insulin resistance. It is thought that both prolonged hyperglycemia and hyperlipidemia are such contributing factors, and they can be detrimental to ß-cell function and mass (6, 7, 8, 14).
In general, glucose has a tendency to increase ß-cell mitogenesis (1, 2). In this study an examination of alternative nutrient fuels that might increase ß-cell growth similar to the effect of glucose found that long chain FFA inhibited glucose-induced and glucose-dependent IGF-I-induced ß-cell DNA synthesis. This in part was attributed to interference by FFAs in IRS-mediated signal transduction pathways downstream of PI3'-K. It is postulated that exposure to FFA leads to a reduction in ß-cell mitogenesis as well as peripheral insulin resistance (7, 15, 16) that would contribute to decreased ß-cell mass in vivo and presentation of type II diabetes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-32P]ATP (3000 Ci/mmol) was obtained from
Amersham Pharmacia Biotech (Piscataway, NJ). Anti-active
phospho-MAPK (Erk1/2), phospho-p38, phospho-c-Jun N-terminal kinase-1/2
(JNK1/2), as well as anti-total MAPK (Erk1/2), p38, and JNK1/2 antisera
were purchased from Promega Corp. (Madison, WI). The
IRS-1- and IRS-2 antisera were generated as previously described
(17). The p70S6K antisera were generated and
used in immunoblot analysis as previously described (18).
Anti-PI3'K p85, protein kinase B (PKB), mammalian Son of
Sevenless (mSOS), growth factor bound protein 2 (Grb2),
Shc, and antiphosphotyrosine antisera were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-protein kinase C
(anti-PKC) antisera was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The PKB activity assay kit
was purchased from New England Biolabs, Inc. (Beverly,
MA), and [Ser159]PKC
-(153–164)-NH2 PKC
peptide substrate was obtained from Upstate Biotechnology, Inc. L-
-Phosphatidylserine and
L--phosphatidylinositol were purchased from Avanti Polar
Lipids, Inc. (Alabaster, AL), 2-bromopalmitate was obtained from
Aldrich Chemical Co., Inc. (Milwaukee, WI),
methyl-palmitate was purchased from Sigma (St. Louis, MO),
and all other FFA were purchased from Alltech (Deerfield, IL). FFA-free
BSA was obtained from Roche Molecular Biochemicals
(Indianapolis, IN), and C2 ceramide,
N-acetyl-D-erythro-sphingosine,
dihydro-N-acetyl-D-erythro-sphingosine, as well
as IGF-I were purchased from Calbiochem-Novabiochem (La
Jolla, CA). Transblot nitrocellulose membrane (0.2-µm pore size) were
obtained from Bio-Rad Laboratories, Inc. (Hercules, CA).
The immunoblot chemiluminescence detection kit was obtained from
NEN Life Science Products (Boston, MA). Unless otherwise
stated all other biochemicals were purchased from either
Sigma (St. Louis, MO) or Fisher Scientific
(Pittsburgh, PA) and were of the highest purity available.
Cell culture
The glucose-sensitive pancreatic ß-cell line, INS-1
(19), was used in the experiments. INS-1 cells were
maintained in RPMI 1640 medium containing 2 mM
L-glutamine, 1 mM sodium pyruvate, 50
µM ß-mercaptoethanol, 100 U/ml penicillin, 100
µg/ml streptomycin, 10% FCS, and 11.2 mM glucose and
incubated at 37 C in 5% CO2 as previously described
(19). Cells were subcultured at 80% confluence.
FFA/BSA complex solution preparation
A 100-mM FFA stock solution was prepared in 0.1
M NaOH by heating at 70 C in a shaking water bath. In an
adjacent water bath at 55 C, a 10% (wt/vol) FFA-free BSA solution was
prepared in H2O. Altering the proportion of these
two solutions when mixed together varied the concentration of FFA
complexed to BSA. For example, a 5-mM FFA/10% BSA stock
solution was prepared by adding 50 µl of the 100 mM FFA
solution dropwise to 950 µl 10% BSA solution at 55 C in a shaking
water bath, then vortex mixed for 10 sec followed by a further 10-min
incubation at 55 C. The FFA/BSA complexed solution is cooled to room
temperature and sterile filtered (0.45-µm pore size membrane filter).
It can be stored at -20 C, where it is stable for 3–4 weeks. Stored
5-mM FFA/10% BSA stock solutions are heated for 15 min at
55 C, then cooled to room temperature before use.
[3H]Thymidine incorporation
Incorporation of [3H]thymidine was
used as an indicator of DNA synthesis and INS-1 cell mitogenesis
(20). A 96-well plate
[3H]thymidine assay was used as previously
described (3, 4). Essentially, INS-1 cells were cultured
on 96-well plates and incubated at 37 C in INS-1 medium to a density of
approximately 2–5 x 104 cells/well. The
medium was removed, and the cells were made quiescent by serum and
glucose deprivation for 24 h in RPMI 1640 containing 0.1% BSA
instead of serum and 0.5 mM glucose. The INS-1 cells were
then incubated for a further 24 h in RPMI 1640 and 0.1% BSA at
different glucose concentrations (0–24 mM glucose) with or
without IGF-I (10 nM) and FFA (0.02–0.5 mM)
complexed to 1% BSA. The last 4 h of this latter incubation
period was carried out in the additional presence of 5 µCi
[3H]thymidine/ml to monitor the degree of DNA
synthesis and assess the ß-cell mitogenesis rate. After this final
incubation period, the cells were centrifuged (3000 x
g, 10 min, 4 C: Sorvall RT7 centrifuge (DuPont Medical Products, Newtown, CT) and RT750 rotor and 96-well plate
holders), washed, and lysed using a semiautomatic cell harvester
(Packard Instrumentation, Meriden, CT), and the cell lysates were
transferred to Whatman glass-fiber micropore filters
(Clifton, NJ). The [3H]thymidine specifically
incorporated into the INS-1 cell DNA trapped on glass-fiber filters was
counted by liquid scintillation counting.
Protein immunoblot and coimmunoprecipitation analysis
INS-1 cells were subcultured on 10-cm plates to about 70%
confluence as previously described (19). The cells were
then incubated for a 24-h period of quiescence by serum and glucose
deprivation in RPMI 1640 medium containing 0.1% BSA instead of serum
and 0.5 mM glucose with or without 0.2–0.4 mM
FFA complexed to 1% BSA. INS-1 cells were then incubated in fresh RPMI
1640 medium containing 0, 3, or 15 mM glucose with or
without 10 nM IGF-I and 0.2–0.4 mM FFA/1% BSA
for between 5 and 30 min as indicated. The cells were lysed in 0.5 ml
ice-cold lysis as previously described (3, 4).
Immunoprecipitation and immunoblot analysis of mitogenic signal
transduction protein expression and protein tyrosine phosphorylation
were performed as previously described (3, 4).
PI3'K assay
PI3'K was assayed as previously described (21, 22). Essentially, INS-1 cells were incubated at 0, 3, or 15
mM glucose with or without 10 nM IGF-I and 0.4
mM FFA/1% BSA for 15 min, then PI3'K was
immunoprecipitated from 750 µg total protein of cell lysate with an
anti-PI3'K p85 antibody. The immunoprecipitates were washed twice in
300 µl PBS (pH 7.4), 1% Nonidet P-40, and inhibitor cocktail
(containing 1 mM
Na3VO4, 20 mM
NaF, 10 mM sodium pyrophosphate, 1 mM EDTA, 10
µM leupeptin, 10 µg/ml aprotinin, and 100
µM phenylmethylsulfonylfluoride); twice with 300 µl 0.1
M Tris-HCl (pH 7.5), 0.5 M LiCl, and inhibitor
cocktail; and finally twice with 300 µl 10 mM Tris-HCl
(pH 7.5), 100 mM NaCl, and inhibitor cocktail.
Immunoprecipitates were then suspended in 70 µl 10 mM
Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 100
µM Na3VO4,
and 100 µM 15 mM
MgCl2 containing 20 µg L--
phosphatidylinositol. The assay was initiated by addition of 5 µl 10
mM Tris-HCl (pH 7.5), 20 mM
MgCl2, and 1 mM ATP containing 20
µCi [-32P]ATP and incubated for 10 min at
30 C in a shaking water bath. The reaction was stopped by
addition 20 µl 8 M HCl, and the phospholipids were
chloroform/methanol (1:1) extracted.
[32P]Phospholipids were then separated by TLC
and detected by autoradiography as previously described (21, 22). Quantification of the
phosphatidylinositol-3'-[32P]phosphate produced
was determined by densitometric scanning of the autoradiographs.
PKB assay
PKB activity was determined using a kit provided by New England Biolabs, Inc. Essentially, INS-1 cells were incubated as
described for the PI3'-K assay described above. PKB was
immunoprecipitated from 1 mg total protein equivalent of cell lysate
with anti-PKB antibody linked to agarose beads overnight at 4 C with
rotary mixing. Agarose beads were then pelleted by centrifugation
(10,000 x g, 30 sec, 4 C), and the supernatant was
removed. Immunoprecipitates were then washed twice in 500 µl ice-cold
lysis buffer, then twice with 500 µl ice-cold PKB assay buffer
consisting of 25 mM Tris (pH 7.5), 5
mM ß-glycerophosphate, 2
mM dithiothreitol, 100 µM mM
Na3VO4, and 10 mM MgCl2
containing 20 µg L--phosphatidylinositol. PKB
immunoprecipitates were suspended in 40 µl assay buffer additionally
containing 200 µM ATP and 1 µg glycogen
synthase-kinase (GSK)-3/ß fusion protein (containing the
Ser21/9 phosphorylation site), then incubated for
20 min at 30° C in a shaking water bath. The reaction was stopped by
the addition of 20 µl 3 x SDS-electrophoresis sample buffer and
then heated at 95 C for 5 min. The sample was run on SDS-PAGE, and the
extent of GSK3 phosphorylation was analyzed with a specific
phospho-GSK-3 antibody.
PKC assay
PKC activity was determined as previously described
(23). Essentially, INS-1 cells were incubated as described
for the PI3'K assay described above. PKC was then immunoprecipitated
from 750 µg total protein equivalent of lysate (in a 500-µl final
volume) with an anti-PKC antibody, followed by protein A-Sepharose
as described previously (3, 4). Immunoprecipitates were
then washed twice in 500 µl ice-cold lysis buffer, then twice with
500 µl ice-cold assay buffer consisting of 20 mM HEPES
(pH 7.5), 1 mM
Na3VO4, 20 mM
NaF, 10 mM sodium pyrophosphate, 1 mM EDTA, 10
µM leupeptin, 10 µg/ml aprotinin, and 100
µM phenylmethylsulfonylfluoride. PKC
immunoprecipitates were then suspended in 50 µl of the assay buffer,
additionally containing 40 µM
[Ser159]PKC--(153–164)-NH2
peptide substrate, 50 µM ATP, and 40 µg/ml
phosphatidylserine. The reaction was started by the addition of 5 µCi
[-32P]ATP and was incubated for 10 min at 30
C in a shaking water bath. A 25-µl aliquot of reaction mixture was
then spotted onto a phosphocellulose p81 filter (Whatman)
presoaked in 10% (vol/vol) acetic acid to trap the
[Ser159]PKC--(153–164)-NH2
peptide substrate. The filters were immediately extensively washed in
10% acetic acid by flow-through on a sample manifold apparatus
(Millipore Corp., Bedford, MA), followed by repeated
washes for an additional 10 min. The filters were then
air-dried. Quantification of
[32P]Ser159PKC--(153–164)-NH2
peptide was performed by liquid scintillation counting.
Other procedures
Protein assay was by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL). Data are presented as the mean
± SE, with the number of individual observations indicated
(n). Statistically significant differences between groups were analyzed
using Student’s t test, where P < 0.05 was
considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-ketoisocaproate, all
established nutrient fuels of pancreatic ß-cells (24),
on INS-1 cell DNA synthesis (with or without 10 nM IGF-I)
was determined by [3H]thymidine incorporation.
INS-1 cells were chosen as a model to examine ß-cell mitogenesis,
because they respond to glucose in the physiologically relevant range
(6–18 mM glucose) (3, 4, 19). A
15-mM glucose concentration significantly increased
[3H]thymidine incorporation in INS-1 cells
between 20- to 25-fold above baseline (P 
0.001;
Figs. 1
-3). In the absence of glucose,
IGF-I (10 nM) had no effect on INS-1 cell DNA
synthesis and had a modest 3- to 4-fold increase at 3
mM glucose (P 0.05), but had a
marked significant 50- to 60-fold increase at a stimulatory 15
mM glucose (
Figs. 1–3; P
0.001), as previously observed (3, 4). The
[3H]thymidine incorporation assay was used as a
marker of INS-1 cell mitogenesis. Cell counting revealed that a 20-fold
increase in [3H]thymidine incorporation in a
24-h period, as instigated by 15 mM glucose, was
equivalent to a 2-fold increase in cell number. A 50-fold in crease in
[3H]thymidine incorporation within 24 h,
as stimulated by 15 mM glucose and 10
nM IGF-I increased cell number 3- to 4-fold.
|
|
|
0.01), and between 25- to 28-fold in the
added presence of 10 nM IGF-I (P
0.001; Fig. 1
). The addition of 1 mM
-cyano-4-hydroxycyanocinnamate, which prevents pyruvate transport
into ß-cells and mitochondria (25, 26), prevented the
pyruvate-induced increase in INS-1 cell DNA synthesis with or without
IGF-I (Fig. 1). Glutamine (10 mM) and leucine (10
mM) had no independent effect on cell
[3H]thymidine incorporation into INS-1 cells,
but in the added presence of 10 nM IGF-I there
was a 3- to 4-fold increase in INS-1 cell DNA synthesis
(P 0.05; Fig. 1). However, when 10
mM leucine or 10 mM
ketoisocaproate (a deaminated product of leucine) was combined with 10
mM glutamine (to activate glutamate dehydrogenase
and increase intramitochondrial -ketoglutarate) (26), a
significant increase in INS-1 cell
[3H]thymidine incorporation was observed (8- to
10-fold; P 0.05) that further increased 25- to
30-fold with 10 nM IGF-I added (P
0.01; Fig. 1).
Long chain FFA inhibit glucose- and IGF-I-induced ß-cell DNA
synthesis
It was examined whether FFAs would affect ß-cell mitogenesis
and/or provide a platform for growth factors to evoke a mitogenic
response as previously observed (3, 4). It was found that
in the absence of glucose or at glucose concentrations of 5
mM or more, long chain FFA (>C12)
tended to slightly increase INS-1 cell
[3H]thymidine incorporation approximately
2-fold, but this was not statistically significant (data not shown). In
contrast, it was found that FFAs inhibited glucose/IGF-I-induced INS-1
cell DNA synthesis (Fig. 2). FFAs of
chain length C8 to C18
[0.4 mM complexed to 1% (wt/vol) BSA] were incubated
with INS-1 cells for 24 h at 15 mM glucose with or
without 10 nM IGF-I. Linoleate
(C18:2), oleate (C18:1),
stearate (C18:0), palmitate
(C16:0), myristate (C14:0),
laurate (C12:0) significantly inhibited
glucose-induced and glucose-dependent IGF-I-induced ß-cell DNA
synthesis by 95% or more (P 0.001) compared with
cells incubated in the absence of FFA (Fig. 2A). Caproate
(C10) also inhibited 15 mM
glucose/IGF-I induced INS-1 cell [3H]thymidine
incorporation, but to a lesser extent (80%; P
0.005; Fig. 2). Capryloate (C8) partially
inhibited 15 mM glucose-induced INS-1 cell
[3H]thymidine incorporation (38.5 ±
3.3%; n = 5; P 0.05), but had a negligible
affect on glucose-dependent IGF-I induced ß-cell DNA synthesis
(20.3 ± 3.4%; n = 5; P = NS; Fig. 2).
In further experiments palmitate was used as a model FFA. Palmitate
significantly inhibited ß-cell DNA synthesis at 15 mM
glucose with or without 10 nM IGF-I by 95% or more at a
concentration of 0.2 mM or more complexed 1% BSA
(P 0.001; Fig. 3A).
Titration inhibition curves indicated a Kd50 of
86.0 ± 9.4 µM palmitate (complexed 1%
BSA; n = 5) for inhibition of 15 mM
glucose-induced INS-1 cell DNA synthesis and a
Kd50 of 64.3 ± 7.7
µM palmitate (complexed to 1% BSA; n = 5)
for inhibition of 15 mM glucose-dependent
IGF-I-induced INS-1 cell [3H]thymidine
incorporation (Fig. 3A). The time course for FFA-mediated inhibition of
glucose-/IGF-I-induced INS-1 cell DNA synthesis was assessed using a
slight modification of the 96-well plate
[3H]thymidine incorporation assay so as to
assess early time points. Quiescent INS-1 cells were incubated in 15
mM glucose with or without 10
nM IGF-I in the added presence or absence of 0.2
mM palmitate complexed to 1% BSA for between
2–24 h, the last 2 h of which was in the presence of 10 µCi
[3H]thymidine/ml. For 0.2
mM palmitate inhibition of 15
mM glucose-induced ß-cell DNA synthesis,
t0.5 = 6.3 ± 0.4 h, and for inhibition
of 15 mM glucose-dependent IGF-I-induced ß-cell
DNA synthesis, t0.5 = 5.8 ± 0.9 h.
(Fig. 3B).
Palmitate inhibition of INS-1 cell DNA synthesis was reversible. If
palmitate was removed from the medium after 24-h exposure to 0.2
mM palmitate/1% BSA and INS-1 cells, then further
incubated in the absence of FFA, inhibition glucose/IGF-I-induced
ß-cell DNA synthesis recovered to 80% of that in control cells by
24 h and was fully recovered by 36 h (data not shown).
Bromopalmitate (0.4 mM complexed to 1% BSA), an analog of
palmitate that is not oxidized (26), significantly
inhibited INS-1 cell [3H]thymidine
incorporation at 15 mM glucose with or without 10
nM IGF-I by 95% or more within 24 h
(P 0.001; data not shown), similar to that of
palmitate (Figs. 2 and 3). This indicated that ß-oxidation of FFA
was not involved in FFA-induced inhibition of ß-cell DNA synthesis.
In contrast, methyl-palmitate (0.4 mM complexed
to a constant 1% BSA), a modified form of palmitate that cannot form a
coenzyme A (CoA) ester, only inhibited INS-1 cell
[3H]thymidine incorporation at 15
mM glucose with or without 10
nM IGF-I by 30% within 24 h
(P = NS; data not shown). This latter observation
suggested that formation of a fatty acyl-CoA moiety was necessary for
FFA-mediated inhibition of glucose/IGF-I induced ß-cell DNA
synthesis. As ceramide can be derived from palmitoyl-CoA (27, 28), it was examined whether ceramide would affect
glucose-induced and/or glucose-dependent IGF-I-induced INS-1 cell DNA
synthesis in a similar fashion to that by FFA. However, addition of
neither 10 µM C2-ceramide
(N-acetyl-D-erythro-sphingosine) nor
10 µM C2-dihydroceramide
(dihydro-N-acetyl-D-erythro-sphingosine;
a biologically inactive analog of C2-ceramide) had any effect on
glucose-induced or glucose-dependent IGF-I-induced INS-1 cell
[3H]thymidine incorporation.
FFA do not inhibit glucose or IGF-I activation of IRS-1/2 or Shc in
INS-1 cells
Activation of IRS-mediated mitogenic signaling transduction
pathways by glucose with or without IGF-I in INS-1 cells that were
previously exposed to 0.4 mM palmitate/1% BSA (or 1% BSA
alone as a control) for 24 h was investigated using
coimmunoprecipitation and immunoblot analysis. Immunoprecipitation of
the 85-kDa regulatory subunit of PI3'-K followed by immunoblot analysis
for IRS-1 (Fig. 4A) or IRS-2 (Fig. 4B)
revealed a significant increased association of PI3'-K with IRS-1 and
IRS-2 instigated by stimulatory 15 mM glucose and IGF-I,
similar to that previously observed (3, 4). However,
palmitate had no adverse affect on gluocse/IGF-I-induced PI3'-K/IRS-1
or PI3'-K/IRS-2 interaction (Fig. 4, A and B). Likewise, immunoblotting
of the p85 PI3'-K immunoprecipitates with antiserum recognizing mSOS
(Fig. 4C) or Grb2 (Fig. 4D) indicated an increased association of mSOS
and Grb2 to a PI3'K/IRS signaling complex by 15 mM glucose,
which was further increased in the presence of 10 nM IGF-I
as previously described (3, 4). However, as for IRS-1/2
association with PI3'-K, palmitate had no adverse affect on
glucose-induced or glucose-dependent IGF-I-induced association of
Grb2/mSOS to the IRS signaling complex in ß-cells (Fig. 4, C and D).
The specific nature of this glucose/IGF-I-induced Grb2/mSOS, IRS-1, and
IRS-2 interaction with PI3'-K was indicated, in that p85 PI3'-K
immunoblot analysis of PI3'-K immunoprecipitates revealed that an
equivalent amount of PI3'-K was present in each sample (Fig. 4E). In
additional experiments, it was found that 15 mM glucose and
IGF-I were able to significantly promote Shc association with Grb2/mSOS
similar to that previously described (3, 4). However,
addition of 0.4 mM palmitate/1% BSA for 24 h had no
affect on the glucose/IGF-I-induced association of Grb2/mSOS to Shc
(data not shown).
|
;
P 0.01). IGF-I also increased Erk-1 and -2
activation at a lower 3 mM glucose (Fig. 5A;
P 0.05). The glucose/IGF-I-induced phosphorylation
activation of Erk-1 and -2 was unaffected by the presence of 0.4
mM palmitate/1% BSA (Fig. 5A). The total amount
of Erk-1 and 2 in INS-1 cells, as ascertained by immunoblot analysis
with antisera recognizing both active and inactive forms of Erk-1 and
-2, was not significantly altered by glucose, IGF-I, or FFA treatment
(Fig. 5A).
|
). At 3 mM
glucose, a p38 phosphorylation state was reduced by 40–50% compared
with zero glucose, and by ore than 90% at 15 mM glucose
(Fig. 5B). As such, the effect of glucose on p38 phosphorylation
activation inversely correlated with glucose-induced activation of
Erk-1 and -2 (Fig. 5A). No significant effect of IGF-I or 0.4
mM palmitate/1% BSA was observed on phosphorylation
activation of p38 at any glucose concentration (Fig. 5B). Total p38 in
INS-1 cells, as ascertained by immunoblot analysis with antisera
recognizing both active and inactive forms of p38, was not
significantly altered by glucose, IGF-I, or FFA treatment (Fig. 5B).
Phosphorylation activation of JNK-1 and -2 was also assessed and was
found to follow the same pattern as that for p38. JNK-1 and -2
phosphorylation inversely correlated to increasing glucose
concentration. As for p38, IGF-I and FFA had no effect on the
phosphorylation state of JNK-1 and -2 (data not shown).
FFA do not inhibit glucose or IGF-I activation of PI3'-K in INS-1
cells
PI3'-K activity was assessed in PI3'-K p85 immunoprecipitates as
previously described (21, 22), derived from INS-1 cells
previously exposed for 24 h with either 0.4 mM
palmitate/1% BSA or 1% BSA, then incubated for 15 min at 3 or 15
mM glucose (with or without 10 nM IGF-I). An
equivalent amount of p85 regulatory subunit of PI3'-K was
immunoprecipitated from the variously treated INS-1 cell lysates (Fig. 6, upper panel). At 3
mM glucose, PI3'-K activity was increased
3.0 ± 0.5-fold (n = 3; P 0.05) above that
at zero glucose, and in the added presence of IGF-I was further
increased 4.6 ± 0.7-fold (n = 3; P 0.05;
Fig. 6). At a stimulatory 15 mM glucose, PI3'-K
activity was increased 4.7 ± 0.6-fold (n = 3;
P 0.05) above that at zero glucose, and in the added
presence of IGF-I was further increased 7.2 ± 0.9-fold (n =
3; P 0.02; Fig. 6). The presence of 0.4
mM palmitate/1% BSA had no effect on glucose- or
IGF-I-induced activation of PI3'-K activity.
|
, middle panel). Total PKB was immunoprecipitated from INS-1
cells previously exposed for 24 h to either 0.4
mM palmitate/1% BSA or 1% BSA, then incubated
for 15 min at 3 or 15 mM glucose (with or without
10 nM IGF-I). An equivalent amount of PKB was
immunoprecipitated from the variously treated INS-1 cell lysates (Fig. 7, upper panel). Extended electrophoresis and subsequent
immunoblot analysis revealed that PKB, PKBß, and, to a lesser
extent, PKB isoforms were present in ß-cells (data not shown). At
3 mM glucose, PKB activity increased 3.6 ±
0.4-fold (n = 6; P 0.01) above that at zero
glucose, and in the added presence of IGF-I was further increased
5.6 ± 0.5-fold (n = 6; P 0.005; Fig. 7).
At a stimulatory 15 mM glucose, PKB activity
increased 5.8 ± 0.5-fold (n = 6; P 0.01)
above that at zero glucose, and in the added presence of IGF-I was
further increased 7 ± 0.8-fold (n = 6; P
0.01; Fig. 7). However, in the presence of 0.4 mM
palmitate/1% BSA, activation of PKB at 3 mM
glucose was significantly reduced by 25% (P 0.05)
in the absence of IGF-I and by 40% (P 0.01) in the
presence of IGF-I (Fig. 7). The addition of 0.4
mM palmitate/1% BSA reduced 15
mM glucose-induced PKB activity by 28%
(P 0.05), but had a more marked effect on
IGF-I-induced PKB activation at 15 mM glucose,
where PKB activity was inhibited by 52% (P 0.01;
Fig. 7).
|
), similar to that previously observed (3, 4).
However, phosphorylation activation of p70S6K in
response to 15 mM glucose was observed, which was further
increased in the presence of IGF-I (Fig. 5C). The presence of 0.4
mM palmitate/1% BSA had no effect on glucose-induced or
glucose-dependent IGF-I-induced phosphorylation activation of
p70S6K.
FFA-induced activation of PKC INS-1 cells
PKC activity was assessed in PKC immunoprecipitates
in vitro as previously described (23). Total
PKC was immunoprecipitated from INS-1 cells previously exposed for
24 h to either 0.4 mM palmitate/1% BSA or
1% BSA, then incubated for 15 min at 3 or 15 mM
glucose (with or without 10 nM IGF-I). An
equivalent amount of PKC was immunoprecipitated from variously
treated INS-1 cell lysates (Fig. 8, upper panel). In the absence of glucose, but in
the presence of 0.4 mM palmitate/1% BSA, PKC
activity was elevated 5.1 ± 0.6-fold (n = 7;
P 0.005) above that in the presence of 1% BSA only
(Fig. 8). This indicated increased activity of PKC during the prior
24-h incubation in the presence of FFA. For INS-1 cells incubated in
the absence of FFA, 3 mM glucose increased PKC
activity 3.2 ± 0.4-fold (n = 6; P 0.05)
above that at zero glucose, and in the added presence of IGF-I it was
further increased to 4.6 ± 0.6-fold (n = 6;
P 0.05; Fig. 8). However, this modest incremental
increase in PKC activity induced by IGF-I at 3
mM glucose was not statistically significant. In
INS-1 cells exposed to 0.4 mM palmitate/1% BSA
and incubated at 3 mM glucose for 15 min, PKC
activity was significantly enhanced 7.5 ± 0.9-fold (n = 5;
P 0.02) above that at zero glucose, 2.3-fold higher
than INS-1 cells incubated in the absence of FFA. Likewise, at 3
mM glucose and 10 nM IGF-I,
0.4 mM palmitate/1% BSA treatment of INS-1
cells, PKC activity was significantly enhanced 11.3 ±
1.7-fold (n = 5; P 0.05) above that at zero
glucose, 2.5-fold higher than equivalent INS-1 cells incubated in the
absence of FFA. At a stimulatory 15 mM glucose,
PKC activity was increased 9.6 ± 1.5-fold (n = 6;
P 0.05) above that at zero glucose, and in the added
presence of IGF-I was further increased to 10.4 ± 1.1-fold
(n = 6; P 0.02; Fig. 8). As at 3
mM glucose, the slight incremental increase in
PKC activity induced by IGF-I at 15 mM glucose
was not statistically significant. Nonetheless, in the presence of 0.4
mM palmitate/1% BSA, activation of PKC by 15
mM glucose was significantly enhanced 13.1
± 1.2-fold (n = 5; P = 0.02) above that at zero
glucose, which was 36% higher than the level in INS-1 cells incubated
in the absence of FFA. Likewise, PKC activation by 15
mM glucose and 10 nM IGF-I
in the presence of 0.4 mM palmitate/1%
BSA-treated INS-1 cells was significantly enhanced 15.3 ±
1.8-fold (n = 5; P 0.05) above that at zero
glucose, which was 47% higher than the level in equivalent INS-1 cells
incubated in the absence of FFA (Fig. 8).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-ketoglutarate (36) independently
increased ß-cell DNA synthesis and provided the environment for
IGF-I-induced ß-cell mitogenesis, similar to the effect of the
glutamine/leucine combination on (pro)insulin synthesis and secretion
(26). As both pyruvate and glutamate/leucine are used as
mitochondrial fuels in ß-cells, these data suggest that ß-cell
mitochondrial metabolism, downstream of glycolysis, is important for
generating secondary coupling signals for glucose-induced ß-cell DNA
synthesis and for providing the permissive environment for
IGF-I-induced ß-cell mitogenesis.
However, not all metabolic fuels act as ß-cell mitogens or provide a
platform of growth factors to induce ß-cell mitogenesis. In the
absence of glucose (or at 5 mM glucose) FFAs were
observed to have only a modest effect on INS-1 cell DNA synthesis
compared with that of 15 mM glucose. Similar observations
of the effect of FFA on ß-cell mitogenesis have been previously made
in islets (37), and INS-1 cells (38). In this
study it was found that long chain (>C10) FFA
prevented 15 mM glucose-induced and glucose-dependent
IGF-I-induced ß-cell DNA synthesis within a 24-h period
(t0.5 = 6 h). FFA-mediated inhibition of
ß-cell DNA synthesis occurred at a relatively low concentration of
FFA (Ki0.5 = 60–90 µM FFA
complexed to 1% BSA) and was reversible upon removal of the FFA. Thus,
it was unlikely that FFA-mediated inhibition of ß-cell mitogenesis
was due to a nonspecific detergent-like effect. FFA-induced inhibition
of ß-cell DNA synthesis might have been acting via FFA-derived
intracellular ceramide formation, but this was unlikely, because
ceramide had no effect on glucose/IGF-I-induced ß-cell DNA synthesis.
This would be consistent with the observation that both palmitate and
oleate inhibited glucose/IGF-I induced ß-cell mitogenesis, as
ceramide is derived from palmitoyl-CoA, but not directly from
oleoyl-CoA (39). Notwithstanding, long chain fatty
acyl-CoA moieties were probably involved, as methyl-palmitate (which
cannot form a CoA ester) was not as effective as other FFAs in
mediating the inhibition of glucose/IGF-I-induced ß-cell DNA
synthesis. However, FFA-mediated inhibition of ß-cell DNA synthesis
was not due to changes in FFA oxidation, as a nonmetabolizable FFA
analog (bromopalmitate) was also effective at inhibiting
glucose/IGF-I-induced ß-cell DNA synthesis. Moreover, it was found
that in INS-1 cells that were incubated for 24 h in the presence
of FFA (0.4 mM palmitate or oleate complexed to 1% BSA),
the rate of glucose oxidation (at either 3 or 15 mM
glucose) was unaffected by FFA (data not shown), as previously shown in
pancreatic islets (40). Therefore, FFA were unlikely to
inhibit glucose metabolism that, in turn, would affect the
glucose-dependent aspect of growth factor-induced ß-cell mitogenesis
(3, 4).
IRS-mediated signal transduction in INS-1 cells (particularly via IRS-2) (9, 10, 11) has previously been shown to be necessary for glucose-induced and glucose-dependent IGF-I-induced ß-cell DNA synthesis (3, 4). It was investigated whether FFA would interfere with glucose/IGF-I-induced signal transduction via IRS in a manner that correlated with FFA-mediated inhibition of glucose/IGF-I-induced ß-cell DNA synthesis. Using palmitate as a model, it was found that FFA did not affect glucose/IGF-I-induced recruitment of PI3'-K or Grb2/mSOS to IRS-1/2, recruitment of Grb2/mSOS to Shc, activation of PI3'K activity, or phosphorylation activation of Erk-1/2 or p70S6K. The stress kinases p38 and JNK-1/2 were phosphorylation activated by decreasing glucose concentrations, but were unaffected by FFA. Thus, it was unlikely that p38 or JNK-1/2 was involved in FFA-mediated inhibition of glucose/IGF-I-induced ß-cell mitogenesis. In contrast, FFA significantly inhibited glucose/IGF-I-induced activation of PKB activity. However, it should be noted that FFA-mediated inhibition of PKB activity in ß-cells was partial, and downstream glucose/IGF-I-induced phosphorylation activation of p70S6K was not affected by FFA. Nonetheless, although residual PKB activity in the presence of FFA was sufficient to promote full activation of p70S6K, it is possible that phosphorylation of alternative protein substrates by PKB is inhibited in the presence of FFA, which, in turn, could lead to decreased ß-cell mitogenesis (41, 42, 43).
It was also found that FFA activated PKC in INS-1 cells during
a 24-h exposure, independently of glucose or IGF-I. In INS-1 cells
previously exposed to FFA for 24 h, subsequent
glucose/IGF-I-stimulated PKC activity was additive to the
FFA-induced PKC activity. This raises the possibility that
glucose/IGF-I and FFA activate ß-cell PKC via distinct mechanisms.
Indeed, PKC can be directly activated by both FFA, and
phosphatidylinositol-3,4,5-trisphosphate can be generated by
glucose/IGF-I induced activation of PI3'-K (44). Also,
PKC activity can be further increased via phosphorylation by
phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that
also lies downstream of PI3'-K (45, 46). Thus, PKC
activity can be chronically activated by FFA-treated ß-cells and
further enhanced by increased glucose/IGF-I levels acutely. However,
signaling proteins such as PKC are only transiently activated
under normal circumstances (44). As such, chronic
FFA-induced activation of PKC may lead to an abnormal
prolonged phosphorylation state of certain protein substrates that
could, in turn, lead to ß-cell dysfunction, including an antagonism
of glucose-induced mitogenesis. Interestingly, it has been shown that
chronic activation of PKC can inhibit PKB activity (47, 48), and one might speculate that chronic FFA-induced activation
of PKC contributed to FFA-mediated inhibition of
glucose/IGF-I-induced activation of PKB in ß-cells. However, although
it is intriguing to contemplate that FFA-mediated inhibition of
glucose/IGF-I-induced activation of PKB and/or chronic activation of
PKC can contribute to FFA-induced inhibition of ß-cell DNA
synthesis, this needs to be demonstrated more directly by further
experimentation. For instance, if this scenario is correct, one might
predict that expression of a dominant negative PKB and/or
constitutively active PKC would mimic FFA-mediated inhibition of
glucose/IGF-I-induced ß-cell DNA synthesis. Likewise, expression of a
constitutively active PKB and/or dominant negative PKC would be
predicted to alleviate FFA-mediated inhibition of glucose/IGF-I-induced
ß-cell DNA synthesis. Notwithstanding, FFA-mediated inhibition and/or
chronic activation of other signal elements downstream of IRS, or in
alternative signal transduction pathways cannot, for the moment,
be ruled out as a mechanism for preventing glucose/IGF-I-induced
ß-cell mitogenesis.
It is currently thought that peripheral insulin resistance in obesity
is compensated by an increase in pancreatic ß-cell mass and
consequential increased insulin production (8). However, a
failure of ß-cell mass to compensate for insulin resistance results
in type II diabetes (6, 7, 11). It has previously been
shown that hyperglycemia and hyperlipidemia contribute to ß-cell
dysfunction (6, 7, 8, 14). Chronic exposure of high
concentrations of glucose can induce ß-cell death (49)
despite the common observation of glucose-induced ß-cell growth
(1, 2). It has also been proposed that FFA and
triglyceride accumulation in ß-cells derived from hyperlipidemia in
obesity-linked type II diabetes contributes to ß-cell dysfunction and
reduction of ß-cell mass (6, 8). Indeed, it has been
previously shown that FFA and subsequent intracellular ceramide
formation instigated ß-cell apoptosis (8). The
FFA-induced inhibition of glucose/IGF-I-induced ß-cell mitogenesis
outlined in this study (via a mechanism independent of ceramide) would
additionally contribute to a FFA-induced decrease in ß-cell mass and
a failure to compensate for peripheral insulin resistance, which are
keys to the pathogenesis of type II diabetes (6, 8). It is
possible that FFA-induced inhibition of glucose/IGF-I-induced ß-cell
mitogenesis could be mediated by FFA-induced inhibition of PKB
activation and/or chronic activation of PKC, although further
experiments will be required to substantiate this observation. However,
it should be noted that FFA also inhibit PKB (50, 51) and
chronically activate certain PKC isoforms (52, 53) in
skeletal muscle cells that, in turn, contribute to peripheral insulin
resistance. Thus, similar adverse affects of FFA on insulin signal
transduction in peripheral tissues and mitogenic signaling pancreatic
ß-cells both contribute to the pathogenesis of obesity-linked type II
diabetes.
|
Footnotes |
|---|
Received June 1, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, ß, and ) by insulin in 3T3/L1 cells.
Transfection studies suggest a role for PKC- in glucose transport.
J Biol Chem 272:2551–2558(MAPK) and JNK/SAPK pathways are important for induction of
nitric-oxide synthase by interleukin-1ß in rat glomerular mesangial
cells. J Biol Chem 274:36200–36206 as a downstream
effector of phosphatidylinositol 3-kinase during insulin stimulation in
rat adipocytes. Potential role in glucose transport. J Biol Chem 272:30075–30082. J Biol Chem 274:8117–8122 by PI 3-kinase and PDK-1. Curr Biol 8:1069–1077[CrossRef][Medline]
is a negative regulator of protein kinase B
activity. J Biol Chem 274:8589–8596
and alterations in the insulin signaling cascade.
Diabetes 48:1270–1274[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  T C. Brelje, N. V Bhagroo, L. E Stout, and R. L Sorenson Beneficial effects of lipids and prolactin on insulin secretion and {beta}-cell proliferation: a role for lipids in the adaptation of islets to pregnancy J. Endocrinol., May 1, 2008; 197(2): 265 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Lai, G. Bikopoulos, M. B. Wheeler, M. Rozakis-Adcock, and A. Volchuk Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic {beta}-cells Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E540 - E550. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. Lin, H.-C. Huang, and J.-K. Lin Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells J. Lipid Res., November 1, 2007; 48(11): 2334 - 2343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Herschkovitz, Y.-F. Liu, E. Ilan, D. Ronen, S. Boura-Halfon, and Y. Zick Common Inhibitory Serine Sites Phosphorylated by IRS-1 Kinases, Triggered by Insulin and Inducers of Insulin Resistance J. Biol. Chem., June 22, 2007; 282(25): 18018 - 18027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yano, M. Ikeda, K.-i. Abe, H. Dansako, S. Ohkoshi, Y. Aoyagi, and N. Kato Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture Antimicrob. Agents Chemother., June 1, 2007; 51(6): 2016 - 2027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Crunkhorn, F. Dearie, C. Mantzoros, H. Gami, W. S. da Silva, D. Espinoza, R. Faucette, K. Barry, A. C. Bianco, and M. E. Patti Peroxisome Proliferator Activator Receptor {gamma} Coactivator-1 Expression Is Reduced in Obesity: POTENTIAL PATHOGENIC ROLE OF SATURATED FATTY ACIDS AND p38 MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION J. Biol. Chem., May 25, 2007; 282(21): 15439 - 15450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Karaskov, C. Scott, L. Zhang, T. Teodoro, M. Ravazzola, and A. Volchuk Chronic Palmitate But Not Oleate Exposure Induces Endoplasmic Reticulum Stress, Which May Contribute to INS-1 Pancreatic {beta}-Cell Apoptosis Endocrinology, July 1, 2006; 147(7): 3398 - 3407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Kim, K. A. Tysseling, J. Rice, M. Pham, L. Haji, B. M. Gallis, A. S. Baas, P. Paramsothy, C. M. Giachelli, M. A. Corson, et al. Free Fatty Acid Impairment of Nitric Oxide Production in Endothelial Cells Is Mediated by IKK{beta} Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 989 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Kapadia and F. V. Chisari Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids PNAS, February 15, 2005; 102(7): 2561 - 2566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Briaud, L. M. Dickson, M. K. Lingohr, J. F. McCuaig, J. C. Lawrence, and C. J. Rhodes Insulin Receptor Substrate-2 Proteasomal Degradation Mediated by a Mammalian Target of Rapamycin (mTOR)-induced Negative Feedback Down-regulates Protein Kinase B-mediated Signaling Pathway in {beta}-Cells J. Biol. Chem., January 21, 2005; 280(3): 2282 - 2293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Eitel, H. Staiger, J. Rieger, H. Mischak, H. Brandhorst, M. D. Brendel, R. G. Bretzel, H.-U. Haring, and M. Kellerer Protein Kinase C {delta} Activation and Translocation to the Nucleus Are Required for Fatty Acid-Induced Apoptosis of Insulin-Secreting Cells Diabetes, April 1, 2003; 52(4): 991 - 997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Wrede, L. M. Dickson, M. K. Lingohr, I. Briaud, and C. J. Rhodes Protein Kinase B/Akt Prevents Fatty Acid-induced Apoptosis in Pancreatic beta -Cells (INS-1) J. Biol. Chem., December 13, 2002; 277(51): 49676 - 49684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Lingohr, L. M. Dickson, J. F. McCuaig, S. R. Hugl, D. R. Twardzik, and C. J. Rhodes Activation of IRS-2--Mediated Signal Transduction by IGF-1, but not TGF-{alpha} or EGF, Augments Pancreatic {beta}-Cell Proliferation Diabetes, April 1, 2002; 51(4): 966 - 976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xiao, S. Gregersen, M. Kruhoffer, S. B. Pedersen, T. F. Orntoft, and K. Hermansen The Effect of Chronic Exposure to Fatty Acids on Gene Expression in Clonal Insulin-Producing Cells: Studies Using High Density Oligonucleotide Microarray Endocrinology, November 1, 2001; 142(11): 4777 - 4784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Dickson, M. K. Lingohr, J. McCuaig, S. R. Hugl, L. Snow, B. B. Kahn, M. G. Myers Jr., and C. J. Rhodes Differential Activation of Protein Kinase B and p70S6K by Glucose and Insulin-like Growth Factor 1 in Pancreatic beta -Cells (INS-1) J. Biol. Chem., June 8, 2001; 276(24): 21110 - 21120. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
