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
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 KH2PO4, 50 beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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).


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Fig. 1.   A: morphological steps in multicellular clustering of adipocytes suspended in basement membrane components. B: bright field photomicrographs of adipocytes in primary culture with Matrigel.

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.


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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.


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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.

                              
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Table 1.   Summary of amino acids in DMEM and experimental groups


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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.


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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).

                              
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Table 2.   Concentrations of individual amino acids in DMEM and in DMEM containing 1× concentrations of amino acids


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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.


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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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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Abstract
Introduction
Procedures
Results
Discussion
References

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