From the Departments of Molecular Biology and
¶ Cell Biology, Parke-Davis Pharmaceutical Research Division
of Warner Lambert Co., Ann Arbor, Michigan 48105
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The insulin-stimulated uptake of
2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable
substrate for system A, in 3T3-L1 adipocytes was investigated. As cells
took on a more adipogenic phenotype, the insulin-stimulated
versus the saturable basal MeAIB uptake increased by
5-fold. The induced transport activity showed properties characteristic
of system A, with a Km value of 190 µM. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and
was not accelerated by ambient amino acids, therefore, it was
mechanistically distinct from the previously described adaptive and
hormonal regulation of system A. Inhibition of mitogen-activated
protein kinase kinase by PD98059, Ras farnesylation by PD152440 and
B581, p70S6K by rapamycin, and phosphatidylinositol
3-kinase (PI 3-K) by wortmannin and LY294002 revealed that only
wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with
IC50 values close to that previously reported for
inhibition of PI 3
-K. These results suggest that the
Ras/mitogen-activated protein kinase and pp70S6K insulin
signaling pathways are neither required nor sufficient for insulin
stimulation of MeAIB uptake, and activation of PI 3
-K or a
wortmannin/LY294002-sensitive pathway may play an important role in
regulation of system A transport by insulin in 3T3-L1 cells.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mammalian cells contain multiple systems for uptake of neutral amino acids. System A is an ubiquitous amino acid transport system that mediates the Na+-dependent uptake of a wide range of neutral amino acids (1-3), many of which are gluconeogenic (4). In a number of cell types system A is regulated by a variety of external stimuli and conditions, such as hormones, amino acid starvation (adaptive regulation), cell growth, differentiation, hyperosmotic stress, and trans-inhibition produced by high levels of endogenous amino acid substrates (1, 2, 4, 5). It is generally believed that hormonal and adaptive regulation of system A occurs at the level of transcription (4, 5). Although the molecular mechanisms of the signal transduction in insulin action have been extensively investigated (6), the mechanism responsible for hormonal regulation of system A transport remains largely unknown. An insulin-insensitive Chinese hamster ovary cell line has been isolated (7). These authors proposed that, when insulin binds to its receptor, it regulates system A activity directly or indirectly by inactivation of a regulatory protein designated r2, but the molecular events leading to neutralizing this protein by insulin have not been resolved (5).
In 3T3-L1 adipocytes, insulin induces the dose- and time-dependent uptake of MeAIB,1 a nonmetabolizable substrate of system A (8). However, the specificity and kinetics of the insulin-induced amino acid uptake in 3T3-L1 adipocytes and the signaling events potentially involved in insulin stimulation of system A transport in mammalian cells have not previously been explored in great detail. In this report, we investigate the regulation of system A transport by insulin in 3T3-L1 adipocytes.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
Cell culture reagents, epidermal growth factor,
PDGF, IGF-I, and IGF-II were purchased from Life Technologies, Inc.
[14C]MeAIB was purchased from American Radiolabeled
Chemicals, Inc., and [-32P]ATP from Amersham Corp.
Wortmannin, LY294002, B518, and rapamycin were purchased from BIOMOL
Research Laboratories, Inc. (Plymouth Meeting, PA). Anti-p42/p44 MAP
kinase antiserum used for Western blotting was prepared from rabbits
immunized with a C-terminal peptide (amino acids 425-445) of p44 MAP
kinase (p44MAPK) expressed as a glutathione
S-transferase fusion protein. PD98059 and PD152440 were
synthesized by Parke-Davis. Phosphatidylinositol was purchased from
Avanti (Birmingham, AL). The S6 kinase kit and the mouse
anti-phosphotyrosine monoclonal antibody were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). The GH and insulin were obtained
from Eli Lilly; IL-11 from R&D Systems (Minneapolis, MN), and all other
chemicals from Sigma.
Cell Culture and Differentiation-- 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum in an atmosphere of 5% CO2, air. Differentiation to adipocytes was induced by incubating confluent monolayers (day 0) for 2 days in DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.4 µg/ml dexamethasone, followed by incubation for 2 more days in DMEM containing 10% fetal bovine serum and 1 µg/ml insulin. Two days after transfer to the same medium without insulin, greater than 90% of the cells expressed the adipocyte phenotype. Unless otherwise stated, experiments were performed on adipocytes 2-4 days after withdrawal from the differentiation medium (days 6-8).
PI 3-K Assay--
This assay was carried out as described
previously (9, 10). In brief, 3T3-L1 adipocytes were serum-starved for
about 16 h. After hormone stimulation for 10 min, the cells (10-cm
culture dishes) were harvested in cold lysis buffer (1% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 20 mM Tris,
pH 7.5, 1 mM Na3VO4, 100 µM phenylmethylsulfonyl fluoride (PMSF), 20 µM leupeptin, 20 µM pepstatin A) and kept
on ice for 30 min. Insoluble materials were removed by centrifugation
and the supernatants were incubated with anti-phosphotyrosine antibody
under agitation at 4 °C for 1 h. Protein A-agarose beads were
added and incubated for another hour. The immunoprecipitates were
sequentially washed with lysis buffer, washing buffer (100 mM Tris, pH 7.5, 0.5 M LiCl, 1 mM
Na3VO4, 100 µM PMSF, 20 µM leupeptin, 20 µM pepstatin A), and PI
3
-kinase buffer (20 mM MgCl2, 10 mM Tris, pH 7.5, 0.2 mM EDTA, 100 mM NaCl, 1 mM Na3VO4,
100 µM PMSF, 20 µM leupeptin, 20 µM pepstatin A). The precipitates were suspended in 50 µl of PI 3
-kinase buffer. The reaction was carried out at 25 °C
for 20 min in the presence of 0.4 mg/ml phosphatidylinositol and 10 µM ATP (containing 0.2 mCi/ml
[
-32P]ATP). The reaction was terminated by adding to
each tube 100 µl of CHCl3:MeOH:HCl (100:200:2). To the
mixture 100 µl of CHCl3 and H2O each were
then added, and the mixture was vortexed. After centrifugation, the
lower phase (organic phase) was spotted on a thin layer chromatography
plate. The plate was developed in CHCl3:MeOH:H2O:NH4OH (43:38:7:5).
The dried plate was visualized by autoradiography on x-ray film.
Protein Kinase Assays--
For the assay of p70 S6 kinase
(p70S6K), 3T3-L1 adipocytes were serum-starved for 16 h. The cells were treated with or without rapamycin (100 ng/ml) for
1 h followed by treatment with insulin for 10 min. The cells were
harvested and the S6 kinase activity of cytosolic extracts was detected
according to the procedure provided by Upstate Biotechnology, Inc.
using a peptide substrate. The S6 kinase activity was obtained by
subtracting the values for unstimulated cells from the values for the
insulin-induced cells. MAP kinase was assayed as described previously
(9). In brief, 3T3-L1 adipocytes were serum-starved overnight and then pretreated or untreated with 50 µM PD98059 for 1 h
before stimulation by 100 nM insulin for 15 min. Cell
lysates were collected in cold buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% deoxycholate, 1%
Nonidet P-40, 50 mM NaF, 10 mM
Na-pyrophosphate, 1 mM p-nitrophenylphosphate, 25 mM -glycerophosphate, 1 mM
Na3VO4, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, and 1 mM benzamidine).
The precleared lysates (10,000 × g, 10 min) containing
10 µg of protein were subjected to 12% SDS-polyacrylamide gel
electrophoresis with 75:1 of acrylamide:bisacrylamide (w/w) and 0.5 M Tris-HCl. After electrophoresis, the gel was transferred to nitrocellulose and probed with anti-p42/p44 MAP kinase antiserum. The immunoreactive proteins were visualized by enhanced
chemiluminescence detection.
Assay of Amino Acid Transport-- The sodium-containing buffer for transport assay was phosphate-buffered saline (PBS) consisting of 137 mM NaCl, 2.7 mM KCl, 10.6 mM Na2HPO4, and 1.5 mM KH2PO4. Prior to use, PBS buffer (pH 7.4) was supplemented with 20 mM D-glucose, 0.49 mM MgCl2, 0.9 mM CaCl2, and 0.2% bovine serum albumin. Before stimulation, the cells were incubated in serum-free DMEM for 3-4 h and then switched to the same medium containing stimuli and incubated for 5-6 h.
The cluster tray transport assay was used as described previously (11). To eliminate trans-inhibition, the intracellular pool of amino acids was depleted by incubation in PBS for 40 min, with a change to fresh PBS at 20 min in the presence or absence of stimuli. An appropriate amount of choline chloride was added to each reaction mixture to keep all solutions at equal osmolarity. Since uptake of MeAIB was linear at 37 °C for at least 15 min, 10-min uptake was used for determining initial uptake rates. Unless otherwise noted, the MeAIB concentration for initial rate of transport measurements was 50 µM, the concentration of insulin was 100 nM, and all transport rates were referred to as saturable uptake rates. The saturable uptake rates were calculated by subtracting the MeAIB uptake rates in the presence of 10 mM excess unlabeled MeAIB from the total uptake rates. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differentiation-dependent Stimulation of MeAIB Transport by Insulin in 3T3-L1 Cells-- The differentiation of 3T3-L1 fibroblasts to adipocytes is accompanied by a dramatic increase in insulin sensitivity (12). To evaluate the regulation of amino acid transport in these cells, the uptake of 50 µM MeAIB was determined for a period of 14 days after induction of differentiation. In preadipocytes insulin produced only about a 2-fold increase over the saturable basal uptake of MeAIB (Fig. 1). The maximal insulin response occurred 3 days after induction of differentiation. The maximal insulin-stimulated transport decreased from days 4 to 5, reaching levels about 2-fold higher than that of preadipocytes. Basal MeAIB uptake was increased 3-4 days after initiation of differentiation. However, the basal uptake rates at these time points were determined 8 h after switching from insulin-containing to insulin-free medium, suggesting that the increased basal uptake may represent residual activity resulting from previous stimulation (see below). As cells took on a more adipogenic phenotype, basal activity was reduced to about 20% of that seen in fibroblasts. Thus the effects of insulin to stimulate amino acid transport increased to 10-fold. This pattern of reduced basal and increased insulin-dependent activity in fully differentiated adipocytes is similar to that observed for glycogen synthesis in these cells (13).
|
|
Insulin-stimulated Amino Acid Transport Is Mediated by System A-- Saturable uptake of MeAIB has been regarded as the simplest indication for mediation by system A (2). To determine that system A mediates the insulin-stimulated amino acid transport in 3T3-L1 adipocytes, the MeAIB transport was characterized. Inhibition of MeAIB uptake by various amino acids produced a typical system A transport pattern (Fig. 3, left). Alanine, serine, cysteine, proline, and histidine inhibited MeAIB uptake by more than 90%, whereas the cationic amino acids, arginine and lysine, had no effect. It is noteworthy that the anionic amino acids, glutamate and aspartate, showed moderate inhibition of MeAIB uptake (30-40%). Inhibition by glutamate was inversely proportional to pH (data not shown), indicating that the anionic amino acids could be interacting with system A as uncharged zwitterions (2). Consistent with the major properties of system A transport, uptake of MeAIB in 3T3-L1 adipocytes was strictly Na+-dependent. More than 90% of the MeAIB entry was eliminated when sodium was replaced with choline. In addition, system A activity decreased by 35% in cells preloaded with 50 µM MeAIB for 40 min (trans-inhibition), and by 65% at pH 5.0 as compared with pH 7.4 (Fig. 3, right). The substrate specificity, hormonal stimulation, Na+ dependence, decreased transport activity at lower pH, and trans-inhibition indicate that the insulin-stimulated MeAIB uptake in 3T3-L1 adipocytes is mediated by a typical A-type transport system (1). In contrast to the insulin-stimulated MeAIB uptake, the basal or unstimulated transport was resistant to inhibition by histidine, leaving open the possibility that the constitutively expressed low transport activity is mediated by a different subtype of system A.
|
Decay of the Insulin-stimulated MeAIB Uptake Is Independent of Protein Synthesis and Ambient Amino Acids-- Instability is a mechanistically important feature of the regulation of system A transport (15-17). To determine the half-life of the stimulated MeAIB transport in 3T3-L1 adipocytes, the time course of its activity was followed after removal of insulin from the adipocyte growth medium. At the onset of adipocyte differentiation (day 2), the insulin-stimulated uptake decayed rapidly with an estimated half-life of 1.5 h (Fig. 4, PBS), similar to what is observed in hepatocytes (15). In fully differentiated adipocytes, the insulin-induced transport activity decayed at much slower rates, with a t1/2 of 4.3 h. To determine if the decay of system A activity was associated with a newly synthesized repressor-type protein or repression by ambient amino acids (15, 16), cycloheximide and actinomycin D (1 µg/ml) were added 1 h before removal of insulin, or the cells were incubated in insulin-free medium in the presence or absence of ambient amino acids. As shown in Fig. 4, these agents had no effect on the half-life. Moreover, the similarity in half-life between amino acid-free and amino acid-containing media (PBS versus DMEM) suggests that the decay is not caused by ambient amino acids. Following decay of the insulin-stimulated uptake, the MeAIB transport activity could be re-established by switching to insulin-containing medium. As with the stimulated transport activity before the decay, most of the re-established MeAIB uptake was also substantially inhibited by cycloheximide (81 ± 2%) and actinomycin D (95 ± 2%) (Fig. 3, right), suggesting that the insulin-induced or the decayed MeAIB uptake is associated, respectively, with de novo synthesis or degradation of protein(s) essential for system A function.
|
Activation of MAP Kinase and p70S6K Is Neither Sufficient Nor Required for the Insulin-stimulated System A Transport-- The regulation of protein phosphorylation is believed to play a central role in insulin action (6). The two best characterized pathways leading to insulin-dependent serine phosphorylation involve either MAP kinase or p70S6K (18). To determine if Ras/MAP kinase pathway is required for the stimulation of system A transport by insulin, PD98059, a specific inhibitor of MEK (19), was examined. At 10 µM concentration, PD98059 is sufficient to block the activation of MEK, MAP kinase, and pp90rsk, and the induction of c-fos transcription (9, 10, 19, 20). However, this agent did not significantly affect the insulin-stimulated MeAIB uptake even at concentrations up to 50 µM, while the same concentration of PD98059 caused nearly complete inhibition of the insulin-induced activation of MAP kinases (Fig. 5). To further exclude involvement of this signaling pathway, inhibition of the farnesylation of Ras, the upstream mediator for activation of the MAP kinase pathway, by specific inhibitors PD152440 (20) and B581 (21) was tested. At concentrations of 10 and 50 µM, respectively, these two compounds have been shown to effectively block activation of MAP kinase (20), but at the same concentrations these two reagents had no effect on the insulin-stimulated transport activity, suggesting further that activation of MAP kinase is not responsible for this action of insulin.
|
Insulin-Stimulated System A Transport Is Highly Sensitive to
Wortmannin and LY294002--
To explore the role of PI 3-K in the
regulation of system A transport, the two structurally distinct
inhibitors of PI 3
-K, wortmannin and LY294002, were employed (24, 25).
As seen in Fig. 5, both compounds were potent inhibitors of the
insulin-stimulated system A activity with IC50 values of 17 nM and 9.8 µM, respectively. These
IC50 values are similar to those reported for inhibition of
insulin-activation of PI 3
-K (20, 26, 27) and phospholipase A2 (28), but are significantly lower than that for
inhibition of phospholipase D and phospholipase C (29), myosin light
chain kinase (30), mammalian target of rapamycin (31), and
phosphatidylinositol 4
-kinase (32).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several models have been proposed to interpret the kinetic and genetic bases for the regulation of system A amino acid transport (5, 15-17). According to these models, a "transport inactivation protein" or an active form of "repressor/inactivator" is thought to be responsible for the decay or adaptive regulation of system A activity. Evidence suggests that the histone-like nuclear proteins (16, 17) or heat shock protein P1-related proteins (33) may serve to inactivate the system A transporter. However, data presented here indicate that the decay of system A transport in adipocytes is not caused by synthesis of a repressor-type protein. Moreover, it also appears to be independent of ambient amino acids. Taken together, these data suggest that the insulin-stimulated system A transport in adipocytes is mediated by a mechanism different from that reported in hepatocytes (15-17). The lack of repressor-type control has been reported for regulation of system A transport by other factors such as osmotic stress (5, 34).
The precise molecular events involved in regulation of system A amino acid transport remain uncertain. The time course of the insulin-stimulated MeAIB uptake and its sensitivity to cycloheximide and actinomycin D suggest that the hormone probably regulates expression of the genes encoding proteins involved in system A transport. We demonstrate here that the Ras/MAP kinase and pp70S6K pathways are not necessary for activation of this transport in 3T3-L1 adipocytes. This finding is consistent with dissociation of Ras/MAP kinase pathway from most of the metabolic responses to insulin, including glucose transport (9, 10, 35), glycogen synthesis (9, 10, 36, 37), and lipogenesis (9, 10). Regulation of gene expression by insulin appears to be differentially sensitive to blockade of the MAP kinase pathway. While c-fos induction clearly requires MAP kinase activation (9), the regulated expression of phosphoenolpyruvate carboxykinase is not MAP kinase-dependent (20). Moreover, dissociation of pp70S6K pathway from stimulation of amino acid transport by insulin is similar to that observed in insulin-induced translocation of GLUT4 (38) and activation of glycogen synthase (23).
In contrast to the MAP kinase and pp70S6K pathway
inhibitors, the two PI 3-K inhibitors, wortmannin and LY294002,
strongly attenuate the insulin-stimulated MeAIB uptake with the
IC50 values close to that for inhibition of PI 3
-K (20,
26, 27). Inhibition by these two agents has been widely applied in
investigations of the importance of PI 3
-K in a number of
insulin-mediated metabolic responses, including glucose transport (18,
39-42), antilipolysis (39, 43), glycogen synthesis (36, 37, 44), and
phosphoenolpyruvate carboxykinase gene expression (45). Wortmannin has
also been used in the studies of insulin-induced
-aminoisobutyric
acid (AIB) uptake in muscle cells (46). In this previous study, AIB uptake was taken as system A transport. However, AIB is a nonspecific probe for system A, and in muscle cells, system A is not the only insulin-inducible amino acid transport system (47). Because the
insulin-induced AIB uptake was totally insensitive to actinomycin D,
its regulatory mechanism could be quite different from the commonly
observed transcriptional regulation of system A.
It should be noted that the specificity of wortmannin and LY294002 for
PI 3-K has been challenged by several recent studies (28, 31, 48). In
addition to inhibition of PI 3
-K, wortmannin also inhibits a number of
other signaling mediators, although the inhibition may occur at
relatively higher concentrations. These wortmannin-sensitive enzymes
include mammalian target of rapamycin (31), phospholipase
A2 (28), phosphatidylinositol 4
-kinase (32), myosin light
chain kinase (30), phospholipase C, and phospholipase D (29). Even
though the effective doses of wortmannin for inhibition of the
insulin-induced amino acid transport occurred at low nanomolar
concentrations, the present study does not exclude PI 3
-K-independent
signaling pathways involved in the regulation of system A. It has been
shown that activation of glycogen synthase by insulin and activation of
MAP kinase by platelet-activating factor are unaffected by introducing overexpressed dominant-negative
p85, but the induced activities remain wortmannin-sensitive (37, 49). These latter results suggest that
there might be an additional wortmannin/LY294002-sensitive mediator(s)
involved (36).
Activation of PI 3-K represents an earlier step in transducing signals
from many receptor tyrosine kinases (50). In addition to insulin,
mitogens, such as PDGF, IL-4, IL-11, and GH, also activate PI 3
-K (10,
49) (this study). In the present report, we demonstrate that activation
of PI 3
-K by GH, PDGF, and IL-11 is not accompanied by coordinate
stimulation of system A transport. This dissociation of increase in
MeAIB uptake from activation of PI 3
-K is consistent with a recent
study showing that PDGF and IL-4 activate PI 3
-K, but fail to induce
glucose transport (51). It has been suggested that activation of PI
3
-K may not be sufficient for some of the metabolic responses to
insulin (51, 52). Alternatively, insulin may activate PI 3
-K in a way
distinct from other growth factors and hematopoietic cytokines. It has recently been shown that insulin and PDGF trigger compartment-specific regulation of PI 3
-K (53). In contrast to insulin stimulation, the
phosphatidylinositol-(3,4,5)P3 synthesis induced by PDGF
has been reported to be barely detectable in 3T3-L1 adipocytes (54). These differences may account for the failure to stimulate amino acid
transport by PDGF observed here. The signaling pathway for activation
of PI 3
-K by IL-11 is unknown. Since Western blots did not show
coimmunoprecipitation of p85 with IRS-1 in IL-11-stimulated 3T3-L1
adipocytes,2 IRS-1 might not
be involved in activation of PI 3
-K by IL-11. Therefore, the
IRS-1-independent activation by PDGF and possibly by IL-11 may trigger
downstream signaling pathways distinct from that by insulin. Although
GH activates PI 3
-K via phosphorylation of IRS-1 (55), GH is
ineffective for stimulation of MeAIB uptake (this study) and glucose
transport (55). For the latter results it was hypothesized that GH and
insulin may not induce phosphorylation of the same subset of tyrosine
residues of IRS-1 or produce the same type of interaction among the
signal mediators that are bound to the phosphorylated IRS-1 (55).
Furthermore, it is possible that the insulin-stimulated system A
transport is mediated by an unique form of PI 3
-K (56, 57). Various
subtypes of PI 3
-K may be differentially regulated. The precise role
of PI 3
-K in insulin action is not fully understood. Recent studies
suggest that phosphoinositide-dependent protein kinase and
protein kinase B may be the two sequential downstream mediators after
activation of PI 3
-K by insulin (58). Interestingly, these authors
showed that activation of protein kinase B was not inhibited by
rapamycin and PD98059, but was prevented by wortmannin. It is unclear
whether these two kinases participate in regulation of system A
transport. These possibilities will require further investigation.
![]() |
FOOTNOTES |
---|
* 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 Molecular Biology, Parke-Davis Pharmaceutical Research Division of Warner Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 313-998-5957; Fax: 313-998-5970.
1
The abbreviations used are: MeAIB,
2-(methylamino)isobutyric acid; PDGF, platelet-derived growth factor;
AIB, -aminoisobutyric acid; GH, growth hormone; IGF, insulin-like
growth factor; IL-11, interleukin 11; IRS, insulin receptor substrate;
MEK, mitogen-activated protein kinase kinase; MAP, mitogen-activated
protein; PI 3
-K, phosphatidylinositol 3
-kinase; p70S6K,
Mr 70,000 S6 kinase; TPA,
12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's
modified Eagle's medium; PMSF, phenylmethylsulfonyl fluoride; PBS,
phosphate-buffered saline.
2 T.-Z. Su, M. Wang, L.-J. Syu, A. R. Saltiel, and D. L. Oxender, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|