Short-Term in Vivo Inhibition of Insulin Receptor Substrate-1 Expression Leads to Insulin Resistance, Hyperinsulinemia, and Increased Adipos
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内分泌学杂志 2005年第3期
Departments of Internal Medicine (E.P.A., C.T.D.S., A.L.G., M.U., M.J.A.S., L.A.V.) and Physiology and Biophysics (A.C.B.), State University of Campinas, Campinas, Sao Paulo, Brazil 13083-970
Address all correspondence and requests for reprints to: Dr. Lício A. Velloso, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Sao Paulo, Brazil 13083-970. E-mail: lavelloso@fcm.unicamp.br.
Abstract
Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF receptors and downstream molecules that participate in insulin and IGF-I signal transduction. Here we employed an antisense oligonucleotide (IRS-1AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days of treatment with IRS-1AS reduced the expression of IRS-1 by 80, 75, and 65% (P < 0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P < 0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P < 0.05) reduction in glucose consumption during a hyperinsulinemic-euglycemic clamp, and a 90% (P < 0.05) increase in basal plasma insulin level. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser (473)] Akt phosphorylation in liver (85%, P < 0.05), skeletal muscle (40%, P < 0.05), and adipose tissue (85%, P < 0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P < 0.05) and skeletal muscle (30%, P < 0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P < 0.05) in adipose tissue of IRS-1AS-treated rats. In rats treated with IRS-1AS for 8 d, a 100% increase (P < 0.05) in relative epididymal fat weight and a 120% (P < 0.05) increase in nuclear expression of peroxisome proliferator-activated receptor- were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a growth-related pathway exclusively in white adipose tissue.
Introduction
INSULIN RECEPTOR SUBSTRATE (IRS)-1 is a 1243-amino-acid protein with an apparent molecular mass of 165–180 kDa, expressed in most mammalian tissues (1). IRS-1 belongs to a family of adaptor proteins composed of at least four members (IRS-1 to IRS-4) (2). All the members of this family possess pleckstrin homology and phosphotyrosine binding (PTB) domains at their N terminus. Moreover, IRS proteins possess several tyrosine residues that may be phosphorylated by upstream kinases generating recognition sites for downstream signal transducers (2). The important role played by IRS-1 and IRS-2 as direct substrates of the insulin receptor (IR) and IGF-I receptor was revealed by generation of cell lines harboring different mutants of these proteins and also the construction of whole-body knockouts of each protein (3, 4). According to these studies, IRS-1 plays a central role in IGF-I-dependent somatic growth, whereas IRS-2 participates mostly in regulation of glucose homeostasis (5). Deletion of the IRS-1 gene leads to an up to 40% reduction in embryonic and neonatal growth (3), whereas deletion of the IRS-2 gene produces a phenotype with only a 10% reduction in neonatal body size (4). In contrast, knockout of IRS-2 leads to insulin resistance accompanied by abnormal ?-cell development (5), and later to type 2 diabetes mellitus, whereas IRS-1 knockout produces insulin resistance accompanied by glucose intolerance with no progression to overt diabetes (6).
The generation of mutants that lack the expression of key proteins involved in insulin and IGF-I signal transduction have contributed enormously to our present understanding of the molecular mechanisms that participate in the genesis of diabetes mellitus and related diseases such as obesity (7), hypertension (8), dyslipidemia (9), and vasculopathy (8). However, in human diabetes the lack of expression of, for example, IRS-1 or IRS-2 or even the expression of functionally compromised mutated proteins, is a rare epidemiological phenomenon (10, 11). In most patients with diabetes mellitus and also most animal models of insulin resistance, the functional impairment of proteins that participate in the transduction of the insulin signal seems to occur progressively, beginning at some time during postnatal life (12, 13, 14, 15, 16). For this reason, modulation of expression or function of proteins belonging to the insulin signaling pathway in adult and nongenetically manipulated and non-diabetes-prone animals may reveal novel insights into the pathogenesis of this complex disorder.
We recently employed a phosphorothioate-modified antisense oligonucleotide to IRS-1 that leads to a 40% reduction in expression of this protein in isolated rat pancreatic islets (17, 18). The objective of the present study was to employ this antisense oligonucleotide to evaluate the effect of short-term in vivo inhibition of IRS-1 expression in non-diabetes-prone Wistar rats. To our surprise, IRS-1 expression inhibition led to glucose intolerance and hyperinsulinemia, and, most interestingly, it led to a selective stimulation of a growth-related signal transduction pathway in white adipose tissue, which was accompanied by increased fat accumulation in this anatomical territory.
Materials and Methods
Antibodies, chemicals, and buffers
Reagents for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Richmond, CA). HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were from Sigma (St. Louis, MO). Protein A-Sepharose 6MB was from Pharmacia (Uppsala, Sweden),125I-protein A was from ICN Biomedicals (Costa Mesa, CA), and nitrocellulose paper (BA85, 0.2 μm) was from Amersham (Aylesbury, UK). Sodium thiopental (Amytal) and human recombinant insulin (Humulin R) were from Lilly (Indianapolis, IN). Antibodies anti-IR (sc-711, rabbit polyclonal), IRS-1 (sc-559, rabbit polyclonal), IRS-2 (sc-8299, rabbit polyclonal), SH2-containing protein (Shc) (sc-1695, rabbit polyclonal), p-ERK (sc-7383, mouse monoclonal, recognizing 42- and 44-kDa ERK phosphorylated at Tyr204), peroxisome proliferator-activated receptor (PPAR) (sc-7196, rabbit polyclonal), glucose transporter-4 (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). Anti-phospho-serine (AB1603, rabbit polyclonal) was purchased from Chemicon International (Temecula, CA), and anti-PTP1B (no. 07-088, rabbit polyclonal) was purchased from Upstate Biotechnology (Lake Placid, NY).
Determination of glucose and insulin
Glucose was determined by the glucose oxidase method, as previously described (19). Insulin was determined by RIA (20).
Phosphorthioate-modified oligonucleotides
Sense and antisense phosphorthioate oligonucleotides specific for IRS-1 (sense, 5'-ACC CAC TCC TAT CCC G-3' and antisense, 5'-CGG GAT AGG AGT GGG T-3') were produced by Invitrogen Corp. (Carlsbad, CA). The sequence was selected among three unrelated pairs of oligonucleotides on the basis of their ability to block IRS-1 protein expression, as evaluated by immunoblot of total protein extracts of isolated pancreatic islets using specific anti-IRS-1 antibodies. The antisense oligonucleotide sequences were submitted to BLAST analyses (www.ncbi.nlm.nih.gov) and matched only for the Rattus norvegicus IRS-1 coding sequence. This oligonucleotide has been used in previous studies (17, 18).
Experimental animals and research protocols
Eight-week-old male Wistar rats (R. norvegicus) from the University of Campinas Central Animal Breeding Center were used in the experiments. The rats were allowed ad libitum access to standard rodent chow and water. Food was withdrawn 12 h before the experiments. All experiments were conducted in accordance with the principles and procedures described by the National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the State University of Campinas Ethical Committee. Rats were randomly sorted into three groups: a control group (C) that received one ip daily dose of 100 μl TE buffer (10 mM Tris-HCl, 1.0 mM EDTA); a sense group that received one daily dose of 100 μl TE buffer containing 0.4 nmol IRS-1 sense oligonucleotide (Sen); and an antisense group that received one daily dose of 100 μl TE buffer containing 0.4 nmol IRS-1 antisense oligonucleotide (AS). Treatment was performed during 3 d, and the experimental procedure was performed on the morning of the fourth day, except for the measurement of epididymal adipose tissue mass and determination of PPAR expression, when treatment was performed during 8 d, and procedure was performed on the morning of the ninth day.
Intraperitoneal glucose tolerance test (ipGTT)
An ipGTT was performed at the end of the experimental period. After an overnight fast, the rats were anesthetized as described above. After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90, and 120 min for determination of glucose and insulin concentrations.
Insulin tolerance test (ITT)
To perform an iv ITT, food was withdrawn 6 h before the test and the rats were anesthetized as described above. Insulin (6 μg) was injected through the tail vein, and blood samples were collected at 0, 4, 8, 12, and 16 min from the tail for serum glucose determination. The constant rate for glucose disappearance (Kitt) was calculated using the formula, 0.693/t1/2. Glucose halftime (t1/2) was calculated from the slope of the least-square analysis of plasma glucose concentrations during the linear decay phase (21).
Clamp studies
All procedures for clamp studies followed previously published descriptions of the method (22). After a 6-h fast, a 2-h hyperinsulinemic euglycemic clamp study was performed in the lower limb. Under sodium thiopental anesthesia and aseptic conditions, a monoocclusive polyethylene catheter was inserted into the femoral artery for infusion of insulin and glucose. A second polyvinyl catheter was inserted into the femoral vein for blood sampling, and the animal was kept in a heated box (37 C) throughout the study. During the first phase of the study (30 min), a priming dose of insulin was infused followed by a rate of glucose infusion necessary to reach a plateau. After glucose equilibration, insulin infusion (3.0 mU·kg–2·min–1) was maintained for 2 h with constant rate (0.20 ml/h), and a variable infusion of glucose (5% solution) was adjusted to maintain the plasma glucose concentration at approximately 120 mg/dl. Blood samples were collected from the femoral vein every 5 min for plasma glucose measurement and every 30 min for plasma insulin determinations. Insulin was measured in duplicate by RIA and oscillated between 4.0 and 9.0 ng/ml in samples collected from animals of both experimental groups.
2-Deoxy-D-[3H] glucose (2-DG) uptake studies
The tissue uptake of 2-DG was measured in vivo according to a method described previously (23). The rats were anesthetized and then injected with 6.0 μCi of 2-deoxy-D-[3H] glucose and [14C]-sucrose in the presence or absence of 0.1 U insulin in 0.4 ml isotonic phosphate buffer (pH 7.4) with 0.1% defatted BSA through the portal vein. After 16 min, slices of liver, gastrocnemius muscle, and epididymal adipose tissue were quickly removed and solubilized in NCS-II tissue solubilizer (Amersham, Buckinghamshire, UK). The radioactivity of 3H in the resulting supernatant was measured in a liquid scintillation fluid (ACS-II Amersham-Japan, Tokyo, Japan), using a scintillation counter (Aloka, model LSC-1000, Kyoto, Japan). Cellular uptake of 2-DG was calculated as the difference between the total tissue radioactivity and the amount of radioactivity present in the tissue extracellular space. The extracellular space volume in microliters per milligram was calculated by dividing the amount of [14C] disintegrations per minute per milligram of tissue by the amount of [14C] disintegrations per minute per microliter of plasma. The concentration of extracellular [3H] disintegrations per minute per milligram of tissue could then be obtained by multiplying the extracellular volume in microliters per milligram of tissue by the concentration of [3H] disintegrations per minute per microliter of plasma. The cellular radioactivity was then converted to picomoles of 2-DG using the specific activity, and the results were expressed per milligram of dry tissue.
Epididymal fat mass determination
Rats were paired by body weight, and one of the animals of the pair was randomly selected for determination of epididymal fat pad at experimental d 0. The other animal of the pair was included in the treatment protocol. After 8 d treatment with one ip daily dose of IRS-1 Sen or AS or TE buffer, rats were anesthetized, the abdominal cavity opened, and the entire epididymal fat pad carefully dissected and weighed. Absolute weight of epididymal fat pad and intrapair variation of epididymal fat pad were used for comparisons.
Tissue extraction, immunoblotting, and immunoprecipitation
The abdominal cavities of anesthetized rats were opened, and the rats received an infusion of insulin (0.2 ml, 10–6 M) or saline (0.2 ml) through the cava vein. After different intervals (described in Results), fragments (3.0 x 3.0 x 3.0 mm) of white adipose tissue, liver, and skeletal muscle were excised and immediately homogenized in 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 orthovanadate, 2.0 mM phenylmethylsulfonyl fluoride (PMSF), 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. Insoluble material was removed by centrifugation for 20 min at 9000 x g in a 70.Ti rotor (Beckman) at 4 C. The protein concentration of the supernatants was determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 5.0 mg total protein were used for immunoprecipitation with antibodies against IR, IRS-1, IRS-2, and Shc at 4 C overnight, followed by SDS-PAGE, transfer to nitrocellulose membranes, and blotting with antiphosphotyrosine, antiphosphoserine, anti-IR, anti-IRS-1, or anti-IRS-2. In direct immunoblot experiments, 0.2 mg of protein extracts obtained from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-IR, IRS-1, IRS-2, anti-phospho[Ser473]-Akt or anti-phospho-ERK antibodies.
Subcellular fractionation
To characterize the expression and subcellular localization of GLUT-4 and PPAR, a subcellular fractionation protocol was employed as described previously (24). Fragments of skeletal muscle and adipose tissue obtained from rats treated, or not, with insulin according to the protocols described above were minced and homogenized in 2 volumes of STE buffer at 4 C [0.32 M sucrose, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1 mM dithiothreitol, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 1 mM PMSF, and 0.1 mg aprotinin/ml]. The homogenates were centrifuged (1000 x g, 25 min, 4 C) to obtain pellets. The pellets were washed once with STE buffer (1000 x g, 10 min, 4 C) and suspended in Triton buffer [1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 200 mM EDTA, 10 mM sodium orthovanadate, 1 mM PMSF, 100 mM NaF, 100 mM sodium pyrophosphate, and 0.1 mg aprotinin/ml], kept on ice for 30 min, and centrifuged (15,000 x g, 30 min, 4 C) to obtain the nuclear fraction. The supernatant was centrifuged (100,000 x g, 60 min, 4 C) to obtain the S100 fraction (which contains most of all intracellular membranes, both low- and high-density microsomes) and the pellet, which was suspended in STE buffer plus 1% Nonidet P-40, kept on ice for 20 min and centrifuged (100,000 x g, 20 min) to obtain the membrane fraction. The fractions 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) were subjected to SDS-PAGE and Western blotting with anti-GLUT-4 antibodies. Samples from adipose tissue total extract and nuclear fraction were separated by SDS-PAGE and transferred to nitrocellulose membrane, which were blotted with anti-PPAR antibodies.
Data presentation and statistical analysis
All numerical results are expressed as the mean ± SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using the Scion Image software (Scion Corp., Frederick, MD). Data were analyzed by the two-tailed unpaired Student’s t test or repeat-measures ANOVA (one-way or two-way ANOVA) followed by post hoc analysis of significance (Bonferroni test) when appropriate, comparing experimental and control groups. The level of significance was set at P < 0.05.
Results
IRS-1AS treatment inhibits IRS-1 without affecting IR and IRS-2 expression
Three days of treatment with IRS-1AS but not IRS-1Sen significantly reduced the expression of IRS-1 protein in liver (80%, P < 0.05), skeletal muscle (75%, P < 0.05), and adipose tissue (65%, P < 0.05) of rats (Fig. 1, A1, B1, and C1), as detected by immunoblot. The inhibiting capacity of the IRS-1AS was already detectable 24 h after the first dose and increased progressively during the experimental period (Fig. 1, A2, B2, and C2). The protein levels of IR (Fig. 1, A3, B3, and C3) and IRS-2 (Fig. 1, A4, B4, and C4) were not affected by IRS-1 inhibition in any of the tissues analyzed.
FIG. 1. Determination of IRS-1 (A1, A2, B1, B2, C1, and C2), IR (A3, B3,and C3), and IRS-2 (A4, B4, and C4) protein expression in liver (A), skeletal muscle (B), and adipose tissue (C) of rats treated for 3 d with Sen or AS IRS-1 phosphorothioate-modified oligonucleotides or with vehicle (C). In A2, B2, and C2, the rats were treated with only AS IRS-1 oligonucleotide for 0, 1, 2 or 3 d. Fragments of liver, skeletal muscle, or adipose tissue were homogenized, and samples (0.2 mg) of total protein extracts obtained were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with specific antibodies. Specific bands were densitometrically quantified. For all conditions, n = 5; *, P < 0.05 vs. C.
Metabolic consequences of short-term inhibition of IRS-1 expression
The treatment with IRS-1AS for 3 d produced no effect on the fasting glucose levels (86 ± 3 vs. 87 ± 4 mg/dl, for control and IRS-1AS, respectively) but led to a significant increase in fasting insulin levels (0.55 ± 0.08 vs. 1.1 ± 0.09 ng/ml, for control and IRS-1AS, respectively, n = 5, P < 0.05). During the ipGTT the levels of blood glucose of IRS-1AS-treated rats were constantly higher than the level of the control and, although no significant differences were obtained when analyzing each time point separately, the area under the glucose curve was significantly higher for IRS-1AS-treated rats (Fig. 2A). The blood insulin levels were higher in IRS-1AS-treated rats at time 0'. At time 30', blood insulin levels of control rats presented a peak reaching 1.48 ± 0.11 ng/ml (170%), whereas in IRS-1AS-treated rats, only a discrete increment from basal was observed (20%). During the remainder of the test, the blood insulin levels were similar between control and IRS-1AS groups, and, again the area under the insulin curve was higher for IRS-1AS-treated rats (Fig. 2A). Furthermore, the treatment with IRS-1AS promoted a reduction of 40% in the Kitt (Fig. 2B) obtained during the ITT and a reduction of 82% in glucose consumption during a hyperinsulinemic-normoglycemic clamp (Fig. 2C).
FIG. 2. A, Glucose (left) and insulin (right) levels during an ipGTT. The areas under the glucose and insulin curves are depicted as insets. Control (C) rats, solid lines; IRS-1AS-treated rats, dashed lines. B, Kitt during the ITT was calculated as described in Materials and Methods. C, Glucose consumption (milligrams per kilograms per minute) during the hyperinsulinemic-euglycemic clamp. The evaluation of insulin action was performed according to the method described in Materials and Methods. In all experiments, n = 6; *, P < 0.05 vs. C.
Insulin signal transduction under IRS-1 expression inhibition
To evaluate the consequences of IRS-1 expression inhibition on the initial and intermediary steps of the insulin signaling pathway, rats were treated with IRS-1AS or IRS-1Sen (not shown) and compared with control. Under anesthesia, a single, acute dose of insulin (0.2 ml, 10–6 M), or saline, was injected through the cava vein, and fragments of liver, skeletal muscle, and visceral adipose tissue were obtained, homogenized, and employed in immunoprecipitation and immunoblotting assays. Figure 3 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in the liver. There was a 50% (P < 0.05) reduction in the insulin-induced tyrosine phosphorylation of the IR (Fig. 3A), a 40% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 3B), a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 3D), an 85% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 3E), and a 20% (P < 0.05) reduction in tyrosine phosphorylation of ERK (Fig. 3F). No significant modulation of insulin-induced IRS-2 phosphorylation was detected in this tissue (Fig. 3C).
FIG. 3. Insulin signal transduction in liver. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the liver were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE transferred to nitrocellulose membranes and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values of the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
Figure 4 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in skeletal muscle. There was a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IR (Fig. 4A), a 50% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 4B), a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 4D), a 40% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 4E), and a 30% (P < 0.05) reduction in tyrosine phosphorylation of ERK (Fig. 4F). Once more no significant modulation in insulin-induced IRS-2 phosphorylation was detected in this tissue (Fig. 4C). Finally, Fig. 5 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in visceral adipose tissue. There was a 60% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IR (Fig. 5A), a 60% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 5B), a 40% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 5D), and an 85% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 4E). Surprisingly, there were significant increases in insulin-induced tyrosine phosphorylation of IRS-2 (50%, P < 0.05; Fig. 5C) and ERK (25%, P < 0.05; Fig. 5F).
FIG. 4. Insulin signal transduction in skeletal muscle. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the gastrocnemius were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values of the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
FIG. 5. Insulin signal transduction in adipose tissue. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the epididymal fat were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values from the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
Inhibition of IRS-1 expression leads to increased serine phosphorylation and PTP1B binding to IR
To evaluate the possible reasons that IRS-1AS treatment led to reduced insulin-induced tyrosine phosphorylation of IR in all tissues studied, rats were treated with IRS-1Sen, IRS-1AS, or saline and employed in experiments for determination of basal IR serine phosphorylation and insulin-induced IR-PTP1B association. As shown in Fig. 6A, inhibition of IRS-1 expression was significantly associated with increased basal serine phosphorylation of IR in liver (90%, P < 0.05), skeletal muscle (55%, P < 0.05), and adipose tissue (110%, P < 0.05). Similarly, as shown in Fig. 6B, the inhibition of IRS-1 was significantly associated with increased insulin-induced binding of PTP1B to IR in liver (45%, P < 0.05), skeletal muscle (55%, P < 0.05), and adipose tissue (80%, P < 0.05).
FIG. 6. Determination of serine phosphorylation (A) and insulin-induced IR-PTP1B association (B) in liver, skeletal muscle, and adipose tissue of rats treated for 3 d with Sen or AS IRS-1 phosphorothioate-modified oligonucleotides or vehicle (C). Fragments of liver, skeletal muscle, or adipose tissue were homogenized, and samples (5.0 mg) of total protein extracts obtained were used in immunoprecipitation (IP) experiments with anti-IR antibodies. Immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with anti-phospho-serine (pSer) antibodies (A) or anti-PTP1B antibodies (B). Specific bands were densitometrically quantified. For all conditions, n = 5.
Insulin-induced membrane localization of GLUT-4 is impaired in muscle and adipose tissue of rats treated with IRS-1AS
To evaluate the functional consequences of the impairment of the insulin signaling through the IR/IRS-1/Akt signal transduction pathway in muscle and adipose tissue of rats treated with IRS-1AS, the experimental animals were injected with a single iv dose of insulin (0.2 ml, 10–6 M) or saline, and after 10 min, fragments of the peri-epididymal fat and gastrocnemius muscle were excised and homogenized. Total extracts, S100, and membrane fractions obtained were separated by SDS-PAGE, transferred to nitrocellulose membranes, and submitted to immunoblotting with anti-GLUT-4 antibodies. As depicted in Fig. 7, the inhibition of IRS-1 expression promoted no significant modulation of total GLUT-4 in muscle and adipose tissue (Fig. 7, A and B, upper blots). There was, however, a tendency to decrease in both tissues. Similarly, the levels of GLUT-4 in S100 fractions of both tissues were not modulated by IRS-1 expression inhibition (Fig. 7, A and B, middle blots). However, IRS-1 expression inhibition led to significant reduction in insulin-induced GLUT-4 expression in the membrane fraction of muscle and visceral adipose tissue (Fig. 7, A and B, lower blots). The levels of GLUT-4 in total extracts and S100 and membrane fractions were similar in control and IRS-1AS rats not stimulated with insulin (saline injected) (not shown).
FIG. 7. Evaluation of insulin-induced GLUT-4 expression (A and B) and 2-DG uptake (C–E) in tissues of rats treated with IRS-1AS or vehicle (C). Protein extract samples (0.2 mg) obtained from skeletal muscle (A) and adipose tissue (B) of rats treated with IRS-1AS or vehicle for 3 d and acutely treated with insulin (200 μl, 10–6 M) were either directly separated by SDS-PAGE (A and B, upper graphs) or initially submitted to a subcellular fractionation protocol, and then the S100 (A and B, middle graphs) and membrane fractions (A and B, lower graphs) obtained were separated by SDS-PAGE. The SDS-PAGE-separated proteins were transferred to nitrocellulose membranes and blotted (IB) with anti-GLUT-4 antibodies. Bands obtained were scanned and densitometrically evaluated. The 2-DG uptake assay was performed in samples obtained from liver (C), skeletal muscle (D), and adipose tissue (E), according to the protocol described in Materials and Methods. In all experiments, n = 5; *, P < 0.05 vs. C+; , P < 0.05 vs. C-.
Effect of IRS-1 inhibition on tissue-specific glucose uptake
To evaluate the effect of inhibition of IRS-1 expression on tissue-specific glucose uptake, we performed a 2-DG uptake assay. In liver, the inhibition of IRS-1 expression exerted no role on basal and insulin-stimulated glucose uptake (Fig. 7C). Conversely and expectedly, in skeletal muscle, the inhibition of IRS-1 expression led to a significant reduction of insulin-induced glucose uptake (25%, P < 0.05; Fig. 7D). Finally, in adipose tissue, IRS-1 expression inhibition promoted a nonsignificant fall in insulin-induced glucose uptake (20%, P = 0.074) and surprisingly a significant increase in basal glucose uptake (80%, P < 0.05; Fig. 7E).
Increased visceral adiposity and increased nuclear expression of PPAR in IRS-1AS-treated rats
The observation that the inhibition of IRS-1 expression leads to increased insulin signaling through the growth-related ERK pathway in adipose tissue prompted us to evaluate the implication of this phenomenon in adipose tissue mass variation during a longer period of treatment with IRS-1AS. For this, rats were treated for 8 d with IRS-1AS, IRS-1Sen, or TE buffer and on the morning of the ninth day were anesthetized, killed, and all adipose tissue in the peri-epididymal region then surgically removed. Another group of rats was submitted to similar treatment regimens, and the periepididymal fat was removed for subcellular fractionation and evaluation of PPAR expression. There were no significant differences in body weight variation (Fig. 8A) and 24-h food intake (Fig. 8B) among the groups. However, there was a significant increase in the rate of body weight gain in IRS-1AS-treated rats (control, 0.635 ± 0.288 g/d; IRS-1Sen, 0.625 ± 0.289 g/d; IRS-1AS, 1.125 ± 0.325 g/d, n = 6, P < 0.05), and a significant increase in intrapair variation of periepididymal fat mass (Fig. 8C). Finally, both total and nuclear expression of PPAR were significantly increased in adipose tissue of IRS-1AS-treated rats (Fig. 8D).
FIG. 8. Effect of an 8-d treatment with IRS-1AS on body weight variation, food intake, adiposity, and subcellular expression of PPAR. Rats treated with Sen (filled circles) or AS (filled triangles) IRS-1 phosphorothioate-modified oligonucleotides or vehicle (C) (open squares) were evaluated for 8-d body weight variation (A), mean 24-h food intake (B), intrapair variation in relative epididymal fat mass (C), and expression of PPAR (D), which was determined in total protein extracts (TE) or nuclear protein extracts (NE) separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with anti-PPAR antibody. In all experiments, n = 6; *, P < 0.05 vs. C.
Discussion
Obesity and type 2 diabetes mellitus have reached epidemic proportions in several regions of the world (25, 26, 27). Resistance to insulin action is the hallmark of type 2 diabetes and is also commonly found in obese patients who have not developed diabetes (28). Most studies concerned with the characterization of the insulin signal transduction in peripheral tissues of humans and animal models of insulin resistance and diabetes have reported a progressive deterioration of the insulin signaling machinery in skeletal muscle, adipose tissue, and liver (12, 15, 29, 30, 31, 32). However, because the inhibition of lyposis and the storage of triglycerides in adipose tissue are both insulin-regulated events, the reason there is a maintenance of fat stores, or even a continuous gain of body weight (mostly due to increased adipose tissue mass), for some time after the installation of insulin resistance has become a matter of intense debate (33, 34, 35, 36, 37, 38). In a recent study, Mosca et al. (33) have shown that insulin-resistant subjects are particularly susceptible to weight gain associated with the ingestion of high-fat food. Accordingly, in two genetically distinct populations, Pima Indians (37) and Chinese Mauritians (36), insulin resistance was a predicting factor for weight gain. However, in two other populations, Asian Indians and Creoles (36), there was an inverse relationship between weight gain and insulin resistance. In the same manner, analyzing patients with variable genetic backgrounds from two distinct studies, Folsom and coworkers (34) reported both direct and inverse association of insulin resistance and weight gain. Finally, in two recent reports (35, 38), an inverse relationship between insulin resistance and development of obesity was determined. Thus, based on clinical data, factors such as genetic background, age and environmental modifiers such as diet composition may favor, or not, the association between insulin resistance and weight gain.
To exert its pleiotropic, multiorganic actions, insulin binds to its cognate receptor (IR) to activate the catalytic, tyrosine kinase activity toward itself and at least nine direct substrates (39). The IRS proteins, particularly IRS-1 and IRS-2, are by far the most studied substrates of the IR. These docking proteins couple insulin/IGF receptors to the phosphatidylinositol 3-kinase (PI 3-kinase) and ERK cascades (2). Through the PI 3-kinase pathway, insulin promotes the activation of Akt leading to metabolic consequences such as control of glucose uptake, glycogen synthesis, and cell survival (40, 41). On the other hand, through the activation of ERK, insulin controls cell growth and mitogenesis (42, 43).
In the first part of the present study, we evaluated the metabolic and molecular outcomes of short-term inhibition of IRS-1 expression in Wistar rats. The treatment with IRS-1AS was extremely effective for inhibiting the expression of the target protein in liver, muscle, and adipose tissue, and as a consequence, the experimental animals became insulin resistant. There were no clinical or laboratorial signs of overt diabetes after 4 or 8 (not shown) d of treatment with IRS-1AS, which was probably due to the capacity of the pancreatic ?-cell to respond to the extra demand. The careful examination of the insulin curve during the ipGTT (particularly the increment from time 0 to time 30 min) suggests that a loss of the first-phase insulin secretion occurs. Defective first-phase insulin secretion is a feature of incipient type 2 diabetes and insulin resistance (44). Thus, we suspect that short-term inhibition of IRS-1 expression leads to insulin resistance, which is accompanied by a compensatory but dysfunctional response by the pancreatic islets. It is interesting to note that in mice with homozygous and heterozygous knockout of the IRS-1, the pancreatic islets exhibit defective response to glucose and arginine (6). However, when pancreatic islets of nongenetically manipulated, non-diabetes-prone rats are treated with IRS-1AS, an increase in glucose-stimulated insulin secretion is observed (17). Nevertheless, this increase occurs after 15 min of exposition of the islets to high glucose, suggesting that inhibition of IRS-1 expression interferes with the first phase of insulin secretion (17). Taken together, all these facts provide further support for the current concept that the loss of first-phase insulin secretion associated with hyperinsulinemia in insulin-resistant states may be the common consequence of peripheral and central (pancreatic islet) defective signaling through IRS-1.
Next, we evaluated the outcomes of inhibition of IRS-1 expression on the regulation of the pathways that lead to Akt and ERK activation. As expected, insulin signaling through IRS-1 and Akt was reduced in all tissues studied. Moreover, signaling through Shc was also reduced. Conversely, we were surprised by the fact that insulin-induced tyrosine phosphorylation of the IR was significantly reduced in liver, skeletal muscle, and adipose tissue, a phenomenon that is not observed in IRS-1 knockout mice (45, 46). In our opinion, there are at least three possible reasons for this finding. First, it could be a matter of stoichiometry. Because IRS-1 and IRS-2 bind to the same domain of the IR, the lower concentration of IRS-1 would allow for higher interaction between IR and IRS-2, which would then lead to higher IRS-2 tyrosine phosphorylation, even in the face of a reduced tyrosine phosphorylation of the IR. Second, the balance between activation and repression of tyrosine phosphatases that shut down IR signal transduction depend on the levels and interactions between the IR and its substrates, IRS-1 and IRS-2 (47, 48, 49). In IRS-1 knockout mice, the absence of IRS-1 is accompanied by an increased expression of IRS-2. However, in IRS-1AS-treated rats, no increase in IRS-2 was detected during the experimental period evaluated in this study. It is possible that the lack in IRS-1 without a compensatory increase in IRS-2 may facilitate phosphatase binding to IR, leading to precocious dephosphorylation.
Another possibility is that the lack of expression of IRS-1, without any compensatory increase in IRS-2, may affect the regulation of insulin-induced activation of serine kinases that catalyze serine phosphorylation of the IR, a phenomenon known to impair insulin-induced activation of its receptor (50, 51, 52). We tested two of these hypotheses by blotting IR immunoprecipitates with antiserine and anti-PTP1B antibodies. Indeed, in all three tissues, there were significant increases in serine phosphorylation and PTP1B binding to the IR. Although we did not investigate the identity of the putative serine kinases involved in IR serine phosphorylation in the present case, we have no reason to believe that they would differ from those serine kinases involved in the control of insulin signaling in other physiological and pathological contexts, such as protein kinase C or C and mammalian target of rapamycin (50, 51, 52, 53). Concerning the association between the IR and PTP1B, it occurs on a similar fashion of that reported in two distinct animal models of obesity, the Goto-Kikazaki and the monosodium glutamate-treated rat (49, 54). In both examples, there is reduced IR tyrosine phosphorylation that is accompanied by increased PTB1B association and catalytic function. Thus, we can conclude that short-term inhibition of IRS-1 expression may affect IR tyrosine phosphorylation status through at least two distinct mechanisms, serine phosphorylation and tyrosine phosphatase targeting.
With respect to the activation of the growth-promoting, mitogenesis-inducing MAPK cascade, which was evaluated by determination of insulin-induced tyrosine phosphorylation of ERK1 and ERK2, we were again surprised by the finding that, although the inhibition of IRS-1 expression led to a reduction in ERK1 and ERK2 activation in liver and skeletal muscle, it also led to a significant and contrasting increase in ERK1 and ERK2 activation in adipose tissue. This was accompanied by an increased insulin-induced tyrosine phosphorylation of IRS-2 only in adipose tissue. When evaluating IRS-1 knockout mice, other groups have observed either impairment or nonmodulation of the activation of ERK by insulin, depending on the tissue studied and whether employing live animals or cultured cells (45, 55). Controversial data exist regarding the possible roles for ERK in adipose tissue growth and differentiation. Some studies have shown that activation of ERK proteins by various effectors, including insulin, leads to a blockade of adipogenesis (56, 57, 58), whereas other studies have shown that ERK promotes preadipocyte differentiation (59, 60, 61). In a recent study, Prusty et al. (62) observed that, in 3T3-L1 preadipocytes, insulin induces an early and transitory activation of ERK, which leads to activation of CCAAT/enhancer-binding protein- and PPAR, transcriptional factors enrolled with proadipocyte differentiation and adipogenesis, respectively (63). The authors conclude that, during incipient adipocyte differentiation, ERK offers a positive signal, participating in the early induction of adiposity. However, in fully differentiated adipocyte it may act by inhibiting PPAR and, thus, participate in the control of further adipogenesis. Interestingly, in a recent clinical study, the gene expression profile of 1152 genes was evaluated in omental adipose tissue of lean and obese subjects. Among others, the genes coding for proteins of the MAPK family were significantly increased in the obese patients (64). According to the authors, activated MAPK proteins in adipose tissue of obese subjects may participate either in further weight gain or control of adipogenesis.
To determine the impact of short-term IRS-1 expression inhibition on functional events controlled by insulin, we evaluated GLUT-4 subcellular expression and glucose uptake. First, it was demonstrated that in skeletal muscle the inhibition of IRS-1 reduces GLUT-4 migration to the cell membrane, a phenomenon that is accompanied by a significant reduction of insulin-induced glucose uptake. In addition, in adipose tissue, there was a reduction of GLUT-4 migration to the cell membrane, but this was accompanied by a tendency of reduction of insulin-induced glucose uptake and a significant increase of basal glucose uptake. In our opinion, these are interesting phenomena, which may contribute to explain the reason that there is fat mass increase in the present model. In a recent study (65), some of us have shown that in an animal model of obesity, there is increased activation of the Cbl (c-Cbl protooncogene)-associated protein/Cbl pathway (66), which occurs in parallel to the inhibition of the IRS-1 signaling pathway. Taken together, these data suggest that the inhibition of insulin signaling through IRS-1 (which may be achieved through a direct inhibition by antisense oligonucleotide, or indirectly, through an unknown mechanism, such as in the animal model of obesity) leads to the activation of mechanisms that sustain glucose uptake exclusively by adipose tissue and allows continuous growth of fat mass, even when the animal is insulin resistant. These effects were further documented in the present model by evaluating body weight variation and expression of the adipogenic transcription factor PPAR. Both the rate of body weight gain and the variation in visceral adipose tissue mass were significantly increased in IRS-1AS-treated rats. Moreover, the total and nuclear expression of PPAR were significantly stimulated by the inhibition of IRS-1.
In our opinion, there are two interesting outcomes of the present study. Initially, it is shown that short-term inhibition of IRS-1 expression is sufficient to produce a phenotype of insulin resistance. Therefore, it provides a costless and easily reproducible method for making a nongenetically manipulated animal resistant to insulin. Second, short-term inhibition of IRS-1 induces a clear molecular and functional resistance to insulin in muscle, which is paralleled by the activation of a growth-promoting pathway in adipose tissue. These findings offer new insights into the complex relationship of insulin resistance and continuously increasing adiposity, placing a defective signal transduction through IRS-1 into a pivotal position in this scenario.
Acknowledgments
We thank Dr. Nicola Conran for English language editing.
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Address all correspondence and requests for reprints to: Dr. Lício A. Velloso, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Sao Paulo, Brazil 13083-970. E-mail: lavelloso@fcm.unicamp.br.
Abstract
Insulin receptor substrate-1 (IRS-1) has an important role as an early intermediary between the insulin and IGF receptors and downstream molecules that participate in insulin and IGF-I signal transduction. Here we employed an antisense oligonucleotide (IRS-1AS) to inhibit whole-body expression of IRS-1 in vivo and evaluate the consequences of short-term inhibition of IRS-1 in Wistar rats. Four days of treatment with IRS-1AS reduced the expression of IRS-1 by 80, 75, and 65% (P < 0.05) in liver, skeletal muscle, and adipose tissue, respectively. This was accompanied by a 40% (P < 0.05) reduction in the constant of glucose decay during an insulin tolerance test, a 78% (P < 0.05) reduction in glucose consumption during a hyperinsulinemic-euglycemic clamp, and a 90% (P < 0.05) increase in basal plasma insulin level. The metabolic effects produced by IRS-1AS were accompanied by a significant reduction in insulin-induced [Ser (473)] Akt phosphorylation in liver (85%, P < 0.05), skeletal muscle (40%, P < 0.05), and adipose tissue (85%, P < 0.05) and a significant reduction in insulin-induced tyrosine phosphorylation of ERK in liver (20%, P < 0.05) and skeletal muscle (30%, P < 0.05). However, insulin-induced tyrosine phosphorylation of ERK was significantly increased (60%, P < 0.05) in adipose tissue of IRS-1AS-treated rats. In rats treated with IRS-1AS for 8 d, a 100% increase (P < 0.05) in relative epididymal fat weight and a 120% (P < 0.05) increase in nuclear expression of peroxisome proliferator-activated receptor- were observed. Thus, acute inhibition of IRS-1 expression in rats leads to insulin resistance accompanied by activation of a growth-related pathway exclusively in white adipose tissue.
Introduction
INSULIN RECEPTOR SUBSTRATE (IRS)-1 is a 1243-amino-acid protein with an apparent molecular mass of 165–180 kDa, expressed in most mammalian tissues (1). IRS-1 belongs to a family of adaptor proteins composed of at least four members (IRS-1 to IRS-4) (2). All the members of this family possess pleckstrin homology and phosphotyrosine binding (PTB) domains at their N terminus. Moreover, IRS proteins possess several tyrosine residues that may be phosphorylated by upstream kinases generating recognition sites for downstream signal transducers (2). The important role played by IRS-1 and IRS-2 as direct substrates of the insulin receptor (IR) and IGF-I receptor was revealed by generation of cell lines harboring different mutants of these proteins and also the construction of whole-body knockouts of each protein (3, 4). According to these studies, IRS-1 plays a central role in IGF-I-dependent somatic growth, whereas IRS-2 participates mostly in regulation of glucose homeostasis (5). Deletion of the IRS-1 gene leads to an up to 40% reduction in embryonic and neonatal growth (3), whereas deletion of the IRS-2 gene produces a phenotype with only a 10% reduction in neonatal body size (4). In contrast, knockout of IRS-2 leads to insulin resistance accompanied by abnormal ?-cell development (5), and later to type 2 diabetes mellitus, whereas IRS-1 knockout produces insulin resistance accompanied by glucose intolerance with no progression to overt diabetes (6).
The generation of mutants that lack the expression of key proteins involved in insulin and IGF-I signal transduction have contributed enormously to our present understanding of the molecular mechanisms that participate in the genesis of diabetes mellitus and related diseases such as obesity (7), hypertension (8), dyslipidemia (9), and vasculopathy (8). However, in human diabetes the lack of expression of, for example, IRS-1 or IRS-2 or even the expression of functionally compromised mutated proteins, is a rare epidemiological phenomenon (10, 11). In most patients with diabetes mellitus and also most animal models of insulin resistance, the functional impairment of proteins that participate in the transduction of the insulin signal seems to occur progressively, beginning at some time during postnatal life (12, 13, 14, 15, 16). For this reason, modulation of expression or function of proteins belonging to the insulin signaling pathway in adult and nongenetically manipulated and non-diabetes-prone animals may reveal novel insights into the pathogenesis of this complex disorder.
We recently employed a phosphorothioate-modified antisense oligonucleotide to IRS-1 that leads to a 40% reduction in expression of this protein in isolated rat pancreatic islets (17, 18). The objective of the present study was to employ this antisense oligonucleotide to evaluate the effect of short-term in vivo inhibition of IRS-1 expression in non-diabetes-prone Wistar rats. To our surprise, IRS-1 expression inhibition led to glucose intolerance and hyperinsulinemia, and, most interestingly, it led to a selective stimulation of a growth-related signal transduction pathway in white adipose tissue, which was accompanied by increased fat accumulation in this anatomical territory.
Materials and Methods
Antibodies, chemicals, and buffers
Reagents for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Richmond, CA). HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and BSA (fraction V) were from Sigma (St. Louis, MO). Protein A-Sepharose 6MB was from Pharmacia (Uppsala, Sweden),125I-protein A was from ICN Biomedicals (Costa Mesa, CA), and nitrocellulose paper (BA85, 0.2 μm) was from Amersham (Aylesbury, UK). Sodium thiopental (Amytal) and human recombinant insulin (Humulin R) were from Lilly (Indianapolis, IN). Antibodies anti-IR (sc-711, rabbit polyclonal), IRS-1 (sc-559, rabbit polyclonal), IRS-2 (sc-8299, rabbit polyclonal), SH2-containing protein (Shc) (sc-1695, rabbit polyclonal), p-ERK (sc-7383, mouse monoclonal, recognizing 42- and 44-kDa ERK phosphorylated at Tyr204), peroxisome proliferator-activated receptor (PPAR) (sc-7196, rabbit polyclonal), glucose transporter-4 (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). Anti-phospho-serine (AB1603, rabbit polyclonal) was purchased from Chemicon International (Temecula, CA), and anti-PTP1B (no. 07-088, rabbit polyclonal) was purchased from Upstate Biotechnology (Lake Placid, NY).
Determination of glucose and insulin
Glucose was determined by the glucose oxidase method, as previously described (19). Insulin was determined by RIA (20).
Phosphorthioate-modified oligonucleotides
Sense and antisense phosphorthioate oligonucleotides specific for IRS-1 (sense, 5'-ACC CAC TCC TAT CCC G-3' and antisense, 5'-CGG GAT AGG AGT GGG T-3') were produced by Invitrogen Corp. (Carlsbad, CA). The sequence was selected among three unrelated pairs of oligonucleotides on the basis of their ability to block IRS-1 protein expression, as evaluated by immunoblot of total protein extracts of isolated pancreatic islets using specific anti-IRS-1 antibodies. The antisense oligonucleotide sequences were submitted to BLAST analyses (www.ncbi.nlm.nih.gov) and matched only for the Rattus norvegicus IRS-1 coding sequence. This oligonucleotide has been used in previous studies (17, 18).
Experimental animals and research protocols
Eight-week-old male Wistar rats (R. norvegicus) from the University of Campinas Central Animal Breeding Center were used in the experiments. The rats were allowed ad libitum access to standard rodent chow and water. Food was withdrawn 12 h before the experiments. All experiments were conducted in accordance with the principles and procedures described by the National Institutes of Health Guidelines for the Care and Use of Experimental Animals and were approved by the State University of Campinas Ethical Committee. Rats were randomly sorted into three groups: a control group (C) that received one ip daily dose of 100 μl TE buffer (10 mM Tris-HCl, 1.0 mM EDTA); a sense group that received one daily dose of 100 μl TE buffer containing 0.4 nmol IRS-1 sense oligonucleotide (Sen); and an antisense group that received one daily dose of 100 μl TE buffer containing 0.4 nmol IRS-1 antisense oligonucleotide (AS). Treatment was performed during 3 d, and the experimental procedure was performed on the morning of the fourth day, except for the measurement of epididymal adipose tissue mass and determination of PPAR expression, when treatment was performed during 8 d, and procedure was performed on the morning of the ninth day.
Intraperitoneal glucose tolerance test (ipGTT)
An ipGTT was performed at the end of the experimental period. After an overnight fast, the rats were anesthetized as described above. After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90, and 120 min for determination of glucose and insulin concentrations.
Insulin tolerance test (ITT)
To perform an iv ITT, food was withdrawn 6 h before the test and the rats were anesthetized as described above. Insulin (6 μg) was injected through the tail vein, and blood samples were collected at 0, 4, 8, 12, and 16 min from the tail for serum glucose determination. The constant rate for glucose disappearance (Kitt) was calculated using the formula, 0.693/t1/2. Glucose halftime (t1/2) was calculated from the slope of the least-square analysis of plasma glucose concentrations during the linear decay phase (21).
Clamp studies
All procedures for clamp studies followed previously published descriptions of the method (22). After a 6-h fast, a 2-h hyperinsulinemic euglycemic clamp study was performed in the lower limb. Under sodium thiopental anesthesia and aseptic conditions, a monoocclusive polyethylene catheter was inserted into the femoral artery for infusion of insulin and glucose. A second polyvinyl catheter was inserted into the femoral vein for blood sampling, and the animal was kept in a heated box (37 C) throughout the study. During the first phase of the study (30 min), a priming dose of insulin was infused followed by a rate of glucose infusion necessary to reach a plateau. After glucose equilibration, insulin infusion (3.0 mU·kg–2·min–1) was maintained for 2 h with constant rate (0.20 ml/h), and a variable infusion of glucose (5% solution) was adjusted to maintain the plasma glucose concentration at approximately 120 mg/dl. Blood samples were collected from the femoral vein every 5 min for plasma glucose measurement and every 30 min for plasma insulin determinations. Insulin was measured in duplicate by RIA and oscillated between 4.0 and 9.0 ng/ml in samples collected from animals of both experimental groups.
2-Deoxy-D-[3H] glucose (2-DG) uptake studies
The tissue uptake of 2-DG was measured in vivo according to a method described previously (23). The rats were anesthetized and then injected with 6.0 μCi of 2-deoxy-D-[3H] glucose and [14C]-sucrose in the presence or absence of 0.1 U insulin in 0.4 ml isotonic phosphate buffer (pH 7.4) with 0.1% defatted BSA through the portal vein. After 16 min, slices of liver, gastrocnemius muscle, and epididymal adipose tissue were quickly removed and solubilized in NCS-II tissue solubilizer (Amersham, Buckinghamshire, UK). The radioactivity of 3H in the resulting supernatant was measured in a liquid scintillation fluid (ACS-II Amersham-Japan, Tokyo, Japan), using a scintillation counter (Aloka, model LSC-1000, Kyoto, Japan). Cellular uptake of 2-DG was calculated as the difference between the total tissue radioactivity and the amount of radioactivity present in the tissue extracellular space. The extracellular space volume in microliters per milligram was calculated by dividing the amount of [14C] disintegrations per minute per milligram of tissue by the amount of [14C] disintegrations per minute per microliter of plasma. The concentration of extracellular [3H] disintegrations per minute per milligram of tissue could then be obtained by multiplying the extracellular volume in microliters per milligram of tissue by the concentration of [3H] disintegrations per minute per microliter of plasma. The cellular radioactivity was then converted to picomoles of 2-DG using the specific activity, and the results were expressed per milligram of dry tissue.
Epididymal fat mass determination
Rats were paired by body weight, and one of the animals of the pair was randomly selected for determination of epididymal fat pad at experimental d 0. The other animal of the pair was included in the treatment protocol. After 8 d treatment with one ip daily dose of IRS-1 Sen or AS or TE buffer, rats were anesthetized, the abdominal cavity opened, and the entire epididymal fat pad carefully dissected and weighed. Absolute weight of epididymal fat pad and intrapair variation of epididymal fat pad were used for comparisons.
Tissue extraction, immunoblotting, and immunoprecipitation
The abdominal cavities of anesthetized rats were opened, and the rats received an infusion of insulin (0.2 ml, 10–6 M) or saline (0.2 ml) through the cava vein. After different intervals (described in Results), fragments (3.0 x 3.0 x 3.0 mm) of white adipose tissue, liver, and skeletal muscle were excised and immediately homogenized in 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 orthovanadate, 2.0 mM phenylmethylsulfonyl fluoride (PMSF), 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. Insoluble material was removed by centrifugation for 20 min at 9000 x g in a 70.Ti rotor (Beckman) at 4 C. The protein concentration of the supernatants was determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 5.0 mg total protein were used for immunoprecipitation with antibodies against IR, IRS-1, IRS-2, and Shc at 4 C overnight, followed by SDS-PAGE, transfer to nitrocellulose membranes, and blotting with antiphosphotyrosine, antiphosphoserine, anti-IR, anti-IRS-1, or anti-IRS-2. In direct immunoblot experiments, 0.2 mg of protein extracts obtained from each tissue were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-IR, IRS-1, IRS-2, anti-phospho[Ser473]-Akt or anti-phospho-ERK antibodies.
Subcellular fractionation
To characterize the expression and subcellular localization of GLUT-4 and PPAR, a subcellular fractionation protocol was employed as described previously (24). Fragments of skeletal muscle and adipose tissue obtained from rats treated, or not, with insulin according to the protocols described above were minced and homogenized in 2 volumes of STE buffer at 4 C [0.32 M sucrose, 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, 1 mM dithiothreitol, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 1 mM PMSF, and 0.1 mg aprotinin/ml]. The homogenates were centrifuged (1000 x g, 25 min, 4 C) to obtain pellets. The pellets were washed once with STE buffer (1000 x g, 10 min, 4 C) and suspended in Triton buffer [1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 200 mM EDTA, 10 mM sodium orthovanadate, 1 mM PMSF, 100 mM NaF, 100 mM sodium pyrophosphate, and 0.1 mg aprotinin/ml], kept on ice for 30 min, and centrifuged (15,000 x g, 30 min, 4 C) to obtain the nuclear fraction. The supernatant was centrifuged (100,000 x g, 60 min, 4 C) to obtain the S100 fraction (which contains most of all intracellular membranes, both low- and high-density microsomes) and the pellet, which was suspended in STE buffer plus 1% Nonidet P-40, kept on ice for 20 min and centrifuged (100,000 x g, 20 min) to obtain the membrane fraction. The fractions 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) were subjected to SDS-PAGE and Western blotting with anti-GLUT-4 antibodies. Samples from adipose tissue total extract and nuclear fraction were separated by SDS-PAGE and transferred to nitrocellulose membrane, which were blotted with anti-PPAR antibodies.
Data presentation and statistical analysis
All numerical results are expressed as the mean ± SEM of the indicated number of experiments. The results of blots are presented as direct comparisons of bands in autoradiographs and quantified by densitometry using the Scion Image software (Scion Corp., Frederick, MD). Data were analyzed by the two-tailed unpaired Student’s t test or repeat-measures ANOVA (one-way or two-way ANOVA) followed by post hoc analysis of significance (Bonferroni test) when appropriate, comparing experimental and control groups. The level of significance was set at P < 0.05.
Results
IRS-1AS treatment inhibits IRS-1 without affecting IR and IRS-2 expression
Three days of treatment with IRS-1AS but not IRS-1Sen significantly reduced the expression of IRS-1 protein in liver (80%, P < 0.05), skeletal muscle (75%, P < 0.05), and adipose tissue (65%, P < 0.05) of rats (Fig. 1, A1, B1, and C1), as detected by immunoblot. The inhibiting capacity of the IRS-1AS was already detectable 24 h after the first dose and increased progressively during the experimental period (Fig. 1, A2, B2, and C2). The protein levels of IR (Fig. 1, A3, B3, and C3) and IRS-2 (Fig. 1, A4, B4, and C4) were not affected by IRS-1 inhibition in any of the tissues analyzed.
FIG. 1. Determination of IRS-1 (A1, A2, B1, B2, C1, and C2), IR (A3, B3,and C3), and IRS-2 (A4, B4, and C4) protein expression in liver (A), skeletal muscle (B), and adipose tissue (C) of rats treated for 3 d with Sen or AS IRS-1 phosphorothioate-modified oligonucleotides or with vehicle (C). In A2, B2, and C2, the rats were treated with only AS IRS-1 oligonucleotide for 0, 1, 2 or 3 d. Fragments of liver, skeletal muscle, or adipose tissue were homogenized, and samples (0.2 mg) of total protein extracts obtained were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with specific antibodies. Specific bands were densitometrically quantified. For all conditions, n = 5; *, P < 0.05 vs. C.
Metabolic consequences of short-term inhibition of IRS-1 expression
The treatment with IRS-1AS for 3 d produced no effect on the fasting glucose levels (86 ± 3 vs. 87 ± 4 mg/dl, for control and IRS-1AS, respectively) but led to a significant increase in fasting insulin levels (0.55 ± 0.08 vs. 1.1 ± 0.09 ng/ml, for control and IRS-1AS, respectively, n = 5, P < 0.05). During the ipGTT the levels of blood glucose of IRS-1AS-treated rats were constantly higher than the level of the control and, although no significant differences were obtained when analyzing each time point separately, the area under the glucose curve was significantly higher for IRS-1AS-treated rats (Fig. 2A). The blood insulin levels were higher in IRS-1AS-treated rats at time 0'. At time 30', blood insulin levels of control rats presented a peak reaching 1.48 ± 0.11 ng/ml (170%), whereas in IRS-1AS-treated rats, only a discrete increment from basal was observed (20%). During the remainder of the test, the blood insulin levels were similar between control and IRS-1AS groups, and, again the area under the insulin curve was higher for IRS-1AS-treated rats (Fig. 2A). Furthermore, the treatment with IRS-1AS promoted a reduction of 40% in the Kitt (Fig. 2B) obtained during the ITT and a reduction of 82% in glucose consumption during a hyperinsulinemic-normoglycemic clamp (Fig. 2C).
FIG. 2. A, Glucose (left) and insulin (right) levels during an ipGTT. The areas under the glucose and insulin curves are depicted as insets. Control (C) rats, solid lines; IRS-1AS-treated rats, dashed lines. B, Kitt during the ITT was calculated as described in Materials and Methods. C, Glucose consumption (milligrams per kilograms per minute) during the hyperinsulinemic-euglycemic clamp. The evaluation of insulin action was performed according to the method described in Materials and Methods. In all experiments, n = 6; *, P < 0.05 vs. C.
Insulin signal transduction under IRS-1 expression inhibition
To evaluate the consequences of IRS-1 expression inhibition on the initial and intermediary steps of the insulin signaling pathway, rats were treated with IRS-1AS or IRS-1Sen (not shown) and compared with control. Under anesthesia, a single, acute dose of insulin (0.2 ml, 10–6 M), or saline, was injected through the cava vein, and fragments of liver, skeletal muscle, and visceral adipose tissue were obtained, homogenized, and employed in immunoprecipitation and immunoblotting assays. Figure 3 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in the liver. There was a 50% (P < 0.05) reduction in the insulin-induced tyrosine phosphorylation of the IR (Fig. 3A), a 40% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 3B), a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 3D), an 85% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 3E), and a 20% (P < 0.05) reduction in tyrosine phosphorylation of ERK (Fig. 3F). No significant modulation of insulin-induced IRS-2 phosphorylation was detected in this tissue (Fig. 3C).
FIG. 3. Insulin signal transduction in liver. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the liver were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE transferred to nitrocellulose membranes and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values of the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
Figure 4 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in skeletal muscle. There was a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IR (Fig. 4A), a 50% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 4B), a 30% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 4D), a 40% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 4E), and a 30% (P < 0.05) reduction in tyrosine phosphorylation of ERK (Fig. 4F). Once more no significant modulation in insulin-induced IRS-2 phosphorylation was detected in this tissue (Fig. 4C). Finally, Fig. 5 depicts the consequences of IRS-1 expression inhibition on the early and intermediary steps of the insulin signaling pathway in visceral adipose tissue. There was a 60% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IR (Fig. 5A), a 60% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of the IRS-1 (Fig. 5B), a 40% (P < 0.05) reduction in insulin-induced tyrosine phosphorylation of Shc (Fig. 5D), and an 85% (P < 0.05) reduction in [Ser473]-phosphorylation of Akt (Fig. 4E). Surprisingly, there were significant increases in insulin-induced tyrosine phosphorylation of IRS-2 (50%, P < 0.05; Fig. 5C) and ERK (25%, P < 0.05; Fig. 5F).
FIG. 4. Insulin signal transduction in skeletal muscle. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the gastrocnemius were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values of the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
FIG. 5. Insulin signal transduction in adipose tissue. Control (C) and IRS-1AS-treated rats were anesthetized and acutely injected with saline (–) or insulin (+) (0.2 ml, 10–6 M) through the cava vein. After 90 sec (A–D) or 5 min (E and F), fragments of the epididymal fat were obtained, homogenized, centrifuged, and aliquots (5.0 mg) of the supernatants used for immunoprecipitation (IP) with anti-IR (A), -IRS-1 (B), -IRS-2 (C), and -Shc (D) antibodies. The immunoprecipitates were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with antiphosphotyrosine (pY) antibodies. In direct Western blot experiments (E and F), samples (0.2 mg) from total extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-phospho-[Ser473] Akt antibodies (E) or anti-phospho ERK (pERK) (F) antibodies. The bands obtained were scanned and evaluated by densitometry. In D, the arbitrary scanning units were obtained from the means of densitometric values from the bands corresponding to the three Shc isoforms (p66, p52, and p46) (inset shows a broader view of the blot). In all experiments, n = 6; *, P < 0.05 vs. C+.
Inhibition of IRS-1 expression leads to increased serine phosphorylation and PTP1B binding to IR
To evaluate the possible reasons that IRS-1AS treatment led to reduced insulin-induced tyrosine phosphorylation of IR in all tissues studied, rats were treated with IRS-1Sen, IRS-1AS, or saline and employed in experiments for determination of basal IR serine phosphorylation and insulin-induced IR-PTP1B association. As shown in Fig. 6A, inhibition of IRS-1 expression was significantly associated with increased basal serine phosphorylation of IR in liver (90%, P < 0.05), skeletal muscle (55%, P < 0.05), and adipose tissue (110%, P < 0.05). Similarly, as shown in Fig. 6B, the inhibition of IRS-1 was significantly associated with increased insulin-induced binding of PTP1B to IR in liver (45%, P < 0.05), skeletal muscle (55%, P < 0.05), and adipose tissue (80%, P < 0.05).
FIG. 6. Determination of serine phosphorylation (A) and insulin-induced IR-PTP1B association (B) in liver, skeletal muscle, and adipose tissue of rats treated for 3 d with Sen or AS IRS-1 phosphorothioate-modified oligonucleotides or vehicle (C). Fragments of liver, skeletal muscle, or adipose tissue were homogenized, and samples (5.0 mg) of total protein extracts obtained were used in immunoprecipitation (IP) experiments with anti-IR antibodies. Immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with anti-phospho-serine (pSer) antibodies (A) or anti-PTP1B antibodies (B). Specific bands were densitometrically quantified. For all conditions, n = 5.
Insulin-induced membrane localization of GLUT-4 is impaired in muscle and adipose tissue of rats treated with IRS-1AS
To evaluate the functional consequences of the impairment of the insulin signaling through the IR/IRS-1/Akt signal transduction pathway in muscle and adipose tissue of rats treated with IRS-1AS, the experimental animals were injected with a single iv dose of insulin (0.2 ml, 10–6 M) or saline, and after 10 min, fragments of the peri-epididymal fat and gastrocnemius muscle were excised and homogenized. Total extracts, S100, and membrane fractions obtained were separated by SDS-PAGE, transferred to nitrocellulose membranes, and submitted to immunoblotting with anti-GLUT-4 antibodies. As depicted in Fig. 7, the inhibition of IRS-1 expression promoted no significant modulation of total GLUT-4 in muscle and adipose tissue (Fig. 7, A and B, upper blots). There was, however, a tendency to decrease in both tissues. Similarly, the levels of GLUT-4 in S100 fractions of both tissues were not modulated by IRS-1 expression inhibition (Fig. 7, A and B, middle blots). However, IRS-1 expression inhibition led to significant reduction in insulin-induced GLUT-4 expression in the membrane fraction of muscle and visceral adipose tissue (Fig. 7, A and B, lower blots). The levels of GLUT-4 in total extracts and S100 and membrane fractions were similar in control and IRS-1AS rats not stimulated with insulin (saline injected) (not shown).
FIG. 7. Evaluation of insulin-induced GLUT-4 expression (A and B) and 2-DG uptake (C–E) in tissues of rats treated with IRS-1AS or vehicle (C). Protein extract samples (0.2 mg) obtained from skeletal muscle (A) and adipose tissue (B) of rats treated with IRS-1AS or vehicle for 3 d and acutely treated with insulin (200 μl, 10–6 M) were either directly separated by SDS-PAGE (A and B, upper graphs) or initially submitted to a subcellular fractionation protocol, and then the S100 (A and B, middle graphs) and membrane fractions (A and B, lower graphs) obtained were separated by SDS-PAGE. The SDS-PAGE-separated proteins were transferred to nitrocellulose membranes and blotted (IB) with anti-GLUT-4 antibodies. Bands obtained were scanned and densitometrically evaluated. The 2-DG uptake assay was performed in samples obtained from liver (C), skeletal muscle (D), and adipose tissue (E), according to the protocol described in Materials and Methods. In all experiments, n = 5; *, P < 0.05 vs. C+; , P < 0.05 vs. C-.
Effect of IRS-1 inhibition on tissue-specific glucose uptake
To evaluate the effect of inhibition of IRS-1 expression on tissue-specific glucose uptake, we performed a 2-DG uptake assay. In liver, the inhibition of IRS-1 expression exerted no role on basal and insulin-stimulated glucose uptake (Fig. 7C). Conversely and expectedly, in skeletal muscle, the inhibition of IRS-1 expression led to a significant reduction of insulin-induced glucose uptake (25%, P < 0.05; Fig. 7D). Finally, in adipose tissue, IRS-1 expression inhibition promoted a nonsignificant fall in insulin-induced glucose uptake (20%, P = 0.074) and surprisingly a significant increase in basal glucose uptake (80%, P < 0.05; Fig. 7E).
Increased visceral adiposity and increased nuclear expression of PPAR in IRS-1AS-treated rats
The observation that the inhibition of IRS-1 expression leads to increased insulin signaling through the growth-related ERK pathway in adipose tissue prompted us to evaluate the implication of this phenomenon in adipose tissue mass variation during a longer period of treatment with IRS-1AS. For this, rats were treated for 8 d with IRS-1AS, IRS-1Sen, or TE buffer and on the morning of the ninth day were anesthetized, killed, and all adipose tissue in the peri-epididymal region then surgically removed. Another group of rats was submitted to similar treatment regimens, and the periepididymal fat was removed for subcellular fractionation and evaluation of PPAR expression. There were no significant differences in body weight variation (Fig. 8A) and 24-h food intake (Fig. 8B) among the groups. However, there was a significant increase in the rate of body weight gain in IRS-1AS-treated rats (control, 0.635 ± 0.288 g/d; IRS-1Sen, 0.625 ± 0.289 g/d; IRS-1AS, 1.125 ± 0.325 g/d, n = 6, P < 0.05), and a significant increase in intrapair variation of periepididymal fat mass (Fig. 8C). Finally, both total and nuclear expression of PPAR were significantly increased in adipose tissue of IRS-1AS-treated rats (Fig. 8D).
FIG. 8. Effect of an 8-d treatment with IRS-1AS on body weight variation, food intake, adiposity, and subcellular expression of PPAR. Rats treated with Sen (filled circles) or AS (filled triangles) IRS-1 phosphorothioate-modified oligonucleotides or vehicle (C) (open squares) were evaluated for 8-d body weight variation (A), mean 24-h food intake (B), intrapair variation in relative epididymal fat mass (C), and expression of PPAR (D), which was determined in total protein extracts (TE) or nuclear protein extracts (NE) separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted (IB) with anti-PPAR antibody. In all experiments, n = 6; *, P < 0.05 vs. C.
Discussion
Obesity and type 2 diabetes mellitus have reached epidemic proportions in several regions of the world (25, 26, 27). Resistance to insulin action is the hallmark of type 2 diabetes and is also commonly found in obese patients who have not developed diabetes (28). Most studies concerned with the characterization of the insulin signal transduction in peripheral tissues of humans and animal models of insulin resistance and diabetes have reported a progressive deterioration of the insulin signaling machinery in skeletal muscle, adipose tissue, and liver (12, 15, 29, 30, 31, 32). However, because the inhibition of lyposis and the storage of triglycerides in adipose tissue are both insulin-regulated events, the reason there is a maintenance of fat stores, or even a continuous gain of body weight (mostly due to increased adipose tissue mass), for some time after the installation of insulin resistance has become a matter of intense debate (33, 34, 35, 36, 37, 38). In a recent study, Mosca et al. (33) have shown that insulin-resistant subjects are particularly susceptible to weight gain associated with the ingestion of high-fat food. Accordingly, in two genetically distinct populations, Pima Indians (37) and Chinese Mauritians (36), insulin resistance was a predicting factor for weight gain. However, in two other populations, Asian Indians and Creoles (36), there was an inverse relationship between weight gain and insulin resistance. In the same manner, analyzing patients with variable genetic backgrounds from two distinct studies, Folsom and coworkers (34) reported both direct and inverse association of insulin resistance and weight gain. Finally, in two recent reports (35, 38), an inverse relationship between insulin resistance and development of obesity was determined. Thus, based on clinical data, factors such as genetic background, age and environmental modifiers such as diet composition may favor, or not, the association between insulin resistance and weight gain.
To exert its pleiotropic, multiorganic actions, insulin binds to its cognate receptor (IR) to activate the catalytic, tyrosine kinase activity toward itself and at least nine direct substrates (39). The IRS proteins, particularly IRS-1 and IRS-2, are by far the most studied substrates of the IR. These docking proteins couple insulin/IGF receptors to the phosphatidylinositol 3-kinase (PI 3-kinase) and ERK cascades (2). Through the PI 3-kinase pathway, insulin promotes the activation of Akt leading to metabolic consequences such as control of glucose uptake, glycogen synthesis, and cell survival (40, 41). On the other hand, through the activation of ERK, insulin controls cell growth and mitogenesis (42, 43).
In the first part of the present study, we evaluated the metabolic and molecular outcomes of short-term inhibition of IRS-1 expression in Wistar rats. The treatment with IRS-1AS was extremely effective for inhibiting the expression of the target protein in liver, muscle, and adipose tissue, and as a consequence, the experimental animals became insulin resistant. There were no clinical or laboratorial signs of overt diabetes after 4 or 8 (not shown) d of treatment with IRS-1AS, which was probably due to the capacity of the pancreatic ?-cell to respond to the extra demand. The careful examination of the insulin curve during the ipGTT (particularly the increment from time 0 to time 30 min) suggests that a loss of the first-phase insulin secretion occurs. Defective first-phase insulin secretion is a feature of incipient type 2 diabetes and insulin resistance (44). Thus, we suspect that short-term inhibition of IRS-1 expression leads to insulin resistance, which is accompanied by a compensatory but dysfunctional response by the pancreatic islets. It is interesting to note that in mice with homozygous and heterozygous knockout of the IRS-1, the pancreatic islets exhibit defective response to glucose and arginine (6). However, when pancreatic islets of nongenetically manipulated, non-diabetes-prone rats are treated with IRS-1AS, an increase in glucose-stimulated insulin secretion is observed (17). Nevertheless, this increase occurs after 15 min of exposition of the islets to high glucose, suggesting that inhibition of IRS-1 expression interferes with the first phase of insulin secretion (17). Taken together, all these facts provide further support for the current concept that the loss of first-phase insulin secretion associated with hyperinsulinemia in insulin-resistant states may be the common consequence of peripheral and central (pancreatic islet) defective signaling through IRS-1.
Next, we evaluated the outcomes of inhibition of IRS-1 expression on the regulation of the pathways that lead to Akt and ERK activation. As expected, insulin signaling through IRS-1 and Akt was reduced in all tissues studied. Moreover, signaling through Shc was also reduced. Conversely, we were surprised by the fact that insulin-induced tyrosine phosphorylation of the IR was significantly reduced in liver, skeletal muscle, and adipose tissue, a phenomenon that is not observed in IRS-1 knockout mice (45, 46). In our opinion, there are at least three possible reasons for this finding. First, it could be a matter of stoichiometry. Because IRS-1 and IRS-2 bind to the same domain of the IR, the lower concentration of IRS-1 would allow for higher interaction between IR and IRS-2, which would then lead to higher IRS-2 tyrosine phosphorylation, even in the face of a reduced tyrosine phosphorylation of the IR. Second, the balance between activation and repression of tyrosine phosphatases that shut down IR signal transduction depend on the levels and interactions between the IR and its substrates, IRS-1 and IRS-2 (47, 48, 49). In IRS-1 knockout mice, the absence of IRS-1 is accompanied by an increased expression of IRS-2. However, in IRS-1AS-treated rats, no increase in IRS-2 was detected during the experimental period evaluated in this study. It is possible that the lack in IRS-1 without a compensatory increase in IRS-2 may facilitate phosphatase binding to IR, leading to precocious dephosphorylation.
Another possibility is that the lack of expression of IRS-1, without any compensatory increase in IRS-2, may affect the regulation of insulin-induced activation of serine kinases that catalyze serine phosphorylation of the IR, a phenomenon known to impair insulin-induced activation of its receptor (50, 51, 52). We tested two of these hypotheses by blotting IR immunoprecipitates with antiserine and anti-PTP1B antibodies. Indeed, in all three tissues, there were significant increases in serine phosphorylation and PTP1B binding to the IR. Although we did not investigate the identity of the putative serine kinases involved in IR serine phosphorylation in the present case, we have no reason to believe that they would differ from those serine kinases involved in the control of insulin signaling in other physiological and pathological contexts, such as protein kinase C or C and mammalian target of rapamycin (50, 51, 52, 53). Concerning the association between the IR and PTP1B, it occurs on a similar fashion of that reported in two distinct animal models of obesity, the Goto-Kikazaki and the monosodium glutamate-treated rat (49, 54). In both examples, there is reduced IR tyrosine phosphorylation that is accompanied by increased PTB1B association and catalytic function. Thus, we can conclude that short-term inhibition of IRS-1 expression may affect IR tyrosine phosphorylation status through at least two distinct mechanisms, serine phosphorylation and tyrosine phosphatase targeting.
With respect to the activation of the growth-promoting, mitogenesis-inducing MAPK cascade, which was evaluated by determination of insulin-induced tyrosine phosphorylation of ERK1 and ERK2, we were again surprised by the finding that, although the inhibition of IRS-1 expression led to a reduction in ERK1 and ERK2 activation in liver and skeletal muscle, it also led to a significant and contrasting increase in ERK1 and ERK2 activation in adipose tissue. This was accompanied by an increased insulin-induced tyrosine phosphorylation of IRS-2 only in adipose tissue. When evaluating IRS-1 knockout mice, other groups have observed either impairment or nonmodulation of the activation of ERK by insulin, depending on the tissue studied and whether employing live animals or cultured cells (45, 55). Controversial data exist regarding the possible roles for ERK in adipose tissue growth and differentiation. Some studies have shown that activation of ERK proteins by various effectors, including insulin, leads to a blockade of adipogenesis (56, 57, 58), whereas other studies have shown that ERK promotes preadipocyte differentiation (59, 60, 61). In a recent study, Prusty et al. (62) observed that, in 3T3-L1 preadipocytes, insulin induces an early and transitory activation of ERK, which leads to activation of CCAAT/enhancer-binding protein- and PPAR, transcriptional factors enrolled with proadipocyte differentiation and adipogenesis, respectively (63). The authors conclude that, during incipient adipocyte differentiation, ERK offers a positive signal, participating in the early induction of adiposity. However, in fully differentiated adipocyte it may act by inhibiting PPAR and, thus, participate in the control of further adipogenesis. Interestingly, in a recent clinical study, the gene expression profile of 1152 genes was evaluated in omental adipose tissue of lean and obese subjects. Among others, the genes coding for proteins of the MAPK family were significantly increased in the obese patients (64). According to the authors, activated MAPK proteins in adipose tissue of obese subjects may participate either in further weight gain or control of adipogenesis.
To determine the impact of short-term IRS-1 expression inhibition on functional events controlled by insulin, we evaluated GLUT-4 subcellular expression and glucose uptake. First, it was demonstrated that in skeletal muscle the inhibition of IRS-1 reduces GLUT-4 migration to the cell membrane, a phenomenon that is accompanied by a significant reduction of insulin-induced glucose uptake. In addition, in adipose tissue, there was a reduction of GLUT-4 migration to the cell membrane, but this was accompanied by a tendency of reduction of insulin-induced glucose uptake and a significant increase of basal glucose uptake. In our opinion, these are interesting phenomena, which may contribute to explain the reason that there is fat mass increase in the present model. In a recent study (65), some of us have shown that in an animal model of obesity, there is increased activation of the Cbl (c-Cbl protooncogene)-associated protein/Cbl pathway (66), which occurs in parallel to the inhibition of the IRS-1 signaling pathway. Taken together, these data suggest that the inhibition of insulin signaling through IRS-1 (which may be achieved through a direct inhibition by antisense oligonucleotide, or indirectly, through an unknown mechanism, such as in the animal model of obesity) leads to the activation of mechanisms that sustain glucose uptake exclusively by adipose tissue and allows continuous growth of fat mass, even when the animal is insulin resistant. These effects were further documented in the present model by evaluating body weight variation and expression of the adipogenic transcription factor PPAR. Both the rate of body weight gain and the variation in visceral adipose tissue mass were significantly increased in IRS-1AS-treated rats. Moreover, the total and nuclear expression of PPAR were significantly stimulated by the inhibition of IRS-1.
In our opinion, there are two interesting outcomes of the present study. Initially, it is shown that short-term inhibition of IRS-1 expression is sufficient to produce a phenotype of insulin resistance. Therefore, it provides a costless and easily reproducible method for making a nongenetically manipulated animal resistant to insulin. Second, short-term inhibition of IRS-1 induces a clear molecular and functional resistance to insulin in muscle, which is paralleled by the activation of a growth-promoting pathway in adipose tissue. These findings offer new insights into the complex relationship of insulin resistance and continuously increasing adiposity, placing a defective signal transduction through IRS-1 into a pivotal position in this scenario.
Acknowledgments
We thank Dr. Nicola Conran for English language editing.
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