Amino acids stimulate phosphorylation of
p70S6k and organization of
rat adipocytes into multicellular clusters
Heather L.
Fox,
Scot R.
Kimball,
Leonard S.
Jefferson, and
Christopher J.
Lynch
Department of Cellular and Molecular Physiology, Pennsylvania
State University College of Medicine, Hershey, Pennsylvania 17033
 |
ABSTRACT |
In previous
studies we have shown that rat adipocytes suspended in Matrigel and
placed in primary culture migrate through the gel to form multicellular
clusters over a 5- to 6-day period. In the present study,
phosphorylation of the insulin-regulated 70-kDa ribosomal protein S6
kinase (p70S6k) was observed
within 30 min of establishment of adipocytes in primary culture. Two
inhibitors of the p70S6k
signaling pathway, rapamycin and LY-294002, greatly reduced
phosphorylation of p70S6k and
organization of adipocytes into multicellular clusters. Of all the
components of the cell culture medium, amino acids, and in particular a
subset of neutral amino acids, were found to promote both
phosphorylation of p70S6k and
cluster formation. Lowering the concentrations of amino acids in the
medium to levels approximating those in plasma of fasted rats decreased
both phosphorylation of p70S6k
and cluster formation. Furthermore, stimulation of
p70S6k phosphorylation by amino
acids was prevented by either rapamycin or LY-294002. These findings
demonstrate that amino acids stimulate the
p70S6k signaling pathway in
adipocytes and imply a role for this pathway in multicellular
clustering.
Matrigel; tissue morphogenesis; LY-294002; rapamycin
 |
INTRODUCTION |
SUSPENDING ADIPOCYTES IN A three-dimensional
extracellular matrix (Matrigel) facilitates the observation of their
capacity to migrate extensively and organize into multicellular
clusters in primary culture (5). Initial morphological changes in the cells include plasma membrane extensions or tubes that form cell-cell junctions between neighboring cells; these can be observed after 2 days
in culture. At the same time, or within 24 h, small asymmetrical clusters of adipocytes appear (5). These so-called "seed
clusters" typically contain four to seven adipocytes in close
proximity. The next event, typically observed on
day 4 or 5, is the formation of roughly
symmetrical spheres of cells, ~0.5-1 mm in diameter, that have
been termed intermediate clusters. These appear to be formed from many
individually migrating adipocytes, lines of two or three adipocytes,
and previously existing smaller colonies migrating as groups. The
intermediate clusters are visible to the naked eye and can be counted
and therefore provide a convenient method for quantitating the
multicellular clustering process. By about
day
6, intermediate clusters and cells
alone or in other groupings join together to form large asymmetrical
structures up to 15 mm in length and 0.5-3 mm in width. Around
these structures there appears to be a thin gelatinous layer or sheath.
By this time, much of the original Matrigel is typically destroyed, and thin threads of fibrillar collagen-like strands connect the structures to one another and the plates. These three-dimensional asymmetrical cell clusters are then suspended floating above the plastic and can be
removed with a forceps by breaking the connecting strands.
We have investigated the formation of multicellular clusters in vitro
as a first step in determining if the process reflects or is associated
with tissue morphogenesis in vivo. Because insulin stimulates
multicellular clustering in vitro (5), we hypothesized that the process
might rely on activation of cell signaling pathways used by insulin or
on inhibition of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathways. To help delineate between these two possibilities, we have examined the effect of cell signaling agonists and inhibitors on multicellular clustering. The results of those studies are reported here; they suggest a role for the FK506- and
rapamycin-associated protein/mammalian target of rapamycin (FRAP/mTOR)
branch of the phosphatidylinositide-3-OH kinase (PI 3-kinase) but not
the cAMP-dependent signaling pathway in multicellular clustering.
Because activation of the FRAP/mTOR signaling pathway is important for
multicellular clustering, we next investigated the stimulus for 70-kDa
ribosomal protein S6 kinase
(p70S6k) phosphorylation in
adipocyte cultures. The present studies indicate that one of the major
stimuli of multicellular clustering and the
p70S6k phosphorylation that
precedes it is the presence of amino acids in the cell culture medium,
Dulbecco's modified Eagle's medium (DMEM), at concentrations
exceeding those found in the plasma of fasting rats in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials.
Matrigel and growth factor-reduced Matrigel were obtained from
Collaborative Biomedical (Bedford, MA). Rapamycin and
2-[4-morpholinyl]-8-phenyl-[4H]-1-benzopyran-4-one (LY-294002) were from Calbiochem (La Jolla, CA). Protein A-agarose beads, calf serum, and DMEM were obtained from GIBCO (Gaithersburg, MD). Fatty acid-free bovine serum albumin (BSA) was purchased from
Miles Pentex/Bayer (New Haven, CT). Collagenase type I was from
Worthington Diagnostics (Freehold, NJ). Amino acids, leupeptin, benzamidine, and microcystin LR were from Sigma Chemical (St. Louis,
MO) or United States Biochemical (Cleveland, OH). Aprotinin was from
Boehringer Mannheim (Indianapolis, IN). The
p70S6k rabbit polyclonal
immunoglobulin G (IgG) antibody was obtained from Santa Cruz Biotech
(Santa Cruz, CA). Polyvinylidene difluoride (PVDF) membrane was from
Bio-Rad (Hercules, CA). Enhanced chemiluminescence (ECL) detection kits
and donkey anti-rabbit IgG were purchased from Amersham
(Arlington Heights, IL).
Primary culture of rat adipocytes.
Unless otherwise indicated, adipocytes were isolated from 7- to
8-wk-old male Sprague-Dawley rats and were suspended in growth factor-reduced Matrigel (unless otherwise indicated) for primary culture in six-well Falcon dishes as previously described (5). The
plating densities ranged from 0.5 × 106 to 1.3 × 106 cells/well, depending on the
type of experiment and yield of adipocytes. Floating adipocyte cultures
were prepared as described by Marshall et al. (22). Both the Matrigel
and the floating cultures were maintained in 3 ml of DMEM supplemented
with 25 mM NaHCO3, 1 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1.5 mM glutamine, 1 U/ml penicillin, 1 µg/ml
streptomycin, and 10% calf serum. Multicellular clustering is not
dependent on serum; however, either serum or 2% BSA was added to the
cell culture medium to facilitate comparisons with previous studies. In
experiments involving cultures of floating cells or comparisons of the
Matrigel and floating cultures, 2% fatty acid-free BSA was added to
cell culture medium. Cell cultures were maintained at 37°C under a
humidified atmosphere of 95% air-5%
CO2. In some experiments DMEM was
replaced with a modified DMEM containing lower concentrations of amino
acids approximating concentrations found in plasma from fasted rats
(i.e., "1×" amino acids, Ref. 24).
Quantitation of intermediate clusters.
Adipocytes were suspended in Matrigel and maintained in primary culture
for at least 4 days. The medium was exchanged every 24-48 h.
Intermediate clusters (spherical clusters of adipocytes, 0.5-1.5
mm in diameter, formed by the migration of cells into seed clusters)
were counted on illuminated plates as previously described (5).
Phosphorylation of p70S6k.
Phosphorylation of p70S6k was
examined using a gel shift assay involving Western blot analysis of
p70S6k immunoprecipitates (2, 6,
8, 9, 16, 29, 32). For this purpose, protein A-agarose beads were
prepared by three washes with phosphate-buffered saline (PBS) and
resuspension in an equivalent volume of PBS. Aliquots (5 µl) of the
p70S6k rabbit polyclonal IgG
antibody were mixed with 100 µl of resuspended beads and incubated at
room temperature for 1 h. The beads were then washed five times with
PBS and once with buffer H [in mM: 100 tris(hydroxymethyl)aminomethane (Tris) base, 10 MgCl2, 1 sodium orthovanadate, 1 dithiothreitol, 1 EDTA, 5 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 10 KH2PO4,
50
-glycerophosphate, 1 benzamidine, and 0.1 phenylmethylsulfonyl
fluoride, as well as 0.2 µM leupeptin, 3 µM aprotinin, and 1 µM
microcystin LR] and then resuspended in 50 µl of buffer
H.
Frozen samples of adipocytes were resuspended in buffer H, sonicated on
ice, and centrifuged at 10,000 g for
30 min at 4°C. A 1-ml aliquot of the supernatant was mixed with 50 µl of p70S6k antibody-protein A
beads and rocked for 1 h at 4°C. The beads were then washed twice
in 0.5 ml of buffer H and resuspended in an equal volume of 2×
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
sample buffer. The unphosphorylated and various phosphorylated forms of
the immunoprecipitated p70S6k
were separated using SDS-PAGE with a 0.75-mm 7.5% acrylamide-0.05% bis-acrylamide resolving gel and a 4% acrylamide-0.11% bis-acrylamide stacking gel run overnight at 4 mA, followed by transfer to PVDF membrane. The membrane was incubated for 1 h in 5% dry milk in Tris-buffered saline with Tween (TBST; 0.1% Tween-20, 20 mM Tris base,
pH 7.6, and 137 mM NaCl) and washed in TBST. The
p70S6k rabbit polyclonal IgG
primary antibody was diluted 1:1,000 in TBST and incubated with the
membrane. After several washes with TBST, the membrane was incubated
with horseradish peroxidase-linked donkey anti-rabbit IgG (Amersham)
diluted 1:4,000 in TBST. The blot was visualized using an ECL detection
kit (Amersham) according to the manufacturer's directions.
 |
RESULTS |
Formation of intermediate clusters.
Matrigel is a fluid at cold temperatures and organizes into a gel at
warmer temperatures. Adipocytes were mixed with ice-cold Matrigel in
the fluid form and applied to six-well Falcon dishes that had
previously been coated with Matrigel. The dishes were transferred to a
37°C cell culture incubator. The Matrigel liquid is viscous enough
that the cells did not rise significantly before it gelled. Thus the
adipocytes became uniformly suspended within a three-dimensional
Matrigel matrix. Cell culture medium was then added to the cells, and
they were maintained in primary culture as previously described (5).
Figure 1 summarizes the previously characterized microscopic events
that occur during the subsequent days in culture (5). In cultures from
7- to 8-wk-old animals essentially all of the adipocytes eventually
organize into large asymmetrical structures (Fig.
1), and during this process
adipocytes maintain postmitotic status as well as
differentiation-dependent and tissue-specific protein expression (5).
Previous findings suggested that multicellular clustering was possibly
associated with the activation of postreceptor components of the same
cell signaling pathways used by insulin or alternatively was due to
inhibition of a cAMP-dependent pathway (5). Standard Matrigel, a
fractionated high-salt/urea extract from
Engelbreth-Holm-Swarm tumor cell cultures, contains
detectable amounts of growth factors, including epidermal growth
factor, insulin-like growth factor I, and platelet-derived growth
factor (e.g., see Ref. 38). To test the hypothesis that one or more of
these contaminating growth factors might be promoting multicellular
clustering, standard Matrigel was compared with a growth factor-reduced
Matrigel prepared as described by Taub et al. (38). Figure
2A shows
that there was no statistically significant difference between the two
forms of Matrigel in terms of intermediate cluster formation. To avoid any possible interference from the growth factors, growth
factor-reduced Matrigel was used for subsequent experiments.

View larger version (33K):
[in this window]
[in a new window]

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of various agents on formation of intermediate clusters in
primary adipocytes suspended in Matrigel.
A: adipocytes were isolated from
Sprague-Dawley rats and maintained in a 3-dimensional matrix of growth
factor-reduced Matrigel. Intermediate clusters were quantitated as
previously described (5) on day 4, day
5, or day 6 of culture
(longer times are required for clusters to be observed from older
animals). Results are means ± SE from replicate experiments in
which control was set to 100%. LY-29004 and rapamycin treatments were
significantly different from control according to Student's
t-test
(P < 0.05).
B: adipocytes were isolated from 7- to
8-wk-old Sprague-Dawley rats, suspended in Matrigel, plated, and
maintained in primary culture as described in
EXPERIMENTAL PROCEDURES in absence or
in presence of indicated concentrations of rapamycin.
|
|
To examine the possible role of cAMP-dependent cell signaling in
multicellular clustering, adipocytes were maintained in the presence or
absence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or
8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP). DPCPX, an A1 adenosine
receptor antagonist, increases cAMP in adipocytes by interfering with
signals arising from the fat cell autocrine adenosine (3). The
permeable, potent, and poorly hydrolyzable cAMP analog CPT-cAMP
stimulates cAMP- and guanosine 3',5'-cyclic
monophosphate-dependent protein kinases. Neither agent had any
significant effect on multicellular clustering (Fig.
2A).
In the next set of experiments, inhibitors of the
p70S6k cell signaling pathways
were examined. A PI 3-kinase inhibitor, LY-294002, caused a
statistically significant decrease in the number of intermediate clusters per dish to 38 ± 5% of control in three different
experiments. LY-294002 inhibits PI 3-kinase and was used instead of
wortmannin because it has a longer half-life in aqueous solution and
may be a more specific inhibitor of PI 3-kinase (33, 40). Rapamycin, which binds to mTOR and prevents activation of
p70S6k by insulin and other
hormones (8, 32, 34), also reduced significantly the formation of
intermediate clusters (29 ± 10% of control) in five
separate experiments (Fig. 2). The inhibition was concentration
dependent, being half-maximal at 0.10 ± 0.04 ng/ml, as shown in
Fig. 2B. The concentration dependency
in these experiments was similar to that reported for rapamycin
inhibition of serum-stimulated
p70S6k activity (8).
Phosphorylation of p70S6k.
The rapamycin-sensitive phosphorylation and activity of the protein
kinase p70S6k is a convenient
indicator of the state of activation of the FRAP/mTOR cell signaling
pathway. The activity of p70S6k
is increased by phosphorylation, which can be detected using a gel
shift assay. Figure 3 shows that
immunoprecipitates from lysates of freshly isolated adipocytes,
prepared in Krebs-Ringer-HEPES buffer (19) containing 2% BSA (KRH),
contained only one or two faster migrating (i.e., less phosphorylated)
forms of p70S6k. In contrast,
adipocytes maintained in primary culture, that is, in Matrigel with
DMEM cell culture medium contained the slower migrating, more
phosphorylated forms. The increase in phosphorylation state was
observed within 24 h (data not shown) and at time points as early as 30 min after beginning culture (Fig. 3). The phosphorylation of
p70S6k induced during cell
culture was sensitive to inhibition by rapamycin (Fig. 3), as indicated
by the increase in the proportion of the protein present in the faster
migrating forms. In the experiments shown in Fig. 3, rapamycin was
added after the Matrigel had gelled. In other experiments (data not
shown), when rapamycin was added to freshly isolated cells, before they
were exposed to Matrigel or cell culture medium, essentially all of the
p70S6k was found in the faster
migrating (i.e., less phosphorylated) form 60 min after the cells were
placed in culture.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
Adipocytes in primary culture exhibit a time-dependent and
rapamycin-sensitive increase in 70-kDa ribosomal protein S6 kinase
(p70S6k) phosphorylation.
Adipocytes were isolated from 7- to 8-wk-old Sprague-Dawley rats in
Krebs-Ringer-HEPES buffer containing 2% BSA (KRH). Aliquots of these
KRH-suspended cells were then either frozen in liquid nitrogen prior to
any manipulations (freshly isolated) or placed in
culture within a 3-dimensional matrix of basement membrane components
(5) on 35-mm plates with serum-free DMEM in presence or absence of 30 ng/ml rapamycin. Plates of cells (cultured) were snap frozen at
indicated times and stored at 84°C and then subjected to
SDS-polyacrylamide gel electrophoresis (PAGE).
p70S6k was immunoprecipitated
from soluble fraction of cell lysates as described in
EXPERIMENTAL PROCEDURES.
Phosphorylation of p70S6k on
several sites leads to at least 4 bands with decreased mobility in
SDS-PAGE gels, as indicated by an upward shift in banding pattern.
|
|
Figure 4A shows that
when adipocytes were maintained floating in KRH for 1 h the
phosphorylation state of p70S6k
was essentially the same as that observed in freshly isolated adipocytes. Conversely, the presence of serum-free DMEM over the same
time period led to the phosphorylation of
p70S6k (Fig.
4A). Because the phosphorylation of
p70S6k was also observed in
experiments with floating cultures of adipocytes and in the absence of
serum, neither Matrigel nor serum appeared to be responsible for the
effect. Instead, the results of Fig. 4A implicate one or more components of
DMEM as the cause of the cell culture-related phosphorylation of
p70S6k.
To identify the component or components of DMEM responsible for the
phosphorylation of p70S6k, KRH
solutions containing different additives found in DMEM were prepared.
In these experiments (Fig. 4B),
cells were incubated in KRH with 1)
dextrose, 2) vitamins and phenol
red, or 3) all of the amino acids.
The concentrations of these components were the same as those found in
DMEM. The phosphorylation state of p70S6k was examined after a
30-min incubation with these different solutions. Of all the components
tested, only amino acids were capable of stimulating
p70S6k phosphorylation. The
stimulation was similar to that observed with DMEM and Matrigel at the
1-h time point in Fig. 3.
To further examine the role of amino acids in the stimulation of
phosphorylation of p70S6k, amino
acids present in DMEM were divided into three subsets referred to as
charged, neutral L, and neutral ASC/A (Table 1). Charged
amino acids included Arg, Gln, His, and Lys; neutral L were neutral
amino acids preferentially transported on the L-type amino acid
transporter; and neutral ASC/A were neutral amino acids preferentially
transported on the ASC- or A-type amino acid transporter (7). The
neutral L subset of amino acids, including Iso, Leu, Met, Phe, Trp,
Try, and Val, partially stimulated
p70S6k phosphorylation, but not
to the same extent as the full mixture of amino acids (Fig.
4B). The decreased efficacy of the
neutral L subset compared with the full set of amino acids was observed in several experiments and suggested that stimulation of
p70S6k phosphorylation by the
amino acids may be complex. No stimulation was detected with either the
charged or neutral ASC/A subsets (Fig.
4B). Figure 5 shows
that stimulation of p70S6k
phosphorylation by amino acids was inhibited by 30 µM LY-294002 or 30 ng/ml rapamycin. These findings demonstrate a role for PI 3-kinase and
the FRAP/mTOR pathway in the response to amino acids.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of KRH, DMEM, and DMEM constituents added to KRH on
p70S6k phosphorylation in
floating adipocytes. A: freshly
isolated adipocytes were adjusted in KRH buffer to a final cytocrit of
50-80%. A 150-µl aliquot of cells was then incubated at
37°C for 1 h in 3 ml of either KRH or DMEM under a humidified
atmosphere of 95% air-5% CO2.
Cells were then isolated from incubating solutions by centrifugation
and frozen in liquid nitrogen. Cell lysates were then prepared in
buffer H, and p70S6k was
immunoprecipitated from aliquots equivalent to 36 × 105 cells (~1 ml).
p70S6k phosphorylation state was
examined as described for Fig. 3. B: a
150-µl aliquot of cells was then incubated floating at 37°C as
above in 3 ml of either KRH or KRH containing additives found in DMEM: + dextrose medium contained 25 mM
D-glucose; + vitamins + phenol
red medium contained (in mg/l) 4 D-calcium pantothenate, 4 choline chloride, 4 folic acid, 7.2 i-inositol, 4 niacinamide, 4 pyridoxal
hydrochloride, 0.4 riboflavin, 4 thiamine hydrochloride, and 15 phenol
red. Table 1 summarizes amino acid categories. Neutral L are neutral
amino acids preferentially transported on L-type amino acid transporter
(7). Neutral ASC/A are neutral amino acids preferentially transported
on ASC- or A-type amino acid transporter (7). After a 30-min
incubation, floating adipocytes were isolated by centrifugation and
were snap frozen. Phosphorylation of
p70S6k in cell lysates was
determined as described for Fig. 3.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of rapamycin and LY-294002 on amino acid-simulated
p70S6k phosphorylation.
Adipocytes were isolated from 7- to 8-wk-old rats and adjusted in KRH
buffer to a final cytocrit of 50-80%. A 150-µl aliquot of cells
was then incubated at 37°C for 30 min in 3 ml of KRH in absence or
presence of 30 ng/ml rapamycin or 30 µM LY-294002. Amino acids were
then added to final concentrations found in DMEM (Table 2). Cells were
then isolated, and p70S6k
phosphorylation was examined as described for Fig. 3.
|
|
The concentrations of amino acids found in DMEM are higher than those
found in plasma during fasting and postabsorptive states (24, 30). When
adipocytes were exposed to DMEM containing lower concentrations of
amino acids (Table
2; referred to as 1×), the distribution of
p70S6k among its various
electrophoretic forms was intermediate between that observed with KRH
and DMEM (Fig. 6).
View this table:
[in this window]
[in a new window]
|
Table 2.
Concentrations of individual amino acids in DMEM and in DMEM
containing 1× concentrations of amino acids
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 1× amino acids on
p70S6k phosphorylation.
Adipocytes were isolated from 7- to 8-wk-old rats and adjusted in KRH
buffer to a final cytocrit of 50-80%. A 150-µl aliquot of cells
was then incubated under a humidified atmosphere of 95% humidified
air-5% CO2 at 37°C for 30 min
in 3 ml of either KRH, KRH with 1× amino acids (see Table 2), or
KRH containing amino acids at concentrations comparable to
concentrations found in DMEM (all amino acids). Cells were processed,
and p70S6k immunoreactivity and
phosphorylation were examined as for Fig. 3.
|
|
Effects of amino acids on formation of intermediate clusters.
Figure 7 shows that the higher amino acid concentrations
found in standard DMEM increased the formation of intermediate clusters compared with DMEM containing the lower 1× concentrations of
amino acids. This result supports the hypothesis that amino acids in DMEM are a major stimulator of multicellular clustering.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of 1× amino acids on formation of intermediate clusters.
Matrigel cultures were maintained in DMEM containing 10% calf serum
(DMEM) or modified DMEM containing 10% calf serum and lower
concentrations of amino acids found in plasma of fasted rats, as
described in Table 2 [1× amino acids (A.A.s)].
Intermediate clusters were counted on
day 5. Results are means ± SE of
triplicate determinations from a representative experiment.
|
|
 |
DISCUSSION |
The studies reported here examined the effects of various cell
signaling agonists and inhibitors on the multicellular clustering of
adipocytes. The results of these studies imply that cell signaling pathways used by the insulin receptor, but not cAMP-dependent pathways,
stimulate this process. The results may help explain how insulin
stimulates clustering of adipocytes (5). To the best of our knowledge
this paper also represents the first report of amino acids stimulating
multicellular clustering of adipocytes and the phosphorylation of
p70S6k. Furthermore, our data
implicate PI 3-kinase and the FRAP/mTOR signaling pathway in the
actions of amino acids on fat
cells.
Multicellular clustering.
Insulin receptor cell signaling involves at least one early bifurcation
that leads to activation of two distinct pathways in which Ras and PI
3-kinase activation play early and critical roles (10, 11, 14, 15, 18,
31, 36, 37, 39). The Ras/mitogen-activated protein kinase
pathway is important for the mitogenic effects of insulin and
c-fos transcription, whereas the PI
3-kinase pathway appears to be involved in glycogen synthesis, glucose
transport, phosphoenolpyruvate carboxykinase transcriptional
regulation, and p70/p85 ribosomal protein S6 kinase regulation. Insulin
stimulates p70S6k through a
rapamycin-sensitive mechanism in a number of tissues (6, 12, 16, 17,
25). The effects of insulin on the initiation of protein synthesis in
fat cells are also at least partially inhibited by rapamycin (10). On
the basis of findings that intermediate cluster formation was inhibited
by rapamycin and LY-294002, it would appear that the multicellular
clustering of adipocytes may require activation of PI 3-kinase and the
FRAP/mTOR elements downstream from PI 3-kinase (Fig. 2). Activation of
PI 3-kinase is also important in other situations that, like the multicellular clustering of adipocytes, involve invasion of basement membrane. For instance, activation of PI 3-kinase is important for
neurite outgrowth that requires invasion of basement membrane (20). A
potential caveat is that the effects of amino acids on PI 3-kinases are
not known. Therefore, an alternative explanation of the results of the
experiments with LY-294002 should be considered, for instance, that the
amino acids are activating some other LY-294002-sensitive step that
leads to activation of mTOR and
p70S6k.
Mechanism of action of amino acids.
Amino acids were found to stimulate the phosphorylation of
p70S6k and multicellular
clustering in adipocytes (Figs. 4-7). Marshall and Monzon (23)
previously reported effects of amino acids on protein synthesis in
adipocytes. Amino acids at concentrations found in DMEM cell culture
medium (~2-4×) caused a 68% increase in insulin
sensitivity, as measured by the 50% effective concentration, and a
157% increase in insulin maximal responsiveness (efficacy). In that
study, effects of amino acids were observed on protein synthesis but
not glucose transport. The exact mechanism of the observed effects was
not determined. Our data may provide an explanation for the findings of
Marshall and Monzon (23). We show that amino acids are capable of
activating one of the same cell signaling pathways used by insulin in
fat cells. Amino acids stimulated the phosphorylation of
p70S6k (Figs. 4-6), as does
insulin (6, 34). The insulin signaling pathway that leads to increased
protein synthesis in adipocytes, i.e., the one studied by Marshall and
Monzon (23), is known to require PI 3-kinase and may also involve a
rapamycin-sensitive mechanism downstream from PI 3-kinase activation
(2, 6, 10, 34). Our data show that the stimulation of multicellular clustering and phosphorylation of
p70S6k by amino acids is in fact
sensitive to a PI 3-kinase inhibitor (LY-29004) and an inhibitor of the
mTOR kinase (rapamycin), as shown in Figs. 2 and 5, respectively.
In rat hepatocytes, some cell signaling effects of amino
acids, like those of glutamine, appear to be mediated by an activation of focal adhesion kinase caused by cell swelling associated with Na+-dependent transport (21).
However, the subset of amino acids we found to be involved in
p70S6k activation is not
transported by Na+-dependent
mechanisms, and the charged amino acids that are transported by this
mechanism do not stimulate p70S6k
phosphorylation (Fig. 4B). Thus
activation of intracellular kinases in response to amino acids may
occur by distinct mechanisms in different cell types.
Others have proposed the existence of a transmembrane receptor for
neutral amino acids whose activation leads to inhibition of autophagy,
stimulation of protein synthesis, or both. For example, Mortimore and
colleagues (26-28) have demonstrated that the effect of leucine on
autophagy can be elicited by an impermeable leucine analog and have
tentatively identified a possible cell surface receptor by
cross-linking. Blommaart et al. (4) have provided data on the mechanism
of the putative receptor by demonstrating that a rapamycin-sensitive
phosphorylation of ribosomal protein S6 is required for the effects of
amino acids on protein synthesis and autophagy in hepatocytes. They
found that the amino acids Phe, Tyr, and Leu inhibited autophagy and
stimulated ribosomal S6 protein phosphorylation and protein synthesis,
but not to the same extent as produced by all of the amino acids. This
is similar to the findings reported here on the phosphorylation of
p70S6k (Fig.
4B), in which the neutral L amino
acid subset stimulated p70S6k
phosphorylation, but not as well as all amino acids together. Other
amino acids, however, had little or no effect on S6 protein phosphorylation. The differences in efficacy between the subset of
amino acids that are active and the complete set of amino acids suggest
that the response may be complex. To explain this, Blommaart et al. (4)
proposed that cell swelling (e.g., from the charged amino acids)
increases the efficacy of the receptor responsible for the effects of
the neutral amino acids. Another possibility is that one or more of the
amino acids may be active but that others, although not active alone,
may need to be present for a complete response to be observed. If the
signaling pathway proposed by Blommaart et al. (4) does exist, our data
may provide further information on steps between activation of the
putative neutral amino acid receptor and ribosomal S6 phosphorylation.
We show that amino acids stimulate the phosphorylation of
p70S6k (Figs. 5-7), a kinase
that is capable of phosphorylating the S6 protein by a
rapamycin-sensitive mechanism. Our data also implicate the PI 3-kinase
pathway in the stimulation of phosphorylation of
p70S6k caused by amino acids
(Fig. 6), as might be expected if a tyrosine kinase activity was
responsible for these effects.
In conclusion, the results reported in this paper show that amino acids
stimulate multicellular clustering of adipocytes in vitro. In adult
humans, concentrations of the amino acids in the subset found to be
important for p70S6k
phosphorylation approximately double after a protein meal (1). If
multicellular clustering in vitro is eventually shown to be reflective
of adipose tissue morphogenesis in vivo, our findings would provide
further support for the emerging idea that elevations in serum nutrient
concentrations act both directly and indirectly, e.g., through insulin,
to promote adipose tissue growth and development (13, 35).
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the American Diabetes
Association (to C. J. Lynch), National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-13499 and DK-15658 (to L. S. Jefferson), and Grant 195058 from the Juvenile Diabetes Foundation
International (to S. R. Kimball).
 |
FOOTNOTES |
Address for reprint requests: C. J. Lynch, Dept. of Cellular and
Molecular Physiology, The Pennsylvania State University College of
Medicine, 500 University Dr., Hershey, PA 17033.
Received 30 July 1997; accepted in final form 1 October 1997.
 |
REFERENCES |
1.
Aoki, T. T.,
M. F. Brennan,
W. A. Muller,
J. S. Soeldner,
J. S. Alpert,
S. B. Saltz,
R. L. Kaufmann,
M. H. Tan,
and
G. F. Cahill, Jr.
Amino acid levels across normal forearm muscle and splanchnic bed after a protein meal.
Am. J. Clin. Nutr.
29:
340-350,
1976[Abstract].
2.
Azpiazu, I.,
A. R. Saltiel,
A. A. DePaoli-Roach,
and
J. C. Lawrence.
Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways.
J. Biol. Chem.
271:
5033-5039,
1996[Abstract/Free Full Text].
3.
Berkich, D. A.,
D. R. Luthin,
R. L. Woodard,
S. J. Vannucci,
J. Linden,
and
K. F. LaNoue.
Evidence for regulated coupling of A1 adenosine receptors by phosphorylation in Zucker rats.
Am. J. Physiol.
268 (Endocrinol. Metab. 31):
E693-E704,
1995[Abstract/Free Full Text].
4.
Blommaart, E. F.,
J. J. Luiken,
P. J. Blommaart,
G. M. van Woerkom,
and
A. J. Meijer.
Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.
J. Biol. Chem.
270:
2320-2326,
1995[Abstract/Free Full Text].
5.
Brown, L. M.,
H. L. Fox,
S. A. Hazen,
K. F. LaNoue,
S. R. Rannels,
and
C. J. Lynch.
Role of the matrixin MMP-2 in multicellular organization of adipocytes cultured in basement membrane components.
Am. J. Physiol.
272 (Cell Physiol. 41):
C937-C949,
1997[Abstract/Free Full Text].
6.
Cheatham, B.,
C. J. Vlahos,
L. Cheatham,
L. Wang,
J. Blenis,
and
C. R. Kahn.
Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.
Mol. Cell. Biol.
14:
4902-4911,
1994[Abstract].
7.
Christiansen, H. N.
Role of amino acid transport and countertransport in nutrition and metabolism.
Physiol. Rev.
70:
43-71,
1990[Free Full Text].
8.
Chung, J.,
C. J. Kuo,
G. R. Crabtree,
and
J. Blenis.
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.
Cell
69:
1227-1236,
1992[Medline].
9.
Cross, D. A.,
D. R. Alessi,
J. R. Vandenheede,
H. E. McDowell,
H. S. Hundal,
and
P. Cohen.
The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf.
Biochem. J.
303:
21-26,
1994[Medline].
10.
Diggle, T. A.,
S. K. Moule,
M. B. Avison,
A. Flynn,
E. J. Foulstone,
C. G. Proud,
and
R. M. Denton.
Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells.
Biochem. J.
316:
447-453,
1996[Medline].
11.
Draznin, B.,
L. Chang,
J. W. Leitner,
Y. Takata,
and
J. M. Olefsky.
Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors.
J. Biol. Chem.
268:
19998-20001,
1993[Abstract/Free Full Text].
12.
Ferrari, S.,
and
G. Thomas.
S6 phosphorylation and the p70s6k/p85s6k.
Crit. Rev. Biochem. Mol. Biol.
29:
385-413,
1994[Abstract].
13.
Flier, J. S.
The adipocyte: storage depot or node on the energy information superhighway?
Cell
80:
15-18,
1995[Medline].
14.
Gabbay, R. A.,
C. Sutherland,
L. Gnudi,
B. B. Kahn,
R. M. O'Brien,
D. K. Granner,
and
R. S. Flier.
Insulin regulation of phosphoenolpyruvate carboxykinase gene expression does not require activation of the ras/mitogen-activated protein kinase signaling pathway.
J. Biol. Chem.
271:
1890-1897,
1996[Abstract/Free Full Text].
15.
Giorgetti, S.,
R. Ballotti,
A. Kowalski-Chauvel,
M. Cormont,
and
E. Van Obberghen.
Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.
Eur. J. Biochem.
207:
599-606,
1992[Abstract].
16.
Han, J.-H.,
R. B. Pearson,
P. B. Dennis,
and
G. Thomas.
Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites.
J. Biol. Chem.
270:
21396-21403,
1996[Abstract/Free Full Text].
17.
Hara, K.,
K. Yonezawa,
H. Sakaue,
K. Kotani,
K. Kotani,
A. Kojima,
M. D. Waterfield,
and
M. Kasuga.
Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase.
Biochem. Biophys. Res. Commun.
208:
735-741,
1995[Medline].
18.
Hausdorff, S. F.,
J. V. Frangioni,
and
M. J. Birnbaum.
Role of p21ras in insulin-stimulated glucose transport in 3T3-L1 adipocytes.
J. Biol. Chem.
269:
21391-21394,
1994[Abstract/Free Full Text].
19.
Hazen, S. A.,
W. A. Rowe,
and
C. J. Lynch.
Monolayer cell culture of freshly isolated adipocytes using extracellular basement membrane components.
J. Lipid Res.
36:
868-875,
1995[Abstract].
20.
Kimura, K.,
S. Hattori,
Y. Kabuyama,
Y. Shizawa,
J. Takayanagi,
S. Nakamura,
S. Toki,
Y. Matsuda,
K. Onodera,
and
Y. Fukui.
Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase.
J. Biol. Chem.
269:
18961-18967,
1994[Abstract/Free Full Text].
21.
Krause, U.,
M. H. Rider,
and
L. Hue.
Protein kinase signaling pathway triggered by cell swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in isolated rat hepatocytes.
J. Biol. Chem.
271:
16668-16673,
1996[Abstract/Free Full Text].
22.
Marshall, S.,
W. T. Garvey,
and
M. Geller.
Primary culture of isolated adipocytes. A new model to study insulin receptor regulation and insulin action.
J. Biol. Chem.
259:
6376-6384,
1984[Abstract/Free Full Text].
23.
Marshall, S.,
and
R. Monzon.
Amino acid regulation of insulin action in isolated adipocytes. Selective ability of amino acids to enhance both insulin sensitivity and maximal insulin responsiveness of the protein synthesis system.
J. Biol. Chem.
264:
2037-2042,
1989[Abstract/Free Full Text].
24.
Meijer, A. J.,
C. Lof,
I. C. Ramos,
and
A. J. Verhoeven.
Control of ureogenesis.
Eur. J. Biochem.
148:
189-196,
1985[Abstract].
25.
Ming, X. F.,
B. M. Burgering,
S. Wennstrom,
L. Claesson-Welsh,
C. H. Heldin,
J. L. Bos,
S. C. Kozma,
and
G. Thomas.
Activation of p70/p85 S6 kinase by a pathway independent of p21ras.
Nature
371:
426-429,
1994[Medline].
26.
Miotto, G.,
R. Venerando,
K. K. Khurana,
N. Siliprandi,
and
G. E. Mortimore.
Control of hepatic proteolysis by leucine and isovaleryl-L-carnitine through a common locus. Evidence for a possible mechanism of recognition at the plasma membrane.
J. Biol. Chem.
267:
22066-22072,
1992[Abstract/Free Full Text].
27.
Miotto, G.,
R. Venerando,
O. Marin,
N. Siliprandi,
and
G. E. Mortimore.
Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala.
J. Biol. Chem.
269:
25348-25353,
1994[Abstract/Free Full Text].
28.
Mortimore, G. E.,
J. J. Wert, Jr.,
G. Miotto,
R. Venerando,
and
M. Kadowaki.
Leucine-specific binding of photoreactive Leu7-MAP to a high molecular weight protein on the plasma membrane of the isolated rat hepatocyte.
Biochem. Biophys. Res. Commun.
203:
200-208,
1994[Medline].
29.
Petritsch, C.,
R. Woscholski,
H. M. Edelmann,
P. J. Parker,
and
L. M. Ballou.
Selective inhibition of p70 S6 kinase activation by phosphatidylinositol 3-kinase inhibitors.
Eur. J. Biochem.
230:
431-438,
1995[Abstract].
30.
Pico, C.,
I. Llado,
A. Pons,
and
A. Palou.
Blood cell to plasma gradients of amino acids in arterial and venous blood in fed and fasted rats.
Comp. Biochem. Physiol. A Physiol.
107:
589-595,
1994.
31.
Porras, A.,
A. R. Nebreda,
M. Benito,
and
E. Santos.
Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.
J. Biol. Chem.
267:
21124-21131,
1992[Abstract/Free Full Text].
32.
Price, D. J.,
J. R. Grove,
V. Calvo,
J. Avruch,
and
B. E. Bierer.
Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase.
Science
257:
973-977,
1992[Medline].
33.
Sanchez-Margalet, V.,
I. D. Goldfine,
C. J. Vlahos,
and
C. K. Sung.
Role of phosphatidylinositol-3-kinase in insulin receptor signaling: studies with inhibitor, LY294002.
Biochem. Biophys. Res. Commun.
204:
446-452,
1994[Medline].
34.
Shepherd, P. R.,
B. T. Nave,
and
K. Siddle.
Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase.
Biochem. J.
305:
25-28,
1995[Medline].
35.
Spiegelman, B. M.,
and
J. S. Flier.
Adipogenesis and obesity: rounding out the big picture.
Cell
87:
377-389,
1996[Medline].
36.
Sutherland, C.,
R. M. O'Brien,
and
D. K. Granner.
New connections in the regulation of PEPCK gene expression by insulin.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
351:
191-199,
1996[Medline].
37.
Sutherland, C.,
R. M. O'Brien,
and
D. K. Granner.
Phosphatidylinositol 3-kinase, but not p70/p85 ribosomal S6 protein kinase, is required for the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin. Dissociation of signaling pathways for insulin and phorbol ester regulation of PEPCK gene expression.
J. Biol. Chem.
270:
15501-15506,
1995[Abstract/Free Full Text].
38.
Taub, M.,
Y. Wang,
T. M. Szczesny,
and
H. K. Kleinman.
Epidermal growth factor transforming growth factor
is required for kidney tubulogenesis in matrigel cultures in serum-free medium.
Proc. Natl. Acad. Sci. USA
87:
4002-4006,
1990[Abstract].
39.
Van den Berghe, N.,
D. M. Ouwens,
J. A. Maassen,
M. G. van Mackelenbergh,
H. C. Sips,
and
H. M. Krans.
Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.
Mol. Cell. Biol.
14:
2372-2377,
1994[Abstract].
40.
Yamamoto-Honda, R.,
K. Tobe,
Y. Kaburagi,
K. Ueki,
S. Asai,
M. Yachi,
M. Shirouzu,
J. Yodoi,
Y. Akanuma,
S. Yokoyama,
Y. Yazaki,
and
T. Kadowaki.
Upstream mechanisms of glycogen synthase activation by insulin and insulin-like growth factor-I. Glycogen synthase activation is antagonized by wortmannin or LY294002 but not by rapamycin or by inhibiting p21ras.
J. Biol. Chem.
270:
2729-2734,
1995[Abstract/Free Full Text].
AJP Cell Physiol 274(1):C206-C213
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society