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Am J Physiol Cell Physiol 291: C1355-C1365, 2006. First published July 19, 2006; doi:10.1152/ajpcell.00125.2006
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

AKT phosphorylation is essential for insulin-induced relaxation of rat vascular smooth muscle cells

Jin Hee Lee1 and Louis Ragolia1,2

1Vascular Biology Institute, Winthrop-University Hospital, Mineola; and 2School of Medicine, State University of New York at Stony Brook, Stony Brook, New York

Submitted 17 March 2006 ; accepted in final form 13 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Insulin resistance, a major factor in the development of type 2 diabetes, is known to be associated with defects in blood vessel relaxation. The role of Akt on insulin-induced relaxation of vascular smooth muscle cell (VSMC) was investigated using siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt to either suppress endogenous Akt or overexpress constitutively active Akt, respectively. siAKTc decreased both basal and insulin-induced phosphorylations of Akt and glycogen synthase kinase 3beta, abolishing insulin-induced nitric oxide synthase (iNOS) expression. cGMP-dependent kinase 1{alpha} (cGK1{alpha}) and myosin-bound phosphatase (MBP) activities, both downstream of iNOS, were also decreased. siAKTc treatment resulted in increased insulin and ANG II-stimulated phosphorylation of contractile apparatus, such as MBP substrate (MYPT1) and myosin light chain (MLC20), accompanied by increased Rho-associated kinase {alpha} (ROK{alpha}) activity, demonstrating the requirement of Akt for insulin-induced vasorelaxation. Corroborating these results, constitutively active Akt upregulated the signaling molecules involved in insulin-induced relaxation such as iNOS, cGK1{alpha}, and MBP activity, even in the absence of insulin stimulation. On the contrary, the contractile response involving the phosphorylation of MYPT1 and MLC20, and increased ROK{alpha} activity stimulated by ANG II were all abolished by overexpressing active Akt. In conclusion, we demonstrated here that insulin-induced VSMC relaxation is dependent on Akt activation via iNOS, cGK1{alpha}, and MBP activation, as well as the decreased phosphorylations of MYPT1 and MLC20 and decreased ROK{alpha} activity.

angiotensin II; myosin-bound phosphatase substrate; inducible nitric oxide synthase; guanosine 3',5'-cyclic monophosphate-dependent kinase 1{alpha}; Rho-associated kinase{alpha}

DEFECTS IN THE RELAXATION mechanisms of smooth muscle within the blood vessels of patients with hypertension, non-insulin-dependent diabetes, and obesity have been reported in many studies (2, 40, 51, 52, 66). Insulin is known to induce relaxation of vascular smooth muscle cell (VSMC) contraction in vivo and in vitro (40, 41). Although insulin resistance has been shown to impair insulin-induced relaxation in the vasculature, the precise signaling mechanisms involved are poorly understood, despite the pathophysiological importance.

Myosin-bound phosphatase (MBP) is a heterotrimer consisting of a protein phosphatase-1 catalytic subunit (PP1C), a 130-kDa regulatory targeting subunit (MYPT1), and a 20-kDa subunit (M20) of unknown function (32). MYPT1 is phosphorylated at threonine-695 by Rho-associated kinase {alpha} (ROK{alpha}) activated by the small GTPase, RhoA, which leads to the inactivation of MBP (25, 46). Because MBP dephosphorylates myosin light chain (MLC20) and induces the relaxation of VSMC without changing intracellular Ca2+ (34), Rho can increase the sensitivity of VSMC contraction to a given intracellular Ca2+ concentration (50). Myosin light chain kinase is activated by intracellular Ca2+ and phosphorylates MLC20 at serine-19 and threonine-20, leading to cell contraction (31, 35, 36). Several downstream signaling pathways that inhibit MBP activity have been discovered recently, including RhoA/ROK{alpha} (46), protein kinase C activation of the inhibitory phosphoprotein CPI-17 (22), and arachidonic acid (30). ROK{alpha} also can phosphorylate the CPI-17 at threonine 38, which thereby becomes a potent inhibitor of MBP (72). MBP binds PP1 and MLC20 at its amino terminus and the M20 subunit and cGMP-dependent protein kinase 1{alpha} (cGK1{alpha}) at its carboxyl terminus. The MBP-cGK1{alpha} interaction is necessary for nitric oxide (NO)/cGMP-mediated activation of MBP (71).

Insulin-induced insulin receptor substrate (IRS)-1 tyrosine phosphorylation activates phosphatidylinositide 3-kinase (PI3-kinase) and the expression of inducible nitric oxide synthase (iNOS; see Refs. 8, 41, and 62), NO, and cGK1{alpha}, resulting in the dephosphorylation of threonine-695 on MYPT1 and inactivation of RhoA and ROK{alpha} (10, 11, 25, 62).

ANG II plays an important role in the contraction process, remodeling cardiovascular structure and tone via AT1/AT2 receptor activation (27, 33, 78). It has been reported that ANG II inhibits insulin signaling and induces insulin resistance. Recent studies have revealed that an ANG II excess may produce a vascular resistance to insulin via the attenuation of insulin signaling at the level of IRS-1 and PI3-kinase (27, 78). Therefore, it is important to understand the cross talk between ANG II and insulin as it pertains to contraction and relaxation in both normal and insulin-resistant states.

Akt protein is a serine/threonine kinase and a downstream effector of PI3-kinase. Akt plays a central role in the metabolic actions of insulin, including glucose transport, and the synthesis of glycogen (18, 23). Mammalian genomes contain three Akt genes, Akt1, Akt2, and Akt3, that encode three widely expressed isoforms of Akt kinase. Akt can be activated by a wide variety of stimuli, such as insulin, insulin-like growth factor I (IGF-I), ANG II, reactive oxygen species (ROS), etc. (81). Glycogen synthase kinase 3beta (GSK3beta) has been identified as a physiological substrate for Akt. Phosphorylation of GSK3beta by Akt subsequently promotes glycogen synthesis (18). Akt activation by insulin is mediated via tyrosine kinase activity of the insulin receptor, IRS-1, and IRS-2 (69, 70). Tyrosyl phosphorylation of IRS-1 and -2 provides binding sites for specific proteins containing SH2 domains, including the 85-kDa regulatory subunit of PI3-kinase (15). Phosphatidylinositol 3-phosphate (PIP3) produced by the catalytic subunit of PI3-kinase, activates Akt by binding to the PH domain of Akt kinases (12), causing Akt translocation to the plasma membrane. PIP3-dependent kinases (PDKs), as well as undefined kinases, activate Akt upon its membrane translocation by phosphorylation on threonine-308 (1) and serine-473 (12).

Many studies report a discrepancy between PI3-kinase and Akt activation (5, 14, 28, 60). Although Akt is involved in glucose transport, the atypical protein kinase C family ({zeta} and {lambda}), both of which are the downstream effectors of PI3-kinase (54), are involved in glucose transport in a different manner (28). Although PI3-kinase and the atypical protein kinase C family ({zeta} and {lambda}) are responsible for insulin-induced GLUT4 translocation in 3T3-L1 adipocytes and L6 myocytes, Akt1 and Akt2 activity may be responsible for activating glycogen synthase (28). This study demonstrates the distinctive separated signaling pathways from the upstream event, PI3-kinase activation, to activation of Akt and an atypical form of protein kinase C in insulin signaling. Moreover, there is a report on wortmannin-sensitive PI3-kinase C2 type, which is responsive to Ca2+, and its role in actin rearrangement and contraction (5, 14). Akt can be activated also in a PI3-kinase independent mechanism through protein kinase A (PKA) (60) and Ca2+/calmodulin-dependent kinase (80) activation.

The aim of this study was to examine the role of the serine/threonine kinase, Akt, signaling pathway in regulation of insulin-induced vasodilation in relation to the insulin-mediating interactions between ANG II in VSMCs. To demonstrate this, siRNA targeting Akt (siAKTc) and adenovirus constructing myristilated Akt (ad-myr-AKT) were used to suppress the endogenous Akt or overexpress the constitutively active Akt in VSMC, respectively. Although previous studies have demonstrated a role for PI3-kinase in insulin-induced VSMC relaxation, it is important to investigate the involvement of Akt in insulin-induced relaxation. This is the first report to demonstrate a mechanism of insulin-induced relaxation signaling pathway in relation to Akt. Here, we show that Akt regulates insulin-stimulated expression of iNOS, resulting in an increase in NO. This caused the activation of cGK1{alpha} and MBP, which were both responsible for the decrease in the phosphorylation status of MYPT1 and MLC20, and the accompanied reduction of ROK{alpha} activity, causing VSMC relaxation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human Insulin (recombinant DNA origin) was from Novo Nordisk Pharmaceuticals (Princeton, NJ). Synthetic human ANG II, sodium orthovanadate, BSA, calmodulin, and antibodies against beta-actin and Flag M2 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-iNOS Antibody was from Transduction Laboratories (Lexington, KY). Rho kinase II/ROK{alpha} positive control, cGK1{alpha} positive control, Rho kinase, and cGMP-dependent protein kinase assay kit were all purchased from Cyclex (Ina Nagano, Japan). Primocin (anti-mycoplasmic), transfection reagent specific for smooth muscle, was purchased from Amaxa Biosystems (Cologne, Germany). siCONTROL nontargeting siRNA and custom siRNA were purchased from Dharmacon (Lafayette, CO). Enhanced chemiluminescence (ECL), anti-rabbit IgG, and anti-mouse IgG [horseradish peroxidase (HRP) linked] were from Amersham Biosciences (Buckinghamshire, UK). Specific antibody targeting MYPT1 and phosphorylated MYPT1 on threonine-696 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody specific for Akt, phospho-Akt (Ser473), and phospho-myosin light chain 20 (Thr18/Ser19) were purchased from Cell Signaling Technology (Beverly, MA). Western blot reagents were from Bio-Rad Laboratories (Hercules, CA). Myosin light chains were prepared from chicken gizzards according to the published protocol (26). [{gamma}-33P]ATP (specific activity ~3,000 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Okadaic acid was from Moana Bioproducts (Honolulu, Hawaii).

Culture of VSMCs and treatment with insulin. VSMCs in primary culture were obtained by enzymatic digestion of the aortic media of male Wistar-Kyoto rats with body weights of 200–220 g as described in our recent publication (8, 9). Unless otherwise indicated, primary cultures of VSMCs were maintained in {alpha}-minimal essential medium (MEM) containing 10% FBS, 1% antibiotic/antimycotic, and antimycoplasmic mixture. Subcultures of VSMCs at passage 5 were used in all experiments. All experiments on MBP activation, Akt, MYPT1, GSK3beta, and MLC20 phosphorylation and Rho kinase and cGK1{alpha} were performed on highly confluent cells at identical passages. Before each experiment, cells were serum starved for 24 h in serum-free {alpha}-MEM containing 5.5 mM glucose and 1% antibiotics. The next day, cells were exposed to insulin (0–100 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II followed by insulin.

Overexpression of Akt with adenovirus (ad-myr-AKT) treatment in VSMC. Adenovirus constructed with myr-AKT-Flag was made at the gene transfer vector core (University of Iowa) as described previously (3). VSMCs were grown to 80% confluency, and cells were washed with serum-free media and then treated with ad-beta-gal or ad-myr-AKT for 4 h with agitation one time every hour. After 4 h, serum was added to the cells and incubated overnight. The next day, cells were washed with fresh media and kept for another 24 h. Cells were serum starved for 24 h before the experiments were performed with insulin or ANG II.

Transfection of VSMC with siAKTc. The sequence of siAKTc targets the homologue site of rat Akt 1 (1040–1058) and rat Akt 2 (1043–1061), which have been shown to abolish Akt 1 and Akt 2 expression (45). This site is common in rats and humans. siCONTROL, which is a nontargeting siRNA no. 1 from Dharmacon, was used to illustrate the nonspecific effect of siRNA transfection. VSMCs were transfected with Amaxa Nucleofector by electroporation with siCON or siAKTc following the manufacturer's instructions. After the transfection (48 h), cells were serum starved for 24 h, and experiments were done as described above.

Preparation of myosin-enriched fractions. Myosin-enriched fractions of VSMCs were prepared by extraction with a high-salt buffer as described previously (38, 67). Okadaic acid at a 1.0 nmol/l concentration was included during the enzyme assay to inhibit any residual protein phosphatase 2A activity (16, 67).

Measurement of MBP activity. Phosphatase activity in myosin-enriched fractions was assayed using [33P]phosphorylase a and 33P-labeled MLC20 as substrates (16). Briefly, equal amounts of proteins (1 µg) were diluted with assay buffer (in mM: 50 Tris·HCl, pH 7.5, 0.1 EDTA, 28 beta-mercaptoethanol, and 30 KCl). The reaction was initiated by the addition of 33P-labeled substrates and stopped after 10 min incubation at 30°C by the addition of 20% TCA. The radioactivity released in the TCA supernatants was counted as detailed in our recent publications (67). [33P]phosphorylase a was prepared by incubating [{gamma}-33P]ATP with purified phosphorylase kinase and phosphorylase b (16). {gamma}-33P-labeled MLC20 was prepared according to the published protocol (38) by incubating MLC20 (0.8 mg/ml) with purified myosin light chain kinase (50 µg/ml), 0.1 mg/ml calmodulin, and 50 µmol/l [{gamma}-33P]ATP.

Western blotting. Cells were lysed in a buffer containing 20 mM Tris·HCl (pH 8.0), 1 mM DTT, 100 mM NaCl, 0.5% SDS, 0.75% deoxycholate, 100 mM NaCl, 100 mM NaF, 50 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 µM microcystin, 50 mM beta-glycerophosphate, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin with phosphatase inhibitors. Lysates were spun down for 30 min at 14,000 g. Equal amounts of proteins were heated with sample buffer containing 2% SDS, 0.2 M Tris·HCl (pH 7.5), 20 mM EDTA, 10% glycerol, and bromphenol blue for 5 min at 95°C and then loaded on SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane and probed with specific antibodies followed by incubation with HRP-conjugated secondary antibodies and detected by ECL. The extent of each protein was quantitated by dividing the intensity of beta-actin. In some cases, the intensity of each protein phosphorylation was normalized to the total protein amount of target protein.

Measurement of Rho kinase and cGK1{alpha} activity. Rho kinase and cGK1{alpha} activities were performed as per the manufacturer's instructions (Cyclex). Rho kinase activity was measured based on the phosphorylation of threonine-695 of MYPT1 with cell lysates. Briefly, equal amounts of cell lysates were incubated with each substrate precoated on plates. Phosphospecific antibody labeled with HRP was then incubated for 1 h before the addition of 3,3',5,5'-tetramethylbenzidine substrate. The absorption was measured at 450 nm with a reference wavelength of 550 nm. Rho kinase II/ROK{alpha} and cGK1{alpha} were used as positive controls for each experiment. Enzyme concentration and time of incubation were adjusted to ensure first-order kinetics.

Statistics. The results are presented as means ± SE of four to six independent experiments, each performed in duplicate at different times. Paired Student's t-test was used to compare the basal vs. insulin-treated preparations. Unpaired t-test or ANOVA was used to compare the mean values between treatments. A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II inhibits insulin-stimulated Akt phosphorylation and induces the phosphorylation of both MYPT1 and MLC20. Akt is phosphorylated at serine-473, known to be important for full Akt activity (81). Contraction of VSMC is accompanied by the dephosphorylation of MYPT1 and phosphorylation of MLC20 (32). To understand the role of Akt on insulin-induced vasodilatation and the effects on the contractile apparatus, such as MYPT1 and MLC20 in VSMC, the phosphorylations of Akt at serine-473, MYPT1, and MLC20 were analyzed in response to various doses of insulin in the presence and absence of ANG II. Insulin, at 1, 10, and 100 nM, stimulated Akt phosphorylation in a dose-dependent manner by 1.3-, 2.7-, and 8.3-fold over basal, respectively (Fig. 1A, lanes 3–5). Preincubation of cells with ANG II (100 nM) reduced insulin-stimulated Akt phosphorylation to 0.7-, 1.7-, and 3.7-fold over basal (Fig. 1A, lanes 6–8). Noteworthy is the fact that ANG II alone induced Akt phosphorylation to 1.6-fold over basal levels (Fig. 1A, lane 2), as reported by others (55, 74). ANG II incubation increased the phosphorylations of MYPT1 and MLC20 to 1.9- and 2.9-fold over basal levels, respectively (Fig. 1, B and C, lane 2 vs. lane 1). Insulin, at 1, 10, and 100 nM, reduced the phosphorylation of MYPT1 by ANG II down to 52, 56, and 41% of levels with ANG II alone (Fig. 1B, lanes 6–8 vs. lane 2). Insulin also decreased MLC20 phosphorylation to 118, 73, and 25% of ANG II alone (Fig. 1C, lanes 6–8 vs. lane 2).


Figure 1
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  		Fig. 1. ANG II inhibits insulin-stimulated Akt phosphorylation and induces the phosphorylation of both myosin-bound phosphatase substrate (MYPT1) and 20-kDa myosin light chain (MLC20). Quiescent vascular smooth muscle cells (VSMCs) at day 9 were stimulated with insulin (1, 10, and 100 nM) for 10 min alone or after pretreatment with ANG II (100 nM; n = 3). Equal amounts of protein from each cell lysate sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and the total and phosphorylated levels of Akt (A), MYPT1 (B), and MLC20 (C) were measured by Western blot analysis. p, Phosphorylated; AT II, ANG II; Ins, insulin; con, control. Intensities of phosphorylated protein were quantitated by densitometric analysis and normalized to the abundance of total protein for Akt and MYPT1 and to beta-actin for MLC20. P < 0.05 vs. basal control (*), vs. corresponding insulin concentration (#), and vs. ANG II ($). I1, Insulin 1 nM; I10, insulin 10 nM; I100, insulin 100 nM; A-I1, ANG-insulin 1 nM; A-I10, ANG-insulin 10 nM; A-I100, ANG-insulin 100 nM.

 
Suppression of endogenous Akt by siRNA reduced Akt-GSK3beta phosphorylation and iNOS expression and increased insulin-stimulated phosphorylation of MYPT1 and MLC20. To investigate the role of Akt on insulin-dependent iNOS expression and inhibition of vasocontractile machinery, such as MYPT1 and MLC20, we used siAKTc. Control (siCON) RNA, which is nontargeting siRNA no. 1 from Dharmacon (Boulder, CO), was used to demonstrate the nonspecific effect of siRNA transfection. siAKTc decreased Akt expression to 82% (Fig. 2A, lanes 5–8) and in turn caused a decrease in the phosphorylation of Akt and GSK3beta (Fig. 2, A and B, lanes 5–8) downstream of Akt. The basal level of Akt phosphorylation was reduced to 60% (Fig. 2A, lane 1 vs. lane 5) and insulin-induced phosphorylation significantly decreased to 46% (Fig. 2A, lane 3 vs. lane 7). ANG II-induced Akt phosphorylation was also reduced by siAKTc to 13% (Fig. 2A, lane 2 vs. lane 6). To confirm the specificity of siAKTc on the effect of Akt phosphorylation, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was also determined. siAKTc did not change the status of ERK 1/2 phosphorylation stimulated by ANG II, insulin, or the combination of both in VSMC and was almost identical to the response in siCON, demonstrating the specific effect of siAKTc on Akt (Fig. 2A, lanes 1–4 vs. lanes 5–8). GSK3beta is a well-known downstream effector for insulin to regulate glycogen synthesis (18) and the cell cycle (20). To confirm the effect of siAKTc in VSMC, the phosphorylation of GSK3beta was determined as a direct Akt substrate. The phosphorylation of GSK3beta by insulin also was decreased by siAKTc in a similar manner (Fig. 2B, lanes 1–4 vs. lanes 5–8). With the use of siAKTc, the role of Akt on MYPT1 phosphorylation was determined. siAKTc did not significantly change the basal level of phosphorylation of MYPT1 (Fig. 2B). siAKTc treatment increased the phosphorylation of MYPT1 by insulin and ANG II followed by insulin by 2.3- and 4-fold, respectively (Fig. 2B, lanes 7 and 8). This result demonstrates the involvement of Akt on the insulin-stimulated decrease in phosphorylation of MYPT1 (Fig. 2B).


Figure 2
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  		Fig. 2. Akt phosphorylation is required for iNOS expression and the decrease in phosphorylation of MYPT1 and MLC20. Quiescent VSMCs transfected with control RNA (siCON) or siRNA targeting constitutive Akt (siAKTc) were starved and treated with insulin (10 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II for 5 min followed by insulin (10 nM). ERK, extracellular signal-regulated kinase; GSK, glycogen synthase kinase. Equal amounts of protein from each cell lysate sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The levels of pAkt/Akt and phospho-ERK 1/2 protein expression compared with beta-actin as an internal control (A), GSK3beta and MYPT1 phosphorylation (B), and the expression of iNOS and MLC20 phosphorylation (C) were measured by Western blot analysis. Densitometric analyses of 4 separate experiments are given below each graph. P < 0.05 vs. basal siCON (*), vs. corresponding value of siCON (**), vs. ANG II in siCON ($), and vs. insulin in siCON (#).

 
iNOS expression, induced by insulin, is related to vasodilatation by the enhanced production of NO in VSMCs. To show the involvement of Akt on insulin-induced iNOS expression, VSMCs were treated with siAKTc to determine whether the regulation of iNOS expression is downstream of Akt. As shown in Fig 2C, insulin induced a 2.6-fold increase in iNOS expression over the basal level (lane 3) that was inhibited by ANG II by 42% (lane 4). siAKTc treatment abolished basal and insulin-induced iNOS expression (Fig. 2C, lanes 5–8). MLC20 phosphorylation and dephosphorylation is recognized as the marker of contraction and relaxation, respectively. The phosphorylation of MLC20 by control, insulin, and ANG II-insulin was increased to 143, 124, and 142%, respectively, by siAKTc (Fig. 2C, lanes 5, 7, and 8), implying increased contraction via the decrease in Akt expression. Although siAKTc treatment caused a decrease in iNOS expression (Fig. 2C, lanes 5–8), phosphorylation of MLC20 and MYPT1 was significantly increased (Fig. 2, B and C, lanes 5–8), implying that Akt plays an important role in iNOS-dependent vasodilatation.

Akt overexpression via ad-myr-AKT increased the phosphorylations of Akt and GSK3beta and the expression of iNOS while reducing the basal and ANG II-induced phosphorylation of MYPT1 and MLC20. To confirm the role of Akt on insulin-induced vasodilatation and its inhibitory action on ANG II-induced contraction, ad-myr-AKT was used to overexpress constitutively active Akt. We used a mutant Akt with a myristilated signal at the carboxy terminus. This mutation targets Akt permanently to the cell membrane, rendering it continuously susceptible to PDK phosphorylation (59). In cells infected with myr-AKT, the basal level and ANG II-induced phosphorylation of Akt were increased to 4- and 3.5-fold over controls, respectively (Fig. 3A, lanes 1 and 2 vs. lanes 4 and 5). Insulin and ANG II- and insulin-stimulated Akt phosphorylation was also higher than in cells infected with ad-beta-gal by 1.5- and 2.5-fold over controls (Fig. 3A, lanes 3 and 4 vs. lanes 7 and 8). The phosphorylation of Akt and GSK3beta increased in parallel with the extent of Akt overexpression (Fig. 3, A and B). The direct downstream effector, GSK3beta, was phosphorylated in response to insulin to 1.7-fold over basal (Fig. 3B, lane 3). The overexpression of Akt caused a 2.7-fold increase of basal GSK3beta phosphorylation and 2.2-fold increase over basal by ANG II (Fig. 3B, lanes 5 and 6). This result implies that Akt overstimulation also caused the enhanced GSK3beta phosphorylation, directly downstream of Akt. To determine the inhibitory role Akt had on the contractile machinery, the effect of ad-myr-AKT on MYPT1 phosphorylation was examined. Ad-myr-AKT decreased the basal level of phosphorylation of MYPT1 compared with that of ad-beta-gal treatment as a control (Fig. 3B, lane 1 vs. lane 5). ANG II-induced phosphorylation of MYPT1 also decreased by the overexpression of Akt to basal levels, demonstrating that the overexpression of Akt leads to dephosphorylation of MYPT1 and enhances the relaxation mechanism (Fig. 3B, lane 2 vs. lane 6).


Figure 3
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  		Fig. 3. Overexpression of constitutively active Akt increased iNOS expression and decreased phosphorylation of MYPT1 and MLC20. Quiescent VSMCs infected with ad-beta-gal or adenovirus constructing myristilated Akt (ad-myr-AKT) were starved and treated with insulin (10 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II for 5 min followed by insulin (10 nM). Equal amounts of protein from each cell lysate sample were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The levels of pAkt/Akt protein expression compared with beta-actin as an internal control (A), GSK3beta and MYPT1 phospshorylation normalized to abundance of total GSK3beta or MYPT1 (B), and expression of iNOS and MLC20 phosphorylation (C) were measured by Western blot analysis. Densitometric analyses of 4 separate experiments are given below each graph. P < 0.05 vs. basal ad-beta-gal (*), vs. corresponding value of ad-beta-gal (**), vs. ANG II in ad-beta-gal ($), and vs. insulin in ad-beta-gal (#).

 
To confirm that the signaling pathway leading to Akt-dependent vasodilatation is via iNOS expression in VSMCs, ad-myr-AKT was employed to overexpress Akt protein. Western blotting with specific antibody targeting iNOS showed that the overexpression of Akt enhanced the basal and ANG II-induced expression of iNOS to more than twofold over those of ad-beta-gal-treated cells (Fig. 3C, lanes 1 and 2 vs. lanes 5 and 6). Insulin-induced iNOS expression was not further increased when compared with the expression level by the same treatment in ad-beta-gal-treated cells (Fig. 3C, lane 3 vs. lane 7). This implies that 10 nM insulin may have induced the maximal level of iNOS protein expression after the given time. These results, along with the results obtained from the siAKTc treatment, strongly demonstrate that iNOS expression is downstream of Akt. Similarly, the effects were observed with MYPT1 phosphorylation (Fig. 3B), where the basal level and ANG II-induced phosphorylation of MLC20 were completely abolished by the overexpression of Akt, confirming the role of Akt on relaxation detected by the dephosphorylation of MLC20 (Fig. 3C, lanes 1 and 2 vs. lanes 5 and 6). Insulin-induced dephosphorylation of MLC20 was further enhanced by Akt overexpression (Fig. 3C, lane 3 vs. 7). These results demonstrate the involvement of Akt on the insulin-stimulated decreased phosphorylation of MYPT1 and MLC20 (Fig. 3, B and C). Thus the insulin-induced relaxation of ANG II-induced contraction in VSMC is dependent on Akt-iNOS expression, which in turn caused the dephosphorylation of MYPT1 and MLC20, resulting in VSMC relaxation.

Akt is responsible for insulin-induced MBP activity. It is recognized that insulin causes MBP activation via the dephosphorylation of MYPT1. To speculate on the involvement of Akt on insulin-induced MBP activation, an MBP activity assay was performed using either siAKTc- or ad-myr-AKT-treated VSMCs. The basal level of Pi released was 3.2 ± 0.1 nmol·min–1·mg protein–1 and the insulin-induced level was 8.5 ± 0.1 nmol·min–1·mg protein–1 (Fig. 4A). Insulin induced a twofold increase of MBP activity over the control, and ANG II decreased it by 20% in VSMC (Fig. 4B). siAKTc, expressing low levels of Akt protein and an accompanying decreased phosphorylation (see Fig 2A, lanes 1–4 vs. lanes 5–8), eliminated insulin-induced MBP activity and returned levels back to near basal, demonstrating a critical role of Akt on insulin-induced MBP activation (Fig. 4B). In contrast, ad-myr-AKT treatment of VSMCs yielded basal and ANG II-induced MBP activities between 2- to 1.7-fold over the ad-beta-gal control, confirming the role of Akt on insulin-induced MBP activity (Fig. 4B).


Figure 4
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  		Fig. 4. Effects of siAKTc and ad-myr-AKT on myosin-bound phosphatase (MBP) activity in VSMC. Quiescent VSMCs infected with siAKTc or ad-myr-AKT were treated with insulin (10 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II for 5 min followed by insulin (10 nM). A: MBP activity measured as Pi released·min–1·mg protein–1 in myosin-enriched fractions. B: MBP activity assayed in myosin-enriched fractions of siAKTc- and ad-myr-AKT-infected cells using 33P-labeled myosin light chain (MLC) as a substrate. The method that delivered Akt for siRNA was siAKTc and for ad-virus was ad-myr-AKT. In the case of siRNA, control was siCON and control for ad-virus was ad-beta-gal. Results are means ± SE of 4 different experiments performed in duplicate. P < 0.05 vs. basal siCON or ad-beta-gal (*), vs. corresponding value of siCON or ad-beta-gal (**), and vs. Insulin in siCON or ad-beta-gal (#).

 
Akt is responsible for insulin-stimulated cGK1{alpha} activity and induces the vasodilatation via the inhibition of ROK{alpha} activity. NO produced by iNOS has been shown to increase cGMP, which in turn increases cGK1{alpha} activity (39). To delineate the signaling pathway from Akt to cGK1{alpha} by insulin, deletion and overexpression of constitutively active Akt using siAKTc and ad-myr-AKT were employed, and the effects on cGK1{alpha} and ROK{alpha} activity were measured. Insulin induced a 1.8-fold increase of cGK1{alpha} over the control, which was inhibited by ANG II back to the basal level (Fig. 5A). siAKTc also abolished the insulin-induced cGK1{alpha} activity and returned levels back to basal (Fig. 5A). In contrast, overexpression of Akt increased both the basal level and ANG II-induced cGK1{alpha} twofold when compared with the results in controls (ad-beta-gal) of the same treatment. However, insulin-induced cGK1{alpha} activity was not further increased by increased active Akt protein overexpression (Fig. 5A). These results demonstrate that Akt plays an important role in induction of cGK1{alpha} activity in response to insulin, causing vasodilatation.


Figure 5
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  		Fig. 5. Effects of siAKTc and ad-myr-AKT on cGMP-dependent kinase 1{alpha} (cGK1{alpha}) and Rho-associated kinase {alpha} (ROK{alpha}) activity. Quiescent VSMCs transfected with either siAKTc or ad-myr-AKT were treated with insulin (10 nM) for 10 min, ANG II (100 nM) for 15 min, or ANG II for 5 min followed by insulin (10 nM). A: insulin-induced cGK1{alpha} activity from equal amounts of protein incubated with cGK1{alpha} substrate and detected by ELISA. B: ROK{alpha} activity. The method that delivered Akt for siRNA was siAKTc and for ad-virus was ad-myr-AKT. In the case of siRNA, control was siCON and control for Ad-virus was ad-beta-gal. Results are means ± SE of 4 different experiments performed in duplicate. P < 0.05 vs. basal siCON or ad-beta-gal (*), vs. corresponding value of siCON or ad-beta-gal (**),vs. ANG II in siCON or ad-beta-gal ($), and vs. insulin in siCON or ad-beta-gal (#).

 
Many contractile reagents activate RhoA, causing translocation of RhoA to the membrane and thus ROK{alpha} activation (46). Activated ROK{alpha} then phosphorylates MYPT1 (25, 46) and subsequent VSMC contraction. Insulin is known to inhibit ROK{alpha} activity and induce vasodilation (11, 62). To more clearly define the role of Akt on inhibition of ROK{alpha} activity, VSMCs were treated with siAKTc and ad-myr-AKT. ANG II induced a twofold increase in ROK{alpha} activity over the control (Fig. 5B). Although insulin alone did not cause any significant increase in ROK{alpha} activity, it did inhibit ANG II-induced ROK{alpha} activity to 32% (Fig. 5B). With the decreased Akt protein levels obtained by siAKTc treatment, ROK{alpha} activity is increased to twofold over the basal levels. ROK{alpha} activity by insulin or ANG II followed by insulin also significantly increased 2.3- to 2.8-fold over similar treatment in the controls (Fig. 5B), suggesting that the absence of inhibition of ROK{alpha} activation by Akt resulted in enhanced ROK{alpha} activity. Furthermore, when Akt is overexpressed by ad-myr-AKT, ANG II-induced ROK{alpha} activity is decreased to basal levels, demonstrating that Akt was responsible for the inhibition of ROK{alpha} activation by a contractile agent (Fig. 5B). These data demonstrate the Akt phosphorylation induced by insulin was responsible for iNOS expression-cGK{alpha} activation-MBP activation, resulting in relaxation of VSMC by lowering ROK{alpha} activity and dephosphorylating MYPT1 and MLC20.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study that provides direct evidence for the role of Akt on insulin-induced vasodilatation in VSMCs. Our main finding is that Akt is required for the insulin-induced relaxation of VSMCs via the induction of iNOS and the activation of cGK1{alpha} and MBP. Akt is also necessary for the activation of MBP activity through the dephosphorylation of MYPT1 (T695) and results in the dephosphorylation of MLC20, a marker of VSMC relaxation. The changes observed in insulin-stimulated Akt phosphorylation correlate well with the extent of MYPT1 and MLC20 dephosphorylation (Fig. 1), the increase in MBP and cGK1{alpha} enzymatic activity (Fig. 4), and the decrease in ROK{alpha} activity (Fig. 5), thereby causing vasodilatation. Pretreatment of cells with ANG II caused the inhibition of insulin-stimulated Akt phosphorylation and the subsequent signaling thereafter. Evidence from either siAKTc or ad-myr-AKT treatment of VSMCs demonstrated that the insulin-induced Akt activation is critical and responsible for the downstream signaling molecules, such as iNOS-cGK1{alpha}-MBP, which are known vasodilatory signals. Also, insulin-induced Akt phosphorylation inhibits the contractile response stimulated by ANG II, thereby causing the dephosphorylation of MYPT1 and MLC20 and inhibition of ROK{alpha} activity, resulting in the inhibition of contraction. Thus the current study showed strong evidence of the role of Akt in stimulation of the vasorelaxation mechanism, causing inhibition on vasoconstriction.

The current study supports the importance of insulin-induced VSMC vasorelaxation on the regulation of vascular tone via Akt-mediated iNOS expression and the inhibition of contraction. Numerous studies examining endothelium-dependent vasodilatation have not demonstrated the vasorelaxation mechanism of insulin in VSMCs. Endothelium-dependent vasorelaxation plays an important role in regulating vascular tone, since endothelial NOS (eNOS)/neuronal NOS (nNOS) are constitutively expressed in endothelium (49, 63). Akt is also known to be important in regulation of endothelium-dependent vasodilation by activating eNOS, which is phosphorylated on Ser1177/1179 and then facilitates association of the enzyme with calmodulin, reducing its inhibitory interaction with caveolin-1 (57, 58). In VSMCs, Ca2+-dependent NOS, such as eNOS or nNOS, is not present; however, iNOS is known to be expressed (9, 37, 63). Even though iNOS is induced by inflammatory cytokines such as interleukin-l1beta, tumor necrosis factor, interferon-{gamma}, endotoxin, and lipopolysaccharide (LPS), it is also known that the iNOS is expressed without these stimulants (63). Acute treatment with insulin for 10 min showed the increase in iNOS expression in the present study. In our study, we found that the induction of iNOS by insulin is dependent on Akt, since siAKTc suppressed iNOS activation and ad-myr-AKT could induce enhanced iNOS expression. Considering that the production of NO by iNOS is 1,000-fold greater than that of eNOS and nNOS (29, 49, 79), insulin may have affected the vasoconstriction of the vasculature directly and more effectively in VSMC than endothelium, since it is where the contraction response occurs. Similarly, iNOS is expressed and activated by insulin and IGF-I in 10 min, and pretreatment of ANG II reduced IGF-I-induced iNOS expression and activity (37). Here, we have shown direct evidence of the role of Akt in iNOS expression.

Maintaining precise physiological levels of Akt/protein kinase B may be critical to avoid insulin resistance. This is evidenced by studies linking impaired Akt expression and activity with type 2 diabetes (47, 48) or the increased activity observed in the renal cortex of db/db mice (24). MBP and cGK1{alpha} activities, as well as phosphorylation of MYPT1 and MLC20, are resistant to insulin after siAKTc treatment (Figs. 4 and 5). Thus it is interesting to investigate whether the defects in Akt may have caused the insulin resistance observed in the vasorelaxation of the diabetic rodent model. In support of this, Akt2 null mice exhibited both fasting hyperglycemia and glucose intolerance (29).

The inhibitory effect of insulin on contraction may be conditional and dependent on the contractile response. Indeed, the order of insulin treatment in combination with the contractile agent may well be critical for the inhibitory action of insulin on contraction. For example, it has been reported that induction of cGMP by insulin, which mediates vasorelaxation, was conditional to the stimulation of the contractile agent serotonin but not on insulin alone (41). Likewise, we have also found that preincubation with the contractile agent ANG II before insulin exposure only primed insulin to inhibit ANG II-induced contraction. A similar finding was presented in a study with ANG II inhibition of IGF-I-induced iNOS expression (37). Thus insulin may have biphasic effects in terms of its inhibition of contraction, a mechanism that needs further investigation. There was a difference in the A-I response in Fig. 5B between siRNA and ad-virus controls. If there were "off target" effects from siCON, other stimulation such as ANG II or insulin may respond in a different manner like ANG-insulin in siCON compared with those in Ad-beta-gal-treated cells. However, only the response of A-I was different, whereas ANG II and insulin responses are very identical. Thus it is only the difference in insulin potency that reduces ANG II-induced ROK{alpha} activity (Fig. 5B).

The critical role of RhoA/ROK{alpha} on vascular contraction and hypertension has been demonstrated in many studies (10, 43, 72, 76). Our present study demonstrated increased ROK{alpha} activity by contractile ANG II and the acute effect of insulin on the inhibition of ROK{alpha} activity causing vasodilatation. Similarly, in vivo, ROK{alpha} inhibitors restore normal blood pressure in several hypertensive rat models, demonstrating their role in contraction (72). Inhibition of ROK{alpha} with a specific inhibitor improved the insulin resistance and hypertension observed in obese Zucker rats (43) by reducing blood pressure and improved serine phosphorylation of IRS-1 and insulin signaling in skeletal muscle (43).

Knowing the inhibitory role of insulin on contraction via inhibition of RhoA/ROK{alpha} activation, one can assume that insulin dependency on RhoA/ROK{alpha} activation on glucose metabolism may be cell type specific. Despite the well-known role of RhoA/ROK{alpha} in vascular contraction (10, 72, 76), several studies have demonstrated that the known vasodilator, insulin, induces the activation of RhoA/ROK{alpha} and its role on glucose homeostasis and insulin resistance in C2C12 skeletal muscle cells and adipocytes (28, 43, 44, 68). It is also known that insulin translocates Rho by a PI3-kinase-dependent mechanism (23). Given these observation, ROK{alpha} activation has been shown to be crucial for insulin activation on glucose homeostasis in C2C12 skeletal muscle cells and adipocytes. Inactivation of ROK{alpha} also reduces insulin-stimulated IRS-1 tyrosine phosphorylation and PI3-kinase activity. Moreover, inhibition of ROK{alpha} activity in mice causes insulin resistance by reducing insulin-stimulated glucose uptake in skeletal muscle in vivo. Thus ROK{alpha} activation by insulin is an important regulator of insulin signaling, specifically in glucose metabolism in skeletal muscle cells and adipocytes (28). However, the role of RhoA/ROK{alpha} activation on insulin-responsive glucose metabolism is still controversial. For example, the C3 toxin from Clostridium botulinum, which inhibits RhoA function, has been reported to mimic (13), inhibit (68), and have no effect (75) on insulin-stimulated glucose uptake and GLUT4 translocation. Moreover, in the vasculature, Rho has been shown to be implicated in the migration (43) and proliferation of VSMCs (64) and the suppression of eNOS expression in vascular endothelial cells (53). Considering the MYPT1 phosphorylation as a direct downstream effector of ROK{alpha}, we also observed the slight increase of MYPT1 phosphorylation by acute insulin (100 nM) in VSMCs (Fig. 1B). Similarly, phosphorylation of MYPT1 was also reported by chronic stimulation of insulin in VSMCs (43). However, the extent of the stimulation was very low compared with that of ANG II observed in the current study, and insulin did not cause VSMC contraction. Also, there is no report of insulin's direct role in contraction, but inhibition of ANG II-induced contraction in VSMC is demonstrated. Thus ROK{alpha} activation by insulin does not play any direct role in contraction of VSMC. Therefore, insulin-induced RhoA/ROK{alpha} activation may have been related to the migration and proliferation signaling in VSMCs rather than insulin-induced relaxation.

Recently, the novel signaling pathway of ANG II-induced Akt activation has been proposed in several studies (55, 60, 73, 74). We found that, in addition to the vasodilator insulin, the contractile agent ANG II can also induce Akt phosphorylation (Fig. 2B). The exact role of ANG II-induced Akt phosphorylation, however, is still not fully understood. Compared with insulin-induced Akt activation, the extent of ANG II-induced Akt phosphorylation was very low (1.6- vs. 8-fold). Indeed, there might be different signaling pathways involved in ANG II-induced Akt phosphorylation compared with that by insulin. ANG II is known to activate Akt both via epidermal growth factor receptor transactivation pathways and via nonreceptor tyrosine kinases, such as Src, in VSMC (21, 56, 77). ANG II-induced Akt activation is mediated by metabolites of arachidonic acid generated via a calmodulin-dependent kinase II-stimulated Ca2+-dependent phospholipase A2 (55) and phospholipase D2 (56). Akt can be activated via a PI3-kinase-independent mechanism through PKA (60) and Ca2+/calmodulin-dependent kinase activation (55). Moreover, wortmannin-sensitive PI3-kinase C2 type, which is responsive to Ca2+, has been shown to play an important role in actin rearrangement and contraction (5, 14). The role of ROS-dependent p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 has also been proposed in ANG II-induced Akt activation in VSMCs (73, 74). Therefore, suppression of ANG II-induced contraction by siAKTc (5 min ANG II stimulation; data not shown) may imply a role for Akt in contraction via a PI3-kinase-independent or Ca2+-responsive PI3-kinase C2 type-dependent and ROS-dependent mechanism, which is separate from the insulin-induced PI3-kinase-dependent vasodilatation pathway in VSMCs. Supporting the role of Ca2+-dependent Akt signaling on contractility, constitutively active Akt enhanced myocardial contractility in vivo in transgenic mice overexpressing Akt via altered sarcoplasmic reticulum Ca2+ release (17). Overexpression of active Akt in our study, however, suppressed the phosphorylation of MYPT1 and MLC20 (Fig. 2) as well as ROK{alpha} activity (Fig. 5B), confirming the inhibitory effect of Akt on contraction and demonstrating a major Akt pathway to be PI3-kinase-dependent vasodilatation.

The importance of ANG II and insulin interaction on insulin resistance has been demonstrated in many studies (4, 27, 65, 66, 78). The mechanism by which ANG II inhibits insulin signaling has shown that ANG II increases serine phosphorylation of the insulin receptor IRS-1 as well as the p85 subunit of the PI3-kinase. At the same time, ANG II inhibits insulin-stimulated tyrosine phosphorylation of IRS-1, preventing the docking between IRS-1 and PI3-kinase (27, 78). In the current study, insulin activates MBP and caused MLC20 dephosphorylation, resulting in vasodilation via Ca2+ sensitization but not actual Ca2+ transient change (7, 62). However, many studies also report that insulin inhibits the contractile response by reducing the Ca2+ transients in VSMC. In these studies, the mechanism by which insulin inhibits Ca2+ transients was via alteration of inositol 1,4,5-trisphosphate-releasable Ca2+ stores (61), via insulin-stimulated glucose transport (42), and via an cGMP- and NOS-dependent manner (41). The latter may have also influenced the Ca2+ sensitization via MBP activation, as recent studies have demonstrated (7, 62). Our study also found that iNOS expression and MBP and cGK1{alpha} activity by insulin are inhibited (Figs. 2C, 3C, 4, and 5A) by ANG II pretreatment, whereas ANG II-induced phosphorylation of MYPT1 and MLC20 (Fig. 1, B and C) and activation of ROK{alpha} (Fig. 5B) are inhibited by insulin. The ANG II-induced inhibition of insulin-induced Akt phosphorylation has been shown in PC-12W cells (19). It has been reported that the production of renal ANG II is increased in the streptozotocin-induced diabetes rat model (6), implying a relationship between ANG II and insulin resistance. Therefore, chronic interaction of hyper-ANG II and hyperinsulin in diabetic subjects may be involved in insulin resistance, which may have an important role in the regulation of vascular physiology and the development of hypertension and diabetes.

The current study demonstrates for the first time the essential involvement of Akt in insulin-responsive signaling proteins such as iNOS-cGK1{alpha}-MBP, related to VSMC relaxation, which was evidenced from either siAKTc or ad-myr-AKT treatment of VSMCs. The contractile response stimulated by ANG II is inhibited by insulin-induced Akt phosphorylation, leading to the dephosphorylation of MYPT1 and MLC20 and inhibition of ROK{alpha} activity, therefore causing the inhibition of contraction. The current study demonstrates the importance of vascular tone regulation by insulin-induced Akt signaling in VSMC. Additional studies focusing on the role Akt plays in defective vasorelaxation among diabetic subjects are needed.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant 5 R01 HL-067953-03.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Nikolai Kholodilov (Department of Neurology, Columbia University) for the generous gift of myr-AKT construct. We thank the University of Iowa, Gene Transfer Vector Core, for preparation of adenovirus constructed with myr-AKT-Flag. We also thank Lisa Urgolites for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Ragolia, Vascular Biology Institute, Winthrop Univ. Hospital, 222 Station Plaza N, Rm. 505B, Mineola, NY 11501 (e-mail: lragolia{at}winthrop.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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