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Endocrinology, doi:10.1210/en.2003-0788
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Endocrinology Vol. 144, No. 12 5604-5614
Copyright © 2003 by The Endocrine Society

The Cross-Talk between Angiotensin and Insulin Differentially Affects Phosphatidylinositol 3-Kinase- and Mitogen-Activated Protein Kinase-Mediated Signaling in Rat Heart: Implications for Insulin Resistance

José B. C. Carvalheira, Vivian C. Calegari, Henrique G. Zecchin, Wilson Nadruz, Jr., Regina B. Guimarães, Eliane B. Ribeiro, Kleber G. Franchini, Lício A. Velloso and Mario J. A. Saad

Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo 13081-970, Brazil

Address all correspondence and requests for reprints to: Mario J. A. Saad, M.D., Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, Campinas, São Paulo 13081-970, Brazil. E-mail: msaad{at}fcm.unicamp.br.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin and angiotensin II (AngII) may act through overlapping intracellular pathways to promote cardiac myocyte growth. In this report insulin and AngII signaling, through the phosphatidylinositol 3-kinase (PI 3-kinase) and MAPK pathways, were compared in cardiac tissues of control and obese Zucker rats. AngII induced Janus kinase 2 tyrosine phosphorylation and coimmunoprecipitation with insulin receptor substrate 1 (IRS-1) and IRS-2 as well as an increase in tyrosine phosphorylation of IRS and its association with growth factor receptor-binding protein 2. Simultaneous treatment with both hormones led to marked increases in the associations of IRS-1 and -2 with growth factor receptor-binding protein 2 and in the dual phosphorylation of ERK1/2 compared with the administration of AngII or insulin alone. In contrast, an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity was induced by both hormones. Insulin stimulated the phosphorylation of MAPK equally in lean and obese rats. Conversely, insulin-induced phosphorylation of Akt in heart was decreased in obese rats. Pretreatment with losartan did not change insulin-induced activation of ERK1/2 and attenuated the reduction of Akt phosphorylation in the heart of obese rats. Thus, the imbalance between PI 3-kinase-Akt and MAPK signaling pathways in the heart may play a role in the development of cardiovascular abnormalities observed in insulin-resistant states, such as in obese Zucker rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SIGNALING mechanism of the angiotensin II (AngII) type 1 (AT1) receptor has traditionally been portrayed as driven by heterotrimeric G proteins (1). The AT1 receptor activates phospholipase Cß (PLCß) via G{alpha}q proteins. This causes the generation of inositol triphosphates as well as diacylglycerol, which, in turn, causes the release of Ca2+ from the intracellular Ca2+ storage sites and activation of protein kinase C (PKC), respectively. The AT1 receptor also couples to G{alpha}i, thereby regulating adenylyl cyclase (2). Besides coupling with the heterotrimeric G proteins, activation of tyrosine kinases is intimately involved in AT1 receptor signaling (3, 4). Both nonreceptor type tyrosine kinases [Src, Fyn, Yes, Pyk2, focal adhesion kinase, and Janus kinase 2 (JAK2)] and receptor-type tyrosine kinases (epidermal growth factor and platelet-derived growth factor receptors) are activated by the AT1 receptor (5, 6, 7, 8). These tyrosine kinases regulate downstream signaling mechanisms, including PLCß, Ras-Raf-MAPK kinase (MEK)-ERK, and signal transducer and activator of transcription (STAT) (6, 9, 10), thereby playing a critical role in cardiac hypertrophy responses to AngII (11).

AngII stimulates the tyrosine phosphorylation of insulin receptor substrates (IRS) (12). Considerable evidence indicates that IRS proteins participate in a common pathway for the regulation of antiapoptotic, metabolic, and growth-related signals (13, 14). After tyrosine phosphorylation on YMXM/YXXM motifs, IRSs act as docking proteins for several Src homology 2 domain-containing proteins, including phosphatidylinositol 3-kinase (PI 3-kinase) and growth factor receptor-binding protein 2 (Grb-2) (15, 16). Survival factors implicated in the activation of PI 3-kinase protect cells against undergoing apoptosis, and Akt, a downstream target of PI 3-kinase, was found to be critical for the prevention of cell death and the induction of cardiac hypertrophy (17, 18, 19, 20, 21). Conversely, Grb-2, a 23-kDa adapter protein constitutively associated with the proline-rich domain of SOS (a guanylnucleotide exchange factor for p21ras), once stimulated, interacts with IRS, and this complex activates MAPK signaling pathways (16, 22) that are likely to have a central regulatory position in the signaling hierarchy of cardiac myocyte hypertrophy (23).

In a previous study we demonstrated that in rat heart AngII inhibits both basal and insulin-stimulated PI 3-kinase activities (24). However, an assumption underlying this finding is that AngII and insulin interactions are selective in the development of type 2 diabetes cardiac myopathy. Otherwise, the growth effects of AngII and insulin on cardiac tissues would also be blunted. Thus, it is possible that there is a selective loss of insulin-induced activation of the PI 3-kinase pathway in type 2 diabetes even though the effectiveness in activating the MAPK pathway by AngII and insulin is maintained or increased. To test this possibility, we have characterized the interactions between insulin and AngII in the MAPK and PI 3-kinase-Akt pathways in vivo directly in cardiac tissues from rats. As this cross-talk may influence cardiac abnormalities in insulin resistance states, we also investigated insulin signaling through Akt and ERK1/2 under AT1 receptor blockade in the heart of obese Zucker rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The reagents for SDS-PAGE and immunoblotting were obtained from Bio-Rad Laboratories (Richmond, CA). Tris, phenylmethylsulfonylfluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, affinity-purified rabbit antimouse immunoglobulin G, BSA (fraction V), and AngII were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Losartan was obtained from Merck Pharmaceutical Co. (Wilmington, DE). Sodium amobarbital and human recombinant insulin (Humulin R) were purchased from Eli Lilly & Co. (Indianapolis, IN). Protein A-Sepharose 6MB, [125I]protein A, and nitrocellulose (Hybond ECL, 0.45 µm) were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Antibodies against IRS-1 (SC-559), IRS-2 (SC-8299), JAK2 (SC-294G), ERK (SC-93), Akt (SC-8312), and phosphotyrosine (SC-508) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against the p85 subunit of PI 3-kinase (06-195) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Akt-Ser473 (9271L) and anti-phospho-p44/42 MAPK (9106L) were purchased from Cell Signaling Technology (Beverly, MA). Anti-AngII antiserum was obtained from Peninsula Laboratories (Belmont, CA). Antiphosphoserine antibodies (AB1603) were obtained from Chemicon International (Temecula, CA). Biotin-conjugated secondary antibodies, streptavidin-Cy2, and rhodamine-conjugated phalloidin were purchased from Sigma-Aldrich Corp.

Animals
Twelve-week-old male Wistar rats and 8-, 10-, and 12-wk-old male Zucker rats were allowed access to standard rodent chow and water ad libitum. All experiments involving animals were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and were approved by the ethics committee at the University of Campinas. Room temperature was maintained between 21–23 C, and a 12-h light, 12-h dark cycle was used.

Determination of plasma glucose and serum insulin concentrations
Plasma glucose was measured using the glucose oxidase method in samples collected from the tail. Serum insulin was detected by RIA using a guinea pig antirat insulin antibody and rat insulin as standard (25).

Surgical procedures and tissue preparation
After a 7-h fast, rats were anesthetized with sodium amobarbital (15 mg/kg body weight, ip) and submitted to the surgical procedure as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the cava vein was exposed, and in vivo stimulation of the heart was obtained by injection of 100 µl normal saline (0.9% NaCl), insulin (10-8 M), AngII (10-8 M), or an equimolar mixture of insulin (10-8 M), and AngII (10-8 M) into the cava vein. Some rats received Losartan (10 mg/kg, ip) 30 min before the experiments. Fragments of the hearts were excised in a time-dependent manner. The tissues were minced coarsely, and homogenized immediately in extraction buffer [1% Triton X-100 and 100 mM Tris (pH 7.4) containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.1 mg aprotinin/ml] at 4 C with a Polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Inc., Westbury, NY) operating at maximum speed for 30 sec. The extracts were centrifuged at 30,000 x g at 4 C in a 70.1 Ti rotor (Beckman Coulter, Inc., Palo Alto, CA) for 20 min to remove insoluble material, and the supernatant was used for immunoprecipitation with the indicated antibodies.

Protein analysis by immunoblotting
Equal amounts of proteins were used for total extracts or for immunoprecipitation with specific antibodies, followed by SDS-PAGE and Western blot analysis with the indicated antibodies and 125I as previously described (12, 15, 24). [125I]Protein A bound to the antipeptide antibodies was detected by autoradiography using preflashed Kodak XAR film (Eastman Kodak Co., Rochester, NY) with Cronex Lightning Plus intensifying screens at -80 C for 12–48 h. Quantitative analysis of the blots was performed using Scion Image software (Scion Corp., Frederick, MD).

JAK2 in vitro kinase assay
JAK2 tyrosine kinase activity was measured by autophosphorylation as described previously (16). AngII was infused into the cava vein to stimulate partial JAK2 autophosphorylation. JAK2 was then immunoprecipitated and allowed to autophosphorylate in vitro in the presence of exogenous ATP. Tyrosine phosphorylation was measured by immunoblotting with antiphosphotyrosine antibody.

PI 3-kinase assay
Aliquots of supernatants containing equal amounts of proteins were incubated overnight at 4 C using antibodies against IRS-1, IRS-2, or p85/PI 3-kinase, and the immunocomplexes were precipitated with a 50% solution of protein A-Sepharose 6MB. In vitro PI 3-kinase assays were performed as previously described (15). 32P-Labeled phosphatidylinositol was quantitated by optical densitometry (Scion Image software).

Laser confocal analysis
AngII localization was measured in frozen sections of Zucker rat hearts. Sections (5 µm) were incubated with primary antibody against AngII (1:75), followed by incubation with biotin-conjugated secondary antibodies and then with streptavidin-Cy2 (1:500) and rhodamine-conjugated phalloidin (1:500). Images were obtained with a laser confocal microscope (LSM510, Zeiss, New York, NY). Secondary antibody specificity was tested in a series of positive and negative control measurements.

Data analysis
Where appropriate, the results were expressed as the mean ± SEM accompanied by the indicated number of experiments. Comparisons among groups were made using parametric two-way ANOVA where F ratios were significant; further comparisons were made using the Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ang-induced tyrosine kinase activity of JAK2 leads to phosphorylation of IRSs in rat heart
To determine whether JAK2 kinase activity is stimulated by AngII, we measured enzyme autophosphorylation in vitro. A low dose of AngII (10-12 M) was infused into the cava vein to obtain limited tyrosine phosphorylation of JAK2, which was then immunoprecipitated and reacted with ATP to allow autophosphorylation. The phosphorylation of tyrosine was quantified by Western blot analysis using an antiphosphotyrosine antibody, which showed that angiotensin induces JAK2 autophosphorylation (Fig. 1AGo, lane 4). The band seen in the heart extract previously infused with a low dose of AngII, but with no exogenous ATP added during the in vitro autophosphorylation step (Fig. 1AGo, lane 3), probably represents JAK2 autophosphorylation using endogenous ATP.



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  		FIG. 1. Effect of AngII on JAK2, IRS-1, and IRS-2 tyrosine phosphorylation and IRS-1 and IRS-2 associations with JAK2 in the hearts of Wistar rats in vivo. A, Upper panel, JAK2 tyrosine kinase activity was measured by autophosphorylation, as described in Materials and Methods, 90 sec after injection of a submaximal dose of AngII in the absence or presence of 15 µM ATP (n = 4). A, Lower panel, Heart extracts from animals treated with AngII for the times indicated were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with anti-JAK2 antibody (IP, immunoprecipitation) and immunoblotted with antiphosphotyrosine antibody (P-Tyr; n = 5). B, Pretreatment with either vehicle or losartan was followed 30 min later by iv administration of vehicle or AngII for 5 min; tissue extracts were then immunoprecipitated with anti-JAK2 antibody and blotted with antiphosphotyrosine antibodies (n = 4). C, Tissue extracts from rats treated with AngII for the times indicated were immunoprecipitated with anti-JAK2 and blotted with anti-IRS-1 (upper panel) and with anti-IRS-2 (lower panel) or antiphosphotyrosine (D; n = 4). Tissue extracts from rats treated with AngII for the times indicated were immunoprecipitated with anti-IRS-1 (E) or IRS-2 (F) and blotted with antiphosphotyrosine (upper panel) or antiphosphoserine (P-Ser) antibody (lower panel; n = 4).

 
To determine AngII-induced tyrosine phosphorylation of JAK2, we performed immunoprecipitation and Western blotting of heart extracts with anti-JAK2 and antiphosphotyrosine antibodies, respectively. The time-course experiments were performed by injecting 10-8 M AngII through the cava vein of rats and collecting heart specimens at different time points. As shown in Fig. 1AGo (lower panel), JAK2 was tyrosine-phosphorylated after 3 min of AngII treatment and continued to increase during the time frame studied. The amount of JAK2 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-JAK2 antibody (data not shown).

Pharmacological inhibitors of AT1 receptor activity, such as losartan, can block AngII activity in vivo (26), suggesting that AT1-dependent signaling is needed for most of the intracellular responses to AngII. We tested whether the stimulatory effects of AngII on JAK2 depend on AT1, by previous ip infusion of losartan into rats (30 min before AngII). Treatment with AngII markedly increased tyrosine phosphorylation of JAK2 (Fig. 1BGo, lane 3), but no AngII-induced JAK2 tyrosine phosphorylation was detected after pretreatment with losartan (Fig. 1BGo, lane 4).

As AngII activated JAK2, we evaluated the ability of AngII to stimulate JAK2/IRS-1 and JAK2/IRS-2 associations. Coimmunoprecipitations between JAK2 and IRS-1 or IRS-2 in heart were detected. In immunoprecipitates of JAK2 blotted with anti-IRS-1 or anti-IRS-2 antibodies, there was an evident association between these proteins after AngII stimulation. These results demonstrate that JAK2 interacts with IRS-1 and IRS-2, developing stable complexes in rat heart (Fig. 1CGo). The amount of JAK2 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-JAK2 antibody (data not shown).

The association between JAK2/IRS-1 and JAK2/IRS-2 after AngII treatment was transient, whereas the tyrosine phosphorylation of JAK2 lasted up to 10 min. To evaluate the association kinetics between JAK2 and IRSs, we reblotted the nitrocellulose membrane previously immunoprecipitated with JAK2 with antiphosphotyrosine antibody. AngII stimulated the tyrosine phosphorylation of a prominent band migrating between 120 and 140 kDa that corresponds to JAK2 and of a faint band of 170 kDa that corresponds to IRSs (Fig. 1DGo). By reblotting the membranes with anti-JAK2, anti-IRS-1, and anti-IRS-2, we confirmed the identity of these bands (data not shown). The band of IRSs demonstrated a different time course of tyrosine phosphorylation compared with the JAK2 band, suggesting that the JAK2/IRSs complex is dissociated after the dephosphorylation of IRSs.

Figure 1EGo (upper panel) shows a clear increase in AngII-stimulated IRS-1 phosphorylation, which was maximal at 5 min and almost vanished after 10 min. Figure 1EGo (lower panel) shows that IRS-1 serine phosphorylation could be observed as early as 1 min after AngII stimulation and lasted up to 10 min. The amount of IRS-1 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-IRS-1 antibody (data not shown). To better define the levels of IRS-2 phosphorylation, we performed Western blot analysis of tyrosyl-phosphorylated proteins in anti-IRS-2 immunoprecipitates before and after stimulation with AngII. Analogously, Fig. 1FGo shows that there was a marked increase in AngII-stimulated IRS-2 tyrosine phosphorylation in the rat heart, which paralleled IRS-1 tyrosine phosphorylation. As shown in Fig. 1FGo (lower panel), AngII stimulated IRS-2 serine phosphorylation in the same fashion as it did IRS-1. The amount of IRS-2 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-IRS-2 antibody (data not shown).

AngII stimulates IRS association with Grb2
To explore AngII signaling to the mitogenic pathways, rats were injected with AngII, and early steps in signal transduction were assessed using heart extracts. Immunoprecipitation of these extracts with anti-Grb2 antisera, followed by immunoblotting with anti-IRS-1 and anti-IRS-2 antibodies, revealed a rapid association of Grb2/IRS-1 and Grb2/IRS-2 (Fig. 2AGo), following a time-dependent pattern. The amount of Grb2 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-Grb2 antibody (data not shown). Using losartan, we tested the hypothesis that treatment with an AT1 inhibitor prevents the tyrosine phosphorylation of IRS-1/2 and their association with Grb2 induced by iv administration of AngII. As shown in Fig. 2Go, B and C (lane 4), losartan prevented AngII-induced tyrosine phosphorylation of IRS-1 and -2 in rat heart.



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  		FIG. 2. Effect of AngII on Grb2 association with IRS-1 and -2 in Wistar rat heart. A, AngII-stimulated IRS/Grb2 associations. Tissue extracts from rats treated with AngII for the times indicated were immunoprecipitated (IP) with anti-Grb2 and blotted with anti-IRS-1 (upper panel) and anti-IRS-2 (lower panel; n = 4). B and C, Pretreatment with either vehicle or losartan was followed 30 min later by iv administration of vehicle or AngII for 5 min. The heart was then lysed, and the proteins were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies, separated by SDS-PAGE on 8% gels, and blotted with antiphosphotyrosine antibodies (upper panel) and anti-Grb2 antibodies (lower panels; n = 4).

 
Effect of simultaneous administration of AngII and insulin on IRS association with Grb2 and on ERK1/2 dual phosphorylation
The association of IRS-1 with Grb2 was first measured in the hearts of rats that were stimulated with AngII, insulin, or both hormones for 5 min. The association of IRS-1/Grb2 in the heart increased by approximately 3.1-fold with AngII stimulation and by approximately 2.8-fold with insulin. AngII and insulin together provided an approximately 5.1-fold increase in IRS-1/Grb2 association, representing an increase of about 160% compared with the effects of AngII alone (Fig. 3AGo). The amount of IRS-1 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-IRS-1 antibody (data not shown). AngII- and/or insulin-stimulated IRS-2/Grb2 association was then evaluated in the hearts of rats that were stimulated with AngII, insulin, or both hormones for 5 min. AngII-stimulated Grb2 association with IRS-2 increased by approximately 2.7-fold compared with basal levels, whereas insulin led to an approximately 2.4-fold increase in IRS-2/Grb2 association. Analogously, simultaneous stimulation with AngII and insulin promoted an additive effect on IRS-2/Grb2 association (Fig. 3BGo). The amount of IRS-2 immunoprecipitated was similar, as confirmed by reblotting the same membrane with anti-IRS-2 antibody (data not shown). Finally, we assessed ERK1/2 dual phosphorylation in the hearts of rats stimulated with AngII, insulin, or both hormones. AngII infusion induced an approximately 3-fold increase in phosphoactive ERK at 3 and 5 min. Similarly, insulin infusion also led to an increase in the dual phosphorylation of ERK1/2 by about 3-fold at 3 and 5 min. The simultaneous infusion of both hormones showed an additive effect with a significant increase in phosphoactive ERK at 3 and 5 min, representing increases of 1.7- and 1.5-fold (P < 0.05), respectively, compared with the effects of AngII alone (Fig. 3CGo). As expected, the protein expression of ERK1/2 did not change after the infusion of insulin, AngII, or both hormones (Fig. 3DGo).



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  		FIG. 3. Effects of AngII and insulin on Grb2 association with IRS-1/2 and dual phosphorylation of ERK in extracts of hearts from rats treated with AngII, insulin, or their combination. Tissue extracts from rats treated with saline, AngII, insulin, or both hormones were immunoprecipitated (IP) with anti-IRS-1 (A) or anti-IRS-2 (B) and blotted with anti-Grb2 antibody. The bar graph shows the quantitative association of IRS-1/Grb2. Data (mean ± SEM; n = 4) are expressed relative to the control. *, P < 0.05 vs. AngII-treated rats. C, Western blots of heart extracts from rats treated with saline, AngII, insulin, or both hormones for 3 and 5 min with dually phosphorylated ERK1/2 antibodies. The bar graph shows the quantitative phosphoactivity of ERK. Data (mean ± SEM; n = 4) are expressed relative to the control. *, P < 0.05 vs. AngII-treated rats.

 
Effect of simultaneous administration of AngII and insulin on PI 3-kinase activation and Akt serine phosphorylation
We next examined whether simultaneous stimulation with AngII and insulin could affect the PI 3-kinase-Akt pathway. IRS-1/2-associated PI 3-kinase activity was measured in the hearts of rats that were stimulated with AngII, insulin, or both hormones for 5 min. Quantitation of multiple experiments revealed that in accordance with our previous studies (24), after AngII injection alone there was an inhibition of PI 3-kinase activity associated with IRS-1 and IRS-2, with decreases of 50% and 60% (P < 0.05) compared with basal values, respectively (Fig. 4Go, A and B, lane 2). Conversely, upon insulin treatment alone, 3.2-fold (P < 0.05) and 3.4-fold (P < 0.05) increases in IRS-1- and IRS-2-associated PI 3-kinase activities occurred in cardiac muscle, respectively (Fig. 4Go, A and B, lane 3). AngII and insulin together resulted in approximately 1.6- and 1.8-fold increases in IRS-1- and IRS-2-associated PI 3-kinase activities in cardiac muscle, respectively, representing increases approximately 50% and 45%, respectively, lower than those produced by insulin alone (Fig. 4Go, A and B, lane 4). AngII alone had no effect on Akt serine phosphorylation in heart. In contrast, insulin induced an increase in Akt serine phosphorylation by about 9-fold at 3 and 5 min. Simultaneous infusion of both hormones showed an antagonistic effect, leading to approximately 40% and 50% lower serine phosphorylation levels, respectively, than those produced by insulin alone (Fig. 4CGo). As expected, protein expression of Akt did not change after the infusion of insulin, AngII, or both hormones (Fig. 4DGo).



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  		FIG. 4. Effects of AngII and insulin on the PI 3-kinase pathway in extracts of hearts from rats treated with AngII, insulin, or their combination. A and B, Rats were injected with the hormones as described, and the heart was extracted and immunoprecipitated overnight using antibodies against IRS-1 and IRS-2. PI 3-kinase assays were performed as previously described (15 ). Fluorographs show the silica thin layer chromatographic plates of IRS-1- and IRS- 2-associated PI 3-kinase activity. PIP3, Migration position of phosphatidylinositol 3-phosphate. The bar graphs show the relative 32P incorporation into PIP3, as determined by Scion Image quantitation. Data (mean ± SEM; n = 4) are expressed relative to the control. *, P < 0.05 vs. AngII- and insulin-treated rats. C, Western blots of heart extracts from rats treated with saline, AngII, insulin, or both hormones for 3 and 5 min with phosphoserine-specific Akt antibodies. The bar graph shows the quantitative phosphorylation of Akt. Data (mean ± SEM; n = 4) are expressed relative to the control. *, P < 0.05 vs. AngII- and insulin-treated rats.

 
Characteristics of lean and obese Zucker rats
As shown in Table 1Go, obese rats had higher body weight, plasma glucose, and fasting serum insulin levels than their age-matched lean controls.


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  		TABLE 1. Characteristics of obese Zucker (fa/fa) rats and their age-matched lean controls

 
AngII expression in heart of Zucker rats
Myocardial fragments, frozen immediately after dissection, were used for the immunocytochemical localization of AngII in myocytes and endothelial cells (Fig. 5Go). Myocytes were stained by antiphalloidin to outline cell profiles. A much more intense staining of AngII was observed in heart sections of Zucker obese rats compared with controls. In both cases AngII staining predominated in the interstitial space (Fig. 5Go). Preabsorption of the primary antibody with AngII resulted in the lack of labeling in Zucker obese rat left ventricle (data not shown).



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  		FIG. 5. Laser confocal analysis of sections of frozen hearts of lean (A) and fat (B) Zucker rats double-labeled with phalloidin (red staining) and anti-AngII (green staining) antibodies.

 
In vivo effect of insulin on early steps of insulin signaling in hearts of Zucker rats
To further explore the interaction between AngII and insulin signaling pathways in rat heart, we evaluated the effect of insulin on insulin signaling activation in fa/fa rats, a known model of hyperinsulinemia and insulin resistance. The effect of in vivo insulin on tyrosine phosphorylation of insulin receptor (IR) was examined in the heart of Zucker lean (Fa/?) and obese (fa/fa) rats. Hearts from insulin- or vehicle-treated rats were submitted to immunoprecipitation with antibody to IR and immunoblotted with antiphosphotyrosine antibody. As shown in Fig. 6AGo, insulin induced increases in IR tyrosine phosphorylation in the hearts of both lean and obese rats. In lean animals the increment was 4.5-fold above the basal level, whereas it was only 1.9-fold in obese rats. Pretreatment with losartan had no effect on IR tyrosine phosphorylation. To determine whether this approximately 75% reduction in IR tyrosine phosphorylation in obese rats was associated with a diminished amount of IRß proteins, immunoprecipitation with anti-IRß was performed in cardiac extracts of lean and obese rats, and immunoblotting was conducted with the same antibody. As shown in Fig. 6BGo, protein levels of IR were not different between lean and obese rats.



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  		FIG. 6. IR, IRS-1 and IRS-2 protein expression, tyrosine phosphorylation, and their association with the p85 subunit of PI 3-kinase and Grb2 in the hearts of lean (L) and obese (F) Zucker rats. A, Insulin-induced IR tyrosine phosphorylation. Pretreatment with losartan or vehicle was followed 30 min later by iv injection of insulin or vehicle. Tissue extracts were immunoprecipitated with anti-IR antibody and immunoblotted with antiphosphotyrosine antibody (pY; n = 6). B, Protein levels of IR. Immunoprecipitation (IP) was conducted with anti-IR and immunoblotted with the same antibody (n = 5). C and D, Insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Pretreatment with losartan or vehicle was followed 30 min later by iv injection of insulin or vehicle. Tissue extracts were immunoprecipitated with anti-IRS-1 or anti-IRS-2 antibodies and blotted with antiphosphotyrosine antibody (pY; n = 5). E and F, The same membranes used for IRS-1 and IRS-2 tyrosine phosphorylation were stripped and reblotted with anti-PI 3-kinase antibodies (n = 5). G and H, The same membranes used for IRS-1 and IRS-2 tyrosine phosphorylation were stripped and reblotted with anti-Grb2 antibodies (n = 5). I and J, Protein levels of IRS-1 and IRS-2. Equal amounts of protein were subjected to immunoprecipitation (IP) with {alpha}IRS-1 or {alpha}IRS-2 and immunoblotted with the same antibody (n = 5). Data are expressed as the mean ± SEM relative to the control, assigning a value of 100% to the lean control. #, P < 0.05, obese vs. respective lean; *, P < 0.05, insulin-plus losartan-treated obese vs. insulin-treated obese.

 
Insulin-stimulated IRS phosphorylation, quantitated by immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies and immunoblotting with {alpha}pY antibody, showed increases of 5.4- and 5.0-fold in the tyrosine phosphorylation of IRS-1 and IRS-2, respectively, in the hearts of lean rats (Fig. 6Go, C and D) compared with increases of 2.7- and 2.3-fold, respectively, in the hearts of obese rats (P < 0.05), representing an approximately 60% lower effect in the obese rats. Pretreatment with losartan partially restored the tyrosine phosphorylation of IRS-1/2 in obese Zucker rats, whereas no effect was observed in lean rats. The same membranes as those used to detect tyrosine phosphorylation of IRS-1 and IRS-2 were reblotted with antibodies against the p85 subunit of PI 3-kinase. In the lean rats, insulin led to increases of 4.9- and 5.3-fold in p85 association to IRS-1 and IRS-2, respectively (Fig. 6Go, E and F), whereas only 2.0- and 1.7-fold increases were observed in the hearts of obese rats, respectively, representing reductions of about 80% in these animals (P < 0.05). Pretreatment with losartan partially restored the association of p85 with IRS-1/2 in obese Zucker rats, whereas no effect was observed in lean rats. To examine the IRS association with Grb2 we reblotted the same membranes used to detect tyrosine phosphorylation of IRS-1 an IRS-2 with antibodies against Grb2. In the lean rats, insulin led to increases of 3.9- and 4.2-fold in Grb2 association with IRS-1 and IRS-2, respectively (Fig. 6Go, G and H), whereas 3.6- and 3.8-fold increases were observed in the hearts of obese rats. Pretreatment with losartan had no effect on IRS/Grb2. The protein expression of IRS-1 and IRS-2 in the hearts of control and obese rats were quantitated by immunoprecipitation and immunoblotting with {alpha}IRS-1 or {alpha}IRS-2 antibodies, and decreases of 33% and 24%, respectively, were detected in the hearts of obese rats compared with lean controls (Fig. 6Go, I and J).

Insulin activation of ERK1/2 and Akt in hearts of Zucker rats
Using antibodies against dually phosphorylated ERK1/2, the phosphoactive ERK1/2 was examined in heart after insulin stimulation. As shown in Fig. 7AGo, insulin stimulated dual phosphorylation of ERK1/2 equally in the hearts of lean and obese rats. Insulin induced a 3.2-fold increase in phosphoactive ERK1/2 in the hearts of lean rats and a 3.8-fold increase in the hearts of obese rats, whereas pretreatment with losartan had no effect on basal (data not shown) or insulin-induced dual phosphorylation of ERK1/2 in the hearts of rats. During in vivo experiments, there was a 10.1-fold increase in insulin-induced serine phosphorylation of Akt in the hearts of lean rats (Fig. 7BGo) compared with an increase of 5.6-fold in the hearts of obese rats, representing an approximately 50% lesser effect on Akt phosphorylation in obese rats. Pretreatment with losartan partially restored insulin-induced serine phosphorylation of Akt in the hearts of obese rats, whereas no effect was observed in lean animals (P < 0.05).



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  		FIG. 7. Insulin-induced phosphoactivation of ERK and Akt in lean (L) and obese (F) Zucker rats. A, Pretreatment with either vehicle or losartan was followed 30 min later by iv administration of insulin for 5 min. The hearts were then excised and lysed, and total protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-pERK1/2 antibodies. The bar graph shows the quantitative phosphoactivation of ERK. Data (mean ± SEM; n = 5) are expressed relative to the control. *, P < 0.05 vs. insulin-treated obese rats. B, Pretreatment with either vehicle or losartan was followed 30 min later by iv administration of insulin for 5 min. The hearts were then excised and lysed, and total protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-p-Akt antibodies. The bar graph shows the quantitative phosphorylation of Akt. Data (mean ± SEM; n = 5) are expressed relative to the control. *, P < 0.05 vs. insulin-treated obese rats.

 
To determine whether alterations in signaling are primary in the development of cardiovascular complications or represent events secondary to the altered metabolic milieu in the obese Zucker rats, we investigated obese Zucker rats at different ages, from 8–12 wk. Figure 7CGo shows no difference in insulin-induced, dually phosphorylated ERK between lean and fat Zucker rats 8, 10, and 12 wk of age, without changes in ERK1/2 expression. On the other hand, there was a progressive decrease in insulin-induced Akt serine phosphorylation in obese Zucker rats 10 and 12 wk of age, whereas no changes were observed in lean rats. The protein expression of Akt did not change in obese Zucker rats from 8–12 wk of age.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we investigated the possibility of direct interactions between AngII and insulin signaling transduction in heart, focusing on some key intermediate steps within these signaling pathways. Our results show that AngII, upon stimulation of JAK2 activity, induced JAK2 coimmunoprecipitation with IRS-1 and IRS-2. These phenomena parallel the AngII-induced tyrosine phosphorylation of IRS-1 and IRS-2. AngII as well as insulin stimulated a moderate increase in the associations of IRS-1/2 with Grb2 in heart, but stimulation with both hormones resulted in a moderate increase in the association of IRS-1/2 with Grb2 compared with isolated administration of AngII or insulin. As previously described, AngII induces an increase in IRS/PI 3-kinase association (12); however, the AngII-induced PI 3-kinase activity associated with this substrate is reduced (24). In contrast, insulin stimulation resulted in a striking increase in activation of the IRS/PI 3-kinase pathways. Whereas there was a clear reduction in maximal phosphorylation of Akt, simultaneous stimulation with both hormones resulted in a marked increase in the dual phosphorylation of ERK1/2 compared with isolated administration of AngII or insulin. Thus, we have demonstrated a selective impairment of insulin activation of the PI 3-kinase-Akt pathway and a potentialized activation of the ERK pathway after simultaneous administration of both hormones.

In cell culture the members of the JAK family of intracellular tyrosine kinases are rapidly phosphorylated on tyrosine and activated in response to AngII, and it is believed that these are some of the kinases responsible for activation of the phosphotyrosine signaling pathway by AngII. However, the relevance of these findings in the intact animal is uncertain. We herein demonstrate that AngII induces JAK2 tyrosine kinase activity in vivo in rat heart. The rapid tyrosine phosphorylation and association of JAK2 with the IRS-1 and IRS-2 proteins suggest that a large signaling complex is formed with the AT1 upon AngII treatment. Coimmunoprecipitation of JAK2 and IRSs could be due to the direct association of JAK2 and IRSs with the AT1 or indirect association of IRSs with JAK2. There are several mechanisms by which JAK2 and IRSs may associate with AT1. One possibility is that JAK2 initially associates with the AT1 and leads to the recruitment of IRSs proteins. A second is that AT1 recruits IRSs proteins, which serve as adapter molecules for binding JAK2. Furthermore, both JAK2 and IRSs associate with the receptor, and upon ligand binding to the receptor, JAK2 phosphorylates the associated IRSs.

Currently, it is thought that AT1 receptor ligand binding causes activation of ERKs through three distinct signaling pathways. One pathway is via the G{alpha}q-PKC-dependent mechanism, another is via the Src-Ras-dependent mechanism, and the third pathway is by trans-activation of the epidermal growth factor receptor (7, 27). In the present study we provide evidence that AngII induces IRS-1/Grb2 and IRS-2/Grb2 associations in a time-dependent fashion; this phenomenon occurs in parallel to IRS tyrosine phosphorylation. As Grb2 occupies a central regulatory position in the activation of the Ras-Raf-MEK-ERK pathway (28), it is reasonable to speculate that the IRS/Grb2 association after AngII treatment may be one additional mechanism by which AT1 activates ERK.

Intracellular interactions between different signaling systems may function as mechanisms for enhancing or counterregulating hormone action. In the case of AngII, the cross-talk with insulin-mediated pathways resulted in positive interactions between AngII and insulin signaling systems at the level of IRS-Grb2 association and ERK. Simultaneous stimulation with both hormones led to increased dually phosphorylated ERK and the association of IRS-1 and IRS-2 with Grb2. In contrast, a reduction of PI 3-kinase activity and Akt serine phosphorylation was observed compared with that after acute insulin administration. The precise reason for this difference is not known; however, possibilities include differential sites of serine and tyrosine phosphorylation on IRS-1 and IRS-2 proteins after AngII plus insulin treatment leading to an impairment of the PI 3-kinase pathway (29, 30), whereas a permissive activation of the IRS-1/Grb2 pathway is maintained. At least part of this hypothesis could be confirmed in our study, as we demonstrated that AngII induces an increase in the serine phosphorylation of IRSs. Another possible, but not excluding, reason for this divergence is a differential AngII/insulin signal amplification through ERK. ERK could be activated either by AngII through the AT1, in an IRS-independent fashion (7, 27), or through the IR by insulin (14).

Cardiomyocyte hypertrophy is dependent on rates of protein synthesis. Cardiomyocytes are regarded as terminally differentiated cells in which adaptive hypertrophic growth involves increases in protein content and cell size and changes in myofibrillar organization and gene expression (31). AngII exerts hypertrophic effects and stimulates protein synthesis in cardiomyocytes (11, 32). Increasing evidence suggests that these phenomena require Ras and MAPK signaling, in particular the MEK/ERK cascade (11, 23, 32, 33). Our results indicate a direct and positive cross-talk between AngII and insulin at the level of dually phosphorylated ERK. This mechanism may serve to potentiate the activity of both AngII and insulin pathways and may be involved in pathological processes, such as the development of cardiac hypertrophy in hyperinsulinemic states.

As the heart is an insulin-responsive tissue (34), AngII-induced insulin resistance may play an important role in the etiology of cardiovascular disease associated with hypertension and type 2 diabetes mellitus, probably through the PI 3-kinase/Akt pathway. Although the specific role of insulin-stimulated PI 3-kinase in the heart has not yet been established, a recent study suggests that this pathway is important for orchestrating putative cell survival-signaling events of the myocardium during reperfusion (35). Thus, the negative cross-talk between AngII and insulin on the PI 3-kinase-Akt pathway may play a role in the development of diabetic cardiac myopathy apoptosis.

Our results show that AngII expression is increased in the hearts of Zucker obese rats. Despite the fact that the mechanism activating the local renin-angiotensin system (RAS) has not been identified, the cross-talk between AngII and insulin may also have a role in the pathogenesis of cardiac hypertrophy in some situations of insulin resistance. In obese Zucker rats, which demonstrate hyperinsulinemia and activation of RAS (36), we found that insulin-induced activation of the IR/Akt pathway in heart was decreased compared with that in lean rats, and that pretreatment with losartan attenuated the reduction of IRS-1/2 tyrosine phosphorylation and of Akt serine phosphorylation, suggesting a role for RAS in the reduction of the IRS/Akt pathway observed in the hearts of obese Zucker rats. The reduction in IRS-1 protein levels in hearts of obese rats may also contribute to the defects in insulin signaling observed in these animals.

The profound insulin resistance to the IRS/Akt pathway contrasts markedly with the ability of insulin to stimulate both the association between IRSs and Grb2 and the dual phosphorylation of ERK1/2 in the hearts of obese rats. Pretreatment with losartan had no effect on insulin-induced IRS/Grb2 association or dually phosphorylated ERK1/2 in the heart of the obese Zucker rat. These data suggest that AngII and insulin signals seem to be independent and additive for ERK1/2 stimulation in the hearts of Zucker rats.

Our results show that Akt serine phosphorylation is progressively reduced as rats become increasingly obese, indicating that the altered metabolic milieu observed in obese rats may account for the alterations observed in insulin signaling. Recently, it was shown that triglycerides accumulate rapidly in the hearts of Zucker obese rats, as they do in other nonadipose tissues, as the animals become obese (37); these alterations may contribute to the impaired insulin signaling that we observed in Zucker obese rats.

In summary, our results show that IRS-1 and IRS-2 can bind to Grb2 after AngII administration, suggesting a new link between AT1 and the MAPK cascade. The results also demonstrate that after simultaneous administration of AngII and insulin, the PI 3-kinase pathway appears to be selectively blunted in heart tissues, compared with MAPK activity. In addition, in hearts of obese Zucker rats, losartan improves the reduced insulin-induced IRS and Akt phosphorylation and has no effect on the dual phosphorylation of ERK. Thus, the imbalance between PI 3-kinase-Akt and the MAPK signaling pathways in the heart may have a role in the development of cardiovascular abnormalities observed in insulin-resistant states, as in obese Zucker rats.


   Acknowledgments
 
We thank Mr. Luiz Janeri for technical assistance.


   Footnotes
 
This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico.

J.B.C.C. and V.C.C. contributed equally to this work.

Abbreviations: AngII, Angiotensin II; AT1, angiotensin II type 1 receptor; Grb2, growth factor receptor-binding protein 2; IR, insulin receptor; IRS, insulin receptor substrate; JAK, Janus kinase; MEK, MAPK kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLCß, phospholipase Cß; RAS, renin-angiotensin system; STAT, signal transducer and activator of transcription.

Received June 25, 2003.

Accepted for publication August 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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