From the Department of Pathology, Washington
University School of Medicine, St. Louis Missouri 63110 and
§ Department of Pharmacology, University of Virginia School
of Medicine, Charlottesville, Virginia 22908
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ABSTRACT |
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Although glucose regulates the biosynthesis of a
variety of beta cell proteins at the level of translation, the
mechanism responsible for this effect is unknown. We demonstrate that
incubation of pancreatic islets with elevated glucose levels results in
rapid and concentration-dependent phosphorylation of
PHAS-I, an inhibitor of mRNA cap-binding protein, eukaryotic
initiation factor (eIF)-4E. Our initial approach was to determine if
this effect is mediated by the metabolism of glucose and activation of
islet cell protein kinases, or whether insulin secreted from the beta
cell stimulates phosphorylation of PHAS-I via an insulin-receptor
mechanism as described for insulin-sensitive cells. In support of the
latter mechanism, inhibitors of islet cell protein kinases A and C
exert no effect on glucose-stimulated phosphorylation of PHAS-I,
whereas the phosphatidylinositol 3-kinase inhibitor, wortmannin, the
immunosuppressant, rapamycin, and theophylline, a phosphodiesterase
inhibitor, promote marked dephosphorylation of PHAS-I. In addition,
exogenous insulin and endogenous insulin secreted by the beta cell
line, TC6-F7, increase phosphorylation of PHAS-I, suggesting that
beta cells of the islet, in part, mediate this effect. Studies with
beta cell lines and islets indicate that amino acids are required for glucose or exogenous insulin to stimulate the phosphorylation of
PHAS-I, and amino acids alone dose-dependently stimulate
the phosphorylation of PHAS-I, which is further enhanced by insulin. Furthermore, rapamycin inhibits by ~62% the increase in total protein synthesis stimulated by high glucose concentrations. These results indicate that glucose stimulates PHAS-I phosphorylation via
insulin interacting with its own receptor on the beta cell which may
serve as an important mechanism for autoregulation of protein synthesis
by translation.
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INTRODUCTION |
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The metabolism of glucose regulates a variety of physiological processes by beta cells of the islets of Langerhans. These processes include insulin biosynthesis and secretion, beta cell replication, and the synthesis of a number of beta cell proteins. Glucose exerts a specific stimulatory effect on insulin gene expression over time intervals of hours (1, 2), whereas the biosynthesis of insulin is significantly controlled within minutes at the level of protein translation (3-5). Although the enhancement of insulin synthesis by glucose at the translational level occurs rapidly and does not require synthesis of new mRNA, limited information is available on processes that regulate translation. More recent studies indicate that insulin is only one of a number of beta cell proteins whose synthesis is regulated by glucose at the level of translation (6). Glucose has been shown to increase the biosynthesis of a secretory granule membrane protein, SGM 110 (7), and also chromogranin A (8) to a similar extent as insulin, suggesting that translational control may extend to a variety of other beta cell proteins.
The initiation phase of mRNA translation is generally rate-limiting
for protein synthesis. Initiation is mediated in part by the
eIF1-4F complex, which is
composed of three subunits, eIF-4, eIF-4A, and eIF-4E (9). eIF-4
is a large subunit (Mr 220,000) that binds
eIF-4A (Mr 45,000) and eIF-4E
(Mr 25,000). eIF-4A is an ATP-dependent helicase, and eIF-4E is the mRNA
cap-binding protein. Thus, eIF-4F is involved in both the recognition
of the capped mRNA and melting of secondary structure in the
5'-untranslated region. eIF-4E is the least abundant of the eIF-4F
subunits, and it is generally believed that the amount of eIF-4E is
limiting for translation initiation.
The availability of eIF-4E is regulated by PHAS-I, a heat- and
acid-stable eIF-4E binding protein first identified in rat adipocytes
(10, 11). The nonphosphorylated form of PHAS-I binds tightly to eIF-4E,
and prevents eIF-4E from binding to eIF-4. When phosphorylated in
the appropriate site(s), PHAS-I dissociates from eIF-4E, allowing the
factor to participate in translation initiation.
PHAS-I is phosphorylated both in vitro and in vivo by a variety of protein kinases. Casein kinase II, protein kinase C, and mitogen-activated protein kinase have been reported to phosphorylate recombinant PHAS-I (12). Insulin-like growth factor-1 and platelet-derived growth factor in smooth muscle cells (13) and insulin in 3T3 L1 adipocytes (14) increase PHAS-I and p70s6k phosphorylation by a rapamycin-sensitive pathway. This mitogen-induced phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin (15). Recent studies on PHAS-I have implicated a signaling pathway involving the mammalian target of rapamycin (mTOR). Thus, mTOR appears to be a site by which the rapamycin·FKBP12 complex mediates the dephosphorylation of PHAS-I (15-18). The phosphorylation of PHAS-I and p70s6k appears to be regulated by mTOR in parallel (18). These studies suggest that PHAS-I has an important role in regulating translation in a variety of cells by signal transduction pathways that involve protein phosphorylation.
Stimulus secretion coupling of insulin secretion from pancreatic beta cells is a complex process involving the transport and metabolism of glucose with the generation of metabolic intermediates including ATP. It is proposed that increases in ATP are associated with the closure of ATP-dependent K+ channels, cell depolarization, and Ca2+ entry through voltage-dependent Ca2+ channels (19). There is also strong evidence that protein kinases play a key role in modulating the insulin secretory process by beta cells. The major serine/threonine kinases that have been studied in insulin secretion include Ca2+ and calmodulin protein kinase (20), Ca2+ and phospholipid protein kinase C (21, 22), cAMP protein kinase A (23), and more recently mitogen-activated protein kinases (24, 25).
Recent evidence also indicates that glucose-stimulated insulin
secretion activates a beta cell surface insulin receptor kinase and the
intracellular effector substrate, insulin receptor substrate-1, from
TC3 cells (26). These results and others (27) have suggested that
insulin released from the beta cell of the islet may bind to its own
cell-surface receptor and serve as an autocrine mechanism for the
regulation of beta cell function. We have therefore examined these
aspects of stimulus secretion coupling of insulin secretion by beta
cells on PHAS-I activation as reflected by its phosphorylation state,
signaling pathways, and proposed functional role to regulate protein
translation by the beta cell of the pancreatic islet.
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EXPERIMENTAL PROCEDURES |
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Materials-- Male Sprague-Dawley rats were purchased from Sasco (O'Fallon, MO) and Harlan Sprague-Dawley (Indianapolis, IN). Collagenase type P was obtained from Boehringer Mannheim. CMRL-1066 tissue culture medium, penicillin, streptomycin, Hanks' balanced salt solution, L-glutamine, MEM amino acids solution, and MEM nonessential amino acids solution were obtained from Life Technologies, Inc. Fetal bovine serum was from Hyclone (Logan, UT). Dulbecco's modified Eagle's medium (DMEM) was supplied by the Washington University Tissue Culture Support Center. Rapamycin and N-6,2'-o-dibutyryladenosine-3':5'-cyclic monophosphate (dibutyryl cAMP) were from Biomol (Plymouth Meeting, PA). Ficoll, theophylline, and CPT-cAMP were from Sigma. Insulin was obtained from Lilly and ICN Biomedicals (Aurora, OH). Pro-mix L-[35S] in vitro cell labeling mix was from Amersham Corp. The primary antibody was generated in rabbit with recombinant His-tagged rat PHAS-I (11). The secondary antibody was peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immunologicals). All other chemicals were from commercially available sources.
Amino Acid Composition-- Krebs-Ringer bicarbonate buffer (KRBB; 25 mM HEPES, 115 mM NaCl, 25 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, and 1 mM MgCl2, pH 7.4, containing 3 mM D-glucose, and 0.1% BSA) was supplemented with MEM amino acids solution, MEM nonessential amino acids solution, and L-glutamine. For these experiments, the 1 × concentration of amino acids was defined as the following (in mM): L-arginine 0.73, L-cystine 0.2, L-glutamine 2.0, L-histidine·HCl·H2O 0.2, L-isoleucine 0.4, L-leucine 0.4, L-lysine HCl 0.5, L-methionine 0.1, L-phenylalanine 0.2, L-threonine 0.4, L-tryptophan 0.05, L-tyrosine 0.2, L-valine 0.4, L-alanine 0.1, L-asparagine 0.1, L-aspartic acid 0.1, L-glutamic acid 0.1, glycine 0.1, L-proline 0.1, L-serine 0.1. The concentrations of amino acids in CMRL-1066 and DMEM are comparable.
Islet Isolation and Culture-- Islets were isolated from male Sprague-Dawley rats (200-250 g) by collagenase digestion as described previously (28). Briefly, pancreases were inflated with Hanks' balanced salt solution, and the tissue was isolated, minced, and digested with 7 mg of collagenase/pancreas for 7 min at 39 °C. Islets were separated on a Ficoll step density gradient and then selected with a stereomicroscope to exclude any contaminating tissues. Islets were cultured overnight in an atmosphere of 95% air, 5% CO2 in "complete" CMRL-1066 tissue culture medium (cCMRL) containing 5.5 mM glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Where "CMRL" is stated, this culture medium does not contain fetal bovine serum.
Pancreatic Beta Cell Lines--
TC3 and
TC6-F7 cells were
kindly provided by Norman Fleischer (Albert Einstein College of
Medicine, Bronx, NY), and RINm5F cells were obtained from the
Washington University Tissue Culture Support Center. RINm5F cells were
grown in cCMRL medium, and
TC3 as well as
TC6-F7 cells were grown
in DMEM containing 25 mM glucose and supplemented with 15%
heat-inactivated horse serum and 2.5% fetal bovine serum as described
by Knaack et al. (29). Cells were cultured in Petri dishes
(60 × 15 mm) at a concentration of 1 × 106
cells/3 ml. RINm5F cells were used after 1 day.
TC3 and
TC6-F7 cells were used after 5 days of culture, and medium was replaced once
during that time.
TC3 and
TC6-F7 cells were pretreated in DMEM
containing 5 mM glucose for 24 h before experiments
were begun.
Incubation of Islets and Cells and Preparation of Extracts for
PHAS-I Determination--
Two separate protocols were used to prepare
tissue for PHAS-I immunoblotting. In the first experimental protocol,
islets (200-300/1 ml or 1500 cells/10 ml) were incubated in CMRL or
DMEM at 37 °C in an atmosphere of 95% air, 5% CO2 as
described in the figure legends. The islets were then centrifuged
(Beckman Microfuge B) for 10 s, the supernatant was removed for
insulin RIA, and the islets were resuspended in 100 µl of
homogenization buffer (50 mM -glycerophosphate, pH 7.3, 100 mM NaF, 5 mM EDTA, 2 mM EGTA, 0.1 mM Na3VO4, 1 mM
benzamidine, 10 µg/ml leupeptin, 0.2 mM
phenylmethylsulfonyl fluoride). The islets were homogenized and
centrifuged at 10,000 × g for 20 min, and the
supernatants were heated at 100 °C for 10 min. Heated supernatants
were then centrifuged at 10,000 × g for 30 min,
Laemmli sample buffer was added to the supernatants, and 50 µl were
subjected to SDS-PAGE. An immunoblot was performed using rabbit
anti-PHAS-I and donkey, anti-rabbit IgG, as primary and secondary
antibodies, respectively. Detection was performed using ECL reagents
from Amersham Corp.
PHAS-I·eIF-4E Binding Assays-- For experiments to determine the binding of PHAS-I to eIF-4E, islet extracts were incubated with an affinity resin of the cap homolog 7-methyl-guanosine triphosphate, proteins were eluted and subjected to SDS-PAGE and immunoblotting (11).
Measurement of Total Protein Synthesis-- Groups of 100 islets were glucose and serum depleted in 1 ml of CMRL (0.1% BSA, 90% methionine-free) for 90 min. Rapamycin (25, 50, or 100 nM) was added to the medium for an additional 30 min. Medium was removed and replaced with 0.1 ml of 90% methionine-free CMRL + 3 or 20 mM glucose ± rapamycin for 1 h with 80 µCi of Pro-mix L-[35S] in vitro labeling mix. Islets were washed twice with ice-cold phosphate-buffered saline (0.1% BSA, 0.02% NaN3), and protein was precipitated with 1 ml of 10% trichloroacetic acid at 4 °C for 30 min. The samples were washed once with 10% trichloroacetic acid and solubilized with 50 µl of Protosol. The scintillation mixture was added and the radioactivity was quantitated.
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RESULTS |
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PHAS-I Phosphorylation by Glucose--
PHAS-I generally appears as
three migrating bands when separated by SDS-PAGE and analyzed by
immunoblotting. These bands are designated as ,
, and
. The
nonphosphorylated
-form migrates most rapidly. Increases in
phosphorylation of the intermediate
-form to the most highly
phosphorylated
-form decreases migration of PHAS-I proportionately
when separated by SDS-PAGE. Under most conditions with islets, the
nonphosphorylated,
-form of PHAS-I is barely detectable. Initial
studies were performed to determine both the effects of glucose
concentration and the time dependence of glucose exposure on
phosphorylation of PHAS-I. As shown in Fig.
1, exposure of pancreatic islets to 20 mM glucose in cCMRL for 10, 30, or 180 min resulted in
enhanced phosphorylation of PHAS-I
(lanes 2,
4, and 6) and the concomitant decrease of the intermediate migrating PHAS-I (
) compared with a basal glucose concentration of 5.5 mM (lanes 1, 3,
and 5). The increased phosphorylation of PHAS-I
in
response to 20 mM glucose occurred rapidly within 10 min or
less and was stable for at least a 3-h period. As a control, PHAS-I
,
, and
obtained from isolated rat adipocytes are shown in
lane 7.
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Identification of Mediators in Glucose-induced Phosphorylation of PHAS-I-- To determine if glucose-induced phosphorylation of PHAS-I is mediated by the metabolism of glucose and activation of major islet cell protein kinases, inhibitors of protein kinases A and C were initially evaluated. The selective protein kinase C inhibitor chelerythrine (10 µM) and phorbol 12-myristate 13-acetate (100 nM), an agonist of protein kinase C, exerted no effects on glucose-induced phosphorylation of PHAS-I. The cAMP analogues, CPT-cAMP (0.2 mM) and dibutyryl-cAMP (1 mM) as well as staurosporine (100 nM), a potent and nonselective inhibitor of protein kinase A and protein kinase C were also ineffective in modulating glucose-induced phosphorylation of PHAS-I, suggesting that protein kinase A and most likely mitogen-activating protein kinase were not involved (30) (data not shown).
Recent studies have indicated a role for the insulin receptor, phosphatidylinositol 3-kinase and mTOR in the signaling pathway involved in phosphorylation of PHAS-I by insulin-sensitive cells (15-18). To determine if insulin secreted from the beta cell stimulates phosphorylation of PHAS-I via an insulin-receptor mechanism, we evaluated the phosphatidylinositol 3-kinase inhibitor, wortmannin, the immunosuppressant, rapamycin, and theophylline, a phosphodiesterase inhibitor that also blocks p70s6k (31, 32), in this signaling pathway by pancreatic islets. As shown in Fig. 2, incubation of islets in CMRL in the presence of 20 mM glucose (lane 2) resulted in increased phosphorylation of PHAS-I
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Amino Acid Dependence of PHAS-I Phosphorylation--
Our previous
studies were performed with islets incubated in CMRL culture medium.
The ability of glucose to stimulate phosphorylation of PHAS-I was also
evaluated in KRBB. Unexpectedly, it was found that glucose did not
stimulate the phosphorylation of PHAS-I in KRBB in the complete absence
of amino acids, even though islets secrete insulin normally in KRBB
under these conditions. As shown in Fig.
4, an elevated glucose concentration of
20 mM stimulated phosphorylation of PHAS-I (lane 2 versus lane 1), and exogenous insulin (200 nM)
produced similar effects from islets incubated in CMRL containing 1 mM glucose (lane 3). In contrast, neither an
elevated glucose concentration (lane 5) nor exogenous
insulin (lane 6) induced PHAS-I phosphorylation by islets
incubated in KRBB in the complete absence of amino acids.
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Beta Cell of the Islet Is a Source of PHAS-I--
To determine if
the beta cell of the islet mediates this effect, conditions that
stimulate insulin secretion from the beta cell line, TC6-F7, were
also evaluated. As shown in Fig.
6A, 20 mM glucose + 0.5 mM carbachol over an amino acid concentration range
of 0.05-0.25 × in KRBB (lanes 4, 6, and
8) significantly enhanced phosphorylation of PHAS-I. Insulin
secretion observed in the absence of these secretagogues (lanes
3, 5, and 8) at amino acid concentrations of
0.05, 0.10, and 0.25 × was 1.2 ± 0.2, 0.9 ± 0.2, and
2.6 ± 0.7 nM, respectively. In the presence of 20 mM glucose + 0.5 mM carbachol, insulin
secretion increased to 8.7 ± 2.0, 9.2 ± 0.5, and 14.8 ± 1.8 nM at these same amino acid concentrations and was
associated with enhanced phosphorylation of PHAS-I. It is interesting
to note that in lane 2 of Fig. 6A in the absence of amino acids, PHAS-I phosphorylation is not increased even though insulin secretion is stimulated with 20 mM glucose + 0.5 mM carbachol (lane 1, 0.9 ± 0.3 nM versus lane 2, 5.8 ± 1.0 nM
insulin). As observed previously with islets in Fig. 4, amino acids are
essential for increased phosphorylation of PHAS-I mediated by glucose
and exogenous insulin.
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Binding of PHAS-I to eIF-4E--
We further explored the ability
of theophylline, a methylxanthine phosphodiesterase inhibitor which
attenuates PHAS-I phosphorylation (Figs. 2 and 5C), to
modulate the binding of PHAS-I to eIF-4E. In this experimental design,
islets were exposed to 3 or 20 mM glucose in DMEM for 30 min, and extracts were prepared as described under "Experimental
Procedures." Islet extracts were then passed over an affinity resin
of the cap homolog 7-methyl-guanosine triphosphate cross-linked to
eIF-4E. Highly phosphorylated PHAS-I and to a lesser extent PHAS-I
(
) do not bind to cross-linked eIF-4E and elute into the
"unbound" fraction. Conversely, unphosphorylated PHAS-I (
) (and
to some degree PHAS-I
) bind to cross-linked eIF-4E and are
designated as "bound."
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Effects of Rapamycin on Total Protein Synthesis-- To evaluate the role of the phosphorylation state of PHAS-I on beta cell function, rapamycin was evaluated for its effects on glucose stimulated protein synthesis by islets. Fig. 8 demonstrates that islets incubated in CMRL in the presence of 20 mM glucose for 60 min increases by 50% the amount of [35S]methionine incorporated into total protein compared with basal conditions of 3 mM glucose. Under these same conditions, rapamycin at 25, 50, and 100 nM significantly inhibited by ~62% glucose stimulated incorporation of [35S]methionine into total protein. Rapamycin at 20 and 100 nM had no effect on glucose-stimulated insulin secretion determined over a 1-h incubation (data not shown). These results suggest that the biosynthesis of a number of proteins may be regulated at the level of translation by the beta cell in an autocrine loop through glucose-induced insulin secretion.
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DISCUSSION |
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This study demonstrates that glucose stimulates in a time- and concentration-dependent manner the rapid phosphorylation of PHAS-I, an inhibitor of mRNA cap-binding protein, eIF-4E, by isolated pancreatic islets. Our initial approach was to determine if this effect was mediated by the metabolism of glucose and subsequent activation of islet cell protein kinases, or whether insulin secreted from the beta cell may stimulate phosphorylation of PHAS-I in a manner analogous to that described for insulin-sensitive cells. Evidence in support of the latter mechanism was that conditions that block insulin exocytosis from beta cells prevented glucose-stimulated phosphorylation of PHAS-I, and exogenous insulin mimicked the effects of elevated glucose concentrations.
During this investigation, in vitro incubations were
routinely performed in CMRL and DMEM tissue culture media. To more
easily manipulate the components of this tissue culture system,
parallel studies were also performed in KRBB in the absence of amino
acids, co-factors, and vitamins. Insulin secretion studies are
routinely performed in KRBB, and islets secrete insulin normally under
these conditions. Unexpectedly, neither elevated glucose concentrations that stimulated insulin secretion from beta cells nor exogenous insulin
altered the basal patterns of PHAS-I,
, or
when islets were
incubated in KRBB in the absence of amino acids. However, addition of a
normal complement of amino acids to KRBB restored the ability of both
endogenous and exogenous insulin to stimulate the phosphorylation of
PHAS-I by islets (data not shown). Additional studies with the
insulinoma-derived cell line, RINm5F, also indicated that this normal
complement of amino acids alone induced in a concentration-dependent manner phosphorylation of PHAS-I,
which was further enhanced by insulin. These results suggested that amino acids were by themselves sufficient and also required in the
presence of insulin to mediate the phosphorylation of PHAS-I. We did
not attempt to determine which amino acids are responsible for these
effects. Studies are in progress to define the mechanism whereby amino
acids exert their effects on PHAS-I phosphorylation. This requirement
for amino acids may explain a recent study by Gilligan et
al. (36), who reported that glucose did not alter the
phosphorylation state of PHAS-I by islets. These negative results are
most likely explained by the fact that these studies were performed in
a buffered salt solution in the absence of amino acids. Recently, Patti
et al. (37) have also reported that amino acids stimulate
the phosphorylation of PHAS-I and at submaximal concentrations
synergize with insulin by FAO hepatocytes.
Our findings with the beta cell line, RINm5F, also suggested that the
beta cell of the islet is responsible for glucose induced phosphorylation of PHAS-I. Thus, amino acids alone stimulated phosphorylation of PHAS-I and this effect was further enhanced with
exogenous insulin by this beta cell line. In addition, conditions which
stimulate insulin secretion, i.e. glucose + carbachol, from the beta cell line, TC6-F7, also increased phosphorylation of PHAS-I
analogous to that observed with isolated islets. Similar results were
also observed with
TC3 cells in relating endogenous insulin
secretion with the phosphorylation of PHAS-I (data not shown).
PHAS-I is phosphorylated both in vitro and in vivo by a variety of protein kinases. Previous studies in smooth muscle cells and 3T3-L1 adipocytes (13, 14) indicated a role for p70s6k, a protein kinase that phosphorylates ribosomal protein S6 in mammalian cells (9), in the phosphorylation of PHAS-I. Recent evidence indicates that mTOR regulates the phosphorylation of both p70s6k and PHAS-I in parallel and that rapamycin is acting on mTOR to prevent phosphorylation of both kinases (17, 18). Similar dephosphorylation of p70s6k and PHAS-I has also been reported with the phosphatidylinositol 3-kinase inhibitor, wortmannin (15, 38), and the phosphodiesterase inhibitor, SQ20006 (31, 32). Wortmannin and rapamycin promoted significant dephosphorylation of PHAS-I by islets exposed to elevated glucose concentrations. These same inhibitors also induced dephosphorylation of PHAS-I by RINm5F cells exposed to amino acids, further indicating that beta cells of the islet mediate this effect in a similar manner as insulin sensitive cells.
The ability of cAMP to regulate PHAS-I phosphorylation was also
evaluated in pancreatic islets. Studies in 3T3-L1 adipocytes and aortic
smooth muscle cells suggested that increasing cAMP levels may promote
dephosphorylation of PHAS-I (13, 14, 39). Phosphodiesterase inhibitors
such as theophylline and isobutylmethylxanthine are commonly used to
modulate cAMP levels in islets. These phosphodiesterase inhibitors
significantly enhance glucose-stimulated proinsulin biosynthesis and
insulin secretion from beta cells by increasing cAMP levels. Our
studies indicated that theophylline results in marked dephosphorylation
of PHAS-I, but unexpectedly, CPT-cAMP and dibutyryl-cAMP exerted no
effects on the phosphorylation state of PHAS-I (data not shown). These
paradoxical results were explained by recent studies indicating that
methylxanthines including theophylline block activation of
p70s6k, similar to rapamycin, by a mechanism independent of
cAMP and cGMP production (31, 32). Our studies demonstrated that
rapamycin mimics the ability of theophylline to block
glucose-stimulated phosphorylation of PHAS-I. Although theophylline
increases cAMP levels and greatly enhances glucose-induced insulin
secretion by islets, its effect on mTOR presumably causes significant
dephosphorylation of PHAS-I in a similar fashion as rapamycin. In
addition, cAMP analogues, CPT-cAMP and dibutyryl-cAMP, had no effect on
the phosphorylation level of PHAS-I by glucose-stimulated islets.
Although insulin secretion increased further in the presence of these
cAMP analogues as anticipated, glucose-induced phosphorylation of
PHAS-I was already maximal, and this additional insulin secretion
caused no further increase in phosphorylation. Overall, our findings suggest that cAMP independent of insulin secretion does not appear to
affect glucose-stimulated phosphorylation of PHAS-I by islets.
Previous studies on the regulation of the biosynthesis of insulin secretory granule proteins by glucose have indicated that a subset of proteins exist whose synthesis is stimulated to a similar extent as insulin (7). These proteins include granule matrix and membrane constituents and possibly other proteins not associated with beta cell granules. Although rapamycin did not affect the acute phase of glucose-stimulated insulin secretion, it inhibited both glucose-stimulated phosphorylation of PHAS-I and total protein synthesis. Even though exogenous insulin stimulated phosphorylation of PHAS-I at basal glucose levels, these conditions did not result in enhanced protein synthesis, suggesting that both elevated glucose concentrations necessary for increased metabolism and the release of insulin are required to mediate increased protein translation by beta cells (data not shown).
Our findings suggest that glucose-stimulated phosphorylation of PHAS-I
by beta cells of the islet is mediated via insulin interacting with its
own insulin receptor, leading to the phosphorylation of PHAS-I. This
raises the possibility that insulin receptor activation may target
downstream insulin signaling proteins such as insulin receptor
substrate-1 and -2 and may provide an important mechanism for the
autoregulation of beta cell function in a manner analogous to
insulin-sensitive cells. Functional parameters that may be regulated by
this autocrine loop in beta cells include protein synthesis, gene
expression, DNA synthesis, and cell proliferation. In support of this
concept, recent studies by Rothenberg et al. (26) have
demonstrated that glucose-induced insulin secretion from TC3 cells
activates the beta cell surface receptor tyrosine kinase and insulin
receptor substrate-1, which then associates with the 85-kDa
-subunit
of phosphatidylinositol 3-kinase. Additional studies by Herbeck
et al. (27) have also demonstrated expression of insulin
receptor mRNA and insulin receptor substrate-1 in single beta cells
obtained from isolated rat islets. Insulin receptors have also been
identified in the beta cell line, RINm5F (40). Further studies are
required to identify specific proteins that may be involved in
translational regulation of protein synthesis and characterize
additional functional aspects of beta cell physiology that may be
affected. Overall, our results support and extend this novel concept at
a functional level that insulin secreted by beta cells activates a beta
cell insulin receptor-signaling pathway, resulting in autoregulation of
protein synthesis. The identification of common insulin signaling
pathways in insulin target cells and beta cells may provide new
insights with regard to possible defects in the development of diabetes
mellitus.
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ACKNOWLEDGEMENTS |
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The authors thank Joan Fink for excellent technical assistance, the Washington University Tissue Culture Support Center, and the Radioimmunoassay Core Facility of the Diabetes Research and Training Center.
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FOOTNOTES |
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* This study was supported in part by an American Diabetes Association Research Grant (to M. L. M.), an American Diabetes Association Mentor-Based Fellowship (to G. X.), and National Institutes of Health Grants DK50628 (to J. C. L.), DK28312 (to J. C. L.), AR41189 (J. C. L.), DK06181 to (M. L. M.), and F32 DK08748 (to G. K.).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 Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid Ave., Saint Louis, MO 63110-1093. Tel.: 314-362-7435; Fax: 314-362-4096; E-mail: mcdaniel{at}pathology.wustl.edu.
1 The abbreviations used are: eIF, eukaryotic initiation factor; mTOR, mammalian target of rapamycin; MEM, minimum essential medium; DMEM, Dulbecco's modified Eagle's medium; KRBB, Krebs-Ringer bicarbonate buffer; BSA, bovine serum albumin; RIA, radioimmunoassay; PAGE, polyacrylamide gel electrophoresis; CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate.
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REFERENCES |
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