Suppressor of Cytokine Signaling-3 Provides a Novel Interface in the Cross-Talk between Angiotensin II and Insulin Signaling Systems
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内分泌学杂志 2005年第2期
Department of Internal Medicine, State University of Campinas, 13081-970 Campinas S?o Paulo, Brazil
Address all correspondence and requests for reprints to: Lício A. Velloso, Departamento de Clínica Médica, Faculdade de Ciencias Medicas–Universidade Estadual de Campinas, 13081-970 Campinas S?o Paulo, Brazil. E-mail: lavelloso@fcm.unicamp.br.
Abstract
Angiotensin II inhibits insulin-induced activation of phosphatidylinositol 3-kinase through a mechanism, at least in part, dependent on serine phosphorylation of the insulin receptor and insulin receptor substrates (IRS)-1/2. Recent evidence shows that suppressor of cytokine signaling-3 (SOCS-3) is induced by insulin and angiotensin II and participates in the negative control of further stimulation of each of these signaling systems independently. In the present study, we evaluated the interaction of angiotensin II-induced SOCS-3 with the insulin signaling pathway in the heart of living rats. A single iv dose of angiotensin II promotes a significant increase of SOCS-3 in heart, an effect that lasts up to 180 min. Once induced, SOCS-3 interacts with the insulin receptor, JAK-2, IRS-1, and IRS-2. The inhibition of SOCS-3 expression by a phosphorthioate-modified antisense oligonucleotide partially restores angiotensin II-induced inhibition of insulin-induced insulin receptor, IRS-1 and IRS-2 tyrosine phosphorylation, and IRS-1 and IRS-2 association with p85-phosphatidylinositol 3-kinase and [Ser473] phosphorylation of Akt. Moreover, the inhibition of SOCS-3 expression partially reverses angiotensin II-induced inhibition of insulin-stimulated glucose transporter-4 translocation to the cell membrane. These results are reproduced in isolated cardiomyocytes. Thus, SOCS-3 participates, as a late event, in the negative cross-talk between angiotensin II and insulin, producing an inhibitory effect on insulin-induced glucose transporter-4 translocation.
Introduction
THE RECIPROCAL SIGNAL transduction modulation that occurs between the insulin and angiotensin II (Ang II) signaling pathways is thought to play an important role in the common clinical association between diabetes mellitus and hypertension (1, 2, 3, 4, 5, 6). The cross-talk between these two systems occurs at distinct levels, beginning at the prereceptor level, as modulation of angiotensin-converting enzyme (ACE) activity leads to simultaneous control of Ang II production and insulin action (7). It then acts at an early postreceptor level because janus kinase (JAK)-2 activity may be modulated by insulin and Ang II (2, 8, 9), and finally it reaches an intermediary postreceptor level because serine kinases activated by Ang II inhibit insulin signal transduction (3). This redundancy may have developed during evolution as a need for tight control of two vital and closely related systems, i.e. the circulatory and metabolic systems.
In recent years, a series of studies have shown that insulin is capable of inducing the expression of suppressor of cytokine signaling (SOCS)-3 (10, 11, 12), a 32-kDa protein that belongs to a family of proteins (SOCS family) originally identified as inducible inhibitors of signal transduction by gp130 cytokines (13, 14). When cotransfected with insulin receptor (IR) and insulin receptor substrate (IRS)-1, SOCS-3 inhibits insulin signal transduction by promoting a reduction of insulin-induced IRS-1 tyrosine phosphorylation (12). Moreover, SOCS-3 may reduce insulin signal transduction by targeting IRS-1 and IRS-2 to proteasomic degradation (15). Recently we demonstrated that Ang II is also capable of inducing SOCS-3 in heart, isolated cardiomyocytes, and the hypothalamus of rats (16, 17). Ang II-induced SOCS-3 inhibits Ang II-stimulated c-jun expression (16) and participates in the mechanisms that lead to refractoriness to Ang II in hypothalamus (17).
In cytokine signaling, proteins of the SOCS family may provide a negative cross-talk between two distinct systems, such as IL-3 and IL-11 (18) and interferon- and IL-4 (19) signaling systems. In addition, SOCS proteins may participate in the cross-talk between a cytokine and hormone signaling pathways, such as IL-1? and GH (20). In the present study, we evaluated whether SOCS-3, induced by Ang II in the heart, interacts with elements of the insulin signaling pathway and exerts any regulatory effect on this system.
Materials and Methods
Antibodies and chemicals
Reagents for SDS-PAGE and immunoblotting were obtained from Bio-Rad Laboratories (Richmond, CA). HEPES, phenylmethylsulfonylfluoride, aprotinin, dithiotreitol, Triton X-100, Tween 20, glycerol, Ang II, and BSA (fraction V) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Protein A-Sepharose 6 MB was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), [125I]-protein A and nitrocellulose membranes were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Phosphatidylinositol was from Avanti Polar Lipids (Alabaster, AL). Chemicals, culture media, Lipofectamine Plus, and sera for isolation and culture of cells were purchased from Life Technologies Inc. (Grand Island, NY). Antibodies against SOCS-3 (sc-9023, rabbit polyclonal and sc-7009, goat polyclonal), SOCS-1 (sc-9021, rabbit polyclonal), JAK-2 (sc-278, rabbit polyclonal), IR? (sc-711, rabbit polyclonal), IRS-1 (sc-559, rabbit polyclonal), IRS-2 (sc-8299, rabbit polyclonal), signal transducer and activator of transcription (STAT)-5b (sc-1656, mouse monoclonal), p85-phosphatidylinositol 3-kinase (PI 3-kinase) (sc-423, rabbit polyclonal), glucose transporter (GLUT)-4 (sc-7938, rabbit polyclonal), and phosphotyrosine (sc-508, mouse monoclonal) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-[Ser473]Akt (9271, rabbit polyclonal) was purchased from Cell Signaling Technology (Beverly, MA). Antiphosphoserine antibody (AB1603, rabbit polyclonal) was purchased from Chemicon Inc. (Temecula, CA). Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies and rhodamine-conjugated phalloidin were purchased from Sigma-Aldrich Corp. Phosphorthioate-modified oligodeoxynucleotides for SOCS-3 (sense, 5'-CAT GGT CAC CCA CAG-3'; antisense, 5'-CTG TGG GTG ACC ATG-3') were synthesized by Life Technologies, Inc. and were previously tested and optimized for in vivo use (16). Sodium amorbital and insulin were obtained from Eli Lilly & Co. (Indianapolis, IN).
Experimental animals
Eight-week-old male Wistar rats from the university’s Central Animal Breeding Center were used in the experiments. The adult rats were allowed access to standard rodent chow and water ad libitum. Food was withdrawn 12 h before the experiments. All experiments were conducted in accord with the principles and procedures described by National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the State University of Campinas ethical committee. For neonatal rat ventricular myocyte (NRVM) preparation, 1- to 3-d-old Wistar rats were employed.
Preparation of NRVM
Ventricular cardiac myocytes were prepared by enzymatic disaggregation following a method previously described (21). After separation the cells were cultured in DMEM at a concentration of 6.0 x 104/cm2 on plates coated with gelatin. The purity of the cell preparations was ascertained by immunofluorescence using tetramethylrhodamine isothiocyanate-phalloidin and evaluation by confocal microscopy.
Hormone stimulation protocols and tissue preparation
Rats were anesthetized by an ip injection of sodium amorbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes. The abdominal cavity was opened, the cava vein was exposed, and in vivostimulation of the heart was obtained by injection of 0.02 ml saline (0.9% NaCl), 10–8 M Ang II, and/or 10–6 M insulin into the cava vein. In some of the time-course experiments the hearts of rats treated with respective hormones were compared with the hearts of rats treated with only saline for similar periods of time. In some cases rats received 6.0 nmol [diluted in 200 μl Tris/EDTA buffer] sense or antisense SOCS-3 oligonucleotide ip 3 h before the experiments. At various time intervals (as shown in Results), the tips of the ventricles were excised and immediately homogenized in approximately five volumes of solubilization buffer at 4 C [1% Triton X-100, 100 mM Tris-HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2.0 mM phenylmethylsulfonylfluoride, and 0.1 mg aprotinin/ml] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 sec.
NRVMs were exposed to oligonucleotides, saline, insulin, and Ang II according to doses and times as stated in Results and following a protocol previously described (16).
Protein analyses by immunoprecipitation and immunoblotting
Insoluble material was removed by centrifugation for 25 min at 11,000 rpm in a 70.Ti rotor (Beckman, Fullerton, CA) at 4 C. The protein concentrations in supernatants were determined by the Bradford dye-binding method (22). Aliquots of the resulting supernatants containing 5.0 mg total protein were used for immunoprecipitation with anti-JAK-2, IR?, IRS-1, IRS-2, STAT-5b, SOCS-1, and SOCS-3 antibodies following a method previously described (23). Unspecific binding of proteins to beads were always tested and observed to be insignificant (not shown). Band intensities were quantified by digital densitometry (Scion Image software, Scion Corp., Frederick, MD) of the developed autoradiographs. For simple immunoblot experiments (not preceded by immunoprecipitation), 0.2 mg total protein from heart was separated by SDS-PAGE (10 or 12% bis-acrylamide), transferred to nitrocellulose membranes, and blotted with the appropriate antibodies (anti-IR, anti-IRS-1, anti-IRS-2, anti-phospho-[Ser473]Akt, and anti-GLUT-4).
PI 3-kinase activity assay
For IRS-1- and IRS-2-associated PI 3-kinase activity assays, 5.0 mg of total protein extracts obtained from the hearts of rats exposed to specific experimental conditions were handled using a method previously described (2, 24). Phosphorylated phosphatidylinositol was separated by thin-layer chromatography, and specific dots were visualized by exposing the thin-layer chromatography plates to RX-films. Dot intensities were quantified by digital densitometry of the developed autoradiographs.
Subcellular fractionation
To characterize the expression and subcellular localization of GLUT-4, a subcellular fractionation protocol was employed as described previously (25). The fractions obtained were treated with Laemmli buffer with 100 mM dithiothreitol, heated in a boiling water bath for 5 min, and aliquots (0.2 mg of protein) subjected to SDS-PAGE and Western blotting with anti-GLUT-4 antibodies as described (23).
Laser confocal microscopy
GLUT-4 localization was determined in frozen sections of Wistar rat hearts and fixed NRVM, following a method described elsewhere (6, 26). Sections (5.0 μm) of heart or microscopy glass-fixed NRVMs were incubated with primary antibody against GLUT-4 (1:20), followed (or not) by incubation with FITC-conjugated secondary antibodies and then 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. All image acquisitions were performed with the same settings of the microscope.
Data presentation and statistical analysis
The results are expressed as the mean ± SD of the indicated number of experiments. The blots are presented as direct comparisons of bands in autoradiographs quantified by densitometry using Scion Image software. The t test for unpaired samples was used for statistical analysis. The level of significance was set at P < 0.05.
Results
Ang II and insulin induce SOCS-3 expression in heart following different time courses
Ang II induced the expression of SOCS-3 beginning at 10 min (1.8-fold, P < 0.05), reaching the maximum expression level at 120 min (4.2-fold, P < 0.05) and returning to basal level at 360 min (Fig. 1A, first blot), confirming a previous report (16). Ang II treatment induced the expected association of SOCS-3 with not only JAK-2 (beginning at time 10 min, 1.8-fold, P < 0.05, and peaking at time 60 min, 2.3-fold, P < 0.05) (Fig. 1A, second blot) but also IR (highest at 120 min, 2.8-fold, P < 0.05), IRS-1 (highest at 60 min, 2.1-fold, P < 0.05), and IRS-2 (highest at 60 min, 2.0-fold, P < 0.05) (Fig. 1A, blots 3–5). As shown in Fig. 1B, insulin induced the expression of SOCS-3 beginning at 120 min (1.7-fold, P < 0.05) and reaching 4.8-fold increase at 360 min (P < 0.05).
FIG. 1. Time course of Ang II-(A) and insulin-induced (B) expression of SOCS-3, Ang II-induced (A) association of SOCS-3 with JAK-2 and other proteins of the insulin signaling pathway and Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway (C). Rats were anesthetized and acutely treated with a single iv dose of Ang II (0.02 ml, 10–8 M) (A and C) or insulin (0.02 ml, 10–6 M) (B). The tips of the hearts were excised at times depicted in the figure and used in immunoprecipitation (IP) assays with anti-SOCS-3 (A, first blot, and B), anti-JAK-2 (A, second blot), anti-IR (A, third blot; C, first blot), anti-IRS-1 (A, fourth blot; C, second blot), and anti-IRS-2 (A, fifth blot; C, third blot) antibodies. Immunocomplexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Anti-SOCS-3 (A and B) or antiphosphoserine (C) antibodies were used for blotting (IB). Specific bands were labeled with [125I] protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5 (*, P < 0.05 vs. time 0). See Materials and Methods for details.
Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway overlaps Ang II-induced SOCS-3 expression and association with proteins of the insulin signaling pathway
Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway is a known mechanism that participates in the negative cross-talk between Ang II and insulin signaling systems (3). To evaluate the temporal relationship between this mechanism and Ang II-induced SOCS-3 association with proteins that belong to the insulin signal transduction pathway, total protein extracts obtained from hearts of rats treated with Ang II (20 μl, 10–8 M) for the times depicted in Fig. 1C were employed in immunoprecipitation and immunoblot experiments. Ang II-induced serine phosphorylation of IR (Fig. 1C, first blot) started at 60 min and lasted for 180 min; Ang II-induced serine phosphorylation of IRS-1 (Fig. 1C, second blot) started at 20 min and lasted for 180 min; and Ang II-induced serine phosphorylation of IRS-2 (Fig. 1C, third blot) started at 20 min and lasted for 180 min. Thus, the time frames of Ang II-induced SOCS-3 association with proteins of the insulin signaling pathway and Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway ran, to a certain degree, in parallel.
Pretreatment with Ang II interferes with insulin signal transduction
Pretreatment with Ang II did not produce significant changes in IR (Fig. 2A, upper blot), IRS-1 (Fig. 2B, upper blot), and IRS-2 (Fig. 2C, upper blot) protein expression and in insulin-induced IR tyrosine phosphorylation (Fig. 2A, second blot). However, Ang II pretreatment led to significant reduction of insulin-induced IRS-1 tyrosine phosphorylation (0.5-fold, P < 0.05), IRS-1/p85 PI 3-kinase association (0.6-fold, P < 0.05), and IRS-1-associated PI 3-kinase activity (Fig. 2B, panels 2–4). Ang II pretreatment also produced a significant fall of insulin-induced IRS-2 tyrosine phosphorylation (0.6-fold, P < 0.05), IRS-2/p85 PI 3-kinase association (0.5-fold, P < 0.05), and IRS-2-associated PI 3-kinase activity (Fig. 2C, panels 2–4). Finally, Ang II pretreatment led to a significant fall of insulin-induced [Ser473]Akt phosphorylation (0.4-fold, P < 0.05) (Fig. 2D).
FIG. 2. Effect of Ang II pretreatment on insulin-induced activation of the IR/IRS-1/IRS-2/PI 3-kinase/Akt pathway. Rats were anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected and, following 2 (IR, IRS-1 and IRS-2) or 5 (PI 3-kinase activity and Akt) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-IR (A, second blot), anti-IRS-1 (B, second and third blots and PI 3-kinase activity assay), and anti-IRS-2 (C, second and third blots and PI 3-kinase activity assay) antibodies or in direct immunoblot (IB) assays for determination of IR (A, first blot), IRS-1 (B, first blot), IRS-2 (C, first blot), and p[Ser473] Akt (D). Specific bands were labeled with [125I] protein A and visualized by autoradiography. For PI 3-kinase activity assay, IRS-1 (B, last panel) and IRS-2 (C, last panel), immunoprecipitates were employed according to the method described in Material and Methods. Band and dot intensities were quantified by digital densitometry. In all experiments n = 5 (*, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated). PIP, 3'Phosphorylated phosphatidylinositol.
Inhibition of SOCS-3 expression abolishes Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway
In a recent study (16), we employed a SOCS-3 antisense oligonucleotide that completely abolished Ang II-induced SOCS-3 expression. In the present study, we observed that SOCS-3 expression was increased by 4.2-fold 120 min after Ang II treatment, preceded by saline or SOCS-3 sense oligonucleotide. However, if Ang II treatment was preceded by SOCS-3 antisense oligonucleotide treatment, a complete abolishment of Ang II-induced SOCS-3 expression was observed (Fig. 3A, upper blot). The effect of SOCS-3 antisense oligonucleotide was specific because no inhibition of SOCS-1 expression was observed (Fig. 3A, lower blot). As shown in Fig. 1, the treatment of living rats with Ang II induced not only SOCS-3 expression but also its association with IR, IRS-1, and IRS-2. When rats pretreated with Ang II (10–8 M, 120 min) received an acute dose of insulin (10–6 M, 10 min) a significant increase of IR (2.1-fold, P < 0.05), IRS-1 (3.8-fold, P < 0.05), and IRS-2 (3.2-fold, P < 0.05) association with SOCS-3 was observed if compared with rats treated with only Ang II (Fig. 3B, left-hand side blots, WO), meaning that an acute insulin treatment enhances Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway. The inhibition of SOCS-3 expression with the antisense oligonucleotide completely abolished Ang II-induced and insulin-enhanced association of SOCS-3 with IR, IRS-1, and IRS-2 (Fig. 3B, right-hand side blots, AS). The same effect was observed on Ang II-induced SOCS-3/JAK-2 association (Fig. 3B).
FIG. 3. Effect of SOCS-3 expression inhibition on Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO), sense (SE), or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, the tips of the hearts were excised (A) or a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected (B), and after 2 (IR, IRS-1 and IRS-2) or 10 (JAK-2) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-SOCS-3 or anti-SOCS-1 antibodies. Anti-SOCS-3 (A, upper blot), anti-SOCS-1 (A, lower blot), anti-JAK-2 (B, first blots), anti-IR (B, second blots), anti-IRS-1 (B, third blots), and anti-IRS-2 (B, fourth blots) antibodies were used for blotting. Specific bands were labeled with [125I] protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. Depicted blots are representative of n = 5. See Materials and Methods for details.
Inhibition of SOCS-3 expression overcomes Ang II inhibition of insulin signal transduction through JAK-2 and STAT-5b
JAK-2 plays a central role in the early intracellular cross-talk between insulin and Ang II signaling systems (2). Both Ang II (9, 27) and insulin (8) rapidly induce tyrosine phosphorylation of JAK-2 in heart muscle. Pretreatment of living rats with Ang II inhibits insulin-induced tyrosine phosphorylation of JAK-2 and STAT-5b (2). To evaluate the participation of SOCS-3 in this phenomenon, rats were pretreated with SOCS-3 antisense oligonucleotide and then treated with a single dose of Ang II. As depicted in Fig. 4, Ang II significantly reduced insulin-induced JAK-2 (0.6-fold, P < 0.05) (upper blot) and STAT-5b (0.2-fold, P < 0.05) (lower blot) tyrosine phosphorylation. Pretreatment with SOCS-3 antisense oligonucleotide almost completely reverted the Ang II-inhibiting effect.
FIG. 4. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin-induced tyrosine phosphorylation of JAK-2 and STAT-5b. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO), or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (0.02 ml) (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, a single dose of saline (0.02 ml) (–) or insulin (0.02 ml, 10–6 M) (+) was injected, and after 10 min the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-SOCS-3 (first blot), anti-JAK-2 (second blot), or anti-STAT-5b (third blot) antibodies. Anti-SOCS-3 (first blot) or antiphosphotyrosine (pY) (second and third blots) antibodies were used for blotting (IB). Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). See Materials and Methods for details.
Inhibition of SOCS-3 expression overcomes Ang II inhibition of insulin signal transduction through IRS-1, -2, and Akt
Pretreatment of rats with Ang II inhibits insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2 and serine phosphorylation of Akt. To evaluate the participation of SOCS-3 in this phenomenon, rats were pretreated with SOCS-3 antisense oligonucleotide and then treated with Ang II. As depicted in Fig. 5, the inhibition of SOCS-3 expression completely reverted Ang II-induced inhibition of insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 5B) and IRS-2 (Fig. 5C) and serine phosphorylation of Akt (Fig. 5D).
FIG. 5. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin signal transduction through IR/IRSs/Akt pathway. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO) or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected, and after 2 (IR, IRS-1 and IRS-2) or 5 (Akt) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-IR (A), anti-IRS-1 (B), or anti-IRS-2 (C) antibodies. Antiphosphotyrosine (pY) antibodies were used for blotting (IB) (A–C). For direct immunoblot (IB) assays, samples containing 0.2 mg total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes and blotted with anti-phospho[Ser473] Akt antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). See Materials and Methods for details.
Inhibition of SOCS-3 expression partially reverts Ang II-induced inhibition of insulin-induced GLUT-4 translocation
To evaluate whether Ang II-induced SOCS-3 interferes with a functional event controlled by insulin, rats were pretreated with Ang II and after 120 min received a single iv dose of insulin to stimulate GLUT-4 migration to cell membrane. In cytosolic fractions, insulin promoted a significant fall in GLUT-4 content, a phenomenon that was partially (not significantly) inhibited by pretreatment with Ang II (Fig. 6A, gray bars). In the membrane fraction, the effect of insulin stimulating the increase of GLUT-4 was remarkable and was significantly inhibited by pretreatment with Ang II (Fig. 6B, gray bars). If rats were pretreated with SOCS-3 antisense oligonucleotide, the effect of Ang II inhibiting insulin-induced GLUT-4 translocation to membrane fraction was partially (significantly) reverted (Fig. 6B, white bars).
FIG. 6. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin-induced GLUT-4 translocation. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (c or i) or Ang II (0.02 ml, 10–8 M) (ai). After 120 min, a single dose of saline (c) or insulin (0.02 ml, 10–6 M) (i or ai) was injected. After 10 min the tips of the hearts were obtained, and total protein extracts were submitted to subcellular fractionation providing cytosol (A) and membrane (B) fractions that were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-GLUT-4 antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry, and results are presented as bar graphs. In all experiments n = 5. *, P < 0.05 vs. saline (c) treated; , P < 0.05 vs. insulin (i) treated; , P < 0.05 vs. Ang II+insulin (ai) treated in rats pretreated with no oligonucleotide. For confocal laser microscopy (C), the rats were submitted to the same protocols as above. Frozen sections of hearts (5.0 μm) were obtained and incubated with primary antibody against GLUT-4, followed by incubation with FITC-conjugated secondary antibodies and then with rhodamine-conjugated phalloidin. Images were obtained with a laser confocal microscope (LSM510, Zeiss), always using the same settings for image acquisition. The images are representative of three independent experiments.
To further explore the role of SOCS-3 in Ang II inhibition of insulin-induced GLUT-4 translocation, we employed double-staining confocal microscopy. As depicted in Fig. 6C, in control rats GLUT-4 is sparsely stained in the cells of the heart ventricle, with only a discrete presence in the cell membrane zone (Fig. 6C, c). Acute treatment with insulin remarkably enhances the staining of GLUT-4 in the periphery of heart cells (Fig. 6C, i), whereas pretreatment with Ang II inhibits the effect of insulin to promote GLUT-4 migration to the peripheral zone of the heart cells (Fig. 6C, ai). Pretreatment with SOCS-3 antisense oligonucleotide partially reverts the negative effect of Ang II on insulin-induced GLUT-4 translocation (Fig. 6C, aiAS).
Ang II-induced SOCS-3 expression interferes with insulin signal transduction in isolated rat cardiomyocytes
Acute injection of Ang II is known to produce systemic effects such as the modulation of heart rate and contractility, increase of blood pressure, and regulation of thirst and renal water balance. To investigate whether the effects of Ang II-induced SOCS-3 expression on insulin signal transduction are independent of the systemic effects of Ang II, we evaluated SOCS-3 expression, activation of insulin signal transduction, and GLUT-4 translocation in NRVMs. The treatment of NRVMs with Ang II led to a significant increase of SOCS-3 expression (4.1-fold, P < 0.05) (Fig. 7A). This effect was completely abolished by SOCS-3 antisense but not SOCS-3 sense oligonucleotide pretreatment (Fig. 7A). Pretreatment of NRVMs with Ang II significantly inhibited insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 7B, WO) and IRS-2 (Fig. 7C, WO) and insulin-induced serine 473 phosphorylation of Akt (Fig. 7D, WO). Inhibition of Ang II-induced SOCS-3 expression by the pretreatment of NRVMs with SOCS-3 antisense oligonucleotide partially reverted the inhibitory effect of Ang II on insulin-induced activation of IRS-1 (Fig. 7B, AS), IRS-2 (Fig. 7C, AS), and Akt (Fig. 7D, AS). Finally, the inhibition of Ang II-induced SOCS-3 expression reverted the inhibitory effect of Ang II on insulin-induced GLUT-4 translocation to the cell membrane of NRVM (Fig. 7E).
FIG. 7. Effect of SOCS-3 expression inhibition on insulin signal transduction and GLUT-4 translocation in NRVMs. Cells were prepared and plated (6.0 x 104/cm2) as described in Materials and Methods. Sense (SE) or antisense (AS) SOCS-3 oligonucleotides were added to the culture medium (1.0 μM) in parallel with the transfection reagent Lipofectamine Plus (10 mg/ml). Some cells were treated with no oligonucleotide (WO) but received the transfection reagent. After 8 h the cells were treated with saline or Ang II (10–10 M), and after another 120 min, the cells were treated with saline or insulin (10–8 M). For evaluation of signal transduction, 5.0 mg of total protein extracts from NRVMs were used in immunoprecipitation (IP) assays with anti-SOCS-3 (A), anti-IRS-1 (B), or anti-IRS-2 (C) antibodies. Anti-SOCS-3 (A) or anti-phosphotyrosine (pY) (B and C) antibodies were used for blotting (IB). For direct immunoblot (IB) assays, samples containing 0.2 mg total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes and blotted with anti-phospho[Ser473] Akt (D) antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). For confocal laser microscopy (E), fixed NRVMs were incubated with primary antibody against GLUT-4, followed by incubation with FITC-conjugated secondary antibodies as described in Materials and Methods. Images were obtained with a laser confocal microscope (LSM510, Zeiss), always using the same settings for image acquisition. The images are representative of three independent experiments. In A–D: c, only saline treatment; i, only insulin treatment; ai, Ang II followed by insulin treatment.
Discussion
Hypertension is one of the most important risk factors for the development of insulin resistance and diabetes mellitus (28, 29). Clinical and experimental data provide evidence for an impairment of the glucose transport stimulated by insulin-dependent mechanisms in muscle of patients and animals with high blood pressure (30, 31). On clinical grounds the association between diabetes and hypertension has long been recognized and is known to play a central role in some of the common macro- and microvasculopathies that develop in subjects with diabetes (28, 29).
Since the early studies on the clinical effects of inhibitors of ACE for the treatment of hypertension, the substantial benefits for patients with diabetes and the occasional occurrence of hypoglycemia have been evident (32, 33, 34). The observation of these effects raised the possibility that Ang II, bradykinin, or both peptides could exert a regulatory role on insulin-dependent and/or -independent mechanisms of glucose mobilization. Exploring the interactions between the Ang II and insulin signaling pathways, we and others have shown that an intracellular molecular cross-talk occurs at an early postreceptor level, which is pivoted by the engagement of JAK-2 and leads to an impairment of the insulin-dependent activation of PI 3-kinase and Akt (2, 9). This phenomenon participates in the inhibition of insulin signal transduction in the heart of animal models of obesity, diabetes, and hypertension that hyperexpress Ang II in the cardiac tissue (6). Besides the intracellular cross-talk that depends mostly on the activation of AT1 (2), a prereceptor mechanism of interaction between Ang II and insulin signaling is evidenced when experimental animals are treated with inhibitors of ACE, which leads to an improved insulin action and signal transduction (7). A bulk of evidence suggest that not only the reduction of Ang II but also the increase of bradykinin participates in this extracellular cross-talk (35). Bradykinin induces the tyrosine phosphorylation of IRS-1 and increases PI 3-kinase activity, but, most importantly, it sensitizes this molecular pathway to insulin action (7). Bradykinin also promotes an increase of intracellular nitric oxide, which can up-regulate GLUT-4 translocation and increase glucose uptake by skeletal muscle (36, 37).
Thus, it becomes clear that the renin-angiotensin system exerts a regulatory role on insulin action. This regulation occurs at distinct levels and engages several extra- and intracellular elements. The gross result of this interaction is mostly inhibitory on insulin action. With the recent evidence of the intrinsic negative regulation of insulin (12, 15) and Ang II (16, 17) signaling by SOCS-3, we decided to investigate whether Ang II-induced SOCS-3 could somehow modulate insulin signal transduction and action.
Initially, a time frame was established during which Ang II and insulin induce the expression of SOCS-3 in heart. According to the present data and a previous report (16), the level of SOCS-3 protein in heart is constitutively low. The treatment with Ang II promotes an increase of SOCS-3 expression that is detectable at 10 min and lasts for approximately 180 min. Conversely, insulin promotes a late stimulation of SOCS-3 expression, beginning at 120 min and lasting for more than 360 min. This finding is similar to that observed by Emanuelli et al. (12) when working with skeletal muscle of mice. The decrease of SOCS-3 protein expression that occurs 360 min after Ang II stimulation seems to be a consequence of a fall of Ang II levels because the stimulation with repetitive doses of the hormone is sufficient to prolong SOCS-3 expression as previously demonstrated (16).
Next, it was shown in heart and NRVMs that Ang II-induced SOCS-3 associates with proteins of the insulin signal transduction pathway and that this phenomenon is paralleled by an impairment of the insulin signal transduction machinery. The property of a priming dose of Ang II to hamper insulin signal transduction through IRS-1, IRS-2, PI 3-kinase, and Akt was previously reported (2) and was shown to be, at least in part, dependent on serine phosphorylation of some of those proteins (3). This was herein confirmed and shown to occur in parallel with the expression/association of SOCS-3. However, when Ang II-induced SOCS-3 expression was inhibited by a previous treatment with an antisense oligonucleotide, a partial reversal of the inhibitory effect of Ang II on insulin stimulated IRS-1, IRS-2, and Akt molecular activation was observed, suggesting that each mechanism acts independently to provide a certain degree of regulation on this pathway.
At least two previous reports have shown the participation of SOCS proteins as candidate intermediaries in the cross-talk between a hormone and a cytokine. Boisclair et al. (20) showed that SOCS-3 mediates an inhibitory cross-talk between IL-1? and GH in hepatoma cells. According to their studies, the pretreatment of the cells with IL-1? induces an increase of up to 8-fold SOCS-3, which interacts with JAK-2/STAT-5 and hampers GH-induced transcription of the acid-labile subunit gene. In another study, Lagathu et al. (38) showed that chronic exposure of 3T3-F442A and 3T3-L1 adipocytes to IL-6 inhibits insulin signal transduction, which is paralleled by an increased expression of SOCS-3. Thus, convincing evidence exists to suggest that proteins of the SOCS family not only modulate signal transduction on an intrapathway basis but also provide a negative feedback for related signaling pathways.
In the last part of the study, we evaluated whether the molecular phenomena controlled by Ang II-induced SOCS-3 would play any regulatory role on a functional event activated by insulin. The activation of GLUT-4 is the rate-limiting step in muscle glucose uptake (39). In the heart, as in skeletal muscle and adipose tissue, insulin stimulates GLUT-4 translocation from an intracellular pool to the cell surface in which it mediates glucose transport (40). Knocking out GLUT-4 expression leads to cardiac dysfunction (41) and progressive heart hypertrophy (42), which illustrates the functional importance of this protein in cardiac tissue. In the present study, we observed that a pretreatment with Ang II significantly reduced insulin-induced GLUT-4 translocation in the heart and cultured NRVMs. The physiological significance of this phenomenon attests the fact that living rats were treated with 0.02 ml Ang II at the concentration of 10–8 M, a dose that provides blood Ang II concentration at the physiological range of 100 ng/liter (43). The inhibition of SOCS-3 expression partially prevented this effect, indicating that SOCS-3 modulates an important function controlled by insulin. In a previous study (15), mice transiently overexpressing SOCS-1 became hyperglycemic and hyperinsulinemic, whereas the levels of the suppressor protein were high. According to this study, both SOCS-1 and SOCS-3 interfere with insulin signal transduction by controlling the ubiquitination and degradation of IRS-1 and IRS-2 (15). Two other recent reports show that insulin-induced SOCS (-1, -3, and -6) expression can modulate further insulin signaling by directly binding to the insulin receptor and early substrates (10, 12, 44). In this context, Krebs and Hilton (45) commented that, besides their classical inhibitory role on cytokine signaling, members of the SOCS family may act as important regulators of signal transduction through members of the family of receptors with intrinsic tyrosine kinase activity.
In conclusion, our data demonstrate that SOCS-3 can mediate a late cross-talk between Ang II and insulin and that this cross-talk reflects on the control of one of the most important physiological events controlled by insulin, i.e. GLUT-4 translocation. It will be interesting to determine whether other hormones and cytokines, known to negatively modulate insulin action, may exert their effect through members of the SOCS family to regulate GLUT-4 function.
Acknowledgments
We thank Dr. N. Conran for English editing.
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Address all correspondence and requests for reprints to: Lício A. Velloso, Departamento de Clínica Médica, Faculdade de Ciencias Medicas–Universidade Estadual de Campinas, 13081-970 Campinas S?o Paulo, Brazil. E-mail: lavelloso@fcm.unicamp.br.
Abstract
Angiotensin II inhibits insulin-induced activation of phosphatidylinositol 3-kinase through a mechanism, at least in part, dependent on serine phosphorylation of the insulin receptor and insulin receptor substrates (IRS)-1/2. Recent evidence shows that suppressor of cytokine signaling-3 (SOCS-3) is induced by insulin and angiotensin II and participates in the negative control of further stimulation of each of these signaling systems independently. In the present study, we evaluated the interaction of angiotensin II-induced SOCS-3 with the insulin signaling pathway in the heart of living rats. A single iv dose of angiotensin II promotes a significant increase of SOCS-3 in heart, an effect that lasts up to 180 min. Once induced, SOCS-3 interacts with the insulin receptor, JAK-2, IRS-1, and IRS-2. The inhibition of SOCS-3 expression by a phosphorthioate-modified antisense oligonucleotide partially restores angiotensin II-induced inhibition of insulin-induced insulin receptor, IRS-1 and IRS-2 tyrosine phosphorylation, and IRS-1 and IRS-2 association with p85-phosphatidylinositol 3-kinase and [Ser473] phosphorylation of Akt. Moreover, the inhibition of SOCS-3 expression partially reverses angiotensin II-induced inhibition of insulin-stimulated glucose transporter-4 translocation to the cell membrane. These results are reproduced in isolated cardiomyocytes. Thus, SOCS-3 participates, as a late event, in the negative cross-talk between angiotensin II and insulin, producing an inhibitory effect on insulin-induced glucose transporter-4 translocation.
Introduction
THE RECIPROCAL SIGNAL transduction modulation that occurs between the insulin and angiotensin II (Ang II) signaling pathways is thought to play an important role in the common clinical association between diabetes mellitus and hypertension (1, 2, 3, 4, 5, 6). The cross-talk between these two systems occurs at distinct levels, beginning at the prereceptor level, as modulation of angiotensin-converting enzyme (ACE) activity leads to simultaneous control of Ang II production and insulin action (7). It then acts at an early postreceptor level because janus kinase (JAK)-2 activity may be modulated by insulin and Ang II (2, 8, 9), and finally it reaches an intermediary postreceptor level because serine kinases activated by Ang II inhibit insulin signal transduction (3). This redundancy may have developed during evolution as a need for tight control of two vital and closely related systems, i.e. the circulatory and metabolic systems.
In recent years, a series of studies have shown that insulin is capable of inducing the expression of suppressor of cytokine signaling (SOCS)-3 (10, 11, 12), a 32-kDa protein that belongs to a family of proteins (SOCS family) originally identified as inducible inhibitors of signal transduction by gp130 cytokines (13, 14). When cotransfected with insulin receptor (IR) and insulin receptor substrate (IRS)-1, SOCS-3 inhibits insulin signal transduction by promoting a reduction of insulin-induced IRS-1 tyrosine phosphorylation (12). Moreover, SOCS-3 may reduce insulin signal transduction by targeting IRS-1 and IRS-2 to proteasomic degradation (15). Recently we demonstrated that Ang II is also capable of inducing SOCS-3 in heart, isolated cardiomyocytes, and the hypothalamus of rats (16, 17). Ang II-induced SOCS-3 inhibits Ang II-stimulated c-jun expression (16) and participates in the mechanisms that lead to refractoriness to Ang II in hypothalamus (17).
In cytokine signaling, proteins of the SOCS family may provide a negative cross-talk between two distinct systems, such as IL-3 and IL-11 (18) and interferon- and IL-4 (19) signaling systems. In addition, SOCS proteins may participate in the cross-talk between a cytokine and hormone signaling pathways, such as IL-1? and GH (20). In the present study, we evaluated whether SOCS-3, induced by Ang II in the heart, interacts with elements of the insulin signaling pathway and exerts any regulatory effect on this system.
Materials and Methods
Antibodies and chemicals
Reagents for SDS-PAGE and immunoblotting were obtained from Bio-Rad Laboratories (Richmond, CA). HEPES, phenylmethylsulfonylfluoride, aprotinin, dithiotreitol, Triton X-100, Tween 20, glycerol, Ang II, and BSA (fraction V) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Protein A-Sepharose 6 MB was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden), [125I]-protein A and nitrocellulose membranes were obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). Phosphatidylinositol was from Avanti Polar Lipids (Alabaster, AL). Chemicals, culture media, Lipofectamine Plus, and sera for isolation and culture of cells were purchased from Life Technologies Inc. (Grand Island, NY). Antibodies against SOCS-3 (sc-9023, rabbit polyclonal and sc-7009, goat polyclonal), SOCS-1 (sc-9021, rabbit polyclonal), JAK-2 (sc-278, rabbit polyclonal), IR? (sc-711, rabbit polyclonal), IRS-1 (sc-559, rabbit polyclonal), IRS-2 (sc-8299, rabbit polyclonal), signal transducer and activator of transcription (STAT)-5b (sc-1656, mouse monoclonal), p85-phosphatidylinositol 3-kinase (PI 3-kinase) (sc-423, rabbit polyclonal), glucose transporter (GLUT)-4 (sc-7938, rabbit polyclonal), and phosphotyrosine (sc-508, mouse monoclonal) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-[Ser473]Akt (9271, rabbit polyclonal) was purchased from Cell Signaling Technology (Beverly, MA). Antiphosphoserine antibody (AB1603, rabbit polyclonal) was purchased from Chemicon Inc. (Temecula, CA). Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies and rhodamine-conjugated phalloidin were purchased from Sigma-Aldrich Corp. Phosphorthioate-modified oligodeoxynucleotides for SOCS-3 (sense, 5'-CAT GGT CAC CCA CAG-3'; antisense, 5'-CTG TGG GTG ACC ATG-3') were synthesized by Life Technologies, Inc. and were previously tested and optimized for in vivo use (16). Sodium amorbital and insulin were obtained from Eli Lilly & Co. (Indianapolis, IN).
Experimental animals
Eight-week-old male Wistar rats from the university’s Central Animal Breeding Center were used in the experiments. The adult rats were allowed access to standard rodent chow and water ad libitum. Food was withdrawn 12 h before the experiments. All experiments were conducted in accord with the principles and procedures described by National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the State University of Campinas ethical committee. For neonatal rat ventricular myocyte (NRVM) preparation, 1- to 3-d-old Wistar rats were employed.
Preparation of NRVM
Ventricular cardiac myocytes were prepared by enzymatic disaggregation following a method previously described (21). After separation the cells were cultured in DMEM at a concentration of 6.0 x 104/cm2 on plates coated with gelatin. The purity of the cell preparations was ascertained by immunofluorescence using tetramethylrhodamine isothiocyanate-phalloidin and evaluation by confocal microscopy.
Hormone stimulation protocols and tissue preparation
Rats were anesthetized by an ip injection of sodium amorbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes. The abdominal cavity was opened, the cava vein was exposed, and in vivostimulation of the heart was obtained by injection of 0.02 ml saline (0.9% NaCl), 10–8 M Ang II, and/or 10–6 M insulin into the cava vein. In some of the time-course experiments the hearts of rats treated with respective hormones were compared with the hearts of rats treated with only saline for similar periods of time. In some cases rats received 6.0 nmol [diluted in 200 μl Tris/EDTA buffer] sense or antisense SOCS-3 oligonucleotide ip 3 h before the experiments. At various time intervals (as shown in Results), the tips of the ventricles were excised and immediately homogenized in approximately five volumes of solubilization buffer at 4 C [1% Triton X-100, 100 mM Tris-HCl (pH 7.4), 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2.0 mM phenylmethylsulfonylfluoride, and 0.1 mg aprotinin/ml] with a Polytron PTA 20S generator (model PT 10/35; Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 sec.
NRVMs were exposed to oligonucleotides, saline, insulin, and Ang II according to doses and times as stated in Results and following a protocol previously described (16).
Protein analyses by immunoprecipitation and immunoblotting
Insoluble material was removed by centrifugation for 25 min at 11,000 rpm in a 70.Ti rotor (Beckman, Fullerton, CA) at 4 C. The protein concentrations in supernatants were determined by the Bradford dye-binding method (22). Aliquots of the resulting supernatants containing 5.0 mg total protein were used for immunoprecipitation with anti-JAK-2, IR?, IRS-1, IRS-2, STAT-5b, SOCS-1, and SOCS-3 antibodies following a method previously described (23). Unspecific binding of proteins to beads were always tested and observed to be insignificant (not shown). Band intensities were quantified by digital densitometry (Scion Image software, Scion Corp., Frederick, MD) of the developed autoradiographs. For simple immunoblot experiments (not preceded by immunoprecipitation), 0.2 mg total protein from heart was separated by SDS-PAGE (10 or 12% bis-acrylamide), transferred to nitrocellulose membranes, and blotted with the appropriate antibodies (anti-IR, anti-IRS-1, anti-IRS-2, anti-phospho-[Ser473]Akt, and anti-GLUT-4).
PI 3-kinase activity assay
For IRS-1- and IRS-2-associated PI 3-kinase activity assays, 5.0 mg of total protein extracts obtained from the hearts of rats exposed to specific experimental conditions were handled using a method previously described (2, 24). Phosphorylated phosphatidylinositol was separated by thin-layer chromatography, and specific dots were visualized by exposing the thin-layer chromatography plates to RX-films. Dot intensities were quantified by digital densitometry of the developed autoradiographs.
Subcellular fractionation
To characterize the expression and subcellular localization of GLUT-4, a subcellular fractionation protocol was employed as described previously (25). The fractions obtained were treated with Laemmli buffer with 100 mM dithiothreitol, heated in a boiling water bath for 5 min, and aliquots (0.2 mg of protein) subjected to SDS-PAGE and Western blotting with anti-GLUT-4 antibodies as described (23).
Laser confocal microscopy
GLUT-4 localization was determined in frozen sections of Wistar rat hearts and fixed NRVM, following a method described elsewhere (6, 26). Sections (5.0 μm) of heart or microscopy glass-fixed NRVMs were incubated with primary antibody against GLUT-4 (1:20), followed (or not) by incubation with FITC-conjugated secondary antibodies and then 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. All image acquisitions were performed with the same settings of the microscope.
Data presentation and statistical analysis
The results are expressed as the mean ± SD of the indicated number of experiments. The blots are presented as direct comparisons of bands in autoradiographs quantified by densitometry using Scion Image software. The t test for unpaired samples was used for statistical analysis. The level of significance was set at P < 0.05.
Results
Ang II and insulin induce SOCS-3 expression in heart following different time courses
Ang II induced the expression of SOCS-3 beginning at 10 min (1.8-fold, P < 0.05), reaching the maximum expression level at 120 min (4.2-fold, P < 0.05) and returning to basal level at 360 min (Fig. 1A, first blot), confirming a previous report (16). Ang II treatment induced the expected association of SOCS-3 with not only JAK-2 (beginning at time 10 min, 1.8-fold, P < 0.05, and peaking at time 60 min, 2.3-fold, P < 0.05) (Fig. 1A, second blot) but also IR (highest at 120 min, 2.8-fold, P < 0.05), IRS-1 (highest at 60 min, 2.1-fold, P < 0.05), and IRS-2 (highest at 60 min, 2.0-fold, P < 0.05) (Fig. 1A, blots 3–5). As shown in Fig. 1B, insulin induced the expression of SOCS-3 beginning at 120 min (1.7-fold, P < 0.05) and reaching 4.8-fold increase at 360 min (P < 0.05).
FIG. 1. Time course of Ang II-(A) and insulin-induced (B) expression of SOCS-3, Ang II-induced (A) association of SOCS-3 with JAK-2 and other proteins of the insulin signaling pathway and Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway (C). Rats were anesthetized and acutely treated with a single iv dose of Ang II (0.02 ml, 10–8 M) (A and C) or insulin (0.02 ml, 10–6 M) (B). The tips of the hearts were excised at times depicted in the figure and used in immunoprecipitation (IP) assays with anti-SOCS-3 (A, first blot, and B), anti-JAK-2 (A, second blot), anti-IR (A, third blot; C, first blot), anti-IRS-1 (A, fourth blot; C, second blot), and anti-IRS-2 (A, fifth blot; C, third blot) antibodies. Immunocomplexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Anti-SOCS-3 (A and B) or antiphosphoserine (C) antibodies were used for blotting (IB). Specific bands were labeled with [125I] protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5 (*, P < 0.05 vs. time 0). See Materials and Methods for details.
Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway overlaps Ang II-induced SOCS-3 expression and association with proteins of the insulin signaling pathway
Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway is a known mechanism that participates in the negative cross-talk between Ang II and insulin signaling systems (3). To evaluate the temporal relationship between this mechanism and Ang II-induced SOCS-3 association with proteins that belong to the insulin signal transduction pathway, total protein extracts obtained from hearts of rats treated with Ang II (20 μl, 10–8 M) for the times depicted in Fig. 1C were employed in immunoprecipitation and immunoblot experiments. Ang II-induced serine phosphorylation of IR (Fig. 1C, first blot) started at 60 min and lasted for 180 min; Ang II-induced serine phosphorylation of IRS-1 (Fig. 1C, second blot) started at 20 min and lasted for 180 min; and Ang II-induced serine phosphorylation of IRS-2 (Fig. 1C, third blot) started at 20 min and lasted for 180 min. Thus, the time frames of Ang II-induced SOCS-3 association with proteins of the insulin signaling pathway and Ang II-induced serine phosphorylation of proteins of the insulin signaling pathway ran, to a certain degree, in parallel.
Pretreatment with Ang II interferes with insulin signal transduction
Pretreatment with Ang II did not produce significant changes in IR (Fig. 2A, upper blot), IRS-1 (Fig. 2B, upper blot), and IRS-2 (Fig. 2C, upper blot) protein expression and in insulin-induced IR tyrosine phosphorylation (Fig. 2A, second blot). However, Ang II pretreatment led to significant reduction of insulin-induced IRS-1 tyrosine phosphorylation (0.5-fold, P < 0.05), IRS-1/p85 PI 3-kinase association (0.6-fold, P < 0.05), and IRS-1-associated PI 3-kinase activity (Fig. 2B, panels 2–4). Ang II pretreatment also produced a significant fall of insulin-induced IRS-2 tyrosine phosphorylation (0.6-fold, P < 0.05), IRS-2/p85 PI 3-kinase association (0.5-fold, P < 0.05), and IRS-2-associated PI 3-kinase activity (Fig. 2C, panels 2–4). Finally, Ang II pretreatment led to a significant fall of insulin-induced [Ser473]Akt phosphorylation (0.4-fold, P < 0.05) (Fig. 2D).
FIG. 2. Effect of Ang II pretreatment on insulin-induced activation of the IR/IRS-1/IRS-2/PI 3-kinase/Akt pathway. Rats were anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected and, following 2 (IR, IRS-1 and IRS-2) or 5 (PI 3-kinase activity and Akt) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-IR (A, second blot), anti-IRS-1 (B, second and third blots and PI 3-kinase activity assay), and anti-IRS-2 (C, second and third blots and PI 3-kinase activity assay) antibodies or in direct immunoblot (IB) assays for determination of IR (A, first blot), IRS-1 (B, first blot), IRS-2 (C, first blot), and p[Ser473] Akt (D). Specific bands were labeled with [125I] protein A and visualized by autoradiography. For PI 3-kinase activity assay, IRS-1 (B, last panel) and IRS-2 (C, last panel), immunoprecipitates were employed according to the method described in Material and Methods. Band and dot intensities were quantified by digital densitometry. In all experiments n = 5 (*, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated). PIP, 3'Phosphorylated phosphatidylinositol.
Inhibition of SOCS-3 expression abolishes Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway
In a recent study (16), we employed a SOCS-3 antisense oligonucleotide that completely abolished Ang II-induced SOCS-3 expression. In the present study, we observed that SOCS-3 expression was increased by 4.2-fold 120 min after Ang II treatment, preceded by saline or SOCS-3 sense oligonucleotide. However, if Ang II treatment was preceded by SOCS-3 antisense oligonucleotide treatment, a complete abolishment of Ang II-induced SOCS-3 expression was observed (Fig. 3A, upper blot). The effect of SOCS-3 antisense oligonucleotide was specific because no inhibition of SOCS-1 expression was observed (Fig. 3A, lower blot). As shown in Fig. 1, the treatment of living rats with Ang II induced not only SOCS-3 expression but also its association with IR, IRS-1, and IRS-2. When rats pretreated with Ang II (10–8 M, 120 min) received an acute dose of insulin (10–6 M, 10 min) a significant increase of IR (2.1-fold, P < 0.05), IRS-1 (3.8-fold, P < 0.05), and IRS-2 (3.2-fold, P < 0.05) association with SOCS-3 was observed if compared with rats treated with only Ang II (Fig. 3B, left-hand side blots, WO), meaning that an acute insulin treatment enhances Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway. The inhibition of SOCS-3 expression with the antisense oligonucleotide completely abolished Ang II-induced and insulin-enhanced association of SOCS-3 with IR, IRS-1, and IRS-2 (Fig. 3B, right-hand side blots, AS). The same effect was observed on Ang II-induced SOCS-3/JAK-2 association (Fig. 3B).
FIG. 3. Effect of SOCS-3 expression inhibition on Ang II-induced association of SOCS-3 with proteins of the insulin signaling pathway. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO), sense (SE), or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, the tips of the hearts were excised (A) or a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected (B), and after 2 (IR, IRS-1 and IRS-2) or 10 (JAK-2) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-SOCS-3 or anti-SOCS-1 antibodies. Anti-SOCS-3 (A, upper blot), anti-SOCS-1 (A, lower blot), anti-JAK-2 (B, first blots), anti-IR (B, second blots), anti-IRS-1 (B, third blots), and anti-IRS-2 (B, fourth blots) antibodies were used for blotting. Specific bands were labeled with [125I] protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. Depicted blots are representative of n = 5. See Materials and Methods for details.
Inhibition of SOCS-3 expression overcomes Ang II inhibition of insulin signal transduction through JAK-2 and STAT-5b
JAK-2 plays a central role in the early intracellular cross-talk between insulin and Ang II signaling systems (2). Both Ang II (9, 27) and insulin (8) rapidly induce tyrosine phosphorylation of JAK-2 in heart muscle. Pretreatment of living rats with Ang II inhibits insulin-induced tyrosine phosphorylation of JAK-2 and STAT-5b (2). To evaluate the participation of SOCS-3 in this phenomenon, rats were pretreated with SOCS-3 antisense oligonucleotide and then treated with a single dose of Ang II. As depicted in Fig. 4, Ang II significantly reduced insulin-induced JAK-2 (0.6-fold, P < 0.05) (upper blot) and STAT-5b (0.2-fold, P < 0.05) (lower blot) tyrosine phosphorylation. Pretreatment with SOCS-3 antisense oligonucleotide almost completely reverted the Ang II-inhibiting effect.
FIG. 4. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin-induced tyrosine phosphorylation of JAK-2 and STAT-5b. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO), or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (0.02 ml) (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, a single dose of saline (0.02 ml) (–) or insulin (0.02 ml, 10–6 M) (+) was injected, and after 10 min the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-SOCS-3 (first blot), anti-JAK-2 (second blot), or anti-STAT-5b (third blot) antibodies. Anti-SOCS-3 (first blot) or antiphosphotyrosine (pY) (second and third blots) antibodies were used for blotting (IB). Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). See Materials and Methods for details.
Inhibition of SOCS-3 expression overcomes Ang II inhibition of insulin signal transduction through IRS-1, -2, and Akt
Pretreatment of rats with Ang II inhibits insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2 and serine phosphorylation of Akt. To evaluate the participation of SOCS-3 in this phenomenon, rats were pretreated with SOCS-3 antisense oligonucleotide and then treated with Ang II. As depicted in Fig. 5, the inhibition of SOCS-3 expression completely reverted Ang II-induced inhibition of insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 5B) and IRS-2 (Fig. 5C) and serine phosphorylation of Akt (Fig. 5D).
FIG. 5. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin signal transduction through IR/IRSs/Akt pathway. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer (WO) or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (–) or Ang II (0.02 ml, 10–8 M) (+). After 120 min, a single dose of saline (–) or insulin (0.02 ml, 10–6 M) (+) was injected, and after 2 (IR, IRS-1 and IRS-2) or 5 (Akt) min, the tips of the hearts were excised and used in immunoprecipitation (IP) assays with anti-IR (A), anti-IRS-1 (B), or anti-IRS-2 (C) antibodies. Antiphosphotyrosine (pY) antibodies were used for blotting (IB) (A–C). For direct immunoblot (IB) assays, samples containing 0.2 mg total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes and blotted with anti-phospho[Ser473] Akt antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments, n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). See Materials and Methods for details.
Inhibition of SOCS-3 expression partially reverts Ang II-induced inhibition of insulin-induced GLUT-4 translocation
To evaluate whether Ang II-induced SOCS-3 interferes with a functional event controlled by insulin, rats were pretreated with Ang II and after 120 min received a single iv dose of insulin to stimulate GLUT-4 migration to cell membrane. In cytosolic fractions, insulin promoted a significant fall in GLUT-4 content, a phenomenon that was partially (not significantly) inhibited by pretreatment with Ang II (Fig. 6A, gray bars). In the membrane fraction, the effect of insulin stimulating the increase of GLUT-4 was remarkable and was significantly inhibited by pretreatment with Ang II (Fig. 6B, gray bars). If rats were pretreated with SOCS-3 antisense oligonucleotide, the effect of Ang II inhibiting insulin-induced GLUT-4 translocation to membrane fraction was partially (significantly) reverted (Fig. 6B, white bars).
FIG. 6. Effect of SOCS-3 expression inhibition on Ang II-induced inhibition of insulin-induced GLUT-4 translocation. Three hours before the beginning of the experiments, the rats received a single ip dose of vehicle buffer or antisense (AS) SOCS-3 oligonucleotide (200 μl, 6.0 nmol). Rats were then anesthetized and acutely treated with a single dose of saline (c or i) or Ang II (0.02 ml, 10–8 M) (ai). After 120 min, a single dose of saline (c) or insulin (0.02 ml, 10–6 M) (i or ai) was injected. After 10 min the tips of the hearts were obtained, and total protein extracts were submitted to subcellular fractionation providing cytosol (A) and membrane (B) fractions that were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-GLUT-4 antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry, and results are presented as bar graphs. In all experiments n = 5. *, P < 0.05 vs. saline (c) treated; , P < 0.05 vs. insulin (i) treated; , P < 0.05 vs. Ang II+insulin (ai) treated in rats pretreated with no oligonucleotide. For confocal laser microscopy (C), the rats were submitted to the same protocols as above. Frozen sections of hearts (5.0 μm) were obtained and incubated with primary antibody against GLUT-4, followed by incubation with FITC-conjugated secondary antibodies and then with rhodamine-conjugated phalloidin. Images were obtained with a laser confocal microscope (LSM510, Zeiss), always using the same settings for image acquisition. The images are representative of three independent experiments.
To further explore the role of SOCS-3 in Ang II inhibition of insulin-induced GLUT-4 translocation, we employed double-staining confocal microscopy. As depicted in Fig. 6C, in control rats GLUT-4 is sparsely stained in the cells of the heart ventricle, with only a discrete presence in the cell membrane zone (Fig. 6C, c). Acute treatment with insulin remarkably enhances the staining of GLUT-4 in the periphery of heart cells (Fig. 6C, i), whereas pretreatment with Ang II inhibits the effect of insulin to promote GLUT-4 migration to the peripheral zone of the heart cells (Fig. 6C, ai). Pretreatment with SOCS-3 antisense oligonucleotide partially reverts the negative effect of Ang II on insulin-induced GLUT-4 translocation (Fig. 6C, aiAS).
Ang II-induced SOCS-3 expression interferes with insulin signal transduction in isolated rat cardiomyocytes
Acute injection of Ang II is known to produce systemic effects such as the modulation of heart rate and contractility, increase of blood pressure, and regulation of thirst and renal water balance. To investigate whether the effects of Ang II-induced SOCS-3 expression on insulin signal transduction are independent of the systemic effects of Ang II, we evaluated SOCS-3 expression, activation of insulin signal transduction, and GLUT-4 translocation in NRVMs. The treatment of NRVMs with Ang II led to a significant increase of SOCS-3 expression (4.1-fold, P < 0.05) (Fig. 7A). This effect was completely abolished by SOCS-3 antisense but not SOCS-3 sense oligonucleotide pretreatment (Fig. 7A). Pretreatment of NRVMs with Ang II significantly inhibited insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 7B, WO) and IRS-2 (Fig. 7C, WO) and insulin-induced serine 473 phosphorylation of Akt (Fig. 7D, WO). Inhibition of Ang II-induced SOCS-3 expression by the pretreatment of NRVMs with SOCS-3 antisense oligonucleotide partially reverted the inhibitory effect of Ang II on insulin-induced activation of IRS-1 (Fig. 7B, AS), IRS-2 (Fig. 7C, AS), and Akt (Fig. 7D, AS). Finally, the inhibition of Ang II-induced SOCS-3 expression reverted the inhibitory effect of Ang II on insulin-induced GLUT-4 translocation to the cell membrane of NRVM (Fig. 7E).
FIG. 7. Effect of SOCS-3 expression inhibition on insulin signal transduction and GLUT-4 translocation in NRVMs. Cells were prepared and plated (6.0 x 104/cm2) as described in Materials and Methods. Sense (SE) or antisense (AS) SOCS-3 oligonucleotides were added to the culture medium (1.0 μM) in parallel with the transfection reagent Lipofectamine Plus (10 mg/ml). Some cells were treated with no oligonucleotide (WO) but received the transfection reagent. After 8 h the cells were treated with saline or Ang II (10–10 M), and after another 120 min, the cells were treated with saline or insulin (10–8 M). For evaluation of signal transduction, 5.0 mg of total protein extracts from NRVMs were used in immunoprecipitation (IP) assays with anti-SOCS-3 (A), anti-IRS-1 (B), or anti-IRS-2 (C) antibodies. Anti-SOCS-3 (A) or anti-phosphotyrosine (pY) (B and C) antibodies were used for blotting (IB). For direct immunoblot (IB) assays, samples containing 0.2 mg total protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes and blotted with anti-phospho[Ser473] Akt (D) antibodies. Specific bands were labeled with [125I]-protein A and visualized by autoradiography. Band intensities were quantified by digital densitometry. In all experiments n = 5. *, P < 0.05 vs. saline treated; , P < 0.05 vs. insulin treated; , P < 0.05 vs. Ang II+insulin treated in rats pretreated with no oligonucleotide (WO). For confocal laser microscopy (E), fixed NRVMs were incubated with primary antibody against GLUT-4, followed by incubation with FITC-conjugated secondary antibodies as described in Materials and Methods. Images were obtained with a laser confocal microscope (LSM510, Zeiss), always using the same settings for image acquisition. The images are representative of three independent experiments. In A–D: c, only saline treatment; i, only insulin treatment; ai, Ang II followed by insulin treatment.
Discussion
Hypertension is one of the most important risk factors for the development of insulin resistance and diabetes mellitus (28, 29). Clinical and experimental data provide evidence for an impairment of the glucose transport stimulated by insulin-dependent mechanisms in muscle of patients and animals with high blood pressure (30, 31). On clinical grounds the association between diabetes and hypertension has long been recognized and is known to play a central role in some of the common macro- and microvasculopathies that develop in subjects with diabetes (28, 29).
Since the early studies on the clinical effects of inhibitors of ACE for the treatment of hypertension, the substantial benefits for patients with diabetes and the occasional occurrence of hypoglycemia have been evident (32, 33, 34). The observation of these effects raised the possibility that Ang II, bradykinin, or both peptides could exert a regulatory role on insulin-dependent and/or -independent mechanisms of glucose mobilization. Exploring the interactions between the Ang II and insulin signaling pathways, we and others have shown that an intracellular molecular cross-talk occurs at an early postreceptor level, which is pivoted by the engagement of JAK-2 and leads to an impairment of the insulin-dependent activation of PI 3-kinase and Akt (2, 9). This phenomenon participates in the inhibition of insulin signal transduction in the heart of animal models of obesity, diabetes, and hypertension that hyperexpress Ang II in the cardiac tissue (6). Besides the intracellular cross-talk that depends mostly on the activation of AT1 (2), a prereceptor mechanism of interaction between Ang II and insulin signaling is evidenced when experimental animals are treated with inhibitors of ACE, which leads to an improved insulin action and signal transduction (7). A bulk of evidence suggest that not only the reduction of Ang II but also the increase of bradykinin participates in this extracellular cross-talk (35). Bradykinin induces the tyrosine phosphorylation of IRS-1 and increases PI 3-kinase activity, but, most importantly, it sensitizes this molecular pathway to insulin action (7). Bradykinin also promotes an increase of intracellular nitric oxide, which can up-regulate GLUT-4 translocation and increase glucose uptake by skeletal muscle (36, 37).
Thus, it becomes clear that the renin-angiotensin system exerts a regulatory role on insulin action. This regulation occurs at distinct levels and engages several extra- and intracellular elements. The gross result of this interaction is mostly inhibitory on insulin action. With the recent evidence of the intrinsic negative regulation of insulin (12, 15) and Ang II (16, 17) signaling by SOCS-3, we decided to investigate whether Ang II-induced SOCS-3 could somehow modulate insulin signal transduction and action.
Initially, a time frame was established during which Ang II and insulin induce the expression of SOCS-3 in heart. According to the present data and a previous report (16), the level of SOCS-3 protein in heart is constitutively low. The treatment with Ang II promotes an increase of SOCS-3 expression that is detectable at 10 min and lasts for approximately 180 min. Conversely, insulin promotes a late stimulation of SOCS-3 expression, beginning at 120 min and lasting for more than 360 min. This finding is similar to that observed by Emanuelli et al. (12) when working with skeletal muscle of mice. The decrease of SOCS-3 protein expression that occurs 360 min after Ang II stimulation seems to be a consequence of a fall of Ang II levels because the stimulation with repetitive doses of the hormone is sufficient to prolong SOCS-3 expression as previously demonstrated (16).
Next, it was shown in heart and NRVMs that Ang II-induced SOCS-3 associates with proteins of the insulin signal transduction pathway and that this phenomenon is paralleled by an impairment of the insulin signal transduction machinery. The property of a priming dose of Ang II to hamper insulin signal transduction through IRS-1, IRS-2, PI 3-kinase, and Akt was previously reported (2) and was shown to be, at least in part, dependent on serine phosphorylation of some of those proteins (3). This was herein confirmed and shown to occur in parallel with the expression/association of SOCS-3. However, when Ang II-induced SOCS-3 expression was inhibited by a previous treatment with an antisense oligonucleotide, a partial reversal of the inhibitory effect of Ang II on insulin stimulated IRS-1, IRS-2, and Akt molecular activation was observed, suggesting that each mechanism acts independently to provide a certain degree of regulation on this pathway.
At least two previous reports have shown the participation of SOCS proteins as candidate intermediaries in the cross-talk between a hormone and a cytokine. Boisclair et al. (20) showed that SOCS-3 mediates an inhibitory cross-talk between IL-1? and GH in hepatoma cells. According to their studies, the pretreatment of the cells with IL-1? induces an increase of up to 8-fold SOCS-3, which interacts with JAK-2/STAT-5 and hampers GH-induced transcription of the acid-labile subunit gene. In another study, Lagathu et al. (38) showed that chronic exposure of 3T3-F442A and 3T3-L1 adipocytes to IL-6 inhibits insulin signal transduction, which is paralleled by an increased expression of SOCS-3. Thus, convincing evidence exists to suggest that proteins of the SOCS family not only modulate signal transduction on an intrapathway basis but also provide a negative feedback for related signaling pathways.
In the last part of the study, we evaluated whether the molecular phenomena controlled by Ang II-induced SOCS-3 would play any regulatory role on a functional event activated by insulin. The activation of GLUT-4 is the rate-limiting step in muscle glucose uptake (39). In the heart, as in skeletal muscle and adipose tissue, insulin stimulates GLUT-4 translocation from an intracellular pool to the cell surface in which it mediates glucose transport (40). Knocking out GLUT-4 expression leads to cardiac dysfunction (41) and progressive heart hypertrophy (42), which illustrates the functional importance of this protein in cardiac tissue. In the present study, we observed that a pretreatment with Ang II significantly reduced insulin-induced GLUT-4 translocation in the heart and cultured NRVMs. The physiological significance of this phenomenon attests the fact that living rats were treated with 0.02 ml Ang II at the concentration of 10–8 M, a dose that provides blood Ang II concentration at the physiological range of 100 ng/liter (43). The inhibition of SOCS-3 expression partially prevented this effect, indicating that SOCS-3 modulates an important function controlled by insulin. In a previous study (15), mice transiently overexpressing SOCS-1 became hyperglycemic and hyperinsulinemic, whereas the levels of the suppressor protein were high. According to this study, both SOCS-1 and SOCS-3 interfere with insulin signal transduction by controlling the ubiquitination and degradation of IRS-1 and IRS-2 (15). Two other recent reports show that insulin-induced SOCS (-1, -3, and -6) expression can modulate further insulin signaling by directly binding to the insulin receptor and early substrates (10, 12, 44). In this context, Krebs and Hilton (45) commented that, besides their classical inhibitory role on cytokine signaling, members of the SOCS family may act as important regulators of signal transduction through members of the family of receptors with intrinsic tyrosine kinase activity.
In conclusion, our data demonstrate that SOCS-3 can mediate a late cross-talk between Ang II and insulin and that this cross-talk reflects on the control of one of the most important physiological events controlled by insulin, i.e. GLUT-4 translocation. It will be interesting to determine whether other hormones and cytokines, known to negatively modulate insulin action, may exert their effect through members of the SOCS family to regulate GLUT-4 function.
Acknowledgments
We thank Dr. N. Conran for English editing.
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