Insulin Induces Upregulation of Vascular AT1 Receptor Gene Expression by Posttranscriptional Mechanisms
Background—An interaction of insulin with angiotensin II effects could be pathophysiologically important for the pathogenesis of atherosclerosis and hypertension.
Methods and Results—We examined the effect of insulin on AT1 receptor gene expression in cultured vascular smooth muscle cells (VSMCs). A 24-hour incubation with insulin (100 nmol/L) produced a 2-fold increase in AT1 receptor density on VSMCs, as assessed by radioligand binding assays. This enhanced AT1 receptor expression was caused by a time- and concentration-dependent upregulation of the AT1 receptor mRNA levels, as assessed by Northern analysis. The maximal effect was detected after a 24-hour incubation of cells with 100 nmol/L insulin (270±20%). AT1 receptor upregulation was caused by a stabilization of the AT1 receptor mRNA, because the AT1 receptor mRNA half-life was prolonged from 5 hours under basal conditions to 10 hours after insulin stimulation. In contrast, insulin had no influence on AT1 receptor gene transcription, as assessed by nuclear run-on assays. The insulin-induced AT1 receptor upregulation was followed by an increased functional response, because angiotensin II evoked a significantly elevated intracellular release of calcium in cells that were preincubated with 100 nmol/L insulin for 24 hours. The insulin-induced AT1 receptor upregulation was dependent on tyrosine kinases, as assessed by experiments with the tyrosine kinase inhibitor genistein. Furthermore, experiments using the intracellular calcium chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester suggest that intracellular calcium release may be involved in AT1 receptor regulation.
Conclusions—Insulin-induced upregulation of the AT1 receptor by posttranscriptional mechanisms may explain the association of hyperinsulinemia with hypertension and arteriosclerosis, because activation of the AT1 receptor plays a key role in the regulation of blood pressure and fluid homeostasis.
There is evidence that hyperinsulinemia is present in diseases other than non–insulin-dependent diabetes mellitus. Among others, essential hypertension and arteriosclerotic heart disease are associated with disorders in the insulin homeostasis.1 It is well established that hypertension is closely correlated with obesity and non–insulin-dependent diabetes mellitus, and therefore it has been suggested that hyperinsulinemia and insulin resistance participate in the pathogenesis of elevated blood pressure.2 This observation is based primarily on epidemiological studies and statistical correlations, but the molecular events involved in the phenomenon are currently unknown. In this context, it has been reported that hyperinsulinemia is associated with reduced renal sodium excretion and stimulation of the sympathetic nervous system.3 Insulin is known to cause an increase of intracellular calcium and sodium in vascular smooth muscle cells (VSMCs), leading putatively to an enhanced vascular tone. Finally, insulin induces growth-promoting effects.4 However, the mechanisms involved in the correlation of hypertension and hyperinsulinemia are still poorly understood.
The renin-angiotensin system plays an important role in the control of cardiovascular function, and in addition, abnormalities in the renin-angiotensin system have been implicated in diseases such as hypertension, heart failure, and arteriosclerosis.5 The angiotensin II (Ang II) type 1 (AT1) receptor is a G protein–coupled receptor expressed in various tissues that mediates most of the known biological effects of Ang II (eg, vasoconstriction, water retention, and vascular and cardiac hypertrophy).6 Because the AT1 receptor expression is highly variable, its expression level may decisively influence the activity of the entire renin-angiotensin system. Indeed, it is well known that the AT1 receptor is regulated in vivo as well as in vitro. Conditions of increased renin-angiotensin system activity cause downregulation of AT1 receptors,7 8 whereas a decrease in the activity of the renin-angiotensin system upregulates the AT1 receptor.9 10 Recently, it has been shown that various growth factors as well as Ang II induce a profound downregulation of AT1 receptor gene expression in cultured VSMCs.11 12 13 With these data in mind, we reasoned that the regulation of the AT1 receptor may also be involved in the mechanisms underlying the insulin-induced elevation of blood pressure. Because smooth muscle cells are the principal target for increments of Ang II and these cells execute the AT1 receptor–mediated vasoconstriction and vascular hypertrophy,14 15 16 we investigated the effect of insulin on AT1 receptor expression in cultured VSMCs and tried to gain an insight into mechanisms participating in these regulative pathways.
Angiotensin peptides, salts, 5,6-dichlorobenzimidazole (DRB), insulin, and other chemicals were purchased from Sigma Chemical Co. [32P]dCTP, Hybond N–nylon membranes, and [125I]-labeled Ang II were obtained from Amersham. Antibiotics, serum, and cell culture medium were purchased from Gibco BRL. RNA-Clean was purchased from AGS. Oligonucleotides were synthesized with Pharmacia Chemicals, with an automated DNA synthesizer (Pharmacia LKB, gene assembler plus). Fura 2-AM was obtained from Calbiochem. Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester (MAPTAM), pertussis toxin (PTX), and genistein were purchased from Calbiochem Novabiochem.
VSMCs were isolated from rat thoracic aorta (strain, female Wistar-Kyoto; 6 to 10 weeks old; Charles River Wega GmbH, Sulzfeld, Germany) by enzymatic dispersion as described previously17 and cultured over several passages according to Ross.18 Cells were grown in a 5% CO2 atmosphere at 37°C in DMEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 1% nonessential amino acids (100×), and 10% FCS. Experiments were performed with cells from passages 5 to 15.
mRNA Isolation and Northern Analysis
After the indicated treatments, culture medium was aspirated and the cells were lysed with 1 mL RNA-Clean (AGS), scraped, and processed according to the manufacturer’s protocol to obtain total cellular RNA. Aliquots (10 μg) were electrophoresed through 1.2% agarose/0.67% formaldehyde gels and stained with ethidium bromide to verify the quantity and quality of the RNA. After capillary transfer onto Hybond N membranes in 20×SSC (3 mol/L sodium chloride, 300 mmol/L sodium citrate), the RNA was cross-linked to the membranes with a Stratalinker 1800 (Stratagene). Northern blots were prehybridized for 2 hours at 42°C in a buffer containing 50% deionized formamide, 0.5% SDS, 6×SSC, 10 μg/mL denatured salmon sperm DNA (Sigma), and 5×Denhardt’s solution and were then hybridized for 15 hours at 42°C with a random-primed, [32P]dCTP-labeled rat AT1 receptor cDNA probe in the same buffer but without Denhardt’s solution. The rat AT1 receptor cDNA probe was an 824-bp fragment generated from an AT1 receptor cDNA template19 by the polymerase chain reaction using the primer pair 5′-GTCATGATCCCTACCCTCTACAGC-3′ and 5′-CCGTAGAACAGAGGGTTCAGGCAG-3′ and Taq polymerase.
Radioligand Binding Assays
Experimental cells were washed 3 times with PBS. Cells were collected, and after a brief centrifugation, the pellet was resuspended in 1 mL ice-cold 50 mmol/L Tris-HCl, pH 8.0, and homogenized by repeated trituration through a 22-gauge needle. The membranes were pelleted by centrifugation at 12 000 rpm for 10 minutes at 4°C. Homogenization and centrifugation were repeated twice. The final pellet was resuspended in an incubation buffer in the absence of DTT (50 mmol/L Tris-HCl, 50 mmol/L NaH2PO4, 10 mmol/L MgCl2, 0.2% BSA). Ang II receptor density and affinity were investigated in saturation experiments using increasing amounts of [125I]-labeled Ang II as radiolabeled ligand (0.125 to 2 nmol/L). Dup753 (10 μmol/L) was used to determine nonspecific binding. The assay was performed in a total volume of 250 μL incubation buffer. The incubation was carried out at 24°C for 60 minutes. These conditions allowed a complete equilibration of the receptor with the radioligand. The reaction was terminated by rapid vacuum filtration through Whatmann GF/C filters; the filters were washed immediately 3 times with 5 mL of ice-cold incubation buffer, and radioactivity was determined in a gamma counter. All experiments were performed in triplicate. The maximal density (Bmax) and apparent affinity (Kd) of binding sites were obtained by nonlinear regression analysis.
Measurement of Free [Ca2+]i
VSMCs were cultured on round glass microscope slides (diameter, 12 mm) and at confluence were incubated with either vehicle or 100 nmol/L insulin for 24 hours. Subsequently, cells were washed with PBS and incubated with 2 μmol/L fura 2-AM at 37°C for 30 minutes in (in mmol/L) HEPES 20, glucose 16, NaCl 130, MgSO4 1, and CaCl2 0.5. Before the measurements, cells were rinsed gently with the same buffer containing 1 instead of 0.5 mmol/L CaCl2. The glass slides were positioned diagonally in the cuvette, and the [Ca2+]i was measured in a Hitachi fluorescence spectrofluorometer at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. Maximum (Rmax) and minimum (Rmin) fluorescence were determined by addition of digitonin at a final concentration of 30 mmol/L followed by the addition of Tris-base/EGTA (final concentration, 0.1 mmol/L/25 mmol/L). Fluorescence was corrected for cellular autofluorescence. Fluorescence signals were calibrated according to Grynkiewicz et al.20
Nuclear Run-On Assays
These assays are a slight modification of recently described protocols.21 After a 24-hour incubation of VSMCs with 1 μg/mL insulin or vehicle, cells were dispersed with trypsin and washed with 150 mmol/L potassium chloride, 4 mmol/L magnesium acetate, and 10 mmol/L Tris-HCl, pH 7.4. After centrifugation in a Beckman GS-GR tabletop centrifuge with a GH 3.8 rotor (1200 rpm, 5 minutes, 4°C), the cell pellet was resuspended in 2 mL of the same buffer containing 0.5% Nonidet P-40 (Sigma). After lysis for 10 minutes on ice, the nuclei were isolated by centrifugation (2000 rpm, 5 minutes, 4°C) through 4 mL of 0.6 mol/L sucrose with the same equipment. The supernatants were carefully removed, and the nuclear pellet was resuspended in a buffer containing 40% glycerol, 50 mmol/L Tris, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA. These were stored at −80°C until used for assays. Nuclei (≈5×108 to 20×108 nuclei per reaction) were used to carry out the transcription in a reaction mixture containing 40% glycerol; 50 mmol/L Tris; 5 mmol/L MgCl2; 0.1 mmol/L EDTA; 0.5 mmol/L levels of CTP, GTP, and ATP; and 0.2 to 0.3 μmol/L [32P]UTP (>300 μCi/mmol) at 30°C for 30 minutes. Reactions were terminated by addition of 800 μL of RNA-Clean, and the radioactive RNA was isolated and purified by collection of the eluate from a Bio-Rad P-30 spin column. [32P]UTP-labeled RNA (≈5×106 to 1×107 cpm) was dissolved in hybridization solution (100 mmol/L TES, pH 7.4, 0.3 mol/L NaCl, and 100 μg/mL Escherichia coli tRNA). Plasmids (5 μg) containing cDNAs for the AT1 receptor (a HindIII-NotI cDNA insert from pCa18b subcloned in pKS+-Bluescript) or GAPDH (rat GAPDH in pIBI30; International Biotechnology) and a plasmid (KS+-Bluescript) without insert were linearized, denatured, and applied to nylon membranes by use of a dot-blot apparatus. These membranes were prehybridized for 2 hours at 42°C in 100 mmol/L TES, 0.3 mol/L NaCl, 100 μg/mL E coli tRNA, and 5×Denhard’s solution and were hybridized at 42°C for 16 hours. Membranes were washed for 10 minutes at room temperature in 2×SSC and for 15 minutes at 50°C in 2×SSC/0.1% SDS. The filters were exposed to film for 12 to 48 hours, and autoradiographic signals were quantified by laser densitometry.
Data are presented as mean±SEM. Statistical analysis was performed with the ANOVA test.
Cells were grown to confluence, and serum was removed from the culture medium 24 hours before initiation of experimental treatments to obviate its effects.
Figure 1⇓ illustrates autoradiographic results from Northern hybridization of a rat vascular AT1 receptor cDNA probe to 10 μg of electrophoretically separated, total cellular RNA extracted from VSMCs at the indicated time points after addition of either 100 nmol/L insulin or vehicle to the culture medium. The probe hybridized to an abundant 2.2-kb transcript and a minor 3.2-kb transcript, as observed previously.19 22 Under basal conditions, the AT1 receptor mRNA remained unchanged over the time course of the experiment. Furthermore, the autoradiogram revealed a time-dependent elevation of the transcript level. The AT1 receptor mRNA signal appeared to be significantly increased 12 hours after exposure to insulin, and this increase was sustained for up to 24 hours. Also shown in Figure 1⇓ is hybridization of a GAPDH cDNA probe to the same Northern blot. GAPDH mRNA appeared to be stable over the time course of the experiment. Autoradiographic data generated from 5 separate experiments were analyzed by laser densitometry. In a set of control experiments, cells were serum-deprived for 24 hours, and subsequently, RNA was isolated after 0, 2, 4, 12, and 24 hours of incubation with vehicle. As demonstrated in Figure 2⇓, top, neither the AT1 receptor mRNA nor the GAPDH mRNA was significantly altered during the time course of the assay, which suggests that the AT1 receptor mRNA expression level remained stable for up to 24 hours after withdrawal of serum. Figure 2⇓, middle, shows the insulin-induced upregulation of AT1 receptor mRNA hybridization signal relative to vehicle-treated control levels at 0 hours. A 12-hour incubation with 100 nmol/L insulin caused an upregulation of AT1 receptor mRNA levels to 231±33%. After 24 hours of insulin stimulation, the AT1 receptor mRNA signals were measured at 270±20% relative to the control level at 0 hours (100%). GAPDH mRNA expression was not significantly regulated by insulin. Figure 2⇓, bottom, demonstrates the concentration-dependent effect of insulin on AT1 receptor mRNA expression. Cells were challenged with vehicle or 0.01 to 100 nmol/L insulin. AT1 receptor upregulation was significant with 0.1 nmol/L insulin (153±12.7% of control) and reached a maximum at 100 nmol/L insulin. These data demonstrate that insulin specifically induces the upregulation of AT1 receptor mRNA in VSMCs.
To assess whether the increased level of AT1 receptor mRNA was translated to an elevation of AT1 receptor protein expression, radioligand binding assays were performed. Therefore, AT1 receptor binding sites were measured in a membrane-binding assay after a 24-hour treatment of VSMCs with either 100 nmol/L insulin or vehicle. Nonlinear regression analysis demonstrated that 125I-labeled Ang II bound to a single population of sites, as expected for VSMCs (data not shown). Figure 3⇓ shows graphically the [125I]-labeled Ang II saturation binding to VSMCs treated with either insulin or vehicle. Binding to vehicle-treated cells revealed a Kd value of 0.33 (0.2 to 0.47) nmol/L and a Bmax value of 389±18.6 fmol/mg protein. Binding to insulin-treated cells showed an increase in the Bmax value to 776±33.5 fmol/mg protein, without changes in the affinity for the radioligand (Kd value, 0.26 [0.15 to 0.36] nmol/L). These binding data indicate that insulin markedly elevates AT1 receptor protein expression as a result of an enhanced level of AT1 receptor mRNA.
Experiments were performed to gain insight into general mechanisms participating in the insulin-induced elevation of AT1 receptor expression. The insulin-induced upregulation of the AT1 receptor mRNA that causes an elevation of AT1 receptor density could mechanistically be based either on an enhanced gene transcription rate or on posttranscriptional modulations of the AT1 receptor mRNA.
To measure the de novo synthesis of the AT1 receptor mRNA, nuclei from VSMCs incubated for 24 hours with either 100 nmol/L insulin or vehicle were isolated and used in a nuclear run-on assay. Figure 4⇓ illustrates a representative autoradiogram of radiolabeled de novo synthesized mRNA to AT1 receptor, GAPDH, and plasmid DNA. Also shown is the densitometric analysis of 3 separate experiments. Incubation with insulin, which had caused significant upregulation of AT1 receptor mRNA, had no effect on AT1 receptor transcription rate. These experiments suggested that the detected enhancement of AT1 receptor mRNA and protein by insulin was not mediated through an increase in de novo AT1 receptor gene synthesis.
Consequently, we assumed that the insulin-induced elevation in AT1 receptor gene expression may be caused by posttranscriptional mechanisms. Therefore, after a 24-hour treatment with either vehicle or 100 nmol/L insulin, gene transcription of VSMCs was inhibited by incubation with 50 μg/mL of the RNA–polymerase II inhibitor DRB. Northern hybridizations were then performed on RNA extracted from VSMCs at the indicated time points. In vehicle-treated cells, AT1 receptor mRNA levels were reduced to 50% of control levels ≈5 hours after the addition of DRB (Figure 5⇓). This rate of AT1 receptor mRNA decay was taken to represent a measure of mRNA stability under otherwise normal conditions. This decay rate contrasted markedly with the degradation for AT1 receptor mRNA in cells pretreated with insulin: preincubation of VSMCs with insulin produced a marked increase in AT1 receptor mRNA stability with an AT1 receptor mRNA half-life calculated at ≈10 hours. These data suggest that insulin-induced enhancement of AT1 receptor mRNA stability rather than modulation of AT1 receptor transcription rate is involved in the insulin-induced upregulation of AT1 receptor gene expression.
Insulin receptor activation by its ligand causes stimulation of various intracellular pathways. To investigate whether involvement of some of these intracellular signals is a prerequisite for insulin-induced AT1 receptor upregulation, cells were preincubated with various agents that selectively block particular signaling pathways of insulin. Incubation with the tyrosine kinase inhibitor genistein (1 μmol/L) for 30 minutes before either 100 nmol/L insulin or vehicle was added to the culture medium effectively inhibited the insulin-induced AT1 receptor upregulation, suggesting that tyrosine phosphorylation is essentially required for this receptor regulation (Figure 6A⇓). Pretreatment with PTX did not influence insulin-induced AT1 receptor regulation (Figure 6B⇓), suggesting that coupling of insulin receptors to Gi proteins is not involved in the observed effects. The intracellular calcium chelator MAPTAM caused a moderate AT1 receptor downregulation. The insulin-induced AT1 receptor upregulation was prevented by MAPTAM (Figure 6C⇓). 18S rRNA expression and viability of VSMCs was monitored throughout the experiments with genistein, PTX, and MAPTAM. There were no significant changes in cell appearance or 18S rRNA expression in this experimental setup (data not shown).
We further reasoned that upregulation of AT1 receptor gene expression should consequently lead to an enhanced functional response of VSMCs on Ang II stimulation. To test this, we examined Ang II–induced elevation of [Ca2+]i. Figure 7⇓ illustrates a representative time course of [Ca2+]i of VSMCs pretreated for 24 hours with either 100 nmol/L insulin or vehicle. Basal [Ca2+]i was measured at ≈30 nmol/L. After 1 minute, the cells were challenged with 100 nmol/L Ang II. Calculation of 4 separate experiments revealed that 100 nmol/L Ang II induced a maximal [Ca2+]i increase of 172.5±20.5 nmol/L in vehicle-treated VSMCs, whereas the same concentration of Ang II caused a maximal [Ca2+]i increase of 292.5±37 nmol/L in insulin-pretreated VSMCs. The Ang II–induced elevation of [Ca2+]i was blocked by addition of an AT1 receptor antagonist (Dup753) but not by an AT2 receptor antagonist (PD123177) (data not shown). These data demonstrate that the insulin-induced upregulation of AT1 receptor mRNA and protein causes the expected elevated functional response of VSMCs with respect to AT1 receptor–mediated Ang II stimulation.
Insulin causes an upregulation of AT1 receptor gene expression through inducible calcium- and MAP kinase–dependent stabilization of AT1 receptor mRNA in cultured VSMCs. The AT1 receptor upregulation results in an enhanced functional response of VSMCs on stimulation with Ang II.
The AT1 receptor gene expression is influenced by various agents and hormones. Ang II causes downregulation of vascular AT1 receptor expression in vitro as well as in vivo.7 8 9 10 12 13 Like Ang II, various growth factors downregulate AT1 receptor gene expression.11 In contrast, LDL leads to an upregulation of the AT1 receptor in vitro as well as in vivo and causes an enhanced biological response to Ang II stimulation.23 24 This observation associated the AT1 receptor regulation for the first time with such metabolic disorders as hypercholesterolemia and the metabolic syndrome with respect to cardiovascular diseases.
In addition, hypertension and arteriosclerosis are frequently associated with hyperinsulinemia and insulin resistance, as shown by epidemiological studies.1 2 Some studies showed that insulin is capable of promoting growth, attenuates sodium excretion, and activates the sympathetic system.3 4 In this context, putative interactions between insulin and the renin-angiotensin system have recently become a prominent subject of interest: Some investigators studied hyperinsulinemia induced by dietary fructose intake in a rat animal model. Interestingly, they found that the vasoconstrictive potency of Ang II is increased during hyperinsulinemia in this model.25 26 Moreover, it has been shown that a selective AT1 receptor antagonist inhibits the elevation of blood pressure induced by the fructose treatment.27 Furthermore, there is a strong correlation between insulin resistance and pressor response to Ang II in hypertensive patients.28 These studies suggested a link between hyperinsulinemia and Ang II with regard to elevated blood pressure but did not establish the molecular events that may have participated in this phenomenon. Because only correlations have been described, it has not been clarified whether insulin itself could cause the observed effects. Our data suggest that insulin induces an upregulation of the AT1 receptor, and therefore the concept of hyperinsulinemia-induced hypertension via upregulation of AT1 receptor gene expression seems to be very reasonable, especially because insulin-induced attenuation of sodium excretion and activation of the sympathetic system,3 4 synergistically with AT1 receptor overexpression, could affect pathophysiological blood pressure regulation.
On the basis of our data, the regulation of the AT1 receptor takes place at the posttranscriptional level. Insulin leads to AT1 receptor upregulation more likely via stabilization of the AT1 receptor mRNA than via activation of AT1 receptor gene transcription. Indeed, the posttranscriptional regulation seems to be of greater relevance than the transcriptionally induced AT1 receptor regulation in the case of Ang II–, growth factor–, or LDL-induced AT1 receptor regulation as well.11 12 13 23 AT1 receptor mRNA binding proteins, which bind at the 3′-untranslated region of the AT1 receptor mRNA, are potentially involved in the inducible (de)stabilization of the AT1 receptor mRNA.13 At present, the intracellular pathways that connect activation of a receptor at the cell surface with this machinery of posttranscriptional regulation are only poorly understood. Insulin receptor activation by its ligand causes stimulation of multiple intracellular pathways, including various phosphorylation processes, G-protein coupling, calcium signaling, phosphatidylinositol-3-kinase and MAP kinase activation, and interference with the cAMP turnover.29 According to our findings, AT1 receptor mRNA is stabilized by insulin through tyrosine phosphorylation and MAP kinase– dependent intracellular pathways. It is attractive to speculate that MAP kinase signaling is involved in the induction of mRNA binding proteins, which then influence AT1 receptor mRNA turnover. This is in good agreement with previous findings that associated MAP kinase activation with posttranscriptional mRNA processing of various genes.30 Intracellular calcium also participates in the regulative processes of AT1 receptor expression, because MAPTAM slightly downregulates the AT1 receptor mRNA. Nevertheless, it is not probable that the insulin-induced AT1 receptor upregulation is specifically blocked by MAPTAM. Moreover, MAPTAM-induced inhibition of the insulin effect on AT1 receptor expression may be due to the initial decrease of AT1 receptor mRNA baseline levels. The results presented provide novel insights into the mechanisms involved in AT1 receptor regulation and will initiate further investigations dealing with this complicated intracellular mechanism governing the modulation of gene expression.
The insulin-induced AT1 receptor regulation may be of fundamental importance for the treatment of patients suffering from hypertension and hyperinsulinemia, because it favors therapy with AT1 receptor antagonists or ACE inhibitors. These therapeutics reduce AT1 receptor activation and may therefore improve the outcome in these syndromes.
This work was supported by the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Marc Wolff is gratefully appreciated.
- Received February 27, 1998.
- Revision received June 25, 1998.
- Accepted July 16, 1998.
- Copyright © 1998 by American Heart Association
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Sirenko OI, Lofquist AK, De Maria CT, Morris JS, Brewer G, Haskill JS. Adhesion-dependent regulation of an A+U-rich element-binding activity associated with AUF1. Mol Cell Biol. 1997;17:3898–3906.Insulin causes a significant upregulation of AT1 receptor expression in vascular smooth muscle cells, as assessed on the protein and mRNA levels. This effect is mediated through tyrosine phosphorylation–mediated posttranscriptional stabilization of AT1 receptor mRNA rather than through modulation of AT1 receptor gene transcription. These data may clarify the mechanisms involved in the association of hyperinsulinemia with the onset of arteriosclerosis and hypertension, because inducible AT1 receptor overexpression may accelerate both cardiovascular diseases.