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Un manque de leucine inhibe l'action anabolique de l'IGF-1

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Un manque de leucine inhibe l'action anabolique de l'IGF-1

Messagepar Nutrimuscle-Conseils » 12 Jan 2009 11:48

Hypoxia and leucine deprivation induce human insulin-like growth factor binding protein-1 hyperphosphorylation and increase its biological activity.
Endocrinology. 2009 Jan;150(1):220-31.
Seferovic MD, Ali R, Kamei H, Liu S, Khosravi JM, Nazarian S, Han VK, Duan C, Gupta MB.

Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)-10 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.
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Messagepar Free » 12 Jan 2009 13:46

Y-a-t il une stratégie préférable concernant la prise de Leucine ?
Plutôt en pré, en post, ou pendant le training ?
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Messagepar Robz » 12 Jan 2009 14:40

Que ce doit être lassant pour Nutrimuscle-Conseils de devoir répéter sans cesse les mêmes choses sur les différentes plateformes internet car la majorité des gens reste passif face à l'information qu'en plus MDG leur mache.

Chapeau !
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Messagepar Free » 12 Jan 2009 14:53

Robz si c'est pour dire cela. tu ferais aussi bien t'abstenir :roll:

Je lis Nutrimuscle-Conseil avec attention (et ses 2 livres que j'ai relu plusieurs fois), je ne pense pas être de ceux qui ne se creusent pas ou ne cherchent pas à comprendre même si parfois ça m'arrangerait qu'on me donne une recette à appliquer car aussi curieux que je sois, j'avoue que ces choses là me dépassent et certaines sont trop complexes pour les appréhender dans leur ensemble. Le corps humain, ce n'est pas une machine qui marche au pétrole ni un organisme binaire...
Il y a beaucoup d'information, et c'est parfois difficile de faire la part des choses...

Si tu sais qqchose tu peux aussi bien aider, mais si c'est pour faire croire que t'es plus malin que les autres (et au passage passer un coup de crème) tu peux aussi bien t'abstenir et trouver autre chose à faire de tes 10 doigts...
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Messagepar Robz » 12 Jan 2009 16:34

Si je ne poste jamais c'est bien parce que je n'ai pas la prétention d'en savoir plus que les autres, je lis les forums comme source d'information, il y a assez de participant qualifié comme cela, aucun besoin de me faire mousser. Ma remarque était sincère envers Nutrimuscle-Conseil, désolé si tu l'as pris pour toi. Je remarque juste dans mes lectures que Nutrimuscle-Conseil rabache souvent la même chose. Ca ne va pas chercher plus loin.

Tu vois je ne suis même pas sur de pouvoir te répondre, il me semble qu'un bon usage de la leucine est son incorporation dans tes shakes pré/pendant/aprés training. En gros il est intéressant d'enrichir sa whey en leucine, la rendant plus efficace.

Voila
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Messagepar Nutrimuscle-Conseils » 12 Jan 2009 19:28

Avant un entraînement
La prise de 5 g de L-LEUCINE Nutrimuscle 30 minutes avant l’effort augmente le niveau de leucine dans le sang et dans les muscles ce qui va préserver du catabolisme et augmenter la force ainsi que la résistance musculaire.

Pendant l’entraînement
Boire 1 g de L-LEUCINE Nutrimuscle toutes les 15 à 20 minutes avec sa boisson glucidique (maltodextrine et/ou dextrose) tout au long de l'effort afin de prévenir la fatigue, le catabolisme et de décupler sa congestion musculaire.

Juste après l’entraînement
Les recherches scientifiques montrent que le renforcement d'une whey protéine par de la L-LEUCINE juste après une séance de musculation augmente de 60 % la riposte anabolique tout en freinant de 24 % le catabolisme par rapport à la whey seule.

Entre les repas
Prendre de 1 à 5 g de L-LEUCINE Nutrimuscle toutes les 2 ou 3 heures en plus des repas et de la supplémentation en protéines afin d'accélérer votre récupération et votre anabolisme musculaire.

Au régime
La L-LEUCINE agit comme un agent de répartition. Ceci signifie que si la L-LEUCINE est naturellement anabolisante pour les muscles, elle est destructrice pour le tissu adipeux. Le renforcement de l’alimentation en L-LEUCINE Nutrimuscle au régime permet d'éliminer sa masse grasse tout en préservant voire en augmentant sa masse musculaire.
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Messagepar NICO-71 » 12 Jan 2009 20:43

Merci! :D 8)
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Messagepar christophe bonnefont » 12 Jan 2009 21:23

:D Merci pour cette réponse complette, que de bonne chose! :idea:
Vive la L-Leucine. :P

Ya-t-il une dose maximal à respecter dans la journée?
(Par exemple peut-on prendre plus de 20gr par jour?)
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Messagepar Nutrimuscle-Conseils » 12 Jan 2009 21:28

plus tu prends le leucine, plus tu peux réduire ta prot
c'est ça qui détermine la quantité totale
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Messagepar Free » 12 Jan 2009 21:34

Merci Nutrimuscle-Conseil (puisqu'on ne peut t'appeler autrement sur ce forum ;-p)
C'est bon à savoir tout ça...
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Messagepar audiomaniac » 13 Jan 2009 12:52

Nutrimuscle-Conseil a écrit:plus tu prends le leucine, plus tu peux réduire ta prot
c'est ça qui détermine la quantité totale


on peut faire un produit en croix avec ca ? :D
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Messagepar Nutrimuscle-Conseils » 14 Jan 2009 11:48

Hypoxia and Leucine Deprivation Induce Human
Insulin-Like Growth Factor Binding Protein-1
Hyperphosphorylation and Increase Its Biological
Activity
Maxim D. Seferovic,* Rashad Ali,* Hiroyasu Kamei, Suya Liu, Javad M. Khosravi,
Steven Nazarian, Victor K. M. Han, Cunming Duan, and Madhulika B. Gupta
Departments of Pediatrics (M.D.S., R.A., V.K.M.H., M.B.G.) and Biochemistry (M.D.S., S.L., S.N., V.K.M.H., M.B.G.),
and Children’s Health Research Institute (M.D.S., V.K.M.H., M.B.G.), University of Western Ontario, London, Ontario,
Canada N6C 2V5; Department of Molecular, Cellular, and Developmental Biology (H.K., C.D.), University of Michigan,
Ann Arbor, Michigan 48109; Diagnostic Systems Laboratories Inc. (J.M.K.), Toronto, Ontario, Canada M5G 1L7; and
Laboratory of Molecular Medicine (C.D.), School of Medicine and Pharmacy, Ocean University of China, Qingdao
266003, China
Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia
and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal
growth restriction has been documented. In this study we tested the hypothesis that hypoxia and
nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting
IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the
deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately
2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the
controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform
is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in
hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry
analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer
169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate
the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated
IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater
affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)10Mand 6.40E09M] relative
to the IGFBP-1 from the controls (dissociation constant 1.54E07 M). Furthermore, the highly
phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation.
These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive
response to hypoxia and nutrient restriction. (Endocrinology 150: 220–231, 2009)
IGF binding protein (IGFBP)-1 is a major IGFBP in pregnancy
that modulates the cellular actions of IGFs (1). IGFBP-1 is
synthesized predominantly by the maternal and fetal liver and by
the maternal decidua during pregnancy (2, 3). Recent in vivo data
show that fetal overexpression of IGFBP-1 inhibits fetal growth
in mice (4, 5) and that IGFBP-1 contributes to fetal growth restriction
(FGR) by inhibiting IGF-mediated fetal growth (6 –9).
IGFBP-1 is a metabolically regulated protein and is suggested
to have an important role in glucose homeostasis (10). The expression
of IGFBP-1 is dynamically influenced by nutritional
status, increasing during fasting, malnutrition, and diabetes
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/en.2008-0657 Received May 5, 2008. Accepted August 22, 2008.
First Published Online September 4, 2008
* M.D.S. and R.A. contributed equally to this work.
Abbreviations: ABC, Ammonium bicarbonate; ACN, acetonitrile; AKP, alkaline phosphatase;
Asp-N, aspartate N-endoproteinases;CM,conditioned media; ECL, Enhanced Chemiluminescence;
FBS, fetal bovine serum; FGR, fetal growth restriction; HEK, human embryonic
kidney; HIF-1, hypoxia-inducible factor 1; HRP, horseradish peroxidase; IGFBP, IGF
binding protein; LC-MS, liquid chromatography-mass spectrometry; LC-MS/MS, liquid
chromatography-tandem mass spectrometry; MS, mass spectrometric; MW, molecular
mass;MWCO,Mr cutoff; pI, isoelectric point; rIGF-I, recombinanthumanIGF-I; SPR, surface
plasmon resonance; 3-D, three-dimensional; TFA, trifluoroacetic acid; TiO2, titanium dioxide;
2-D, two-dimensional.
G E N E R A L E N D O C R I N O L O G Y
220 endo.endojournals.org Endocrinology, January 2009, 150(1):220–231
Downloaded from endo.endojournals.org at Bibl Interuniv de Medecine Series AGR Douane 1 115 on January 13, 2009
while decreasing upon insulin treatment (11–13). Inhibition of
IGFBP-1 production by insulin (14, 15) is one of the potential
mechanisms in regulation of fetal growth (16–18).
Recent studies also suggest that induction of the IGFBP-1
expression under hypoxia and other catabolic conditions is an
evolutionarily conserved mechanism. The biological significance
of IGFBP-1 induction is to reduce the availability of IGFs to their
receptors, and to divert the limited energy resources away from
growth and development toward those metabolic processes essential
for survival (7, 9, 19, 20).
The biological effect of IGFBP-1 depends not only on the total
protein levels, but also on its proteolysis (21) and phosphorylation
state (22, 23). It has been reported that phosphorylation of
IGFBP-1 at certain sites can increase its binding affinity for IGF-I
and, thus, restricts IGF-I’s bioavailability for binding its receptor
(24). In addition, phosphorylation makes IGFBP-1 more resistant
to proteolysis (25), therefore, accentuating its inhibitory
effect on IGF-I.
Exposing HepG2 cells to hypoxia and leucine derivation
treatment significantly induced IGFBP-1mRNAand protein expression
(26–28), however, it is not clear whether hypoxia and
leucine deprivation treatments also affect the phosphorylation
states and biological activity of IGFBP-1. The objectives of this
study were to examine possible changes in IGFBP-1 phosphorylation
status induced by hypoxia and leucine deprivation, determine
the major phosphorylation sites, and investigate the biological
and physiological relevance.
Materials and Methods
Materials
All chemicals used were of electrophoresis or analytical grade. Human
hepatocellular carcinoma cell line HepG2 and human embryonic
kidney (HEK) 293 cells were purchased from American Type Culture
Collection (Manassas, VA). Antihuman IGFBP-1 monoclonal antibody
(Mab 6303) was from Medix Biochemica (Kauniainen, Finland), and
antihuman IGFBP-1 polyclonal was a gift from Dr. R. Baxter of the
Kolling Institute of Medical Research (Sydney, Australia). Horseradish
peroxidase (HRP)-conjugated secondary antibodies were goat antirabbit
or goat antimouse (Bio-Rad Laboratories, Inc., Hercules, CA). ELISA
kits for total and serine phosphorylated IGFBP-1 were from Diagnostic
Systems Laboratories, Inc. (Webster, TX). The total albumin ELISA kit
was from Bethyl Laboratories, Inc. (Montgomery, TX). The total protein
was measured by Bradford assay (Bio-Rad Laboratories).
Phosphopeptide enrichment was performed using titanium dioxide
(TiO2) (Titansphere TiO; GL Sciences Inc., Tokyo, Japan). The interaction
of IGF-I and IGFBP-1 was analyzed using surface plasmon resonance
(SPR) using Biacore X instrument (Biacore, Inc., Piscataway, NJ)
with sensor chips CM5. The amine coupling was performed using
N-hydroxysuccinimide, N-ethyl-N-(3-diethylaminopropyl) carbodiimide,
and ethanolamine hydrochloride. The sensor chips and all the chemicals
for Biacore were from GE Healthcare Bio-Sciences AB (Piscataway,
NJ). Recombinant human IGF-I (rIGF-I) was a gift from Dr. George
Bright of Tercica Inc. (Brisbane, CA).
HepG2 cell culture and treatment conditions
HepG2 cells were grown at 37Cunder 95% air,5%CO2 inDMEM/
F-12 with 10% (vol/vol) fetal bovine serum (FBS) (Life Technologies,
Inc.; Invitrogen Corp., Carlsbad, CA). Cells grown to approximately
90% confluence were trypsinized, counted, replated on 100  20-mm
plates (Falcon; BD Biosciences, Franklin Lakes, NJ) at a density of 1.4
104 cells per ml, and incubated in DMEM/F-12 containing 10% FBS for
24 h until approximately 70% confluence.
Hypoxic treatments
Before treatment, the cells were rinsed twice and incubated for 3 h in
FBS-free DMEM/F-12. The media were then replaced with new FBS-free
DMEM/F-12 and cells immediately placed in a modular incubator chamber
(Billups-Rothenberg Inc., Del Mar, CA) that was flushed with 1% O2,
and 5% CO2 with the bulk N2. The cells in the sealed hypoxic chamber
were placed in the incubator (20% O2) with cells cultured normally
(controls). Oxygen content in the hypoxic chamber was monitored at
12-h intervals with a Hudson 5590 Oxygen Monitor (Hudson, Ventronics
Division, Temecula, CA). The partial pressure measurements for pO2
and pCO2, as well as pH evaluations, were made using an ABL700 series
blood gas analyzer (Radiometer, Copenhagen, Denmark). After treatment,
conditioned media (CM) were collected at 48 h (26, 27). Samples
were centrifuged at 1200 rpm for 10 min and aliquots of the supernatants
stored at 20 C.
Leucine deprivation treatments
Media containing various concentrations of the essential amino acid
leucine were prepared from DMEM/F-12 lacking methionine, leucine,
lysine, and glutamine, and various salts (Sigma-Aldrich Corp., St. Louis,
MO). Cell media were formulated by adding the missing amino acids and
salt components to make it consistent with normal DMEM/F-12, except
for leucine, which was added in 450 (equivalent to DMEM/F-12), 140,
70, and 0 M concentrations. Once cells were approximately 70% confluent,
they were washed and incubated for 3 h with the specially formulated
FBS-free DMEM/F-12 (with 450 M leucine). Cells were then
rinsed with FBS-free DMEM/F-12 (0 M leucine) and finally incubated
with FBS-free DMEM/F-12 containing various concentrations of leucine
as described earlier. The CM were collected after 16 h incubation (27),
centrifuged at 1200 rpm for 10 min, and stored at 20 C.
Western immunoblot and ligand blot analysis for
IGFBP-1
All protein separations were conducted using 1.5 mm 12% sodium
dodecyl sulfate polyacrylamide gels using MagicMark XP (Invitrogen)
Mr marker. Crude amniotic fluid from a healthy pregnancy was used as
a positive control. For IGFBP-1 expression, equal volumes (10 or 15 l)
of direct CM samples were obtained from cells grown in incubator air
(2O% O2) and hypoxia (1% O2), and from cells cultured with leucine
(450 M leucine) and leucine deprived (0 M leucine) conditions in all
analysis unless specified otherwise. Immunoblot analysis was performed
using wet transfer (29), and membranes were blocked using 4% BSA.
IGFBP-1 Mab 6303 (1:10,000 dilution) was used as the primary antibody
and HRP-conjugated goat antimouse IgG (1:8,000 dilution) as the
secondary antibody. Western Lighting Enhanced Chemiluminescence
(ECL) Reagent Plus (PerkinElmer, Boston, MA) and KodakXOMATLS
films (Eastman Kodak Co., Rochester, NY) were used for detection of
proteins.
To detect other IGFBPs, 5lCMsample from HepG2 cells was used
for ligand blot analysis using biotin-labeled rIGF-I (10 ng/ml) (30). Crude
amniotic fluid was used as a positive control, and proteins were detected
using HRP-conjugated streptavidin (1:1000 dilution) and the ECL Reagent
Plus kit.
Two-dimensional (2-D) immunoblot analysis and
evaluation of IGFBP-1 variants
Equal volumes (100l) ofCMwere desalted and concentrated 10-fold
using 10-kDa Mr cutoff (MWCO) Centricon tubes (PALL Life Sciences,
Ann Arbor, MI). Desalted samples were reconstituted with rehydration
buffer {8 M urea, 2% (3-[(3-cholamidopropyl) dimethylammonio]-
1-propanesulfonate (CHAPS) (Bio-Rad), 50 mM dithiothreitol, 0.2%
Biolyte (Bio-Rad), (pH 3–10 ampholyte), and 0.001% bromophenol
Endocrinology, January 2009, 150(1):220–231 endo.endojournals.org 221
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blue} and transferred onto a polyvinylidene fluoride membrane by wet
transfer (31). Membranes were blocked in 4% nonfat dry milk and then
incubated overnight with IGFBP-1 polyclonal antibody (1:10,000 dilution).
The goat antirabbit HRP-conjugated antibody (1:8000 dilution)
was used as a secondary antibody, and proteins were visualized using the
ECL Plus system.
The 16-bit digital images of the gels and immunoblots were acquired
in the linear range under white light using the FluoroChem 8800 imaging
system (Alpha Innotech Corp., San Leandro, CA). Three-dimensional
(3-D) qualitative evaluation of IGFBP-1 isoforms on the blots was performed
using PG220 software (Nonlinear Dynamics Ltd., Newcastle
upon Tyne, UK).
The phosphorylation state of IGFBP-1 inCMwas confirmed by pretreatment
of the samples (100 l) with calf intestinal alkaline phosphatase
(AKP) (Sigma-Aldrich) (200 U) for 6 h at 37 C. The reaction was
stopped by addition of rehydration buffer. The dephosphorylated protein
samples were further analyzed for IGFBP-1 isoforms using 2-D immunoblotting
as described previously.
Immunoassays for total and phosphorylated IGFBP-1
The phosphorylated IGFBP-1 ELISA is based on first capturing total
phosphorylated and nonphosphorylated IGFBP-1 with an anti-IGFBP-1
monoclonal antibody (32); the captured serine phosphorylated IGFBP-1
is then selectively detected by a specific antiphosphoserine antibody labeled
with HRP (33). Total IGFBP-1 levels in CM were normalized to
total protein by the Bradford method. The total albumin levels in the
same set of CM samples were analyzed by ELISA as per the manufacturer’s
instructions.
Mass spectrometric (MS) analysis of IGFBP-1
phosphorylation
Sample preparation
For MS analysis of IGFBP-1 phosphorylation, samples were from
control (2O% O2) and hypoxia (1% O2) and from cells cultured with
leucine treatments (450 and 0MLeu) conditions. Equal volumes ofCM
(400 l) were desalted (10 kDa MWCO) at 4 C with ammonium bicarbonate
(ABC) buffer (pH 8.0). Samples were then separated on onedimensional
gels. To identify the band corresponding to IGFBP-1 on the
gel, the lane with amniotic fluid (positive control) was excised for immunoblot
analysis using Mab 6303. The remaining gel was fixed for 30
min (10% methanol and 7% acetic acid) and stained overnight with
SYPRO Ruby (Invitrogen) stain. Gel images were captured under UV
excitation with aSYPRO-500filter. Using the immunoblot as a guide, the
specific band on the gel corresponding to IGFBP-1 [28 kDa molecular
mass (MW)] from different treatments was manually excised under
UV light.
For in-gel digestion, the gel slices were cut into small cubes (1mm3),
transferred to siliconized Eppendorf tubes (Hamburg, Germany), and
sequentially washed with 100 mM ABC buffer (pH 8.0), followed by
acetonitrile (ACN). The samples were dried in a vacuum centrifuge. For
reduction and alkylation, the gel pieces were treated with 10 mM dithiothreitol,
followed by 100 mM iodoacetamide. Subsequently, proteins
were digested with aspartate N-endoproteinase (Asp-N) (Sigma-Aldrich)
(25 ng/l), followed by sequencing grade trypsin (12.5 ng/l) (Promega
Corp., Madison, Whey Isolat) at 37 C overnight. The gel was extracted with 30
l ABC, 50% ACN, and 5% formic acid sequentially. The extracted
peptides were dried and stored at 80 C.
Enrichment of IGFBP-1 phosphopeptides
Phosphorylated IGFBP-1 peptides were enriched using TiO2. In brief,
the pelleted IGFBP-1 peptides after digestion were dissolved in 20 l
loading buffer [80% ACN and 1% trifluoroacetic acid (TFA)] and incubated
with 1 l TiO2 slurry (5 m, 1 mg, in 50% ACN) for 20 min
at room temperature on a shaker. The solution and TiO2 particles were
transferred to a pipette tip with a piece of filter paper inserted at its end
to serve as a frit. The tip was placed in a microcentrifuge tube and centrifuged
for 5 min. The particles were washed using 20 l loading buffer
(50 mg/ml dihydroxybenzoic acid and 0.2% TFA in 40% ACN) and
centrifuged again. The phosphopeptides were eluted using 20 l elution
buffer[5%ammoniumhydroxide(pH11.0)] and centrifuged for 10 min.
To the receiving tube, 5 l 5% TFA was added before eluting the bound
phosphopeptides. The samples were dried in SpeedVac and reconstituted
in 0.1% formic acid in water or in 50 mM EDTA in water before liquid
chromatography-mass spectrometry (LC-MS) or liquid chromatography-
tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS and LC-MS analysis for IGFBP-1 phosphoresidue
identification
The enriched phosphopeptides were analyzed on a CapLC (Waters
Corp., Milford, MA) coupled with a Quadrupole Time-of-Flight mass
spectrometer (Global Ultima; Micromass, Manchester, UK) using a 5
m0.5mmC18 precolumn and a 75m150mmanalytical column
(LC Packings, Amsterdam, The Netherlands) with a 300-nl/min flow rate
through the analytical column.LC-MS/MSanalysis was performed using
a gradient elution and the data-dependent acquisition function (34). For
estimations of the phosphorylation changes upon treatments, LC-MS
analysis were performed on the same instrument setting. The selected ion
chromatograms for different phosphopeptide peaks were plotted, and
the spectra were summed. The intensities of the phosphopeptide peaks
in the summed spectra were used for the semiquantitative determination
of the relative amounts of phosphopeptide in the samples from control
to treatment conditions.
LC-MS/MS spectra were processed using the Maxnt 3 function in
Masslynx software (version 4.0; Waters). Mascot (http://mascot.bio.nrc.
ca/search_e.php; Matrix Science, Boston, MA) and PEAKS software
were used to search Swiss-prot database for protein identification. Peptide
mass/charge (m/z) tolerance was set to 1.2 and the peptide fragment
ion tolerance to 0.1 Da. Asp-N and/or trypsin was designated as the
protease, and up to one missed cleavage was allowed. Carbamidomethylation
on cysteine residue was included as a fixed modification, whereas
oxidation of methionine and phosphorylation of serine/threonine/tyrosine
and tyrosyl residues were selected as a variable modification.
Phosphopeptides identified were manually inspected to verify that the
majority of high abundance peaks were y or b sequence ions, or y 
H2O/H3PO4 or b  H2O/H3PO4 ions when appropriate. For all the
phosphopeptides, their phosphorylation sites were verified manually.
SPR for binding characteristics of IGFBP-1 with IGF-I
The comparative measurements of the binding rate constants characteristic
of IGFBP-1 and rIGF-I were performed using a Biacore X instrument.
Seventy microliters of rIGF-I (10 g/ml) diluted in 100 mM
acetate buffer (pH 4.0) were immobilized to the Sensor Chip CM5 surface
by amine coupling as per the manufacturer’s protocol. The rIGF-I
immobilization was performed on a “sample flow cell,” that achieved
approximately 4000–5000 resonance unit signals in three different
experiments.
AllCMsamples were buffer exchanged with HBS EP buffer (pH 7.4)
[10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant
P20 (pH 7.4)], concentrated 10-fold, and serially diluted at analysis in the
HBS EP buffer. The overabundance of IGFBP-1 and negligible IGFBP-3
secreted by HepG2 cells (35) discounted interference if any in Biacore
analysis. IGF-I binding assay was additionally performed on dephosphorylated
IGFBP-1, obtained by AKP treatment of hypoxia (1% pO2)
and leucine deprivation (0MLeu) treated CM. The AKP reactions were
performed as described earlier, except here the reactions were terminated
using EDTA (final concentration, 50mM) followed by immediate buffer
exchange with HBS EP buffer.
In a typical binding experiment, 70 l CM with various concentrations
of analyte (70–700 Nutrimuscle) was injected for a 60-sec association phase
in both reference and sample cells. The interaction of IGFBP-1 with the
immobilized IGF-I was monitored until equilibrium was attained. The
dissociation phase was initiated by passage of HBS EP buffer for a period
222 Seferovic et al. IGFBP-1 Hyperphosphorylation Endocrinology, January 2009, 150(1):220–231
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of 1–3 min. The biosensor surfaces were regenerated by a 60-sec injection
of 10–30 l glycine buffer [50 mM (pH 2.0)] after each injection.
CM samples from three independent cell culture experiments for
hypoxic and leucine deprivation treatments were analyzed in triplicate in
random order and tested on at least three different sensor chips. A low
immobilization level as well as a high flow rate (50 l/min) and analyte
concentrations limited the mass transport phenomenon. Furthermore,
the resonance unit response was always reported as the difference between
signals occurring from the sample and the reference cell (with no
ligand). Therefore, bulk refractive index, background and nonspecific
binding of the soluble ligands were always subtracted. Representative
curves were generated for the association and dissociation phases, and
kinetic data were analyzed using the BIAevaluation software version 3.0
(Biacore) as per 1:1 Langmuir binding model.
Biological assay of IGFBP-1 activity
The biological activity of IGFBP-1 inCMsamples from HepG2 cells
was studied using MTS assay (CellTiter 96 AOueous Non-Radioactive
Cell Proliferation Assay; Promega). HEK293 cells were cultured in
DMEM supplemented with 10% FBS, penicillin, and streptomycin in a
humidified-air atmosphere containing5%CO2.CMwere collected from
HepG2 cells grown in incubator air (2O% O2) and hypoxia (1% O2).
The total IGFBP-1 concentrations in CM samples were estimated by
ELISA as described previously. To eliminate possible effects of other
factors in the CM from HepG2 cells, IGFBP-1 was depleted and used as
a control. For this purpose,CMwere incubated with a polyclonal rabbit
antihuman IGFBP-1 antibody overnight at 4 C (1:500 dilution); 50 l
protein A-Sepharose was then added and rocked for another 4 h at 4 C.
The CM were centrifuged, and the supernatants collected were used as
controls. This IGFBP-1 depletedCMsample was also added to the IGF-I
(25 Nutrimuscle) group. Various concentrations of IGFBP-1 (8.3, 25, or 75 Nutrimuscle)
were added singly or with 25 nMIGF-I. The assays were terminated after
48 h following the manufacturer’s instructions.
Statistical evaluation
Statistical significance among each experimental group was determined
by the unpaired t test. Values are represented as means  SD.
Statistical analysis was performed using GraphPad Prism 3.0 software
(GraphPad Software Inc., San Diego, CA), and significance was accepted
at P  0.05.
Results
Effect of hypoxia and leucine deprivation on IGFBP-1
expression and phosphorylation in HepG2 cells
Air monitoring of the hypoxic chambers ensured desired levels
of O2 in hypoxic treatments. Upon completion of the treatments
(48 h), pO2 tension levels ofCMshowed an average (SEM)
43.3 (1.53) and 133.3 (7.36) torr (mm Hg) for 1 and 20% O2
levels, respectively. The levels of pC02 remained relatively stable
within time points tested, and pHs of the media between the
hypoxic and control treatments were also comparable.
In agreement with previous reports (17, 36, 37), immunoblot
analysis qualitatively indicates (Fig. 1A, lanes 1 and 2) that IGFBP-
1 expression was induced in hypoxia (26, 27). Similarly,
FIG. 1. One-dimensional Western immunoblot. A, Samples are equal volumes of FBS-free CM from HepG2 cells cultured in incubator air (control, 20%
O2) (lane 1) or under hypoxic (1% O2) (lane 2) conditions for 48 h. Lane 3 is amniotic fluid as a positive control. B, CM from HepG2 cells treated with 450
M leucine (lane 1) and without (0 M) leucine (lane 2). C, IGF-I ligand blot of CM from HepG2 cells (lane 1) and amniotic fluid as a positive control (lane
2). IGFBPs identified by their Mr are indicated. ELISA data indicating concentration of albumin as percentage (%) of total protein in samples from cells in
incubator air (control, (20% O2) and hypoxic (1% O2) conditions, and with leucine (control, 450 M) (D) and without (0 M) leucine (E). Decreased levels
of albumin in CM confirmed the effectiveness of the treatment conditions.
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leucine deprivation (0 M leucine) also increased IGFBP-1 levels
compared with cells cultured with high concentrations of leucine
(450 M leucine) (Fig. 1B, lanes 1 and 2). Modest increases in
IGFBP-1 levels were found (data not shown) in cells cultured in
lower leucine concentrations (70 and 140 M leucine). Furthermore,
ligand blot analysis results in Fig. 1C show detection of
mainly IGFBP-1 (lane 1), suggesting a predominance of this protein
and negligible levels of other IGFBPs in HepG2 CM (35).
ELISA estimations in Fig. 1, D and E, show reduced concentration
of albumin for both hypoxia and leucine deprivation.
Albumin is a major mediator of the acute phase response to
disturbances of homeostasis, mainly due to altered hepatic metabolism
(38). Decreased plasma albumin being characteristic of
a negative acute phase reaction and mimicked by HepG2 cells
(37, 39) in hypoxia confirmed effectiveness of the treatment,
physiologically.
Identification of various IGFBP-1 phosphoisoforms
isoforms in hypoxia and leucine deprivation
2-D immunoblot analysis was performed qualitatively to examine
possible differential phosphorylation states in IGFBP-1
induced under hypoxic and leucine deprivation conditions. Data
with CM from the control HepG2 cells show three spots (28
kDa) betweenpH4.5 and 5.5, representing a mixture of non and
variably phosphorylated IGFBP-1 variants (Fig. 2, A1 and B1).
The 3-D densitometric view of the 2-D image shown in Fig. 2, A
and B (panel 2), showed three major peaks. The change in IGFBP-
1 isoelectric point (pI) caused by phosphorylation is estimated
to be pH 0.09 for the first phosphorylation, 0.08 for
the second and third, and0.07 for the fourth subsequent (ScansiteM
r and pI calculator; http://scansite.mit.edu). The pI difference
between units illustrated in the 3-D view was manually
estimated aspHapproximately 0.1. Greater intensity toward the
higher pH is indicative of less phosphorylated states (40). The
peak in the most alkaline region is the nonphosphorylated variant.
The other two spots represent medium and highly phosphorylated
IGFBP-1 isoforms. It is evident from these results that
HepG2 cells secrete a mixture of three major forms of IGFBP-1
with the nonphosphorylated isoform as the dominant one. To
confirm this, the same CM sample was treated with AKP. As
shown in panel 3, AKP treatment resulted in a single nonphosphorylated
form. The results of the hypoxia group are shown in
Fig. 2A, panels 4 and 5. There was an intense spot/peak in the
more acidic region, representing the dominance of a highly phosphorylated
isoform of IGFBP-1. AKP treatment shifted the majority
of the IGFBP-1s to the alkaline region (panel 6), confirming
the phosphorylation status.
Figure 2B shows representative 2-D immunoblots of IGFBP-1
isoforms under the control (450 M Leu) with three distinct
IGFBP-1 isoforms (panel 1); the least or nonphosphorylated isoform
is clearly the dominant form (Fig. 2B, panel 2). AKP treatment
resulted in shifting of the other spots to the alkaline region
(Fig. 2B, panel 3). In the leucine-deprived group (0 M leucine),
a higher proportion of medium and highly phosphorylated variants
were observed. Two of the three spots found at around pH
4.5 had higher intensity (Fig. 2B, panels 4 and 5) compared with
those of the control (Fig. 2B, panels 1 and 2). Although there was
no marked reduction in the levels of nonphosphorylated isoform
as seen in hypoxic experiments, a significant increase in intensity
of some spots suggests higher levels of phosphorylated isoforms.
AKP treatment shifted these phospho-IGFBP-1s to the alkaline
region (Fig. 2B, panel 6). These data suggest that hypoxia and
leucine deprivation increase the phosphorylation of IGFBP-1 in
HepG2 cells.
It should be noted that a polyclonal IGFBP-1 is used in 2-D
immunoblot analysis. The use of this polyclonal was essential
because the monoclonal 6303 antibody was not efficient in detecting
IGFBP-1 on 2-D immunoblots, possibly due to harsh
sample preparation conditions in 2-D gel analysis.
Total and phosphorylated IGFBP-1 concentrations by
ELISA
The effects of hypoxia and leucine deprivation on total IGFBP-
1 and serine phosphorylated IGFBP-1 levels were determined
and are represented as fold change in Fig. 3. As anticipated,
the concentrations of total IGFBP-1 increased in both
hypoxia and in leucine deprivation group. The levels of serine
phosphorylated IGFBP-1 relative to their respective controls
showed proportional increases (Fig. 3, A and B). The data indicate
that an induction of total IGFBP-1 is accompanied by a
proportional increase in IGFBP-1 phosphorylation.
Mass spectrometry for identification of phosphorylation
sites of IGFBP-1
Three phosphorylation sites, Ser 101, Ser 119, and Ser 169,
have been reported for IGFBP-1 (41). Upon sequential Asp-N
and trypsin digestions, the following three phosphopeptides
were detected, and the amino acid sequences [phosphoserine
(pS) shown in parentheses] were confirmed by LC-MS/MS:
DASAPHAAEAGSPESPEpS(101)TEITEEELL, 949.73 m/z,3;
DNFHLMAPpS(119)EE, 685.30 m/z, 2; and AQETpS(169)
GEEISK, 629.78 m/z, 2. The phosphopeptides shown with their
mass to charge ratios (m/z) were detected in samples from hypoxic
(control, 20%O2 and hypoxia,1%O2) and in leucine deprivation
(450 and 0 M leucine) treatments.
A new doubly phosphorylated peptide, DASAPHAAEAGSPEpS
(98)PEpS(101)TEITEEELL, 976.42 m/z.3 was also
detected but only when analyzed with EDTA added to the sample.
The modified protocol (34) increases the detection sensitivity
of multiphosphorylated peptides. Using the CM from hypoxia
treatments, the LC-MS/MS spectra are shown with a peak at
949.73 m/z for the single phosphorylated peptide at Ser 101 (Fig.
4A) and at 976.42 m/z for Ser 98 with Ser 101 (Fig. 4B). This
doubly phosphorylated phosphopeptide was detected in two
out of three samples from hypoxia but not with the controls
(20% O2), or leucine deprivation (450 and 0 M leucine)
treatments.
Semiquantitation of the phosphorylation changes of
IGFBP-1 induced by hypoxia and leucine deprivation
We next performed LC-MS analysis of TiO2 enriched IGFBP-
1 phosphopeptides in CM from different treatments. IGFBP-
1 phosphopeptide peak intensity ratios were calculated
from the relative phosphopeptide peak intensities between the
224 Seferovic et al. IGFBP-1 Hyperphosphorylation Endocrinology, January 2009, 150(1):220–231
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control and treated groups. The data from these experiments are
summarized in Table 1. For the hypoxia experiment, pSer101,
pSer 119, and pSer 169 all showed equal to or more than double
increases in peak signal intensities relative to the control. Similarly,
fold increases were also recorded for leucine deprivation
from two out of three samples.
The doubly phosphorylated peptide with pSer98 and pSer 101
was detected in the samples from hypoxia(1%O2) as shown in Fig.
4B. Due to the absence of pSer98 in the controls (20%O2), the fold
increase upon treatment was not discernable. However, these results
clearly indicate that pSer 98 was hyperphosphorylated in hypoxia.
Being adjacent to the major site, it is possible that pSer98 acts
with pSer101 in hypoxic stress to contribute to changes in IGFBP-1
functions.
It should be noted that the MS analysis was performed using
three independent preparations from three separate cell culture
experiments. Furthermore, the LC-MS analyses were performed
using equal volumes ofCMat different times; therefore, the data
should be considered an estimate. Despite this caveat, the outcome
ofMSanalysis is highly consistent with the results obtained
FIG. 2. 2-D Western immunoblot showing separation of the IGFBP-1 phosphoisoforms based on pI using CM from HepG2 cells. The samples were concentrated
in 10 kDa MWCO centrifugal tubes and loaded on 7-cm immobilized pH gradient strips (pH 4–7). A, CM from cells grown in incubator air (control, 20% O2) (1)
or hypoxic (1% O2) (4) conditions. The blot shown in A4 appears to have a higher intensity of IGFBP-1 with a single dominant phosphoisoforms shifted in pI
toward acidic end compared with the control (A1). Shown in A2 and A5 are the 3-D densitometric views of the specified area. Blots in A3 and A6 are the same
sample of A2 and A4, after treatment with AKP. B1 shows CM from cells grown in control (450 M leucine), B4 in leucine deprived cells (0 M leucine). Shown in
B2 and B5 are the 3-D densitometric views of the specified spot areas. Blots in B3 and B6 are the same samples in B1 and B4 after treatment with AKP.
Endocrinology, January 2009, 150(1):220–231 endo.endojournals.org 225
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by immunoblotting (Fig. 2,Aand B) and ELISA (Fig. 3,Aand B).
Altogether, our LC/MS data clearly demonstrate (Table 1) that
the phosphorylation of IGFBP-1 was consistently increased in
hypoxia and in leucine deprivation treatment, but more prominently
in hypoxia.
IGF-I binding kinetics using SPR analysis
Biacore biosensor measurements were performed to gain insight
into the influences of phosphorylation on the ligand binding
kinetics of the IGFBP-1 molecule. Hypoxia and leucine deprivation
treatment lowered the equilibrium dissociation
constant (KD) value of IGFBP-1 (Table 2). A representative comparison
of association and dissociation phases of the interaction
of IGFBP-1 with IGF-I, in control vs. the hypoxia group, isshown
in Fig. 5A, and with leucine (450MLeu) and leucine deprivation
(0M Leu) groups in Fig. 5B. Compared with IGFBP-1 prepared
from the control cells, IGFBP-1 prepared from the hypoxic cells
and the leucine-deprived cells exhibited slower dissociation
rates, and the resultant complexes were more stable. Kinetic
analysis of the biosensorgram curves demonstrates that the IGFBP-
1 binding affinity for IGF-I under basal conditions was comparable
to those reported previously using Biacore analysis (42).
The estimations of the off rates suggest that binding interactions
of IGFBP-1 with IGF-I were affected by both hypoxia and leucine
deprivation but more significantly by hypoxia (Table 2). To ascertain
that the changes in ligand binding kinetics were indeed
due to elevated phosphorylation, these preparations were dephosphorylated
and analyzed. The results showed that KD values
were returned to the control levels (Table 2), suggesting
that the changes in phosphorylation states are responsible for
the changes in IGF binding affinity.
Hypoxia treatment increases the biological potency of
IGFBP-1 in inhibiting IGF actions
We next determined the functional significance of the hypoxia-
induced IGFBP-1 hyperphosphorylation. Addition of
IGF-I (25 Nutrimuscle) to cultured HEK293 cells resulted in a significant
increase in cell number. Total IGFBP-1 isolated from direct CM
from HepG2 cells grown in either incubator air (20% O2), or
hypoxia (1% O2) inhibited IGF-I activity in a dose-dependent
manner (Fig. 6). The IGFBP-1 sample derived from the hypoxia
group was more potent than that from the20%O2 group. At the
highest dosage (75 Nutrimuscle) tested, it caused a 20% reduction in
IGF-I-induced cell proliferation. In comparison, the less phosphorylated
IGFBP-1 derived from the20%O2 group only caused
a 9% reduction. The difference was statistically significant (P 
0.05). A similar trend was also observed at low doses (25 Nutrimuscle),
although the difference was not statistically significant. These
functional data together with the binding kinetics data indicate
that the hypoxia-induced IGFBP-1 phosphorylation increases its
ability to inhibit IGF actions.
Discussion
Increased expression of IGFBP-1 has been considered a marker
of metabolic irregularities in fetal nutrition (12, 43, 44) and in
oxygen delivery (9, 45) that are strongly linked to FGR (46–48).
By subjecting human HepG2 cells to hypoxia and leucine deprivation
(27, 35, 49–51), we demonstrated that hypoxia and
leucine deprivation lead to altered phosphorylation states of IGFBP-
1. We have identified pSer 169 as a major site of phosphorylation
that may, along with pSer 101, be responsible for altering
the affinity of hepatic IGFBP-1 with IGF-I. We have provided
data suggesting that elevated phosphorylation of the IGFBP-1
molecule increases its IGF binding affinity. The highly phosphorylated
IGFBP-1 also has greater biological activity in inhibiting
IGF-I-stimulated cell proliferation. These findings suggest that
IGFBP-1 phosphorylation may be a novel mechanism of fetal
adaptive response to hypoxia and nutrient restriction.
Regulation of IGFBP-1 and modulation of IGF-I actions are
highly dynamic and complex, particularly in human FGR (52).
Besides the endocrine factors, IGFBP-1 is induced by a variety
of catabolic conditions. For example, fasting, malnutrition,
and protein restriction rapidly induce IGFBP-1 at the transcription
level (19). The depletion of a single amino acid (arginine,
cysteine, and all essential amino acids) is sufficient to
induce IGFBP-1 expression in vitro. Other catabolic conditions
regulating IGFBP-1 expression include endoplasmic reticulum
stress and hypoxic stress (19).
Induction of IGFBP-1 to reduce IGF action is considered to be
part of a regulatory mechanism during fetal development (17,
53). The stress signaling events that alter IGFBP-1 expression
have thus far been shown to be highly significant in signal transduction
events (54). The impacts of chronic hypoxia (7, 8, 17, 35,
FIG. 3. ELISA data indicating the change in total and phosphorylated
IGFBP-1 isoforms from incubator air (control, 20% O2) to hypoxic (1% O2)
condition (A) and control [450 M leucine (Leu)] to leucine deprived (0 M
Leu) conditions (B).
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55) and poor nutrient transfer (20) to the fetus on regulation of
the IGFBP-1 gene at the transcription level are well studied.
Tazuke et al. (35) have identified a hypoxia response element
located in intron 2 of the human IGFBP-1 gene responsible for
the hypoxia response in cultured human HepG2 cells. Likewise,
recent studies using zebrafish embryo show that the induction of
IGFBP-1 gene expression by hypoxia is mediated through hypoxia-
inducible factor 1 (HIF-1) both in vitro and in vivo, and
the HIF-1 pathway is established in early embryonic stages (56).
The functional importance of the HIF-1 pathway in hypoxiainduced
IGFBP-1 gene expression (8, 9) has been suggested to be
a mechanism that in the human fetus could restrict IGF-mediated
growth in utero.
The physiological role of IGFBP-1 depends not only on the
levels of IGFBP-1 but also on its phosphorylation (57). Phosphorylation
and glycosylation of proteins usually result in specific
functional consequences or may be caused by a disease (58,
59). IGFBP-1 phosphorylation is suggested as a key mechanism
in modulation of cellular responses to IGFs (60) and subsequently
in restriction of IGF-I mediated fetal growth (25, 61).
HepG2 cells represent fetal liver metabolism in vitro (62–64)
and have successfully been used in studies with IGFBP-1 involving
fetal hypoxia (17, 36, 65). Furthermore, IGFBP-1 phosphoisoforms
purified from HepG2 show similar IGF-I binding
characteristics to that of the plasma (66). Considering that vari-
TABLE 1. Ratios of IGFBP-1 phosphopeptide peak intensity in
hypoxia (1% O2) and leucine-deprived (0 M leucine) samples
relative to controls (20% O2 and 450 M leucine)
Treatment
Average fold change in IGFBP-1
phosphopeptide peak intensity
Hypoxia/incubator
air
pSer 101 pS119a pS169a
Mean 3.17 2.07 4.22
SD 0.66 0.65 0.96
0/450 M leucine pSer 101a pS119b pS169a
Mean 1.86 4.43 2.45
SD 1.10 0.28
The relative peak intensity measurements for each set (control and treatment
condition) were done sequentially to ensure identical analytical conditions in an
individual experiment. Three MS analyses were done separately using samples
collected from three independent cell culture experiments for both hypoxic and
leucine-deprived treatments.
a Two out of three experiments.
b SD not determined because the intensity in one of the two control samples
(450 M Leu) was lower than the detection level.
FIG. 4. LC-MS/MS spectra showing newly identified phosphorylation site pSer 98 for IGFBP-1 secreted from HepG2 cells under hypoxic (1% O2) condition. The
deconvoluted spectra of ion at 949.73 m/z are for the singly phosphorylated pSer 101 with peptide sequence shown in A and 976.42 m/z is for the doubly
phosphorylated pSer 101 together with pSer 98 in B. Both ions were observed as triply charged ions. In spectrum A, intense b ions confirm the amino acid
sequence of the peptide; the observed b18 ion at 1771.68 and the b18–98 ion at 1673.75 that is derived from b18 ion with a loss of H3PO4 indicate the
phosphorylation on Ser (101) residue. In spectrum B, the precursor ion is 80 Da heavier than the ion in the spectrum A, indicating an additional
phosphorylation; the observed b15 ion at 1458.63 and the b15–98 ion at 1360.59 indicate the phosphorylation on the Ser (98) in addition to the Ser 101.
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able phosphorylation of IGFBP-1 could modulate IGF-I bioavailability
in hypoxia, we selected HepG2 cells for the current
study.
ELISA data revealed that the total serine phosphorylation of
IGFBP-1 increased for hypoxia and leucine deprivation, proportional
to the overall increases in IGFBP-1 secretion. Although
total protein was decreased, qualitatively, IGFBP-1 and its phosphorylation
were concomitantly induced with both treatments.
2-D immunoblotting combined with the ELISA results suggest
that despite modest changes detected in overall phosphorylation,
the proportion of multiphosphorylated isoforms is substantially
increased. The affinity of IGFBP-1 for IGF-I was also increased
for both hypoxic and leucine deprivation treatments, but more
so, consequent to hypoxia.
The relative phosphopeptide intensity for pSer 169 increased
most dramatically under hypoxia. This was followed by pSer
101. Protein phosphorylation being a complex dynamic process
often involves multiple phosphorylation sites (67). Although
technical challenges limit the identification of multiply phosphorylated
peptides (34, 68), a doubly phosphorylated peptide
was identified in hypoxia. With close proximity of pSer 98 with
the major functional site, pSer101 (41), it is conceivable that in
hypoxia, interactions between the adjacent phosphoserines may
be significant in the mechanism of kinase actions (69). Future
mutagenesis study should clarify the importance of pSer 98 in
IGFBP-1 structure and in the functional responses (70, 71).
In the case of leucine deprivation, pSer 119 appeared to increase
the greatest proportion, followed by pS169. In addition,
the effects on IGFBP-1 induction and its phosphorylation were
not solely dose dependent (data not shown) when tested under
physiologically relevant concentrations (72). These results suggest
that possibly extreme nutrient restriction may be necessary
to induce IGFBP-1 phosphorylation to exert any potential metabolic
effects in regulation of IGF-I. The differences in IGFBP-1
phosphorylation sites induced by hypoxic and leucine deprivation
are of potential interest; distinct variations in IGFBP-1 phosphorylation
were also consistent with its IGF-binding kinetics.A
greater binding affinity of IGFBP-1 for IGF-I in hypoxia is also
associated with stronger inhibitory activity to IGF actions.
During pregnancy, IGFBP-1 in plasma is in the non and lesserphosphorylated
forms (66, 73). The putative mechanisms leading
to hyperphosphorylated IGFBP-1 in stress conditions are
unknown. IGFBP-1 is a substrate of multiple protein kinases
(74–76). We speculate that the balance of kinases regulated by
environmental stimuli is altered (35, 77, 78). As a result, the
production and/or the activity of one or multiple protein kinases
(79) may be induced (80). Alternatively, reduced dephosphorylation
by AKP isoforms (81), such as due to aberrant glycosylation
(58), may broaden action potentials resulting in hyperphosphorylated
IGFBP-1. Assuming that hypoxic modulation of
IGFBP-1 phosphorylation in cell culture reflects the in vivo situation,
further investigations should have important implications
for the mechanisms through which the fetus responds to
low oxygen supply. Proteolysis and dephosphorylation of
IGFBP-1 are two physiological processes that may have complementary
roles in regulating the bioavailability of IGF-I. The
widespread oxygen-sensing (82) and signaling mechanisms (83)
FIG. 5. The association and dissociation phases of concentrationdependent
binding of IGFBP-1 to immobilized rIGF-I, comparing hypoxic
or leucine depriving treatment of the HepG2 cells with controls. Analyte
(IGFBP-1) is CM from HepG2 cells grown in incubator air (control, 20% O2)
or hypoxia (1% O2) for 48 h (A) and control (450 M Leu) and leucine
deprived (0 M leucine) treatments (B) for 16 h. The kinetic analysis shows
alterations in dissociation phases for both hypoxic and leucine (Leu)
treatments. Resp. Diff., Response unit differences.
TABLE 2. The kinetics of the affinity of IGFBP-1 for IGF-I
assessed in triplicate by Biacore analysis for CM in hypoxia (1%
O2) and leucine-deprived (450 M leucine) samples relative to
controls (incubator air, 20% O2 and 450 M leucine)
Samples
Ka
(1/msec)
KD
(1/sec) KA (1/M) KD(M)
Incubator air
Mean 4.89E03 7.46E04 6.57E06 1.54E07
SD 1.00E01 1.05E06 2.52E04 1.00E09
Hypoxia
Mean 5.09E03 2.97E06 1.73E09 5.83E10
SD 1.00E01 1.00E08 1.00E07 2.00E12
Dephosphorylated
Mean 1.50E03 1.05E03 1.38E06 7.09E07
SD 5.86E00 4.16E05 1.22E03 1.29E08
450 M Leucine
Mean 7.64E02 1.05E03 6.50E05 1.40E07
SD 2.65E00 5.03E05 9.47E04 4.93E08
0 M Leucine
Mean 1.56E05 9.98E04 1.57E08 6.40E09
SD 4.04E02 1.80E06 2.89E05 5.7735E12
Dephosphorylated
Mean 2.43E03 1.13E03 1.08E06 4.56E07
SD 2.50E01 2.00E05 1.00E01 7.21E09
CM from hypoxic or leucine-deprived conditions were dephosphorylated by AKP
and the kinetics reassessed. E, Times 10 to the power.
228 Seferovic et al. IGFBP-1 Hyperphosphorylation Endocrinology, January 2009, 150(1):220–231
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together with increased phosphorylation in hypoxia could also
affect IGFBP-1 proteolysis (25, 84).
Several factors have so far been associated with fetal stress
that could contribute to elevated levels of IGFBP-1 mRNA and
protein (85–87). This study provides the first biochemical and
physiological evidence of altered IGFBP-1 phosphorylation as a
regulatory mechanism of fetal adaptive response to hypoxia and
possibly to severe undernutrition in utero. Whether IGFBP-1
phosphorylation may be a potential mechanism in other catabolic
conditions that lead to elevated IGFBP-1 production (88–
91) needs to be investigated.
Acknowledgments
Wethank Dr. Gillis Lajoie, Director, Biological Mass Spectrometry Laboratory,
University of Western Ontario, for his interest and invaluable
discussions throughout the project. We also thank Ms. Majida Abushehab
for her technical advice in proteomic analysis, and Ms. Sylvia Katzer,
who diligently proofread the manuscript.
Address all correspondence and requests for reprints to: Madhulika B.
Gupta, Departments of Pediatrics and Biochemistry, and Children’s
Health Research Institute, University of Western Ontario, VRL Room
A5-136 (WC), 800 Commissioners Road East, London, Ontario, Canada
N6C 2V5. E-mail: mbgupta@uwo.ca.
M.B.G. received a Natural Sciences and Engineering Research Council
of Canada (NSERC)-Discovery Grant for financial support. R.A. was
the recipient of a NSERC Undergraduate Student Research Award. Research
conducted in C.D.’s laboratory is supported by National Institutes
of Health Grant 2RO1HL60679 and National Science Foundation Research
Grant IOB 0110864.
Disclosure Statement: The authors have nothing to disclose.
References
1. IwashitaM1994 [Physiological significance of IGF-I and its binding proteins
on fetal growth and maturation]. Nippon Sanka Fujinka Gakkai Zasshi 46:
660–672 (Japanese)
2. Martina NA, Kim E, Chitkara U, Wathen NC, Chard T, Giudice LC 1997
Gestational age-dependent expression of insulin-like growth factor-binding
protein-1 (IGFBP-1) phosphoisoforms in human extraembryonic cavities, maternal
serum, and decidua suggests decidua as the primary source of IGFBP-1
in these fluids during early pregnancy. J Clin Endocrinol Metab 82:1894–1898
3. Sugawara J, Tazuke SI, Suen LF, Powell DR, Kaper F, Giaccia AJ, Giudice LC
2000 Regulation of insulin-like growth factor-binding protein 1 by hypoxia
and 3,5-cyclic adenosine monophosphate is additive in HepG2 cells. J Clin
Endocrinol Metab 85:3821–3827
4. Ben Lagha N, Seurin D, Le Bouc Y, Binoux M, Berdal A, Menuelle P, Babajko S
2006 Insulin-like growth factor binding protein (IGFBP-1) involvement in intrauterine
growth retardation: study on IGFBP-1 overexpressing transgenic mice.
Endocrinology 147:4730–4737
5. Watson CS, Bialek P, Anzo M, Khosravi J, Yee SP, Han VK 2006 Elevated
circulating insulin-like growth factor binding protein-1 is sufficient to cause
fetal growth restriction. Endocrinology 147:1175–1186
6. Giudice LC, de Zegher F, Gargosky SE, Dsupin BA, de las Fuentes L, Crystal
RA, Hintz RL, Rosenfeld RG 1995 Insulin-like growth factors and their binding
proteins in the term and preterm human fetus and neonate with normal and
extremes of intrauterine growth. J Clin Endocrinol Metab 80:1548–1555
7. Maures TJ, Duan C 2002 Structure, developmental expression, and physiological
regulation of zebrafish IGF binding protein-1. Endocrinology
143:2722–2731
8. Huang ST, Vo KC, Lyell DJ, Faessen GH, Tulac S, Tibshirani R, Giaccia AJ,
GiudiceLC2004 Developmental response to hypoxia.FASEBJ 18:1348–1365
9. Kajimura S, Aida K, DuanC2005 Insulin-like growth factor-binding protein-1
(IGFBP-1) mediates hypoxia-induced embryonic growth and developmental
retardation. Proc Natl Acad Sci USA 102:1240–1245
10. Rajkumar K, Krsek M, Dheen ST, Murphy LJ 1996 Impaired glucose homeostasis
in insulin-like growth factor binding protein-1 transgenic mice.
J Clin Invest 98:1818–1825
11. Finlay D, Patel S, Dickson LM, Shpiro N, Marquez R, Rhodes CJ, Sutherland C
2004 Glycogen synthase kinase-3 regulates IGFBP-1 gene transcription through
the thymine-rich insulin response element. BMC Mol Biol 5:15
12. Langford K, Blum W, Nicolaides K, Jones J, McGregor A, Miell J 1994 The
pathophysiology of the insulin-like growth factor axis in fetal growth failure:
a basis for programming by undernutrition? Eur J Clin Invest 24:851–856
13. Tapanainen PJ, Bang P, Wilson K, Unterman TG, Vreman HJ, Rosenfeld RG
1994 Maternal hypoxia as a model for intrauterine growth retardation:
effects on insulin-like growth factors and their binding proteins. Pediatr Res
36:152–158
14. Unterman TG, Oehler DT, Murphy LJ, Lacson RG 1991 Multihormonal
regulation of insulin-like growth factor-binding protein-1 in rat H4IIE hepatoma
cells: the dominant role of insulin. Endocrinology 128:2693–2701
15. Lee PD, Giudice LC, Conover CA, Powell DR 1997 Insulin-like growth factor
binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med
216:319–357
16. Gibson JM, Westwood M, Lauszus FF, Klebe JG, Flyvbjerg A, White A 1999
Phosphorylated insulin-like growth factor binding protein 1 is increased in
pregnant diabetic subjects. Diabetes 48:321–326
17. Popovici RM, Lu M, Bhatia S, Faessen GH, Giaccia AJ, Giudice LC 2001
Hypoxia regulates insulin-like growth factor-binding protein 1 in human fetal
hepatocytes in primary culture: suggestive molecular mechanisms for in utero
fetal growth restriction caused by uteroplacental insufficiency. J Clin Endocrinol
Metab 86:2653–2659
18. Lindsay RS, Westgate JA, Beattie J, Pattison NS, Gamble G, Mildenhall LF,
Breier BH, Johnstone FD 2007 Inverse changes in fetal insulin-like growth
factor (IGF)-1 and IGF binding protein-1 in association with higher birth
weight in maternal diabetes. Clin Endocrinol (Oxf) 66:322–328
19. Kajimura S, Duan C 2007 An evolutionarily conserved fine tuner of IGF actions
under catabolic conditions. J Fish Biol 94:E46–E54
20. Fowden AL, Giussani DA, Forhead AJ 2006 Intrauterine programming of
physiological systems: causes and consequences. Physiology (Bethesda) 21:
29–37
21. Firth SM, Baxter RC 2002 Cellular actions of the insulin-like growth factor
binding proteins. Endocr Rev 23:824–854
22. Westwood M, Gibson JM, Davies AJ, Young RJ, White A 1994 The phosphorylation
pattern of insulin-like growth factor-binding protein-1 in normal
plasma is different from that in amniotic fluid and changes during pregnancy.
J Clin Endocrinol Metab 79:1735–1741
23. Loukovaara M, Leinonen P, Teramo K, Nurminen E, Andersson S, Rutanen
EM 2005 Effect of maternal diabetes on phosphorylation of insulin-like
growth factor binding protein-1 in cord serum. Diabet Med 22:434–439
24. Jones JI, Busby JrWH,Wright G, Smith CE, KimackNM,ClemmonsDR1993
Identification of the sites of phosphorylation in insulin-like growth factor
FIG. 6. Hypoxia-induced IGFBP-1 phosphorylation increases its ability to
inhibiting IGF actions. IGFBP-1 prepared from HepG2 cells grown under
20% O2 (solid column) and 1% O2 (open column) and was added to
cultured HEK293 cells with or with IGF-I at indicated concentrations.
Values are represented as means  SD of two independent assays, each
performed in triplicates. Significance was accepted at P  0.05 as shown
in asterisk (*).
Endocrinology, January 2009, 150(1):220–231 endo.endojournals.org 229
Downloaded from endo.endojournals.org at Bibl Interuniv de Medecine Series AGR Douane 1 115 on January 13, 2009
binding protein-1. Regulation of its affinity by phosphorylation of serine 101.
J Biol Chem 268:1125–1131
25. Gibson JM, Aplin JD, White A, Westwood M 2001 Regulation of IGF bioavailability
in pregnancy. Mol Hum Reprod 7:79–87
26. Eghbali-Fatourechi G, Conover CA, Sieck GC, Gores GJ, Fitzpatrick LA 1994
Secretion of insulin-like growth factor binding protein-1 from individual hepatocytes.
Res Commun Mol Pathol Pharmacol 85:243–259
27. Averous J, Maurin AC, Bruhat A, Jousse C, Arliguie C, Fafournoux P 2005
Induction of IGFBP-1 expression by amino acid deprivation of HepG2 human
hepatoma cells involves both a transcriptional activation and an mRNA stabilization
due to its 3UTR. FEBS Lett 579:2609–2614
28. El Khattabi I, Remacle C, Reusens B 2006 The regulation of IGFs and IGFBPs
by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal
malnutrition. Am J Physiol Endocrinol Metab 291:E835–E842
29. Seferovic MD, Krughkov V, Pinto D, Han VK, Gupta MB 2008 Quantitative
2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted
plasma. J Chromatogr B Analyt Technol Biomed Life Sci 865:
147–152
30. Grulich-Henn J, Spiess S, Heinrich U, Schonberg D, BettendorfM1998 Ligand
blot analysis of insulin-like growth factor-binding proteins using biotinylated
insulin-like growth factor-I. Horm Res 49:1–7
31. Gupta MB, Seferovic MD, Liu S, Gratton RJ, Doherty-Kirby A, Lajoie GA,
Han VKM2006 Altered proteome profiles in maternal plasma in pregnancies
with fetal growth restrictions. Clin Proteomics 2:169–184
32. Khosravi MJ, Diamandi A, Mistry J 1997 Immunoassay of insulin-like growth
factor binding protein-1. Clin Chem 43:523–532
33. Khosravi J, Krishna RG, Bodani U, Diamandi A, Khaja N, Kalra B, Kumar A
2007 Immunoassay of serine-phosphorylated isoform of insulin-like growth
factor (IGF) binding protein (IGFBP)-1. Clin Biochem 40:86–93
34. Liu S, Zhang C, Campbell JL, Zhang H, Yeung KK, Han VK, Lajoie GA 2005
Formation of phosphopeptide-metal ion complexes in liquid chromatography/
electrospray mass spectrometry and their influence on phosphopeptide detection.
Rapid Commun Mass Spectrom 19:2747–2756
35. Tazuke SI, Mazure Nutrimuscle, Sugawara J, Carland G, Faessen GH, Suen LF, Irwin
JC, Powell DR, Giaccia AJ, Giudice LC 1998 Hypoxia stimulates insulin-like
growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a
possible model for IGFBP-1 expression in fetal hypoxia. Proc Natl Acad Sci
USA 95:10188–10193
36. Scharf JG,Unterman TG, Kietzmann T 2005 Oxygen-dependent modulation
of insulin-like growth factor binding protein biosynthesis in primary cultures
of rat hepatocytes. Endocrinology 146:5433–5443
37. Wenger RH, Rolfs A, Marti HH, Bauer C, Gassmann M 1995 Hypoxia, a
novel inducer of acute phase gene expression in a human hepatoma cell line.
J Biol Chem 270:27865–27870
38. Gabay C, Kushner I 1999 Acute-phase proteins and other systemic responses
to inflammation. N Engl J Med 340:448–454
39. Sonna LA, Cullivan ML, Sheldon HK, Pratt RE, Lilly CM 2003 Effect of
hypoxia on gene expression by human hepatocytes (HepG2). Physiol Genomics
12:195–207
40. Halligan BD, Ruotti V, Jin W, Laffoon S, Twigger SN, Dratz EA 2004 Pro-
MoST (Protein Modification Screening Tool): a web-based tool for mapping
protein modifications on two-dimensional gels. Nucleic Acids Res 32:W638–
W644
41. Jones JI, Busby Jr WH, Wright G, Clemmons DR 1993 Human IGFBP-1 is
phosphorylated on 3 serine residues: effects of site-directed mutagenesis of the
major phosphoserine. Growth Regul 3:37–40
42. Wong MS, Fong CC, YangM1999 Biosensor measurement of the interaction
kinetics between insulin-like growth factors and their binding proteins. Biochim
Biophys Acta 1432:293–301
43. ChardT1994 Insulin-like growth factors and their binding proteins in normal
and abnormal human fetal growth. Growth Regul 4:91–100
44. El Khattabi I, Remacle C, Reusens B 2006 The regulation of IGFs and IGFBPs
by prolactin in primary culture of fetal rat hepatocytes is influenced by maternal
malnutrition. Am J Physiol Endocrinol Metab 291:E835–E842
45. Verhaeghe J, Van Herck E, Billen J, Moerman P, Van Assche FA, Giudice LC
2003 Regulation of insulin-like growth factor-I and insulin-like growth factor
binding protein-1 concentrations in preterm fetuses. Am J Obstet Gynecol
188:485–491
46. Giudice LC, Martina NA, Crystal RA, Tazuke S, DruzinM1997 Insulin-like
growth factor binding protein-1 at the maternal-fetal interface and insulin-like
growth factor-I, insulin-like growth factor-II, and insulin-like growth factor
binding protein-1 in the circulation of women with severe preeclampsia. Am J
Obstet Gynecol 176:751–757
47. GrobmanWA,KazerRR2001 Serum insulin, insulin-like growth factor-I, and
insulin-like growth factor binding protein-1 in women who develop preeclampsia.
Obstet Gynecol 97:521–526
48. Ingec M, Gursoy HG, Yildiz L, Kumtepe Y, Kadanali S 2004 Serum levels of
insulin, IGF-1, and IGFBP-1 in pre-eclampsia and eclampsia. Int J Gynaecol
Obstet 84:214–219
49. Takenaka A, Komori K, Morishita T, Takahashi SI, Hidaka T, Noguchi T
2000 Amino acid regulation of gene transcription of rat insulin-like growth
factor-binding protein-1. J Endocrinol 164:R11–R16
50. Casanello P, Torres A, Sanhueza F, Gonzalez M, Farias M, Gallardo V, Pastor-
Anglada M, San Martin R, Sobrevia L 2005 Equilibrative nucleoside transporter
1 expression is downregulated by hypoxia in human umbilical vein
endothelium. Circ Res 97:16–24
51. Ahmad S, Ahmed A 2004 Elevated placental soluble vascular endothelial
growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ Res 95:
884–891
52. Underwood LE, Thissen JP, Lemozy S, Ketelslegers JM, Clemmons DR 1994
Hormonal and nutritional regulation of IGF-I and its binding proteins. Horm
Res 42:145–151
53. Maulik D, Frances Evans J, Ragolia L 2006 Fetal growth restriction: pathogenic
mechanisms. Clin Obstet Gynecol 49:219–227
54. Leu JI, George DL 2007 Hepatic IGFBP1 is a prosurvival factor that binds to
BAK, protects the liver from apoptosis, and antagonizes the proapoptotic
actions of p53 at mitochondria. Genes Dev 21:3095–3109
55. Neerhof MG, Thaete LG 2008 The fetal response to chronic placental insufficiency.
Semin Perinatol 32:201–205
56. Kajimura S, Aida K, Duan C 2006 Understanding hypoxia-induced gene expression
in early development: in vitro and in vivo analysis of hypoxia-inducible
factor 1-regulated zebra fish insulin-like growth factor binding protein 1
gene expression. Mol Cell Biol 26:1142–1155
57. Coverley JA, Baxter RC 1997 Phosphorylation of insulin-like growth factor
binding proteins. Mol Cell Endocrinol 128:1–5
58. Liu F, Zaidi T, Iqbal K, Grundke-Iqbal I, Gong CX 2002 Aberrant glycosylation
modulates phosphorylation of tau by protein kinase A and dephosphorylation
of tau by protein phosphatase 2A and 5. Neuroscience 115:829–837
59. Freeze HH, Aebi M 2005 Altered glycan structures: the molecular basis of
congenital disorders of glycosylation. Curr Opin Struct Biol 15:490–498
60. Baxter RC 2000 Insulin-like growth factor (IGF)-binding proteins: interactions
with IGFs and intrinsic bioactivities. Am J Physiol Endocrinol Metab
278:E967–E976
61. Westwood M 1999 Role of insulin-like growth factor binding protein 1 in
human pregnancy. Rev Reprod 4:160–167
62. Kelly JH, Darlington GJ 1989 Modulation of the liver specific phenotype in the
human hepatoblastoma line Hep G2. In Vitro Cell Dev Biol 25:217–222
63. Wilkening S, Stahl F, Bader A 2003 Comparison of primary human hepatocytes
and hepatoma cell line Hepg2 with regard to their biotransformation
properties. Drug Metab Dispos 31:1035–1042
64. Maruyama M, Matsunaga T, Harada E, Ohmori S 2007 Comparison of basal
gene expression and induction of CYP3As in HepG2 and human fetal liver
cells. Biol Pharm Bull 30:2091–2097
65. Sugawara J, Suh DS, Faessen GH, Suen LH, Shibata T, Kaper F, Giaccia AJ,
Giudice LC 2000 Regulation of insulin-like growth factor-binding protein-1
by nitric oxide under hypoxic conditions. J Clin Endocrinol Metab 85:2714–
2721
66. Westwood M, Gibson JM, White A 1997 Purification and characterization of
the insulin-like growth factor-binding protein-1 phosphoform found in normal
plasma. Endocrinology 138:1130–1136
67. Raggiaschi R, Gotta S, Terstappen GC 2005 Phosphoproteome analysis. Biosci
Rep 25:33–44
68. Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP,
Choudhary JS, Grant SG 2005 Proteomic analysis of in vivo phosphorylated
synaptic proteins. J Biol Chem 280:5972–5982
69. Beck EJ, Sorensen RG, Slater SJ, Covarrubias M 1998 Interactions between
multiple phosphorylation sites in the inactivation particle of a K channel.
Insights into the molecular mechanism of protein kinaseCaction. JGenPhysiol
112:71–84
70. Johnson LN, Barford D 1993 The effects of phosphorylation on the structure
and function of proteins. Annu Rev Biophys Biomol Struct 22:199–232
71. Johnson LN, O’ReillyM1996 Control by phosphorylation. Curr Opin Struct
Biol 6:762–769
72. Jauniaux E, Gulbis B, Acharya G, Gerlo E 1999 Fetal amino acid and enzyme
levels with maternal smoking. Obstet Gynecol 93:680–683
73. Bhatia S, FarssenGH,Carland G, Balise RL, Gargosky SE, DruzinM,El-Sayed
Y, Wilson DM, Giudice LC 2002 A longitudinal analysis of maternal serum
insulin-like growth factor (IGF-I) and total and nonphosphorylated IGF-bind-
230 Seferovic et al. IGFBP-1 Hyperphosphorylation Endocrinology, January 2009, 150(1):220–231
Downloaded from endo.endojournals.org at Bibl Interuniv de Medecine Series AGR Douane 1 115 on January 13, 2009
ing protein-1 in human pregnancies complicated by intrauterine growth restriction.
J Clin Endocrinol Metab 87:1864–1870
74. Frost RA, Tseng L 1991 Insulin-like growth factor-binding protein-1 is phosphorylated
by cultured human endometrial stromal cells and multiple protein
kinases in vitro. J Biol Chem 266:18082–18088
75. Koistinen R, Angervo M, Leinonen P, Seppala M 1993 Phosphorylation of
insulin-like growth factor-binding protein-1 from different sources. Growth
Regul 3:34–37
76. Ankrapp DP, Jones JI, Clemmons DR
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Messagepar Free » 14 Jan 2009 11:54

Une question aux experts,
je suis tombé sur qqs études indiquant le lien entre IGF-1 et cancer. Qu'en est-il ?

Certaines études pronnent une alimentation hypoprotéiné pour vivre plus longtemps...
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Messagepar audiomaniac » 14 Jan 2009 13:28

tu enrichis toutes tes prot en leucine Nutrimuscle-Conseil ?
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Messagepar Nutrimuscle-Conseils » 14 Jan 2009 13:52

non, juste après l'entraînement seulement
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