Glucose Regulates EF-2 Phosphorylation and Protein
Translation by a Protein Phosphatase-2A-dependent Mechanism
in INS-1-derived 832/13 Cells*
Limei
Yan
,
Angus C.
Nairn§,
H. Clive
Palfrey¶, and
Matthew J.
Brady
From the Departments of
Medicine and
¶ Neurobiology, Pharmacology and Physiology, University of
Chicago, Chicago, Illinois 60637 and § Department of
Psychiatry, Yale University School of Medicine, New Haven,
Connecticut 06508
Received for publication, February 3, 2003, and in revised form, March 17, 2003
 |
ABSTRACT |
The role of elongation factor (EF)-2
phosphorylation in the regulation of pancreatic
-cell protein
synthesis by glucose was investigated in the INS-1-derived cell line
832/13. Incubation of cells in media containing 1 mM
glucose resulted in a progressive increase in EF-2 phosphorylation that
was maximal by 1-2 h. Readdition of 10 mM glucose promoted
a rapid dephosphorylation of EF-2 that was complete in 10 min and
maintained over the ensuing 2 h. Similar results were obtained
using primary rat islets or Min-6 insulinoma cells. The glucose effect
in 832/13 cells was replicated by addition of pyruvate or
-ketocaproate, but not 2-deoxyglucose, suggesting that mitochondrial
metabolism was required. Accordingly, glucose-mediated dephosphorylation of EF-2 was completely blocked by the mitochondrial respiratory antagonists antimycin A and oligomycin. The hyperglycemic effect was not mimicked by incubation of cells in 100 nM
insulin, 30 mM potassium chloride, or 0.25 mM
diazoxide, indicating that insulin secretion and/or depolarization of
cells was not required. The locus of the high glucose effect
appeared to be protein phosphatase-2A, the principal phosphatase acting
on EF-2. Protein phosphatase-2A activity was stimulated by glucose
addition to 832/13 cells, but neither protein phosphatase-1 nor
calmodulin kinase III (EF-2 kinase) activity was affected under these
conditions. The slower rephosphorylation of EF-2 during the transition
from high to low glucose may involve effects on EF-2 kinase activity.
Addition of 5-aminoimidazole-4-carboxamide
1-
-D-ribofuranoside in high glucose led to a marked
stimulation of EF-2 phosphorylation, consistent with the possibility
that increased AMP kinase activity in low glucose stimulates EF-2
kinase. In parallel with the effects on EF-2 dephosphorylation,
addition of high glucose to 832/13 cells markedly increased the
incorporation of [35S]methionine into total protein.
Taken together, these results suggest that modulation of
extracellular glucose impacts protein translation rate in
cells at
least in part through regulation of the elongation step, via
phosphorylation/dephosphorylation of EF-2.
 |
INTRODUCTION |
Glucose exerts many effects on pancreatic islets in addition to
the familiar enhancement of insulin secretion. Several studies have
shown that hyperglycemia results in both transcriptional and
translational activation in
cells or their derivative cell lines
(e.g. Refs. 1-5). For example, proinsulin production is controlled by glucose at both levels, and acetyl-CoA carboxylase gene transcription is activated. Measures of total protein synthesis using [35S]methionine labeling and two-dimensional
SDS-PAGE reveal substantial increases in many proteins after glucose
stimulation of isolated islets, particularly the components of
secretory granules (6, 7). Mechanistic studies of the glucose effect on
protein synthesis have been ambiguous. Early studies in isolated islets
suggested that the initiation step was primarily affected (1), but
subsequent studies proposed that both initiation and elongation were
involved (2). However, attempts to identify the specific initiation and/or elongation factors that might contribute to changes in translational control have been inconclusive. The situation is complicated by the fact that glucose causes insulin secretion, and
insulin itself can exert profound effects on protein synthesis in
responsive cells, including
cells themselves. Thus, some of the
effects of glucose on translation in
cells may well be mediated in
an autocrine manner (8).
Regulation at the level of initiation is often dependent on the
phosphorylation of the limiting accessory factors eIF-2B and eIF4E-BP
(4E-BP, Phas-1) (for review, see Ref. 9). Whereas insulin is known to
alter the activity of 4E-BP via the phosphatidylinositol 3-kinase-Akt-mTOR1 pathway in
many target cells, glucose appears not to directly affect this system
in
cells (10) but can induce 4E-BP phosphorylation via the
autocrine pathway, provided that amino acids are present (8). Glucose
does appear to increase the activity of eIF-2B, but not by altering the
phosphorylation state of the eIF-2
factor commonly found to be
involved in translational initiation control (10). Protein translation
can also be regulated at the level of elongation, principally by
secondary modification of both elongation factors 1 and 2. EF-1 is a
substrate for several protein kinases, and its activity can be
modulated by insulin in target cells (11). Phosphorylation of EF-2 by
the enzyme Ca2+/CaM-dependent protein kinase
III (EF-2 kinase), primarily at Thr-56, blocks the ability of this
factor to participate in protein synthesis (12, 13). EF-2
phosphorylation state has subsequently been shown to vary in a wide
variety of cell types in response to diverse stimuli (for review, see
Ref. 14). Frequently, these stimuli elevate intracellular
Ca2+, thereby activating the kinase and increasing
phospho-EF-2 (EF-2P) levels. However, other mechanisms of altering
EF-2P levels may be even more important, including phosphorylation and
activation or inactivation of EF-2 kinase by calcium-independent
kinases, as well as effects on the dephosphorylating arm of the cycle.
In this study we examined the effects of glucose on the EF-2
phosphorylation system in
cells or cell lines derived from
cells. For the most part, we used the INS-1-derived 832/13 cell line
that shows robust insulin secretory responses to glucose and appears to
mimic
-cell behavior in several ways (15). We found that glucose
addition to cells maintained in low glucose medium induced a marked and
rapid dephosphorylation of EF-2 that was accompanied by a significant
increase in overall protein synthesis. The effect on EF-2
phosphorylation seemed to be principally mediated by stimulation of
protein phosphatase-2A (PP2A) activity rather than by inhibition of the
kinase reaction. Together, our data suggest that promotion of protein
synthesis by glucose involves modification of the elongation step of
protein translation.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture reagents were supplied by Mediatech,
Inc., except for cysteine/methionine-free media, which was from
Invitrogen. All chemicals were purchased from Sigma, with the exception
of AICAR (Toronto Research Chemicals) and rapamycin, PD98059, and wortmannin (Calbiochem). Tran35S-label (1000 Ci/mmol) and
[
-32P]ATP (4500 Ci/mmol) were obtained from ICN.
Anti-EF-2 and anti-phospho-EF-2 antibodies were generated as described
previously (16); the phospho-acetyl-CoA carboxylase antibody was from
Upstate Cell Signaling Solutions. Horseradish peroxidase-conjugated
goat anti-rabbit and goat anti-mouse IgG was from Bio-Rad, whereas ECL
reagent was obtained from Amersham Biosciences.
Cell Culture and Experimental Treatment--
The INS-1-derived
cell lines 832/1 and 832/13 were generously provided by Dr. C. Newgard
(Duke University) and cultured as described previously (15). Multiwell
cultures (75-90% confluent) were rinsed twice with phosphate-buffered
saline and incubated for 2 h in Krebs-Ringer buffer supplemented
with 25 mM Hepes (pH 7.4) and 0.5% bovine serum albumin
(KRBH) and either 1 or 10 mM glucose. Sample wells were
then switched to KRBH containing the same or different glucose
concentrations for various periods as indicated in the figures. Cells
were then rapidly washed three times on ice with phosphate-buffered
saline, scraped into lysis buffer (50 mM Hepes (pH 7.4),
150 mM NaCl, 10 mM NaF, 10 mM EDTA, 10% glycerol, 0.5% Triton X-100, and protease inhibitors), and centrifuged at 14, 000 × g for 10 min at 4 °C.
Supernatants were removed, and equal amounts of lysate protein were
analyzed by SDS-PAGE and immunoblotting. Rat islets were prepared as
described previously (17), and treated samples were kindly provided by Drs. J. Corbett and P. Hansen (St. Louis University).
Enzyme Assays--
Ca2+/CaM-dependent
EF-2 kinase activity was measured as described previously (18), using
purified rat liver EF-2 as substrate. Reactions contained 5 µg of
lysate protein, 3 µg of EF-2, and 25 µM
[
-32P]ATP. Reactions were terminated by the addition
of SDS sample buffer and boiling; 32P-EF-2 was resolved by
SDS-PAGE, identified by autoradiography, and counted by liquid
scintillation. Protein phosphatase assays were performed as described
previously (19). After treatment, cells were collected in phosphatase
homogenization buffer (50 mM Hepes (pH 7.4), 2 mM EDTA, 2 mg/ml glycogen, 0.2%
-mercaptoethanol, 0.5%
Triton X-100, and protease inhibitors). Samples were then centrifuged
at 14,000 × g for 10 min at 4 °C, and ~5 µg of
supernatant protein was used for assays.
[32P]Phosphorylase was used as substrate and prepared as
described previously (19), with the modification that recombinant
phosphorylase kinase catalytic subunit was used. Phosphatase activity
inhibited by addition of 3 nM okadaic acid is defined as
PP2A, whereas phosphatase activity lost between 3 and 500 nM okadaic acid is defined as protein phosphatase-1.
Addition of 500 nM okadaic acid completely inhibited all
cellular phosphatase activity measured under these conditions.
[35S]Methionine/Cysteine Incorporation into Total
Protein--
832/13 cells were plated in 12-well dishes and
serum-starved as described above. Cells were pretreated in triplicate
for 10 min as indicated, and then 10 µCi of Tran35S-label
was added to all wells. After a 10-min incubation at room temperature,
the dishes were washed thrice with ice-cold 10% trichloroacetic acid; precipitated proteins were then dissolved in 0.5 ml of 0.1 N NaOH and transferred to scintillation vials. The samples
were neutralized by addition 10 µl of 10% acetic acid, and
35S incorporation into proteins was measured by
scintillation counting. In parallel, a replicate well for each
condition was washed three times with phosphate-buffered saline, and
protein lysates were analyzed by SDS-PAGE and autoradiography.
 |
RESULTS |
Glucose Increases Protein Translation in 832/13
Cells--
Shifts in extracellular glucose markedly affect insulin
secretion and protein biosynthesis in
-cell lines and primary islets (1-5). To confirm that glucose levels also regulate protein synthesis in the INS-1-derived 832/13 cells, we preincubated cells for 2 h
in 1 mM glucose and then added back either glucose (3 or 10 mM), pyruvate (5 mM), or insulin (100 nM) for 10 min and measured incorporation of
[35S]methionine into total cellular protein. Addition of
glucose caused a ~4-fold increase in total incorporation (Fig.
1A), and analysis of labeled
proteins by SDS-PAGE showed that this effect involved numerous species
(Fig. 1B). The effect was reproduced by pyruvate, but
insulin in the presence of 1 mM glucose had little effect
on labeling. These results are comparable with those found previously
with isolated islets (7) and demonstrate that the 832/13 cell line
exhibits typical glucose responsiveness with respect to protein
translation.

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Fig. 1.
Stimulation of protein synthesis in
INS-1-derived 832/13 cells by glucose. 832/13 cells were
maintained in KRBH/10 mM glucose (con) or
glucose-restricted in KRBH/1 mM glucose for 2 h.
Replicate wells kept in low glucose were stimulated with glucose (3 or
10 mM), 5 mM pyruvate, or 100 nM
insulin (in 1 mM glucose) for 10 min, and then all samples
were pulse-labeled in [35S]methionine-containing medium
for an additional 10 min. Samples were collected for measurement of
total protein synthesis by trichloroacetic acid precipitation
(A) or for analysis of synthesized proteins by SDS-7.5%
PAGE and autoradiography (B). Results in A are a
summary of three experiments, each point performed in duplicate (data
are means ± S.D.). B, lane 1, 1 mM glucose; lane 2, 3 mM glucose;
lane 3, 10 mM glucose; lane 4, 5 mM pyruvate; lane 5, 100 nM insulin.
Autoradiograph in B is representative of three independent
experiments.
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Extracellular Glucose Levels Regulate EF-2 Phosphorylation in
-Cell Lines and Primary Islets--
Because EF-2 phosphorylation
state has been correlated with protein synthetic capacity in several
studies (for review, see Ref. 14), we investigated whether ambient
glucose affected EF-2P levels in the 832/13 cell line, as well as in
Min-6 insulinoma cells and isolated pancreatic islets. Cells were
preincubated for 2 h in buffer containing either 1 or 10 mM glucose, and lysates were prepared and analyzed by
immunoblot using a phospho-specific EF-2 antibody (raised against a
peptide containing phospho-Thr-56; Ref. 16). Cultures maintained in low
glucose exhibited relatively high levels of EF-2P, whereas those
maintained in high glucose had low levels of EF-2P (Fig.
2A). When samples were shifted
from low to high glucose, EF-2P declined within 10 min to the level found in cells maintained in high glucose throughout (Fig. 2, A and B). In contrast, there was no change in
total EF-2 protein with any treatment (Fig. 2, A and
B). Importantly, the glucose-dependent phosphorylation/dephosphorylation of EF-2 was also manifest in Min-6
cells and primary rat islets (Fig. 2A), indicating that this
effect may be physiologically relevant in
cells from a variety of
sources. However, in high glucose, EF-2P levels were elevated in the
islets as compared with the cell lines, perhaps reflecting the presence
of non-regulated EF-2P in
and
cells in the islet preparations.
To investigate the reversibility of the phenomenon, we verified that
after a 2-h incubation in high glucose, samples returned to low glucose
exhibited a rephosphorylation of EF-2, albeit with slower kinetics as
described below.

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Fig. 2.
Glucose levels regulate the state of EF-2
phosphorylation in INS-1-derived 832/13 and pancreatic
cells. A, 832/13 cells (left
panel), Min-6 cells (center panel), or freshly isolated
rat pancreatic islets (right panel) were preincubated in
KRBH containing 10 mM glucose (hi) or 1 mM glucose (lo) for 2 h. Some of the cells
in KRBH/1 mM glucose were then switched back to KRBH/10
mM glucose for 10 min (lo hi). Extracts
were made, and equal amounts of lysate protein were separated by
SDS-7.5%PAGE, transferred to nitrocellulose, and incubated in rabbit
polyclonal anti-EF-2P IgG (0.5 µg/ml), followed by secondary antibody
and development using ECL. After exposure, blots were stripped and
reprobed with anti-EF-2 IgG as a loading control (bottom
panels; signals varied <10% by densitometric analysis).
B, 832/13 cells were preincubated for 2 h in KRBH
containing 10 mM (hi) or 1 mM
glucose; 10 mM glucose was added to individual wells for
the indicated times (lo hi). Fluorograms are
representative of three to five independent experiments. Note that
EF-2P levels were somewhat elevated in islets cultured in high glucose,
probably due to the presence of and cells in addition to cells.
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Mitochondrial Metabolism of Glucose Is Required for Reduction in
EF-2P Levels--
To determine whether the hyperglycemic effect on
EF-2 phosphorylation was mediated by breakdown and mitochondrial
utilization of the sugar, we tested the effects of non-metabolizable
glucose analogs, as well as permeant compounds that can bypass glucose catabolism. Addition of 2-deoxyglucose to cells maintained in low
glucose had no effect on EF-2P level, indicating that metabolism beyond
the glucokinase step is required (Fig.
3A). By contrast, addition of
5 mM of either pyruvate or
-ketocaproic acid, both of
which can enter mitochondria and be directly metabolized to produce
ATP, could substitute for glucose in mediating EF-2 dephosphorylation (Fig. 3B). Further evidence for mitochondrial involvement
came from the use of respiratory chain inhibitors such as antimycin A
or oligomycin, either of which blocked the effect of glucose on EF-2
dephosphorylation (Fig. 3C). These data indicate that production of ATP or some catabolite derived from glucose may be an
important intermediary in promoting EF-2
dephosphorylation.

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Fig. 3.
Glucose metabolism is required for EF-2
dephosphorylation in 832/13 cells. A, 832/13 cells were
glucose-restricted for 2 h (lo), and then 10 mM glucose (hi) or 2-deoxyglucose
(lo + 2-DOG; 10 mM) was added for
10 min. B, replicate wells were pretreated as described in
A, and then 10 mM glucose (hi), 5 mM pyruvate, or 5 mM -ketocaproate was added
for 10 min. C, cells were glucose-deprived for 2 h
(lo) and stimulated with 10 mM glucose for 10 min (lo hi). The indicated samples were preincubated
with the mitochondrial inhibitors antimycin A or oligomycin (both 1 µM) for 10 min before changing glucose levels. All
autoradiographs are representative of three to four experiments.
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Glucose-stimulated EF-2 Dephosphorylation Is Not Mediated by
Insulin Secretion or by Insulin-activated Signaling
Pathways--
INS-1 cells and islet
cells themselves have insulin
receptors, and it is likely that some effects of glucose are mediated by released insulin, acting in an autocrine or paracrine manner (20).
Moreover, insulin treatment of some other cell types has been reported
to reduce EF-2P levels via inactivation of EF-2 kinase (e.g.
see Ref. 21). To investigate the potential role of insulin
release/signaling on EF-2P levels, 832/13 cells were preincubated for
2 h in buffer containing 1 mM glucose.
Insulin-dependent effects on EF-2P were estimated by direct
addition of 100 nM insulin or by depolarizing the cells
with either 30 mM KCl or the ATP-sensitive potassium
channel blocker tolbutamide. Exogenous insulin did lead to a small
decrease in EF-2P levels at 10 min (Fig.
4A) but was unable to mimic
the glucose effect, even after 2 h (data not shown). Additionally,
stimulation of endogenous insulin release from 832/13 cells by addition
of 100 µM tolbutamide or 30 mM KCl had no
effect on EF-2P levels (Fig. 4B; data not shown). EF-2
dephosphorylation resulting from glucose addition was also unaffected
by inclusion of 0.25 mM diazoxide, an inhibitor of insulin
exocytosis (data not shown). Finally, the glucose effect was evinced in
832/1 cells (Fig. 4B), INS-1-derived cells that exhibit
meager glucose-stimulated insulin secretion (15).

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Fig. 4.
The glucose effect on EF-2P does not involve
insulin-dependent signal transduction pathways.
A, 832/13 cells were maintained in KRBH/1 mM
glucose (lo) for 2 h to elevate EF-2P. Half the samples
were preincubated for 15 min with 100 nM wortmannin; 10 mM glucose (hi) or 100 nM insulin
(lo + Ins) was then added for 10 min. EF-2P levels were
analyzed by immunoblotting; loading controls using total EF-2
immunoblotting showed <10% variation between lanes (data not shown).
B, left panel, cells were glucose-deprived
for 2 h (lo) and then pretreated for 15 min without or
with 10 µM tolbutamide (T). 10 mM
glucose (hi) was then added to half the wells for 10 min
before preparation of cell lysates. Right panel, a variant
INS-1-derived cell line (832/1), selected for poor insulin secretory
responses to glucose, was treated as described in the Fig. 2 legend.
Note that an increase in glucose also lowers EF-2P in these cells.
C, inhibitors of the Ras-mitogen-activated protein kinase
and phosphatidylinositol 3-kinase-mTOR pathways do not block
glucose-induced dephosphorylation of EF-2P. After 2 h of glucose
restriction, 832/13 cells were maintained in either 1 mM
glucose (lo; lanes 1-5) or 10 mM
glucose (hi; lanes 1'-5') for 10 min.
Lanes 1, control; lanes 2, PD98059 (20 µM); lanes 3, rapamycin (100 nM);
lanes 4, wortmannin (200 nM); lanes
5, PD98059 + wortmannin. All drugs were preincubated with cells
for 15 min before shift to high glucose. EF-2P levels were analyzed by
immunoblotting. Bottom panel, reprobe of blot with EF-2 IgG
as loading control. All results are representative of three independent
experiments.
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To investigate potential signaling pathways mediating the regulation of
EF-2P levels by glucose, a variety of well-characterized kinase
inhibitors were used. 832/13 cells were glucose-restricted for 2 h
in KRBH containing 1 mM glucose and preincubated for 15 min
with the indicated compounds, and then 10 mM glucose was
added for a final 10-min period. Pretreatment of 832/13 cells with 100 nM of the phosphatidylinositol 3-kinase inhibitor
wortmannin had no effect on the glucose-dependent
dephosphorylation of EF-2, whereas the modest insulin effect on EF-2P
levels was completely blocked by the drug (Fig. 4A). Neither
the mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase inhibitor PD98059 (20 µM) nor the mTOR
inhibitor rapamycin (100 nM), alone, in combination, or
together with wortmannin, had any effect on the glucose-mediated
reduction in EF-2P levels (Fig. 4C; data not shown). The
efficacy of these compounds on their respective signaling pathways was
confirmed in parallel experiments using insulin-stimulated 3T3-L1
adipocytes (data not shown). Cumulatively, these results indicate that
the regulation of EF-2P levels by extracellular glucose occurs
independently of insulin secretion/signaling and activation of several
well-characterized kinase signaling cascades.
Stimulation of Protein Phosphatase-2A Plays a Major Role in the
High Glucose Effect on EF-2 Dephosphorylation--
The regulation of
EF-2P levels by extracellular glucose must involve alteration of kinase
and/or phosphatase activities. EF-2 kinase is the major and perhaps
only kinase that phosphorylates EF-2, whereas PP2A is the most active
cellular phosphatase acting on EF-2P (12, 14, 18, 22). Direct assays of
kinase activity in lysates derived from cells shifted from low to high
glucose revealed no significant difference in either basal or
Ca2+/CaM-stimulated activity (using activity in 1 mM glucose as 100%, cells maintained in 10 mM
glucose had 113% of this activity, whereas cells shifted for 10 min
from 1 to 10 mM glucose had 97% of this activity),
suggesting that this enzyme is not the primary target of the
hyperglycemic effect. In parallel, both protein phosphatase-1 and PP2A
activities were measured in cellular lysates using
[32P]phosphorylase as substrate. Extracts from cells
incubated with 10 mM glucose contained a high level of PP2A
activity (Fig. 5A). Reducing
extracellular glucose to 1 mM for 2 h reduced PP2A
activity by ~2-fold. Conversely, raising extracellular glucose from 1 to 10 mM caused a substantial activation of PP2A activity
within 10 min; by contrast, protein phosphatase-1 activity measured in the same lysate remained constant during these transitions (Fig. 5A). If stimulation of PP2A is involved in the effect of
high glucose, then the response should be sensitive to permeant
inhibitors of this enzyme. Accordingly, preincubation of 832/13 cells
with calyculin A (50 nM), an inhibitor of both protein
phosphatase-1 and PP2A but not of protein phosphatase-2B or protein
phosphatase-2C, markedly impaired EF-2 dephosphorylation in response to
glucose addition (Fig. 5, B and C). Taken
together, these data suggest that the hyperglycemic reduction in EF-2
phosphorylation state appears to be mediated primarily by an increase
in PP2A rather than a decrease in EF-2 kinase activity.

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Fig. 5.
Glucose-induced dephosphorylation of EF-2 is
mediated by PP2A. A, lysates were prepared from 832/13 cells
that were treated as described in the Fig. 2 legend. Cells were
collected in phosphatase homogenization buffer, and protein
phosphatase-1 and PP2A activities against
[32P]phosphorylase were determined as detailed under
"Experimental Procedures." Results represent mean ± S.D. of
three independent experiments, each performed in duplicate. *,
p < 0.01; NS, p > 0.3 by
Student's t test. B, phosphatase inhibition
reduces the glucose effect on EF-2P. Replicate wells of 832/13 cells
were maintained in KRBH with 1 mM (lo) or 10 mM (hi) glucose for 2 h. Some samples were
then preincubated for 10 min with 50 nM calyculin A before
the addition of 10 mM glucose (lo hi) for
the indicated times. Lysates were analyzed by sequential anti-EF-2P and
anti-EF-2 immunoblotting and quantitated by densitometry
(C). Results are representative of three
independent experiments. Note in B that calyculin A
treatment of cells in high glucose does not increase EF-2P
significantly within 10 min; other experiments show that this is also
true at 1 h (data not shown).
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Phosphorylation of EF-2 in Low Glucose May Involve AMP
Kinase--
When cells were shifted from high to low glucose media,
there was a relatively slow reappearance of EF-2P (Fig.
6A). The marked kinetic
difference between dephosphorylation in high glucose and rephosphorylation in low glucose suggested that the two processes might
be mediated by distinct mechanisms. Indeed, whereas calyculin A
attenuated the dephosphorylation of EF-2 on glucose addition, the
phosphatase inhibitor did not substantially raise the already low EF-2P
levels of 832/13 cells maintained in high glucose (e.g. see
Fig. 5B; data not shown). Thus, a simple reversal of PP2A stimulation cannot account for the effect of glucose restriction. This
result implies that stimulation of EF-2 kinase, rather than a decrease
in PP2A activity with no change in EF-2 kinase, is somehow involved in
the phosphorylation phenomenon. It seemed highly unlikely that such a
stimulation could occur via changes in intracellular Ca2+
because low glucose hyperpolarizes
cells, resulting in the closure
of plasma membrane Ca2+ channels and a consequent
lowering of cytoplasmic [Ca]. Additionally, a 15-min treatment
with 10 µM forskolin caused only a slight increase in
EF-2 phosphorylation, suggesting that changes in cAMP levels were not
involved in the phosphorylation response to low glucose (Fig.
6B).

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Fig. 6.
Phosphorylation of EF-2 in low glucose:
possible role of AMP kinase. A, 832/13 cells were
transferred from 10 mM to 1 mM
glucose-containing KRBH for the indicated times, and EF-2
phosphorylation was determined by immunoblotting (inset).
The graph shows densitometric quantitation of EF-2P
immunoblots, indicating a half-time to maximum phosphorylation of ~20
min. B, 832/13 cells were incubated in KRBH plus 10 mM (hi) without AICAR (con) or in the
presence of 1 mM AICAR (+AICAR) for the
indicated times. A replicate well was incubated in KRBH with 1 mM glucose (lo) for 2 h. Lysates were
analyzed by anti-EF-2P immunoblotting. A 15-min exposure to 10 µM forskolin (forsk) caused only a slight
increase in EF-2P. C, acetyl-CoA carboxylase
phosphorylation. As control, cells were incubated in KRBH and 1 mM (lo) or 10 mM (hi)
glucose for 2 h. Additionally, cells were incubated in 1 mM glucose (hi lo), or 1 mM
AICAR was added to the high glucose media (+AICAR), for the
indicated times. Lysates were analyzed by anti-phospho-acetyl-CoA
carboxylase (ACC-P) immunoblotting. All results are
representative of three to four independent experiments.
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Shifts in extracellular glucose have been reported to alter the ATP/AMP
ratio in
cells, and it has been proposed that AMP kinase might act
as a sensor to transmit signals from metabolism to various effector
pathways (23). Accordingly, we tested whether AICAR, a nucleotide
analog activator of AMP kinase (24), would affect EF-2 phosphorylation
in 832/13 cells. AICAR treatment of cells maintained in high glucose
led to an increase in EF-2P that peaked at 30 min (Fig. 6B).
The effects of the nucleotide were not additive with glucose
restriction (data not shown), suggesting that AMP kinase may play a
role in the calcium-independent activation of EF-2 kinase in
glucose-restricted cells. This conclusion was bolstered by the
observations that glucose restriction increased the phosphorylation of
the AMP kinase substrate acetyl-CoA carboxylase (25), with a time
course similar to that of EF-2 (Fig. 6, compare C with
A), and that AICAR stimulation of EF-2 and acetyl-CoA
carboxylase phosphorylation was also temporally correlated (Fig. 6,
compare C with B).
 |
DISCUSSION |
The acceleration of protein synthesis by glucose in pancreatic
cells was established many years ago, yet the mechanisms underlying the
increase in translational capacity are still obscure. Initiation is the
rate-limiting step in translation under many circumstances, thus it had
been anticipated that glucose stimulation would primarily impact this
step. One report does claim an effect of glucose stimulation on eIF-2B
activity, but the mechanism is unknown (10). We show here that, as in
islets themselves, the 832/13
-cell line exhibits a robust increase
in overall protein synthetic rates in response to glucose. Our data
strongly suggest that glucose-induced EF-2 dephosphorylation plays a
major role in this response. A potential route by which this might take
place is by an autocrine/paracrine mechanism whereby secreted insulin activates
-cell insulin receptors. Insulin is known to affect
-cell function, as strikingly shown in the differential effects of
glucose or insulin on insulin gene transcription in control versus
-cell-specific, insulin receptor-deficient
(
IRKO) mice (20). However, the mechanism of glucose action on EF-2
dephosphorylation in the present study is definitively not via secreted
insulin and is not mediated by either the phosphatidylinositol
3-kinase-mTOR pathway or Ras-mitogen-activated protein kinase, both of
which are downstream of the activated insulin receptor. Neither insulin itself nor stimuli that evoke a glucose-independent secretion of
insulin had a profound effect on EF-2 phosphorylation state. Conversely, glucose-stimulated dephosphorylation of EF-2 was unaffected by inhibition of cellular depolarization and insulin secretion. Nevertheless, as with the insulin secretory response, catabolism of
glucose was required, and it appears likely that a mitochondrial metabolite might be pivotal to the signaling pathway required for the
effect on EF-2 phosphorylation state.
The failure to find an obvious effect of glucose addition on EF-2
kinase activity raised the possibility that the high glucose control of
EF-2P resided primarily on the phosphatase arm of the cycle.
Hyperglycemic conditions evoked an activation of protein phosphatase
activity in 831/13 cells, confirming a previous preliminary report in
islets (26). Differential measurement of protein phosphatase activities
revealed that PP2A was the likely locus of the effect in 832/13 cells.
With respect to EF-2 dephosphorylation, this makes sense because PP2A
is the most potent phosphatase acting on EF-2P (12, 22).
Dephosphorylation of EF-2P upon glucose addition was attenuated by
calyculin A, supporting the notion that control is exercised at the
phosphatase level in the glucose signaling pathway. PP2A is a key
member of the serine/threonine-protein phosphatase family and is
responsible for a substantial fraction of the dephosphorylating
activity present in all mammalian cells. In this group of enzymes, a
conserved catalytic subunit (C) is linked to variable anchoring (A) and
regulatory subunits (B) to form a variety of heterotrimeric enzymes,
although AC dimers may also exist in cells (27). A novel regulatory
mechanism involving the TAP42/
4 protein may also play an important
role in PP2A specificity. TAP42 is a yeast protein implicated in the
TOR pathway, and its mammalian equivalent (
4) has been found in
association with the PP2A catalytic subunit in vivo. Indeed,
overexpression of
4 has been reported to affect EF-2P levels in
COS-7 cells without influencing other phosphoproteins relevant to
protein synthesis (28). It is possible that the effects of glucose in
cells will be mediated by some factor that modulates the
association of PP2Ac with a specific regulatory subunit. Molecular
identification of PP2A subspecies in
cells is clearly warranted and
will be required before the details of the glucose signaling pathway
can be elucidated. Our data are not consistent with a recent report
claiming that glucose might exert some of its effects in
cells
through the inhibition of protein phosphatase activity (29). In that
study, several metabolites of glucose such as
phosphoenolpyruvate were found to inhibit total protein
serine/threonine phosphatase activity in
-cell extracts. However,
very high concentrations of these intermediates were used, and no
attempt was made either to discriminate between different phosphatases
or to show that enzyme activity was modulated in intact cells. Glucose
has also been reported to increase protein phosphatase-2B (calcineurin)
activity in islets through a calcium-dependent mechanism,
and this may be involved in transcriptional and secretory control in
cells (30). Protein phosphatase-2B is unlikely to be involved in
EF-2P dephosphorylation because depolarization had no effect on EF-2P
state, and protein phosphatase-2B has minimal activity against EF-2P
in vitro.
EF-2 kinase is a member of an atypical family of protein kinases that
differ in the primary structure of the catalytic domain from the
"conventional" protein kinases, such as protein kinase A or CaM
kinase I (31, 32). The enzyme is conserved from Caenorhabditis elegans to man and presumably plays a critical role in cell
behavior because it is expressed in most tissues of the body. The
purified enzyme is activated by Ca2+/CaM, but other
calcium-independent mechanisms of activation as well as inhibition of
activity have been demonstrated. For example, as pointed out above,
insulin appears to inhibit the enzyme in some cells, leading to a
decrease in EF-2P (21). The mechanism of this effect is still poorly
defined but may involve mTOR-dependent phosphorylation of
EF-2 kinase because it is reportedly blocked by rapamycin (33). In the
present study, we did find a small effect of exogenous insulin on EF-2P
levels, consistent with an inhibition of EF-2 kinase, and this was
blocked by wortmannin and rapamycin. In addition, the stress-activated
pathway initiated by treatments such as protein synthesis inhibitors or
ligands such as tumor necrosis factor
can also significantly
inhibit EF-2 kinase in epithelial cells (34). This effect is mediated by direct phosphorylation of the enzyme on serine residues by stress-activated protein kinase/p38
(35). Our results suggest that
glucose addition does not lead to an acute inhibition of EF-2 kinase
activity that would contribute significantly to the dephosphorylation
of EF-2. Nevertheless, it seems likely that the increase in
phosphorylation of EF-2 during glucose restriction involves stimulatory
effects on kinase activity, rather than just a reversal of PP2A
stimulation, because inhibition of PP2A activity by calyculin A in high
glucose did not substantially raise EF-2P levels. An attractive
possibility is that AMP kinase is responsible for the indirect
stimulation of EF-2 kinase under hypoglycemic conditions. Although
further work is needed on this question, the fact that AICAR treatment
resulted in elevated EF-2P levels in cells maintained in high glucose
suggests that a connection between AMP kinase and EF-2 phosphorylation
exists. Indeed, low glucose levels do increase AMP kinase in islets
(23), and EF-2 phosphorylation levels are sensitive to AMP kinase
activation in other cell types (36). Although it is too early to rule
out an effect of AMP kinase on EF-2 phosphorylation by a more
circuitous route in
cells (e.g. via inhibition of PP2A),
an AMP kinase stimulation of EF-2 kinase and a consequent increase in
EF-2P have been proposed to account for the rapid, reversible shut-off in protein synthesis found in hepatocytes under hypoxic conditions (36). Evidently, the complex regulation of EF-2 phosphorylation at both
the kinase and phosphatase levels expands the options available in
cells for modulating protein synthetic capacity in response to a
variety of inputs (see Fig. 7 for
summary).

View larger version (29K):
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|
Fig. 7.
Model of translational regulation in
cells. Extracellular glucose impacts protein
synthesis by a variety of mechanisms (dashed lines indicate
undefined mechanisms, whereas solid lines represent
established pathways). Increased glucose uptake and metabolism alter
translational initiation through modulation of eIF-2B activity (10). As
shown here, extracellular glucose levels also regulate protein
translation rate through the bidirectional modulation of EF-2
phosphorylation. Elevation of metabolites in high glucose causes the
PP2A-mediated dephosphorylation of EF-2, contributing to increased
protein translation rate. Conversely, reduced metabolites in low
glucose favor EF-2 phosphorylation, potentially through an AMP kinase
(AMPk)-EF-2 kinase (EF-2k) link, which
contributes to the suppression of protein synthesis. Additionally,
glucose stimulates insulin secretion and autocrine signaling to protein
synthesis through the phosphatidylinositol 3-kinase-Akt-mTOR pathway
(8).
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. C. Newgard for providing the
INS-1-derived 832/1 and 832/13 cells and Drs. P. Hansen and J. Corbett
for providing the rat islet samples.
 |
FOOTNOTES |
*
This work was supported by a Career Development Award (to
M. J. B.) and a Research Award (to H. C. P.) from the American
Diabetes Association.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 Medicine,
University of Chicago, MC1027, 5841 S. Maryland Ave., Chicago, IL
60637. Tel.: 773-702-2346; Fax: 773-834-0486; E-mail:
mbrady@medicine.bsd.uchicago.edu.
Published, JBC Papers in Press, March 18, 2003, DOI 10.1074/jbc.M301116200
 |
ABBREVIATIONS |
The abbreviations used are:
mTOR, mammalian
target of rapamycin;
CaM, calmodulin;
EF, elongation factor;
EF-2P, phospho-elongation factor 2;
PP2A, protein phosphatase-2A;
KRBH, Krebs-Ringer buffer supplemented with 25 mM Hepes and 0.5%
bovine serum albumin;
AICAR, 5-aminoimidazole-4-carboxamide
1-
-D-ribofuranoside.
 |
REFERENCES |
1.
|
Permutt, M. A.
(1974)
J. Biol. Chem.
249,
2738-2742[Abstract/Free Full Text]
|
2.
|
Welsh, M.,
Scherberg, N.,
Gilmore, R.,
and Steiner, D. F.
(1986)
Biochem. J.
235,
459-467[Medline]
[Order article via Infotrieve]
|
3.
|
Skelly, R. H.,
Schuppin, G. T.,
Ishihara, H.,
Oka, Y.,
and Rhodes, C. J.
(1996)
Diabetes
45,
37-43[Abstract]
|
4.
|
Goodge, K. A.,
and Hutton, J. C.
(2000)
Semin. Cell Dev. Biol.
11,
235-242[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Webb, G. C.,
Akbar, M. S.,
Zhao, C.,
and Steiner, D. F.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
97,
5773-5778[Abstract/Free Full Text]
|
6.
|
Grimaldi, K. A.,
Siddle, K.,
and Hutton, J. C.
(1987)
Biochem. J.
245,
567-573[Medline]
[Order article via Infotrieve]
|
7.
|
Guest, P. C.,
Bailyes, E. M.,
Rutherford, N. G.,
and Hutton, J. C.
(1991)
Biochem. J.
274,
73-78[Medline]
[Order article via Infotrieve]
|
8.
|
Xu, G.,
Marshall, C. A.,
Lin, T. A.,
Kwon, G.,
Munivenkatappa, R. B.,
Hill, J. R.,
Lawrence, J. C., Jr.,
and McDaniel, M. L.
(1998)
J. Biol. Chem.
273,
4485-4491[Abstract/Free Full Text]
|
9.
|
Rhoads, R. E.
(1999)
J. Biol. Chem.
274,
30337-30340[Free Full Text]
|
10.
|
Gilligan, M.,
Welsh, G. I.,
Flynn, A.,
Bujalska, I.,
Diggle, T. A.,
Denton, R. M.,
Proud, C. G.,
and Docherty, K.
(1996)
J. Biol. Chem.
271,
2121-2125[Abstract/Free Full Text]
|
11.
|
Traugh, J. A.
(2001)
Prog. Mol. Subcell. Biol.
26,
33-48[Medline]
[Order article via Infotrieve]
|
12.
|
Nairn, A. C.,
and Palfrey, H. C.
(1987)
J. Biol. Chem.
262,
17299-17303[Abstract/Free Full Text]
|
13.
|
Ryazanov, A. G.,
and Davydova, E. K.
(1989)
FEBS Lett.
251,
187-190[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Nairn, A. C.,
Matsushita, M.,
Nastiuk, K.,
Horiuchi, A.,
Mitsui, K.,
Shimizu, Y.,
and Palfrey, H. C.
(2001)
Prog. Mol. Subcell. Biol.
27,
91-129[Medline]
[Order article via Infotrieve]
|
15.
|
Hohmeier, H. E.,
Mulder, H.,
Chen, G.,
Henkel-Rieger, R.,
Prentki, M.,
and Newgard, C. B.
(2000)
Diabetes
49,
424-430[Abstract]
|
16.
|
Marin, P.,
Nastiuk, K. L.,
Daniel, N.,
Girault, J. A.,
Czernik, A. J.,
Glowinski, J.,
Nairn, A. C.,
and Premont, J.
(1997)
J. Neurosci.
17,
3445-3454[Abstract/Free Full Text]
|
17.
|
McDaniel, M. L.,
Colca, J. R.,
Kotagal, N.,
and Lacy, P. E.
(1983)
Methods Enzymol.
98,
182-200[Medline]
[Order article via Infotrieve]
|
18.
|
Mitsui, K.,
Brady, M.,
Palfrey, H. C.,
and Nairn, A. C.
(1993)
J. Biol. Chem.
268,
13422-13433[Abstract/Free Full Text]
|
19.
|
Brady, M. J.,
Nairn, A. C.,
and Saltiel, A. R.
(1997)
J. Biol. Chem.
272,
29698-29703[Abstract/Free Full Text]
|
20.
|
Kulkarni, R. N.,
Bruning, J. C.,
Winnay, J. N.,
Postic, C.,
Magnuson, M. A.,
and Kahn, C. R.
(1999)
Cell
96,
329-339[Medline]
[Order article via Infotrieve]
|
21.
|
Redpath, N. T.,
Foulstone, E. J.,
and Proud, C. G.
(1996)
EMBO J.
15,
2291-2297[Abstract]
|
22.
|
Redpath, N. T.,
and Proud, C. G.
(1990)
Biochem. J.
272,
175-180[Medline]
[Order article via Infotrieve]
|
23.
|
Salt, I. P.,
Johnson, G.,
Ashcroft, S. J.,
and Hardie, D. G.
(1998)
Biochem. J.
335,
533-539[Medline]
[Order article via Infotrieve]
|
24.
|
Corton, J. M.,
Gillespie, J. G.,
Hawley, S. A.,
and Hardie, D. G.
(1995)
Eur. J. Biochem.
229,
558-565[Abstract]
|
25.
|
Dyck, J. R.,
Kudo, N.,
Barr, A. J.,
Davies, S. P.,
Hardie, D. G.,
and Lopaschuk, G. D.
(1999)
Eur. J. Biochem.
262,
184-190[Abstract/Free Full Text]
|
26.
|
Murphy, L. I.,
and Jones, P. M.
(1996)
Mol. Cell. Endocrinol.
117,
195-202[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Janssens, V.,
and Goris, J.
(2001)
Biochem. J.
353,
417-439[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Chung, H.,
Nairn, A. C.,
Murata, K.,
and Brautigan, D. L.
(1999)
Biochemistry
38,
10371-10376[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Sjoholm, A.,
Lehtihet, M.,
Efanov, A. M.,
Zaitsev, S. V.,
Berggren, P. O.,
and Honkanen, R. E.
(2002)
Endocrinology
143,
4592-4598[Abstract/Free Full Text]
|
30.
|
Donelan, M. J.,
Morfini, G.,
Julyan, R.,
Sommers, S.,
Hays, L.,
Kajio, H.,
Briaud, I.,
Easom, R. A.,
Molkentin, J. D.,
Brady, S. T.,
and Rhodes, C. J.
(2002)
J. Biol. Chem.
277,
24232-24242[Abstract/Free Full Text]
|
31.
|
Ryazanov, A. G.
(2002)
FEBS Lett.
514,
26-29[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Yamaguchi, H.,
Matsushita, M.,
Nairn, A. C.,
and Kuriyan, J.
(2001)
Mol. Cell
7,
1047-1057[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Wang, X.,
Li, W.,
Williams, M.,
Terada, N,
Alessi, D. R.,
and Proud, C. G.
(2001)
EMBO J.
20,
4370-4379[Abstract/Free Full Text]
|
34.
|
Knebel, A.,
Haydon, C. E.,
Morrice, N.,
and Cohen, P.
(2002)
Biochem. J.
367,
525-532[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Knebel, A.,
Morrice, N.,
and Cohen, P.
(2001)
EMBO J.
20,
4360-4369[Abstract/Free Full Text]
|
36.
|
Horman, S.,
Browne, G.,
Krause, U.,
Patel, J.,
Vertommen, D.,
Bertrand, L.,
Lavoinne, A.,
Hue, L.,
Proud, C.,
and Rider, M.
(2002)
Curr. Biol.
12,
1419-1423[CrossRef][Medline]
[Order article via Infotrieve]
|
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