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J. Biol. Chem., Vol. 277, Issue 7, 5667-5674, February 15, 2002
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,,
,,**,,, and
From the Third Division and Second Division,
Department of Medicine and the § Department of Basic Allied
Medicine, Kobe University School of Medicine, Kobe, 650-0017, the
** College of Nursing Art and Science, Hyogo, Akashi
673-8588, and the Department of
Biochemistry, National Cardiovascular Center Research Institute, Suita,
Osaka 565-8565, Japan
Received for publication, May 1, 2001, and in revised form, October 26, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Ghrelin was identified in the stomach as an
endogenous ligand specific for the growth hormone secretagogue receptor
(GHS-R). GHS-R is found in various tissues, but its function is
unknown. Here we show that GHS-R is found in hepatoma cells. Exposure
of these cells to ghrelin caused up-regulation of several
insulin-induced activities including tyrosine phosphorylation of
insulin receptor substrate-1 (IRS-1), association of the adapter
molecule growth factor receptor-bound protein 2 with IRS-1,
mitogen-activated protein kinase activity, and cell proliferation.
Unlike insulin, ghrelin inhibited Akt kinase activity as well as
up-regulated gluconeogenesis. These findings raise the possibility that
ghrelin modulates insulin activities in humans.
Growth hormone (GH)1 is
synthesized and secreted from the anterior pituitary under complex
regulation mechanisms. The two hypothalamic peptides, GH-releasing
hormone and somatostatin, coordinately exert the positive and negative
control of GH release, respectively (1). On the other hand, GH
secretagogues (GHSs) were discovered as a series of small peptide
derivatives of pentapeptides Leu- and Met-enkephaline, which
selectively stimulated GH secretion from pituitary cells. The prototype
of this class of GHSs, GHRP-6, was found to be extremely potent and
specific in mammals and particularly in humans (2). Non-peptidyl GHSs,
L-692,429 (3) and L-163,191 (MK-0677) (4), were also manufactured to
improve oral bioavailability. The GHS receptor (GHS-R) was cloned by
the robust expression system that pig pituitary poly(A)+
RNA was coinjected into Xenopus oocytes together with
cDNA encoding G Recently, an endogenous ligand for GHS-R, named ghrelin, was purified
from the extracts of the stomach and found to be abundant exclusively
in the stomach (7). GHS-R mRNA is expressed not only in the
pituitary and brain but also in other tissues such as pancreas (8),
suggesting that ghrelin may have other physiological functions in
addition to the regulation of GH release. Furthermore, a very recent
report showed that ghrelin caused hyperphagia and obesity (9). These
findings let us to explore the possibility that ghrelin may play some
role in glucose homeostasis and metabolism and modulate insulin action.
Materials--
The sources of materials used in this study were
as follows. Horseradish peroxidase-conjugated monoclonal
antiphosphotyrosine antibody (RC20) was purchased from Transduction
Laboratories. Anti-insulin receptor substrate-1 (IRS-1,
anti-phosphatidylinositol 3-kinase (PI3K) p85 Cell Culture--
HepG2 cells, human hepatocellular carcinoma
cell line, were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, penicillin (200 units/ml),
and streptomycin (50 µg/ml). All hormone treatments were started
after maintaining the cells for 12 h in Dulbecco's modified
Eagle's medium with 0.1% bovine serum albumin (BSA). Then the cells
cultured in Dulbecco's modified Eagle's medium with 0.1% BSA and
varying concentrations of test substances were collected for Western
blot analysis, MAPK assay, and Akt kinase assay. H4-II-E cells, rat
hepatoma cell line, were maintained in Reverse Transcriptase (RT)-PCR--
Total RNA was prepared from
cultured HepG2 cells and H4-II-E cells using Trizol according to the
manufacturer's instructions (Invitrogen). One µg of total RNA was
reverse-transcribed for 1 h at 37 °C in 20 µl of reaction
medium made up of 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, pH
8.3, 1 mM dNTPs, 15 pmol of antisense primer, and 200 units
of Moloney murine leukemia virus RT (M-MLV, Promega). The PCR
amplifications were performed in 50 µl of medium containing 50 mM Tris-HCl, 50 mM KCl, 1 mM MgCl2, pH 9.0, 0.2 mM dNTPs, 15 pmol of sense
and antisense primers, and 2.5 units of Taq DNA polymerase
(Promega). The reaction mixtures were subjected to 40 cycles of PCR
amplification of GHS-R cDNA consisting of denaturation for 60 s at 94 °C, annealing for 60 s at 45 °C, and
elongation for 90 s at 72 °C. The oligonucleotide primers
(5'-CTCTGCATGCCCCTGGACCTCGTTCGC-3' sense and
5'-CTGCCGATGAGACTGTAGAGGACCGTGAGAC-3' antisense) allowed amplification
of a 429-bp GHS-R cDNA product and were verified with DNA
sequencing (model 310, PerkinElmer Life Sciences). The reaction
mixtures were subjected to 40 cycles of PCR amplification of PEPCK
cDNA consisting of denaturation for 60 s at 94 °C,
annealing for 60 s at 56 °C, and elongation for 120 s at
72 °C. The oligonucleotide primers (5'-AGCCTCGACAGCCTGCCCCAGG-3' sense and 5'-CCAGTTGTTGACCAAAGGCTTTT-3' antisense) allowed
amplification of a 575-bp PEPCK cDNA product (10), and the product
was verified with DNA sequencing. The Western Blot Analysis--
Cells were lysed in 20 mM
Tris-HCL, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Nonidet
P-40, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml aprotinin
for 30 min on ice and cleared by centrifugation for 20 min at 4 °C.
Immunoprecipitation was performed by incubating the supernatant
fraction with antibodies specific for IRS-1, IR MAPK in Vitro Kinase Assays--
MAPK activity of HepG2 cells
was measured by using a MAPK assay kit according to the manufacturer's
instructions. In short, the cells were washed with phosphate-buffered
saline, lysed in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
Akt in Vitro Kinase Assays--
Akt kinase activity of HepG2
cells was measured by using Akt kinase assay kit according to the
manufacturer's instructions. In short, the cells were washed with
phosphate-buffered saline, lysed in lysis buffer (20 mM
Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM Phosphatidylinositol 3-Kinase Activity--
In vitro
phosphorylation of PtdIns was carried out in the immune complexes as
described previously (11). Subconfluent HepG2 cells grown in 100-mm
dishes were made quiescent by overnight incubation in Dulbecco's
modified Eagle's medium with 0.1% BSA. The quiescent cells were
incubated with ghrelin for 20 min, insulin for 3 min, or ghrelin for 20 min and then insulin for 3 min, and washed once with ice-cold
phosphate-buffered saline and twice with 20 mM Tris-HCl, pH
7.5, containing 137 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, and 100 µM Na3VO4 (buffer A). The cells
were solubilized in 1 ml of buffer A containing 1% Nonidet P-40, 10%
glycerol, and 0.35 mg/ml phenylmethylsulfonyl fluoride, and
insoluble material was removed by centrifugation at 13,000 × g for 10 min. Supernatant was incubated with anti-IRS-1
antibody for 4 h at 4 °C, and immune complexes were
precipitated from the supernatant with protein A-Sepharose and washed
successively in PBS containing 1% Nonidet P-40 and 100 µM Na3VO4 (three times); 100 mM Tris-HCl, pH 7.5, containing 500 mM LiCl and
100 µM Na3VO4 (three times); and
10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl,
1 mM EDTA, and 100 µM
Na3VO4 (two times). The pellets were
resuspended in 50 µl of 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 1 mM EDTA and combined with 10 µl of 100 mM MnCl2 and 10 µl of 2 µg/µl PtdIns sonicated in 10 mM
Tris-HCl (pH 7.5) containing 1 mM EGTA. The phosphorylation
reaction was started by adding 10 µl of 440 µM ATP
containing 30 µCi of [ Northern Blot Analysis--
Total RNA was prepared from cultured
H4-II-E cells using Trizol. Twenty µg of total RNA was heat-denatured
for 5 min at 70 °C in buffer containing 50% formamide, 2.4 M formaldehyde, 1× 0.02 MOPS, 50 mM sodium
acetate, and 10 mM Na2 EDTA and then loaded onto a 1.2% agarose/formaldehyde gel made up in 1× MOPS, pH7.4, at 50 V for 3-4 h. After electrophoresis, RNA was transferred onto a Hybond
N membrane by capillary action and fixed onto the filters by
ultraviolet light cross-linking. The blot was probed with either a rat
PEPCK or a rat Cell Proliferation Assays--
Cell proliferation assays were
performed by CellTiter 96 AQueous One Solution Cell Proliferation Assay
kit according to the manufacturer's instructions (Invitrogen).
Briefly, ~48-72 h before the assay, HepG2 cells were seeded into
96-well plates at 1-3 × 105 cells/well. When cells
reached 60-70% confluence, the medium was replaced with Dulbecco's
modified Eagle's medium containing 0.1% BSA and then with 100 nM ghrelin, 100 nM insulin, both 100 nM ghrelin and 50 µM PD98059, or both 100 nM insulin and 50 µM PD98059. The cultures
were incubated in a CO2 incubator for 48 h at
37 °C, and then 20 µl/well of CellTiter 96 AQueous One Solution Reagent, which contains a tetrazolium compound,
methanethiosulfonate, was added to the culture medium. The
methanethiosulfonate tetrazolium compound is bioreduced by cells into a
colored formazan product that is soluble in tissue culture medium. This
conversion is presumably accomplished by NADPH or NADH produced by
dehydrogenase enzymes in metabolically active cells. After incubating
the plate 2-4 h in a CO2 incubator, the absorbance at 490 nm was recorded with a 96-well plate reader. All samples were assayed
in duplicate, and each experiment was repeated at least three times.
Statistical Analysis--
The data were expressed as the
mean ± S.E. unless noted otherwise. Statistics were analyzed by
one-way repeated measures analysis of variance with a significance
level of 0.05.
To investigate the possible effects of ghrelin on
insulin-regulated responses, we looked for cell lines expressing a
GHS-R. Various cell lines derived from the liver, adipose tissue, and muscle were screened by RT-PCR with oligonucleotides that have been
reported previously (12). A human hepatocellular carcinoma cell line,
HepG2 cells, provided one PCR product, for which identity with human
GHS-R mRNA was confirmed by DNA sequencing. The same product was
obtained by RT-PCR from a human pituitary cDNA library, a human
liver cDNA library, and a rat hepatoma cell line, H4-II-E cells
(Fig. 1A), and its identity
was confirmed by sequencing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11 (5) and subsequently in rats (6).
GHS-R was prominently expressed in several hypothalamic nuclei and also
in the dentate gyrus and CA2 layers of the hippocampus (5). In
searching an endogenous ligand for GHS-R, however, all efforts to use
the brain extracts proved fruitless.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, anti-adapter molecule
growth factor receptor-bound protein 2 (GRB2), anti-insulin receptor
(IR), and horseradish peroxidase-conjugated anti-rabbit IgG were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Anti-IRS-2 was purchased from Upstate Biotechnology, Inc. (Lake Placid,
NY). PD98059, p44/42 mitogen-activated protein kinase (MAPK) assay kit,
and Akt kinase assay kit were purchased from New England Biolabs.
CellTiter 96 AQueous One Solution Cell Proliferation Assay was
purchased from Promega. Phosphatidylinositol (PtdIns), and 8-CPT-cAMP
were purchased from Sigma. Gene Images random prime labeling module,
Gene Images CDP-star detection module and [
-32P]ATP
were purchased from Amersham Biosciences Inc. TLC plates were purchased
from Merck.
-modified Eagle's medium
containing 10% fetal bovine serum, penicillin (200 units/ml), and
streptomycin (50 µg/ml). All treatments were started after
maintaining the cells for 12 h in -modified Eagle's medium
with 0.1% BSA. Cells were pretreated first with 8-CPT-cAMP for 3 h and then with insulin, ghrelin, or both insulin and ghrelin and
collected for Northern blot analysis.
-actin sequence of the
upstream primer was 5'-TGACCCAGATCATGTTTGAGAGACC-3' and of the
downstream primer was 5'-CCATACCCAAGAAGGAAGGC-3'. After an
initial denaturation for 30 s at 94 °C, PCR was performed for
40 cycles The conditions for PCR were 94 °C for 60 s, 60 °C
for 60 s, 72 °C for 60 s. The rat -actin-coding region
was obtained by RT-PCR and verified with DNA sequencing.
, IRS-2, GRB2, or p85
PI3K overnight at 4 °C, followed by binding to protein A-agarose
(Amersham Life Sciences). After a 2-h incubation at 4 °C, the
incubation was centrifuged briefly at 10000 × g, and
the agarose was washed with 20 mM Hepes, pH 7.0, 10%
glycerol, 0.1% Triton X-100, 150 mM NaCl, 1 mM
Na3VO4 and recentrifuged. This was repeated
three times after which Laemmli medium was added, and the agarose was
heated at 100 °C for 5 min. Immunoprecipitated proteins were
fractionated on 7.5% acrylamide gel and blotted to polyvinylidene
difluoride membrane for 30 min. Immunoblot was blocked by incubation in
TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% Tween 20) containing 5% skim milk for 1 h. Blots were
incubated with horseradish peroxidase-conjugated antiphosphotyrosine
antibodies RC20 for 1 h to detect tyrosine-phosphorylated IRS-1,
IRS-2, and IR. Blots were incubated with anti-IRS-1 for 1 h and
then with horseradish peroxidase-conjugated anti-rabbit IgG for
1 h to detect association of GRB2 or PI3K with IRS-1. A
chemiluminescent peroxidase substrate (ECL, Amersham Biosciences, Inc.)
was applied according to the manufacturer's instructions, and the
membranes were exposed briefly to x-ray film.
-glycerophosphate, 1 mM Na3VO4,
1 µl/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), scraped off the dishes, transferred to microcentrifuge tubes, sonicated, and microcentrifuged for 10 min at 4 °C. The supernatant was used for immunoprecipitation of activated MAPKs. The supernatant was incubated with immobilized phospho-p44/42 MAPK
(Thr202/Tyr204) monoclonal antibody for
4 h and microcentrifuged for 30 s at 4 °C. The pellets
were washed twice with ice-cold lysis buffer and twice with kinase
buffer (25 mM Tris (pH 7.5), 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). The pellets were incubated with 200 µM ATP and 2 µg of Elk-1 fusion protein for 30 min at
30 °C. The reaction was terminated by 25 µl of 3× SDS sample
buffer (187.5 mM Tris-HCl (pH 6.8), 6% w/v SDS, 30% glycerol, 150 mM dithiothreitol, 0.3% w/v bromphenol
blue). Samples were boiled, separated by electrophoresis through a 10%
SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride
membrane for 30 min. Immunoblot was blocked by incubation in
TBST containing 5% skim milk at room temperature for 1 h
and then probed with 1:1000 dilution of anti-phospho-Elk-1 antibody.
The membranes were washed three times in TBST, incubated with a 1:1000
dilution of horseradish peroxidase-linked anti-rabbit IgG antibody, and then reacted with LumiGLO reagent according to the manufacturer's instructions. The membranes were exposed briefly to x-ray film.
-glycerophosphate, 1 mM
Na3VO4, 1 µl/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), scraped off the dishes, transferred to
microcentrifuge tubes, sonicated, and microcentrifuged for 10 min at
4 °C. The supernatant was used for immunoprecipitation of activated
Akt kinase. The supernatant was incubated with immobilized Akt antibody
for 2-3 h and microcentrifuged for 30 s at 4 °C. The pellets
were washed twice with ice-cold lysis buffer and twice with kinase
buffer (25 mM Tris (pH 7.5), 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM
MgCl2). The pellets were incubated with 200 µM ATP and 1 µg of GSK-3 fusion protein for 30 min at
30 °C. The reaction was terminated by 20 µl of 3× SDS sample
buffer (187.5 mM Tris-HCl (pH 6.8), 6% w/v SDS, 30% glycerol, 150 mM dithiothreitol, 0.03% (w/v) bromphenol
blue). Samples were boiled, separated by electrophoresis through a 12% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membrane for 30 min. Immunoblot was blocked by incubation in TBST containing 5% skim milk at room temperature for 1 h and then
probed with 1:1000 dilution of anti-phospho-GSK-3 antibody. The
membranes were washed three times in TBST, incubated with a 1:1000
dilution of horseradish peroxidase-linked anti-rabbit IgG antibody, and then reacted with LumiGLO reagent according to the manufacturer's instructions. The membranes were exposed briefly to x-ray film.
-32P]ATP. After 10 min at
22 °C, the reaction was stopped with 20 µl of 8 N HCl
and 160 µl of CHCl3:methanol (1:1). The samples were
centrifuged, and the lower organic phase was removed and applied to a
silica gel TLC plate that had been coated with 1% potassium oxalate.
TLC plates were developed in
CHCl3:CH3OH:H2O:NH4OH (60:47:11.3:2), dried, and visualized by autoradiography.
-actin probe labeled by a Gene Images random prime
labeling module and detected by a Gene Images CDP-star detection module
according to the manufacturer's instructions, and the membranes were
exposed to x-ray film for 30-60 min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The ghrelin receptor GHS-R is found in
hepatoma cells, and ghrelin up-regulates tyrosine phosphorylation of
IRS-1. A, RT-PCR of mRNA from human pituitary
(lane 1), human liver (lane 2), HepG2 cells
(lane 3), H4-II-E cells (lane 4), 3T3-L1
adipocytes (lane 5), and L6 myocytes (lane 6).
B, time-dependent effects of ghrelin on tyrosine
phosphorylation of IRS-1. Serum-starved HepG2 cells were treated with
100 nM ghrelin for 0, 3, 10, and 20 min. Cell extracts were
immunoprecipitated (IP) with antibodies to IRS-1 and
immunoblotted with anti-Tyr(P) (Anti-pTyr). The
representative result is shown in the upper panel. In the
lower panel, all values are expressed as the mean ± S.E. of tyrosine-phosphorylated IRS-1 after densitometric analysis
(n = 5). HepG2 cells were treated with 100 nM ghrelin ( 
) or untreated (
). An arbitrary value of
100 was assigned to the basal level before treatment. Statistical
significance is shown by asterisks: *, p < 0.01 versus vehicle control. C,
dose-dependent increase by ghrelin of tyrosine
phosphorylation of IRS-1. HepG2 cells were treated with 0.1-100
nM ghrelin for 20 min. Control cells (lane 1)
gave 96 ± 6 DU. Cells treated with 0.1, 1, 10, and 100 nM ghrelin gave 102 ± 7, 104 ± 8, 215 ± 20, and 324 ± 16 DU, respectively. The representative result is
shown in the upper panel. In the lower panel, all
values are the mean ± S.E. of tyrosine phosphorylation of IRS-1
after densitometric analysis (n = 5). Statistical
significance is shown by asterisks: *, p < 0.01 versus control, **, p < 0.01 versus 10 nM ghrelin-treated cells.
D, HepG2 cells were treated for 20 min with vehicle alone
(103 ± 10 DU), 100 nM ghrelin (178 ± 15 DU),
100 nM ghrelin + 25 µM
[D-Lys-3]GHRP-6 (97 ± 10 DU), and 25 µM [D-Lys-3]GHRP-6 alone (97 ± 6 DU).
The representative result is shown in the upper panel. In
the lower panel, all values are the mean ± S.E. of
tyrosine phosphorylation of IRS-1 after densitometric analysis
(n = 5). Statistical significance is shown by
asterisks: *, p < 0.01 versus
vehicle alone. The amount of IRS-1, determined in the same blot by
anti-IRS-1, was not changed in panels B-D.
We next investigated the effect of ghrelin on the profile of tyrosine-phosphorylated cellular proteins. HepG2 cells were treated with 100 nM ghrelin for 0-20 min, and cellular proteins were analyzed by immunoblot analysis with antibodies to phosphotyrosine (anti-Tyr(P)). Ghrelin treatment for 10-20 min caused a significant increase in the amount of tyrosine-phosphorylated IRS-1 in HepG2 cells compared with those without ghrelin treatment (354 ± 42 versus 100 ± 5% basal level at 10 min, p < 0.01; 364 ± 47 versus 106 ± 5% basal level at 20 min, p < 0.01) (Fig. 1B). When we cultured HepG2 cells for 20 min with varying concentrations of ghrelin, 10-100 nM ghrelin caused a significant and dose-dependent increase in the amount of tyrosine-phosphorylated IRS-1 as shown in Fig. 1C. Furthermore, ghrelin-induced tyrosine phosphorylation of IRS-1 was canceled by an antagonist for GHS-R, [D-Lys-3]GHRP-6 (Fig. 1D).
HepG2 cells were treated with 100 nM ghrelin for 20 min
followed by stimulation with 100 nM insulin for 1 min, and
cellular proteins were analyzed by immunoblot analysis with
anti-Tyr(P). Ghrelin and insulin significantly increased
tyrosine-phosphorylated IRS-1 levels up to 172 ± 10 and 262 ± 15 densitometric units (DU), respectively, compared with vehicle
alone (99 ± 9 DU). Combined stimulation with ghrelin and insulin
resulted in an additive increase of tyrosine-phosphorylated IRS-1
levels up to 442 ± 23 DU (p < 0.01, n = 5) (Fig.
2A). The tyrosine
phosphorylation of IR chain was not stimulated by the presence of
ghrelin (Fig. 2B). Ghrelin also increased tyrosine
phosphorylation of IRS-1 in H4-II-E cells (Fig. 2D). To
investigate whether the effect of ghrelin on tyrosine phosphorylation
is specific for IRS-1, we tested the effect of ghrelin on tyrosine
phosphorylation of IRS-2. HepG2 cells were treated with 100 nM ghrelin for 20 min followed by stimulation with 100 nM insulin for 1 min. Ghrelin did not increase tyrosine phosphorylation of IRS-2 (Fig. 2C).
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Downstream signaling of IRS-1 is mediated by several associated
proteins including GRB2 and PI3K (13). We therefore tested the effect
of ghrelin on the interaction of GRB2 or PI3K with IRS-1. When HepG2
cells were pretreated with 100 nM ghrelin for 20 min
followed by stimulation with 100 nM insulin for 3 min, the
amount of GRB2-associated IRS-1 (basal, 114 ± 10 DU) was
increased either by ghrelin (256 ± 34 DU, p < 0.01) or by insulin (288 ± 33 DU, p < 0.01).
Combined treatment with ghrelin and insulin resulted in an additive
increase (418 ± 44 DU, p < 0.01, n = 5) (Fig.
3A). Similarly treated cells
were analyzed to examine the association of PI3K with IRS-1.
PI3K-associated IRS-1 in vehicle-treated cells (114 ± 10 DU) was
increased either by ghrelin (220 ± 28 DU, p < 0.01) or by insulin (275 ± 32 DU, p < 0.01).
However, no additive increase in PI3K associated with IRS-1 was found
by a combined treatment with ghrelin and insulin (211 ± 24 DU, p < 0.01, n = 5) (Fig.
3B).
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Furthermore, because MAPKs and Akt are the downstream substrates for
GRB2 and PI3K, respectively, we tested whether MAPKs and Akt are
involved in cellular responses to ghrelin. Phosphorylated active MAPK
was collected from cell lysates using anti-phospho-MAPK antibody, and
its enzyme activity was determined by the amount of phosphorylated
Elk-1 fusion protein. HepG2 cells were pretreated with 100 nM ghrelin for 20 min followed by stimulation with 100 nM insulin for 3 min and were then analyzed for MAPK
activity. MAPK activity in vehicle-treated cells (75 ± 14 DU) was
increased either by ghrelin (246 ± 24 DU, p < 0.01) or by insulin (389 ± 36 DU, p < 0.01).
Combined treatment with ghrelin and insulin caused an additive increase
(546 ± 58 DU, p < 0.01, n = 5)
(Fig. 4A). These response
patterns of MAPK activity to either insulin, ghrelin, or the
combination of both were compatible with those of GRB2-associated IRS-1
(Fig. 3A). We also measured the Akt kinase activity
determined by the amount of phosphorylated glycogen synthase kinase-3
(GSK-3) fusion protein. Similarly treated cells were analyzed for Akt
kinase activity. Akt kinase activity in vehicle-treated cells (154 ± 24 DU) was increased by insulin (524 ± 126 DU,
p < 0.01) and, in contrast, decreased by ghrelin
(29 ± 8 DU, p < 0.01) as well as by a combined
treatment with ghrelin and insulin (82 ± 17 DU,
p < 0.01, n = 5) (Fig.
4B). These findings were not correlated with the association
of PI3K with IRS-1. We therefore measured the amount of PI3K activity
in IRS-1 immunoprecipitates. IRS-1-associated PI3K activity in
vehicle-treated cells (64 ± 10 DU) was increased either by
ghrelin (200 ± 19 DU, p < 0.01) or by insulin
(203 ± 24 DU, p < 0.01). However, no additive
increase in IRS-1-associated PI3K activity was found by combined
treatment with ghrelin and insulin (226 ± 7 DU, p < 0.01, n = 4) (Fig. 4C).
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We also investigated whether ghrelin affects glucose homeostasis in
cell culture. Hepatic and renal gluconeogenesis is crucially important
in maintaining glucose homeostasis. The rate-limiting enzyme of
gluconeogenesis is phosphoenolpyruvate carboxykinase (PEPCK). This
enzyme has no known allosteric control and is down-regulated by insulin
at the transcriptional level. The rat hepatoma cell line, H4-II-E
cells, has been used successfully to study the regulation of PEPCK
expression, whereas HepG2 cells do not express PEPCK efficiently
(14-16). The amount of PEPCK mRNA in H4-II-E cells treated first
with 8-CPT-cAMP and then with insulin was reduced compared with cells
treated with 8-CPT-cAMP alone. Surprisingly, incubation of the cells
with ghrelin for 1 to 2 h before adding insulin partially reversed
the down-regulating effect of insulin on PEPCK mRNA levels (Fig.
5). Another main regulator of PEPCK gene
expression is cAMP. Hence, we investigated the intracellular cAMP using
the enzyme immunoassay for cAMP. Ghrelin did not increase the
intracellular cAMP in H4-II-E cells (data not shown).
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Next we tested the effects of ghrelin on cell proliferation. On the
basis of the analogous data between ghrelin and insulin with regard to
the stimulation of MAPK activity in HepG2 cells, we hypothesized that
ghrelin causes cell proliferation in HepG2 cells via MAPK pathway.
Ghrelin stimulated proliferation of HepG2 cells, and a MAPK
kinase-1-specific inhibitor, PD98059, completely blocked both ghrelin-
and insulin-induced cell proliferation (Fig. 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have demonstrated for the first time that ghrelin treatment causes stimulation of the IRS-1-GRB2-MAPK pathway as well as cell proliferation and that ghrelin inhibits Akt kinase activity as well as up-regulates PEPCK gene expression.
The effect of ghrelin on IRS-1 phosphorylation is unlikely
to be mediated via IR, because tyrosine phosphorylation of the IR
chain was not stimulated by the presence of ghrelin. However, it would
be exerted independently by an activation of a GHS-R signaling cascade.
We found that ghrelin-induced tyrosine phosphorylation of IRS-1 was
blunted by an antagonist for GHS-R, [D-Lys-3]GHRP-6, and
that other GHSs, GHRP-6 and GHRP-2, induced a significant increase in
the amount of tyrosine-phosphorylated IRS-1 in HepG2 cells (data not
shown). Therefore, the downstream molecules of GHS-R signaling
can cross-talk with the insulin-signaling pathway. Indeed, it has been
reported that IRS-1 is phosphorylated by growth factors and cytokines,
including insulin-like growth factor-I, interferon-, interleukin-4,
and interleukin-9 as well (11, 17-20). It is unique, however, that
GHS-R is the G protein-coupled receptor that cross-talks with the
insulin-signaling pathway. Hence, it remains to be elucidated what
molecules in the GHS-R signaling pathway affect the tyrosine
phosphorylation of IRS-1 (Fig. 7).
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IRS-1 is characterized to possess the 20-22 potential tyrosine phosphorylation sites that are conserved between IRS-1 homologs. The surrounding amino acid residues are also highly conserved, and several of these represent potential binding sites for proteins that contain Src homology 2 (SH2) domains (21, 22). IRS-1 interacts with many SH2 proteins with diverse phosphotyrosine motif requirements including PI3K and GRB2 (13, 21). In the present study, ghrelin increased the tyrosine phosphorylation of IRS-1, association of GRB2 with IRS-1, and MAPK activity, indicating up-regulation of the IRS-1-GRB2-MAPK pathway. Furthermore, ghrelin-stimulated proliferation of HepG2 cells and PD98059 completely blocked ghrelin-induced cell proliferation, indicating that MAPKs were essential in HepG2 cell proliferation caused by ghrelin. However, ghrelin suppressed Akt kinase activity, despite of the presence of insulin, as well as up-regulating the amount of PEPCK mRNA expression, although it increased not only the association of PI3K with IRS-1 but also IRS-1-associated PI3K activity.
The phospholipid kinase PI3K is activated by virtually all receptor tyrosine kinases. Activated PI3K phosphorylates PtdIns(4)P and PtdIns(4,5)P2 to generate the membrane-embedded second messengers PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These lipids play a crucial role in the activation of Akt. PtdIns(3,4,5)P3 mediated membrane translocation of a variety of signaling proteins, including the protein-serine/threonine kinases, 3'-phosphoinositide-dependent kinase-1 (PDK-1), and Akt. Akt is phosphorylated by PDK-1 on Thr-308 in its activation loop. Phosphorylation of Thr-308 is a prerequisite for kinase activation, but phosphorylation of the C-terminal hydrophobic residue is required as well for full activation of Akt kinase. The Akt Ser-473 kinase (hypothetical PDK-2) remains to be identified (23-25).
Furthermore, the activity of effector proteins that are dependent on PI3K activation can be negatively regulated by PTEN (phosphate and tensin homolog deleted on chromosome ten) and SHIP (SH2-containing inositol 5'-phosphatase), two phosphoinositide-specific phosphatases that dephosphorylate the 3' and 5' positions of the inositol ring of phosphoinositides, respectively, leading to inhibition of cellular responses mediated by PI3K products (26, 27). In the present study, the dissociation of the downstream molecules in the IRS-1-PI3K-Akt pathway remains difficult to explain, but the presence of a potent inhibiting mechanism of Akt kinase activity by ghrelin, even under full activation by the PI3K-IRS-1 association, is likely. It is possible that these enzymes, such as PDK-1, PDK-2, SHIP, and PTEN, may be affected by GHS-R-mediated signaling molecules. Hence, it remains to be elucidated what molecules in the GHS-R signaling pathway affect the IRS-1-PI3K-Akt pathway (Fig. 7).
The repression of the PEPCK gene by insulin has been studied in detail. There is conflicting evidence as to which signaling pathways may be involved in insulin repression of PEPCK gene expression, the activation of the PI3K pathway, or the activation of the MAPK pathway in which GRB2 engages IRS-1, IRS-2, Shc, or SHP2 (28-30). Recently, however, accumulating evidence suggests that the signaling pathways in insulin repression of PEPCK gene expression may involve the activation of PI3K pathway but not the MAPK pathway (30-32). Our findings that ghrelin inhibits Akt activity may be in agreement with the recent data that PI3K pathway is involved in the insulin-induced repression mechanism of PEPCK expression. Another main regulator of PEPCK gene expression is cAMP. However, ghrelin did not increase the intracellular cAMP in H4-II-E cells (data not shown). Therefore, the signaling pathway by which ghrelin stimulates PEPCK gene expression remains as yet unknown. We propose that ghrelin can affect gluconeogenesis, at least in the H4-II-E cells, by attenuating the effect of insulin on the expression of PEPCK. Considering the effect of ghrelin on glucose homeostasis, it is of note that GHSs are diabetogenic in rats (33). Physiological significance of ghrelin in vivo animals is to be clarified.
In conclusion, we found two novel actions of ghrelin in addition
to its GH-releasing action: one is insulin-like action stimulating the
IRS-1-GRB2-MAPK pathway, which in turn activates cell proliferation; and the other is anti-insulin action suppressing Akt activity and
up-regulation of gluconeogenesis. Although the mechanism by which
ghrelin affects insulin signaling pathways remains not fully understood, our findings obtained in hepatoma cells strongly
implicate the peripheral actions of ghrelin in glucose
homeostasis and in mitogenic processes in humans.
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ACKNOWLEDGEMENT |
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We are grateful to Chika Ogata for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Japanese ministry of Education, Science, and Culture and by grants from the Japanese Ministry of Health, Labor, And Welfare and the Foundation for Growth Science in Japan.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.
¶ To whom correspondence should be addressed: Dept. of Basic Allied Medicine, Kobe University School of Medicine, 7-10-2 Tomogaoka, Kobe 654-0142, Japan. Tel.: +81-78-796-4540; Fax: +81-78-796-4540; E-mail: okimura@ams.kobe-u.ac.jp.
Published, JBC Papers in Press, November 27, 2001, DOI 10.1074/jbc.M103898200
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ABBREVIATIONS |
|---|
The abbreviations used are:
GH, growth hormone;
GHS, growth hormone secretagogue;
GHS-R, growth hormone secretagogue
receptor;
GHRP, growth hormone-releasing peptide;
IRS, insulin
receptor substrate;
PI3K, phosphatidylinositol 3-kinase;
GRB2, growth
factor receptor-bound protein 2;
GSK-3, glycogen synthase kinase-3;
IR, insulin receptor ;
MAPK, mitogen-activated protein kinase;
PtdIns, phosphatidylinositol;
8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate;
BSA, bovine
serum albumin;
RT, reverse transcriptase;
PEPCK, phosphoenolpyruvate
carboxykinase;
MOPS, 4-morpholinepropanesulfonic acid;
anti-Tyr(P), antibodies to phosphotyrosine;
DU, densitometric units;
PDK-1, phosphoinositide-dependent kinase-1;
PTEN, phosphate and
tensin homolog deleted on chromosome ten;
SH2, Src homology 2.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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