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Circulation Research. 1996;79:1167-1176

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(Circulation Research. 1996;79:1167-1176.)
© 1996 American Heart Association, Inc.

Articles

Insulin Signaling and Its Regulation of System A Amino Acid Uptake in Cultured Rat Vascular Smooth Muscle Cells

Toshiyuki Obata, Atsunori Kashiwagi, Hiroshi Maegawa, Yoshihiko Nishio, Satoshi Ugi, Hideki Hidaka, Ryuichi Kikkawa

the Third Department of Medicine, Shiga University of Medical Science, Otsu, Japan.

Correspondence to Atsunori Kashiwagi, MD, PhD, the Third Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-21, Japan. E-mail kasiwagi@belle.shiga-med.ac.jp.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [3H]thymidine incorporation but did stimulate S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that >=1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1–dependent phosphatidylinositol 3'-kinase (PI 3'-kinase) and p70 S6 kinase (p70S6K) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90RSK). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated {alpha}-aminoisobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 µg/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 µg/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3'-kinase and p70S6K pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90RSK. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70S6K pathways.


Key Words: insulin • wortmannin • {alpha}-aminoisobutyric acid • vascular smooth muscle cell


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Many epidemiological studies have demonstrated that hyperinsulinemia is an independent risk factor for atherosclerosis in patients with non–insulin-dependent diabetes mellitus.1 2 3 4 5 However, its exact role in the pathogenesis of atherosclerosis remains to be clarified. In addition to this, whether hyperinsulinemia in the physiological range directly potentiates the growth of VSMCs is still debated. We previously reported that insulin at a concentration of 1 nmol/L stimulated autophosphorylation of the insulin receptor in VSMCs.6 However, there are no reports concerning insulin signalings after autophosphorylation of insulin receptors in VSMCs.

According to reports about insulin signalings in another cell type, IRS-1 is phosphorylated by the insulin receptor7 and binds to both GRB28 9 and PI 3'-kinase.10 Although Shc-GRB2 association is the dominant pathway to activate the p21ras–MAP kinase cascade in many cells, IRS-1–GRB2 association also activates p21ras,11 resulting in the activation of MAP kinase12 13 and p90RSK, which mediate mitogenic and/or growth-promoting actions.14 On the other hand, IRS-1–p85 association stimulates PI 3'-kinases, which participate in various insulin-mediated actions, such as mitogenesis,15 glucose transport,15 16 and activation of p70S6K.15 17 18 There are two types of S6 kinases,19 p90RSK, which is activated by MAP kinase,20 and p70S6K, which is regulated by a pathway independent of both p21ras and MAP kinase.21 22 p70S6K is inhibited by rapamycin23 24 and may be activated through IRS-1 and PI 3'-kinase.15 17 18 In our previous study,6 hyperinsulinemic levels of insulin (<=10 nmol/L) did stimulate S6 kinase activity but failed to stimulate either MAP kinase activity or thymidine incorporation in VSMCs. On the other hand, IGF-I was able to stimulate these pathways. Furthermore, we found that insulin activation of S6 kinase might be mediated through its own receptor but not the IGF-I receptor.6 However, we did not address which pathway is responsible for insulin stimulation of S6 kinase in VSMCs.

Furthermore, it is well known that insulin stimulates AIB uptake, a paradigm of system A amino acid transport, and that system A activity is correlated with cellular growth in many cell types.25 Thus, the activation of system A may possibly participate in the progression of atherosclerosis. So, it is important to clarify which signal of insulin may mediate the stimulation of AIB uptake in VSMCs.

In the present study, we characterized the signaling pathways of insulin in VSMCs with regard to (1) phosphorylation of IRS-1 under the influence of 1 nmol/L insulin, (2) activation of IRS-1–dependent PI 3'-kinase, (3) differential activation of p70S6K and/or p90RSK, (4) involvement of the activation of PI 3'-kinase and/or p70S6K in insulin-stimulated AIB uptake, and (4) mechanisms of insulin-stimulated AIB uptake in VSMCs.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Porcine insulin and human IGF-I were generously provided by Novo Nordisk and Fujisawa Pharmaceuticals, respectively. A GST–IRS-1 antibody was raised in a rabbit against a corresponding GST fusion protein.26 A polyclonal antibody raised against a synthetic peptide corresponding to amino acids 478 to 502 of p70S6K (for immunoblotting) was kindly provided by Dr George Thomas (Friedrich Miescher Institute, Basel, Switzerland).27 A monoclonal antibody against phosphotyrosine was purchased from ICN Biomedicals Inc. Polyclonal antibodies of p70S6K (for immunoprecipitation),28 p90RSK (for immunoblotting and immunoprecipitation),29 and rsk kinase substrate peptide (RRLSSLRK)30 were from Upstate Biotechnology Inc. A monoclonal antibody against Erk 2 was from Santa Cruz Biotec. All other chemicals and reagents were of reagent grade and purchased from Nakarai Chemicals or Sigma Chemical Co, unless otherwise indicated.

Cell Culture
VSMCs were isolated from the aortas of male Sprague-Dawley rats (200 to 300 g) by enzymatic digestion, and they were maintained in MEM (GIBCO BRL) supplemented with 10% FCS (GIBCO BRL), 80 U/mL penicillin G, and 80 µg/mL streptomycin in 100-mm plates ({approx}5x106 cells per dish) as described previously.6 Culture media were changed every third day, and VSMCs were mainly used between the 4th and 10th passages. Cell growth was arrested for 72 hours in MEM supplemented with 0.4% FCS before the experiments.

Binding Assays
Binding assays were performed on cells attached on six-well plates as described previously.31 The cells were incubated in 1 mL of binding buffer containing MEM, 25 mmol/L HEPES-HCl (pH 7.8), and 0.5% BSA with or without 8.3 pmol/L [125I]insulin (2000 Ci/mmol, Amersham) or 6.7 pmol/L [125I]IGF-I (2000 Ci/mmol, Amersham) and the indicated concentrations of unlabeled peptides at 15°C for 4 hours. After incubation, cells were washed five times with ice-cold PBS and then dissolved in 1N NaOH for measuring the radioactivity. The peptide binding is expressed as a percentage of maximum. Nonspecific bindings were measured by incubating cells with either 3.3 µmol/L unlabeled insulin or 133 nmol/L unlabeled IGF-I together with radiolabeled peptide.

Immunoblotting
Quiescent cells seeded on 100-mm plates in MEM supplemented with 0.4% FCS were pretreated with or without 20 nmol/L rapamycin (Calbiochem) for 15 minutes and then stimulated with the indicated concentration of insulin or IGF-I at 37°C for the indicated periods. After stimulation, cells were washed with ice-cold PBS and lysed in a solubilizing buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 140 mmol/L NaCl, 1% Nonidet P-40, 1 mmol/L sodium orthovanadate, 1 mmol/L PMSF, 50 mmol/L NaF, and 100 U/mL aprotinin at 4°C for 20 minutes. The cell lysates were centrifuged at 15 000g for 20 minutes. The supernatants were incubated with indicated specific antibody for 3 hours and then incubated for a further 2 hours with protein G–Sepharose (Pharmacia). The bound proteins in the immunoprecipitates or aliquots from the soluble fractions of the lysates were then resolved by SDS-PAGE, transferred onto polyvinylidenedifluoride membranes (Immobilon-P, Millipore) by electroblotting, and subsequently immunoblotted with the indicated specific antibodies. Bound antibodies were detected with horseradish peroxidase–conjugated anti-mouse IgG (Amersham) and visualized with an ECL detection system (Amersham).

PI 3'-Kinase Activity
PI 3'-kinase activity associated with IRS-1 was measured according to the methods described by Okamoto et al.32 Quiescent cells in MEM supplemented with 0.4% FCS were pretreated with or without the indicated concentrations of wortmannin (Wako) for 5 minutes, then stimulated with the indicated concentrations of insulin or IGF-I for 5 minutes at 37°C, lysed in 20 mmol/L Tris-HCl buffer (pH 7.5) containing 1% Nonidet P-40, 10% glycerol, 137 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 100 µmol/L sodium orthovanadate, 1 mmol/L PMSF, 100 U/mL aprotinin, and 1 µg/mL leupeptin, kept on ice for 20 minutes, and centrifuged at 15 000g for 30 minutes. The resulting supernatants were incubated with the GST–IRS-1 antibody for 3 hours and then incubated for 1 hour with protein G–Sepharose at 4°C. The immunoprecipitates were washed three times with PBS containing 1% Nonidet P-40 and 100 µmol/L sodium orthovanadate and then washed three times with 100 mmol/L Tris-HCl buffer (pH 7.5), 500 mmol/L LiCl, and 100 µmol/L sodium orthovanadate and twice with 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, and 100 µmol/L sodium orthovanadate. The pellets were suspended in 50 µL of 10 mmol/L Tris-HCl (pH 7.5). Kinase assay was measured by incubating the suspensions with 200 µmol/L ATP, 30 µCi of [{gamma}-32P]ATP (>5000 Ci/mmol, DuPont NEN), 10 mmol/L MgCl2, and 10 µg phosphatidylinositol at 22°C for 10 minutes. The reaction was terminated by the addition of 20 µL of 8N HCl. After extraction with chloroform/methanol (1:1), the lower phases were removed and applied to silica gel TLC plates (Merck). The TLC plates were developed in methanol/chloroform/ammonia/water (100:70:15:25), dried, and subjected to autoradiography. The radioactivity of the phosphatidylinositol phosphate was quantified by Cerenkov counting.

S6 Kinase Assay
S6 kinase activities in cell lysates or immune complexes were measured using rat 40S ribosomal protein and rsk substrate peptide as substrates, as described previously.6 Quiescent cells seeded on 100-mm plates in MEM supplemented with 0.4% FCS were pretreated with or without 20 nmol/L rapamycin for 15 minutes and then stimulated with the indicated concentrations of either insulin or IGF-I at 37°C for the indicated periods. The cells were rinsed, scraped, and then homogenized by ultrasonication in 100 mmol/L ß-glycerophosphate buffer (pH 7.3) containing 10 mmol/L MgCl2, 10 mmol/L EGTA, 5 µg/mL leupeptin, 100 U/mL aprotinin, 1 mmol/L phenanthroline, and 5 µg/mL pepstatin at 4°C. The homogenates were centrifuged at 100 000g for 1 hour, and the supernatants were assayed for S6 kinase activity. To measure p70S6K activity in the immune complex, the supernatants were incubated with 2 µg of anti-p70S6K antibody28 for 2 hours and then incubated with protein G–Sepharose (Pharmacia) at 4°C. The immunoprecipitates were washed three times with the ß-glycerophosphate buffer and once with kinase buffer containing 20 mmol/L HEPES (pH 7.4) and 10 mmol/L MgCl2 and resuspended with 10 µL of the kinase buffer. To measure the activity in cell lysate, 5 µg/10 µL of the supernatant was applied to kinase buffer assay. The kinase reaction was performed for 30 minutes at 37°C in a final volume of 25 µL containing 20 mmol/L HEPES (pH 7.4), 10 mmol/L MgCl2, 50 µmol/L unlabeled ATP, 0.2 µCi of [{gamma}-32P]ATP, and 5 µL of the 40S ribosomal fraction (0.4 optical density unit). The reaction was terminated by the addition of SDS-PAGE sample buffer33 and then analyzed by 12.5% SDS-PAGE and autoradiography. The radioactivity of the excised 32-kD band was measured by Cerenkov counting.

To measure p90RSK activity in the immune complex, quiescent cells in MEM supplemented with 0.4% FCS were pretreated with 20 nmol/L rapamycin for 15 minutes and then stimulated with 10 nmol/L insulin or 10 nmol/L IGF-I at 37°C for 5 minutes. Cell lysates were immunoprecipitated with a specific antibody against p90RSK.29 An in vitro kinase assay was performed using rsk substrate peptide30 as a substrate, as described above. After kinase reaction, the reaction was terminated by adding 8N HCl and spotted onto P81 paper. These papers were washed with 0.5% orthophosphate, and the radioactivity was measured.

AIB Uptake
AIB uptake was determined as described previously.34 Briefly, cells were preincubated with Earle's balanced salt solution containing 25 mmol/L NaHCO3 and 0.1% BSA for 2 hours at 37°C. The medium was replaced with the same buffer and pretreated with or without either 20 nmol/L rapamycin or 100 nmol/L wortmannin for 15 minutes. The cells were stimulated with the indicated concentrations of either insulin or IGF-I at 37°C for 3 hours. The uptake of [3H]AIB (8 µmol/L, 0.5 µCi per tube, DuPont NEN) into the cells over a 12-minute period was determined by liquid scintillation counting. The effect of 10 µg/mL cycloheximide or 5 µg/mL actinomycin D on either insulin-stimulated or IGF-I–stimulated AIB transport was also measured by coincubating cells with both an inhibitor and the growth factors for 3 hours before the initiation of [3H]AIB uptake.

To confirm the positive transcriptional inhibition by 5 µg/mL actinomycin D, we examined the effect of actinomycin D on serum-stimulated ODC mRNA expression in VSMCs by Northern blot analysis. Cells were cultured in MEM supplemented with 10% FCS, and then 5 µg/mL actinomycin D was added into the medium and incubated for 3 or 6 hours. After incubation, total mRNA was analyzed by Northern blotting using 700-bp of the Pst I fragment of ODC-cDNA.

Statistics
The data are expressed as mean±SE, unless otherwise stated. Student's t tests and Scheffe's multiple comparison tests were used to determine the significance of any difference between the two groups. Values of P<.05 were considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Insulin and IGF-I Binding on VSMCs
At first, to investigate the relative insulin and IGF-I receptor number and their binding affinity, we performed a binding study, as shown in Fig 1Down. The concentration of unlabeled insulin that inhibited the specific radiolabeled insulin binding by 50% (IC50) was 0.33±0.02 nmol/L, which was {approx}200 times less than that of unlabeled IGF-I, as shown in Fig 1ADown. From radiolabeled IGF binding, the IC50 of unlabeled IGF-I was 6.6±0.8 nmol/L, which was at least 100 times less than that of unlabeled insulin. According to the Scatchard analysis of the competition curve, the binding capacities for insulin and IGF-I were 12.8±0.86 and 1200±170 fmol/0.5 mg protein, respectively, as we reported previously.6



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  		Figure 1. Displacement curves of [125I]insulin and [125I]IGF-I binding on VSMCs. Displacement curves of [125I]insulin (A) and [125I]IGF-I binding (B) in the presence or absence of indicated concentrations of either unlabeled insulin or IGF-I on VSMCs are shown. The cells were incubated in 1 mL of binding buffer in the absence or presence of 8.3 pmol/L radiolabeled insulin or 6.7 pmol/L radiolabeled IGF-I and the indicated concentrations of unlabeled peptides at 15°C for 4 hours. After incubation, cells were washed and dissolved in 1N NaOH, and the radioactivity was measured. The peptide binding is expressed as percentage of maximum. The maximum bindings of insulin and IGF-I on VSMCs were 6.6±0.8 (1.35±0.2% of total counts) and 1200±175 fmol/0.5 mg protein (29±3% of total counts), respectively (mean±SE). Nonspecific bindings of insulin and IGF-I on cells were 2.1±0.1 and 130±20 fmol/0.5 mg protein, respectively (mean±SD). The radiolabeled insulin binding and IGF-I binding were indicated by solid circles and solid squares, respectively. Data are expressed as mean±SE.

Insulin-Stimulated Tyrosine Phosphorylation of IRS-1 in VSMCs
To investigate the kinetics of insulin-stimulated tyrosine phosphorylation of IRS-1, we studied the time course for the tyrosine phosphorylation of IRS-1 after exposing VSMCs to 10 nmol/L insulin. As shown in Fig 2ADown, immunoprecipitates were subjected to SDS-PAGE and immunoblotted with phosphotyrosine. Phosphorylation of IRS-1 was observed 30 seconds after insulin stimulation and reached a peak level within 2.5 minutes, which was maintained for at least 10 minutes. Next, we studied the dose dependence for the tyrosine phosphorylation of IRS-1 after exposing VSMCs to the indicated concentration of insulin, as shown in Fig 2BDown. We observed that >=1 nmol/L insulin tyrosine-phosphorylated IRS-1 in a dose-dependent manner. The level of phosphorylated IRS-1 stimulated by 1 nmol/L insulin showed a 2.98±0.25-fold increase (P<.05) compared with the basal level (1.0±0.40). IGF-I (10 nmol/L) also phosphorylated the tyrosine residue of IRS-1, and the level of phosphorylated IRS-1 showed a 7.54±0.55-fold increase compared with the basal level.



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  		Figure 2. Insulin-stimulated tyrosine phosphorylation of IRS-1 in VSMCs. A, Quiescent cells were stimulated with 10 nmol/L insulin at 37°C for the indicated periods, and cell lysates were immunoprecipitated with anti–IRS-1 antibody. The bound proteins were resolved by SDS-PAGE, blotted, analyzed with anti-phosphotyrosine antibody, and quantified by ECL as described in "Materials and Methods." Phosphorylated IRS-1 is indicated by an arrow. A representative result of three separate experiments is shown. B, Quiescent cells were stimulated with the indicated concentrations of insulin or IGF-I for 2.5 minutes at 37°C. Phosphorylation of IRS-1 in the immunoprecipitates was detected as described above. IP indicates immunoprecipitation. Phosphorylated IRS-1 is indicated in the figure by an arrow. C, Insulin-stimulated phosphorylation of IRS-1 was quantitatively analyzed by densitometry. Data are expressed as arbitrary units (1100 densitometric units at the basal level are regarded as 1 arbitrary unit). IGF-I–stimulated phosphorylation of IRS-1 was 7.54±0.55 arbitrary units. Data are expressed as mean±SE (n=3). *P<.05 and **P<.01 compared with the basal level by Student's t test.

Insulin Stimulated IRS-1–Dependent PI 3'-Kinase Activity in VSMCs
After tyrosine-phosphorylation of IRS-1, IRS-1 binds to PI 3'-kinase and activates the kinase activity. We observed the increase in IRS-1–associated PI 3'-kinase activity in response to insulin in a dose-dependent manner, and 1 nmol/L insulin could significantly stimulate PI 3'-kinase activity (1.88±0.26 versus 1.00±0.14 arbitrary units at basal state, P<.05), as shown in Fig 3Down. IGF-I (10 nmol/L) stimulated PI 3'-kinase activity (6.82±0.69 arbitrary units).



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  		Figure 3. Insulin-stimulated IRS-1–dependent PI 3'-kinase activity in VSMCs. A, Quiescent cells were stimulated with the indicated concentrations of insulin or IGF-I at 37°C for 5 minutes. Cells were lysed, and 1500 µg of total cell lysates was immunoprecipitated with 30 µL of anti–GST–IRS-1 antibody. The PI 3'-kinase activity in the immune complex was measured by an in vitro kinase assay and detected by TLC. [{gamma}-32P]Phosphatidylinositol phosphate (PIP) and origin are indicated by arrows. B, IRS-1–dependent PI 3'-kinase activity was quantitatively analyzed by Cerenkov counting and is shown in Fig 2BUp. Data are expressed as mean±SE (n=3) arbitrary units (189 cpm at the basal level is regarded as 1 arbitrary unit). The PI 3'-kinase activity that was stimulated by 10 nmol/L IGF-I was 6.82±0.69 arbitrary units. *P<.05 and **P<.01 compared with the basal level by a Student's t test.

Effect of Insulin on S6 Kinase in VSMCs
We previously reported that insulin stimulated S6 kinase activity in VSMCs.6 However, it still remains unclear which S6 kinase, p70S6K or p90RSK, is involved in insulin-stimulated S6 kinase activation. Thus, we first measured insulin-induced activation of both rapamycin-sensitive and rapamycin-resistant S6 kinases by in vitro kinase assays using the 40S ribosomal protein as a substrate. Panels A and B of Fig 4Down show the time courses of rapamycin-sensitive and rapamycin-resistant S6 kinase activities, respectively, in response to growth factors. Rapamycin-sensitive S6 kinase activities in Fig 4ADown were determined by subtracting the rapamycin-resistant activities from the total activities. Both insulin and IGF-I at 10 nmol/L stimulated rapamycin-sensitive S6 kinase activity, which peaked 30 minutes after the stimulation, and more than one third of their maximum activities remained after 60 minutes. In contrast, rapamycin-resistant S6 kinase was not significantly stimulated by 10 nmol/L insulin. However, IGF-I induced a transient stimulation of this enzyme activity, reached a peak level after 5 minutes, and declined to the basal activity after 10 minutes.



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  		Figure 4. Effects of insulin on rapamycin-sensitive and rapamycin-resistant S6 kinase in VSMCs. A, Quiescent cells were pretreated with or without 20 nmol/L rapamycin and then stimulated with 10 nmol/L insulin ({bullet}) or 10 nmol/L IGF-I ({blacksquare}) at 37°C for the indicated periods. Cell extracts were obtained and assayed for their ability to phosphorylate ribosomal S6 protein in vitro. They were then analyzed by SDS-PAGE and autoradiography. The radioactivity of the excised 32-kD band was measured by Cerenkov counting. The data for rapamycin-sensitive S6 kinase were determined by subtraction of rapamycin-resistant activities from the total activities. B, Quiescent cells were pretreated with 20 nmol/L rapamycin for 15 minutes and then stimulated with 10 nmol/L insulin ({bullet}) or 10 nmol/L IGF-I ({blacksquare}) at 37°C for the indicated periods. After the stimulation, rapamycin-resistant S6 kinase activities were measured as described above. Data are expressed as mean±SE (n=3). *P<.05 and **P<.01 compared with the basal level by Student's t test.

Next, we examined the phosphorylation status of MAP kinase (Erk 2), p70S6K, and p90RSK by retarded mobility assay, as shown in Fig 5Down. After exposing cells to 10 nmol/L insulin or 10 nmol/L IGF-I for indicated periods, cell lysates were resolved by SDS-PAGE, transferred onto Immobilon-P membranes, and examined with monoclonal anti–Erk 2 antibody (Fig 5ADown), polyclonal anti-p90RSK antibody29 (Fig 5BDown), and anti-p70S6K antibody28 (Fig 5CDown), respectively. The MAP kinases are phosphorylated/activated by MAP kinase kinase, and serine phosphorylates/activates p90RSK. These phosphorylated enzymes were readily detected by a retarded migration during SDS-PAGE.17 As shown in Fig 5A and 5B, 10DownDownDown nmol/L insulin did not induce any significant effects on either Erk 2 or p90RSK. On the other hand, it caused a mobility change in p70S6K at 30 minutes, as shown in Fig 5C-1Down. In contrast, 10 nmol/L IGF-I caused mobility changes in not only p70S6K at 30 minutes but also p90RSK at 5 and 30 minutes.



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  		Figure 5. Insulin stimulates p70S6K but not MAP kinase (Erk 2) or p90RSK. A, Quiescent cells were stimulated with either 10 nmol/L insulin or 10 nmol/L IGF-I for 10 minutes, and cell lysates were resolved by SDS-PAGE, blotted, analyzed with a monoclonal anti–Erk 2 antibody, and then detected by ECL reading. Lanes are as follows: 1 and 3, control; 2, insulin stimulation; and 4, IGF-I stimulation. B and C, Quiescent cells were pretreated with or without 20 nmol/L rapamycin for 15 minutes and then stimulated with 10 nmol/L insulin or 10 nmol/L IGF-I treatments for 5 or 30 minutes. Cell lysates were analyzed by immunoblotting with either anti-p90RSK or anti-p70S6K antibody as described above. For panels B-1 and C-1, lanes are as follows: 1 and 4, control; 2, insulin stimulation (5 minutes); 3, insulin stimulation (30 minutes); 5, IGF-I stimulation (5 minutes); and 6, IGF-I stimulation (30 minutes). For panel B-2, lanes are as follows: 1 and 4, control; 2, insulin stimulation (5 minutes); 3, pretreatment with rapamycin and insulin stimulation (5 minutes); 5, IGF-I stimulation (5 minutes); and 6, pretreatment with rapamycin and insulin stimulation (5 minutes). For panel C-2, lanes are as follows: 1 and 4, control; 2, insulin stimulation (30 minutes); 3, pretreatment with rapamycin and insulin-stimulation (30 minutes); 5, IGF-I stimulation (30 minutes); and 6, pretreatment with rapamycin and insulin stimulation (30 minutes). The bands corresponding to Erk 2, p90RSK, and p70S6K are indicated by arrows. Data are representative of at least three independent experiments.



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  		Figure 10. Effects of either cycloheximide or actinomycin D on insulin-stimulated AIB uptake in VSMCs. Cells were pretreated with or without either 10 µg/mL cycloheximide (solid bars) or 5 µg/mL actinomycin D (hatched bars) for 2 hours and then stimulated with 10 nmol/L insulin for 3 hours. After the incubation, uptake of [3H]AIB in the cells for 12 minutes was determined as described above. Data are expressed as mean±SE (n=4). ***P<.001 compared with the control level by Scheffe's multiple comparison test.

Furthermore, in order to confirm the effects of insulin on activation of S6 kinases, we examined the kinase activities in p70S6K- and p90RSK-specific immunoprecipitates in VSMCs, as shown in Figs 6Down and 7. As shown in Fig 6ADown, like phosphorylation of IRS-1 or stimulation of PI 3'-kinase, >=1 nmol/L insulin stimulated p70S6K activity in a dose-dependent manner. More than 0.1 nmol/L IGF-I also significantly stimulated p70S6K. Activity of p90RSK in anti-p90RSK antibody29 –immune complex in the absence (Fig 7ADown) or presence (Fig 7BDown) of 20 nmol/L rapamycin was examined. IGF-I (10 nmol/L) induced significant activation of p90RSK, but 10 nmol/L insulin did not stimulate it at all. The findings were in accordance with other data, in that 10 nmol/L insulin failed to stimulate rapamycin-resistant S6 kinase activity and induced a mobility change in neither MAP kinase (Erk 2) nor p90RSK.



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  		Figure 6. Effects of either insulin or IGF-I on activity of p70S6K. A and B, Quiescent cells were stimulated with the indicated concentration of either insulin (A) or IGF-I (B) at 37°C for 30 minutes. The obtained 200 µg of cell lysates was incubated with 2 µg of anti-p70S6K antibody for 2 hours and then incubated with protein G–Sepharose. The immunoprecipitates were washed, resuspended with 10 µL of kinase buffer, subjected to in vitro kinase assay, and detected by autoradiography, as described above. C, Effects of rapamycin on growth factor–stimulated p70S6K, as described. Quiescent cells were pretreated with or without 20 nmol/L rapamycin for 15 minutes and then stimulated with 10 nmol/L growth factors for 30 minutes for kinase assay, as described above. Phosphorylated 40S ribosomal proteins are indicated by an arrow. Lanes are as follows: 1, control (vehicle); 2, pretreatment with rapamycin and vehicle; 3, insulin stimulation; 4, pretreatment with rapamycin and insulin stimulation; 5, IGF-I stimulation; and 6, pretreatment with rapamycin and IGF-I stimulation. D, Growth factor–stimulated p70S6K activities were quantified (corresponding to those in Fig 5CUp). Data are expressed as mean±SE (n=3) in arbitrary units (1.25x102 cpm at the control level is regarded as 1 arbitrary unit). *P<.05 and **P<.01 compared with the basal level by Student's t test.



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  		Figure 7. Effects of either insulin or IGF-I on activity of p90RSK. Quiescent cells were pretreated without (A) or with (B) 20 nmol/L rapamycin for 15 minutes and then stimulated with 10 nmol/L insulin or 10 nmol/L IGF-I at 37°C for 5 minutes. Cell lysates were immunoprecipitated with a specific antibody against p90RSK. In vitro kinase assay was performed using rsk substrate peptide as a substrate, as described in "Materials and Methods." After kinase reaction, the reaction was terminated by adding 8N HCl and spotted onto P81 paper. These papers were washed with 0.5% orthophosphate, and the radioactivities were measured. Data were expressed as mean±SE (n=3) in arbitrary units (2.15x103 cpm at the control level is regarded as 1 arbitrary unit). *P<.05 compared with the control level by Scheffe's multiple comparison test.

Moreover, in order to confirm the specificity of rapamycin to inhibit p70S6K but not p90RSK, we examined the effect of rapamycin on p70S6K or p90RSK using the mobility change assay (Fig 5B-2 and 5C-2UpUp) and the kinase assay in the immune complex (Figs 6C and 7UpUp). As shown in Figs 5C-2 and 6C, 20UpUp nmol/L rapamycin completely inhibited mobility changes of p70S6K and activities of p70S6K in the immune complex stimulated by either insulin or IGF-I, whereas rapamycin failed to inhibit the IGF-I–induced mobility change of p90RSK and IGF-I–stimulated p90RSK activity in the immune complex as shown in Figs 5B-2 and 7UpUp.

Effect of Either Rapamycin or Wortmannin on Insulin-Stimulated AIB Uptake in VSMCs
Insulin (10 nmol/L) significantly stimulated AIB uptake (1.47±0.04 at the basal level, 1.82±0.09 nmol AIB/mg protein for 12 minutes when stimulated with 10 nmol/L insulin [P<.05]), as shown in Fig 8ADown. To clarify the mechanism for these activations, we examined the effects of coincubation of insulin with either rapamycin or wortmannin on AIB uptake. As shown in Fig 8B, 20Down nmol/L rapamycin did not significantly affect insulin-stimulated AIB uptake, although the same dose of rapamycin completely inhibited rapamycin-sensitive S6 kinase in VSMCs. On the other hand, 100 nmol/L wortmannin completely inhibited insulin-stimulated AIB uptake from 1.82±0.09 to 1.26±0.03 nmol AIB/mg protein for 12 minutes (P<.01). Similarly, IGF-I also stimulated AIB uptake in VSMCs (1.47±0.04 at the basal level, 2.16±0.13 nmol AIB/mg protein for 12 minutes [P<.01] when stimulated with 10 nmol/L IGF-I). Furthermore, we observed that wortmannin also inhibited the IGF-I–stimulated AIB uptake in VSMCs from 2.16±0.13 to 1.19±0.13 (P<.001) nmol AIB/mg protein for 12 minutes, but rapamycin did not affect IGF-I–stimulated AIB uptake in VSMCs.



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  		Figure 8. Insulin-stimulated AIB uptake and the effects of either rapamycin or wortmannin on insulin-stimulated AIB uptake in VSMCs. Insulin-stimulated AIB uptake (A) and the inhibitory effects of either rapamycin or wortmannin on this stimulation (B) were measured as described in "Materials and Methods." Cells were pretreated with or without 20 nmol/L rapamycin (hatched bars) or 100 nmol/L wortmannin (solid bars) for 15 minutes and then stimulated by the indicated concentrations of either insulin or IGF-I at 37°C for 3 hours. After the incubation, the uptake of [3H]AIB in the cells was determined for 12 minutes. Data are expressed as mean±SE (n=4). *P<.05 and **P<.01 compared with the basal level by Scheffe's multiple comparison test.

Dose-Dependent Effect of Wortmannin on Both Insulin-Stimulated PI 3'-Kinase Activity and AIB Uptake
Wortmannin, a potent PI 3'-kinase inhibitor, inhibited insulin-stimulated AIB uptake in VSMCs, as shown in Fig 9Down. Thus, we evaluated the sensitivity of wortmannin on inhibition of both insulin-stimulated PI 3'-kinase activity and insulin-stimulated AIB uptake. We found that wortmannin inhibited both insulin-stimulated PI 3'-kinase activities in anti–IRS-1 immunoprecipitates and insulin-stimulated AIB uptake in VSMCs in a similar dose-dependent manner with an IC50 between 5 and 10 nmol/L.



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  		Figure 9. Dose effect of wortmannin on insulin-stimulated IRS-1–dependent PI 3'-kinase activity or insulin-stimulated AIB uptake in VSMCs. IRS-1–dependent PI 3'-kinase activity ({blacksquare}) was measured as described below. Quiescent cells were pretreated with the indicated concentrations of wortmannin at 37°C for 15 minutes and then stimulated with 10 nmol/L insulin at 37°C for 5 minutes. Cells were lysed and immunoprecipitated with anti-GST–IRS-1 antibody. The PI 3'-kinase activities in the immunoprecipitates were measured by an in vitro kinase assay and detected by TLC. The radioactivity of the position on the TLC corresponding to phosphatidylinositol phosphate was measured by Cerenkov counting. The percent inhibitions of the means from two separate experiments are shown. The effect of wortmannin on insulin-stimulated AIB uptake ({bullet}) in VSMCs was measured as described below. Cells were pretreated with the indicated concentration of wortmannin for 15 minutes and then stimulated by the indicated concentration of insulin for 3 hours. After incubation, uptake of [3H]AIB in the cells was determined for 12 minutes. Data are expressed as mean±SE (n=3).

Effect of Either Cycloheximide or Actinomycin D on Insulin-Stimulated AIB Uptake
To further clarify the mechanisms of insulin-stimulated AIB uptake, we studied the effect of either cycloheximide or actinomycin D on insulin-stimulated AIB uptake, as shown in Fig 10Up. Cycloheximide (10 µg/mL), a translation inhibitor, completely inhibited insulin-stimulated AIB uptake in VSMCs. On the other hand, 5 µg/mL actinomycin D, a transcription inhibitor, failed to inhibit the uptake in VSMCs. Similarly, IGF-I–stimulated AIB uptake was also inhibited by 10 µg/mL cycloheximide from 2.16±0.13 to 1.25±0.02 (P<.001) nmol AIB/mg protein for 12 minutes, but it was not inhibited by 5 µg/mL actinomycin D.

To confirm the positive transcriptional inhibition by 5 µg/mL actinomycin D, we examined the effect of actinomycin D on serum-stimulated ODC mRNA expression in VSMCs by Northern blot analysis. The intensity of band was 53.4% and 3.4% reduction of the basal level (supplemented with FCS) after exposing cells to actinomycin D (supplemented with FCS) for 3 and 6 hours, respectively.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we demonstrated that insulin at a concentration of 1 nmol/L significantly phosphorylated the tyrosine residues of IRS-1 and subsequently activated PI 3'-kinase and p70S6K in cultured rat VSMCs. As in our present and previous studies,6 VSMCs expressed both receptors for insulin and IGF-I. Thus, it is possible that insulin may transmit its signal through IGF-I receptors. To rule out this possibility, we first examined the IGF-I signal using the cells desensitized to insulin signaling by preincubating them in 10 nmol/L insulin for 12 hours. In desensitized cells, the insulin receptor was downregulated, and insulin-stimulated S6 kinase activity was almost attenuated.6 However, IGF-I–stimulated S6 kinase activity was unchanged,6 suggesting that insulin and IGF-I may act through their own receptors. Furthermore, in the present study, we performed a precise dose-response study of insulin and IGF-I stimulation on p70S6K activation as shown in Fig. 6Up. Based on the present and previous binding studies, relative receptor number of insulin/IGF-I is {approx}1/100,6 and the affinity of insulin to the IGF-I receptor was {approx}1/200 in VSMCs, as shown in Fig 1Up. Moreover, affinity of IGF-I to the insulin receptor was {approx}1/300. Thus, cross-reactivity of insulin to IGF-I receptor was at most <1/100, indicating that 1 nmol/L insulin was equal to, at most, 0.01 nmol/L IGF-I. As shown in Fig 6B, 0Up.01 nmol/L IGF-I failed to stimulate p70S6K activity, even though 1 nmol/L insulin significantly stimulates IRS-1 phosphorylation and the activation of PI 3'-kinase and p70S6K, as shown above. Therefore, these data indicate that insulin stimulates p70S6K activity through the specific insulin receptors.

Insulin-mediated tyrosine phosphorylation of IRS-1 can bind several SH2-containing molecules within its phosphotyrosine motifs, including p85 of PI 3'-kinase,10 leading to activation of PI 3'-kinase.10 On the other hand, generally, GRB2 is known to bind Shc in response to insulin and subsequently activates the p21ras pathway, including MAP kinase and p90RSK, and association of GRB2 with IRS-1 is not a major pathway for activation of the p21ras pathway. In the present study, we observed that insulin at concentrations of >=1 nmol/L is enough to activate PI 3'-kinase activity via association with IRS-1, as shown in Fig 2Up. However, we did not observe association of GRB2 with IRS-1 by immunoblotting (data not shown). In contrast, we did observe tyrosine phosphorylation of Shc and binding of GRB2 to Shc in response to 10 nmol/L IGF-1 or 10 nmol/L epidermal growth factor but not 10 nmol/L insulin in VSMCs. These findings were in accordance with our observation that MAP kinase activity was stimulated in response to either 10 nmol/L IGF-1 (Fig 5AUp) or 10 nmol/L epidermal growth factor (data not shown) but not 10 nmol/L insulin (Fig 5AUp).

In our present study, we found discrepancy of the time course between activation of S6 kinase and mobility change of S6 kinase, such that IGF-I–stimulated rapamycin-resistant S6 kinase reached a peak at 5 minutes, as shown in Fig 4BUp, but the IGF-I–induced mobility change of p90RSK during SDS-PAGE was stronger at 30 minutes than at 5 minutes, as shown in Fig 5BUp. It is generally accepted that p90RSK is inactivated by phosphatase such as protein phosphatase 1 or protein phosphatase 2A. However, it has been reported that the changes in mobility and activity are not always correlated.35 Thus, we suppose that further phosphorylation of p90RSK may contribute to the reduction of the activity.

It is also interesting that IGF-I could stimulate p90RSK only in a transient fashion, with a small change that was concomitant with a sustained and greater increase in p70S6K activity in VSMCs. According to previous reports,36 37 a growth factor that stimulates both MAP kinase and p90RSK only transiently regulates those activities, which are thought to be associated with regulated mitogenesis or gene expression.36 38 In contrast, it is also reported that the metabolic effect of insulin is maintained for much longer periods.36 In our present study, we also observed a sustained activation of p70S6K. The magnitude of rapamycin-resistant S6 kinase (p90RSK) activity was smaller than that of rapamycin-sensitive S6 kinase (p70S6K) activity, as shown in Fig 4Up. Although p90RSK has an ability to phosphorylate ribosomal S6 protein in vitro, p70S6K is responsible for S6 phosphorylation in intact cells.39

There are many reports indicating that the PI 3'-kinase cascade is one of the most important pathways in the stimulation of cell growth,15 and we found that physiological hyperinsulinemia stimulated this PI 3'-kinase cascade in VSMCs. To evaluate the biological meanings of insulin-stimulated PI 3'-kinase activity in VSMCs, we measured both p70S6K activity and the AIB uptake, which were stimulated by 10 nmol/L insulin. We showed that 10 nmol/L insulin significantly stimulated AIB uptake, as shown in Fig 8AUp. Furthermore, wortmannin inhibited both insulin-stimulated AIB uptake and PI 3'-kinase in a similar dose-dependent fashion, with an IC50 of {approx}5 to 10 nmol/L, as shown in Fig 9Up. With regard to AIB uptake, Ferrer-Martinez et al40 indicated that insulin stimulated AIB uptake, which was associated with an increase in the number of transporter into the plasma membrane in rat hepatocytes. In the present study, we observed that cycloheximide completely inhibited both insulin-stimulated and IGF-I–stimulated AIB uptake in VSMCs. Thus, protein synthesis may be necessary for their stimulation of AIB uptake. According to many reports, 5 µg/mL actinomycin D inhibits transcription in cultured rat VSMCs.41 42 43 44 Furthermore, we also observed that the same dose of actinomycin D completely inhibited the production of serum-stimulated ODC mRNA in VSMCs. On the other hand, the same dose of actinomycin D failed to inhibit the growth factor–stimulated AIB uptake, suggesting that transcription may not be involved in insulin-stimulated AIB uptake in VSMCs. In the present study and our previous work,6 we found that 10 nmol/L insulin failed to stimulate the MAP kinase/p90RSK pathway. Furthermore, inhibition of p70S6K activity by 20 nmol/L rapamycin did not affect insulin-stimulated AIB uptake. Therefore, the pathway through p70S6K activation is not involved in the insulin-stimulated AIB uptake. Recently, there has been a report indicating that wortmannin inhibits another enzyme,45 so it may not be true that insulin-stimulated AIB uptake is mediated through only the activation of PI 3'-kinase at this point. However, we at least conclude that pathways involving MAP kinase/p90RSK and p70S6K are not necessary for the stimulation of AIB transport, whereas wortmannin-sensitive pathways may play an important role in the insulin-stimulated system A amino acid transport via some kind of translational control. To clarify the control mechanisms for this activation, further studies are needed.

Finally, it is interesting that p70S6K, but not p90RSK, is activated by insulin in VSMCs, whereas IGF-I stimulated both S6 kinases, as shown in Figs 4 through 7UpUpUpUp. We confirmed these different types of regulation of S6 kinases between insulin and IGF-I using three different methods (eg, by assessment of S6 kinase activities in the immune complex, by assay of retarded mobility, and by determination of the effect of rapamycin on S6 kinase activation). The activated p70S6K phosphorylates the S6 protein of the ribosomal 40S protein subunits21 and is associated with stimulation not only of protein synthesis but also of cell proliferation.28 46 Recently, various functions of p70S6K were reported, including stimulation of the glycogen synthesis,47 control of PHAS,48 control of eEF-2 synthesis,49 control of PDGF-stimulated leucine uptake,50 and phosphorylation/activation of the CRE modulator.51 Phosphorylation of PHAS in response to insulin activates initiation factors and subsequently initiates translation. eEF-2 is an essential factor for the extension of polypeptide chains on ribosomes, so it is also associated with both protein synthesis and cell growth. Recently, it has been reported that PDGF stimulates leucine uptake and activates p70S6K; these effects were concomitantly inhibited by rapamycin.50 Those results indicate that activation of PI 3'-kinase via growth factors can stimulate various amino acid transport systems via different pathways, including p70S6K-dependent and -independent pathways. Phosphorylated CRE modulators may affect the expression of other growth factor genes and/or extracellular matrix genes that have CREs on their promoter regions.51 These modified gene expressions, mediated through activation of p70S6K, should be further evaluated in a future study to confirm the biological significance of hyperinsulinemia in terms of atherosclerosis.


*    Selected Abbreviations and Acronyms
 
AIB = {alpha}-aminoisobutyric acid
CRE = cAMP-responsive element
ECL = enhanced chemiluminescence
eEF-2 = eukaryotic elongation factor 2
Erk 2 = extracellular signal-regulated kinase 2
GRB2 = growth factor receptor-bound protein 2
GST = glutathione S-transferase
IGF-I = insulin-like growth factor I
IRS-1 = insulin receptor substrate-1
MAP kinase = mitogen-activated protein kinase
ODC = ornithine decarboxylase
p70S6K = p70 S6 kinase
p90RSK = p90 S6 kinase
PDGF = platelet-derived growth factor
PHAS = phosphorylated heat- and acid-stable protein
PI 3'-kinase = phosphatidylinositol 3'-kinase
PMSF = phenylmethylsulfonyl fluoride
Shc = Src homologous and collagen
SH2 = Src homology 2
TLC = thin-layer chromatography
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This study was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture, Japan (grant 07671127) and a grant from Sankyo Co Ltd, Japan. We are deeply indebted to Dr Yoshifumi Takagi for help with the binding assay. We are grateful to Dr George Thomas (Friedrich Miescher Institute, Basel, Switzerland) and Dr Ryuhei Kanamoto (Kyoto Prefectural University, Kyoto, Japan) for the gift of anti-p70S6K antibody and ODC probe, respectively.

Received October 20, 1995; accepted September 18, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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