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Insulin Receptor Substrate-4 Is Expressed in Muscle Tissue without Acting as a Substrate for the Insulin Receptor

Molecular Cardiology (S.S., D.L., I.R., J.E.), Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes Research Institute, D-40225 Düsseldorf, Germany; and Institute of Pathophysiology (I.K.), University of Greifswald, D-17495 Karlsburg, Germany

Address all correspondence and requests for reprints to: Professor Dr. Jürgen Eckel, German Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail: eckel{at}uni-duesseldorf.de.

	Abstract

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Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
INSULIN RECEPTOR SUBSTRATE (IRS) proteins exert key functions as signaling intermediates for the insulin and the IGF-1 receptor (1, 2). This involves the tyrosine phosphorylation of IRS proteins within short motifs that bind to Src homology 2 domains of intracellular signaling proteins, including phosphatidylinositol 3-kinase (3), growth factor receptor-binding protein 2 (4), and the protein tyrosine phosphatase SHP-2/Syp (5), finally activating specific signaling cascades. So far, four members of the IRS family (IRS-1, -2, -3, and -4) have been identified with a similar general architecture and domain structure (6). Despite an apparent functional redundancy in the IRS family, these proteins are thought to play distinct roles in mediating insulin action. This is based on their specific tissue and cellular expression (7) and the unique structural features of each IRS protein, which affect the interaction with upstream and downstream proteins (8, 9).

IRS-4 is the most recently characterized member of the IRS family that was initially detected in human embryonic kidney (HEK) 293 cells (10, 11). In vitro studies have shown that IRS-4 binds to phosphatidylinositol 3-kinase and growth factor receptor-binding protein 2 (11) and that overexpression of IRS-4 in rat adipocytes leads to the translocation of GLUT4 to the cell surface (12). However, IRS-4 has a different signaling capacity, compared with the other IRS proteins. Thus, IRS-4 does not interact with SHP2 (11) and overexpression in 32D cells failed to promote cell survival, in sharp contrast to IRS-1 and IRS-2 (13). Furthermore, Tsuruzoe et al. (14) recently suggested that IRS-3 and IRS-4 may even act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins.

In contrast to the in vitro studies, the in vivo function of IRS-4 has remained elusive. Interestingly, IRS-4 exhibits a more limited tissue expression, and it was not possible to detect IRS-4 protein in any mouse tissue so far (15). This may explain the absence of a discernible phenotype in knockout mice lacking IRS-4 (16). However, mRNA expression of IRS-4 was recently demonstrated in different human and rodent tissues including heart and skeletal muscle (17). In the present study, we assessed the protein expression of IRS-4 in rat muscle of different fiber type composition and in human skeletal muscle cells. The data show specific expression of IRS-4 in soleus and cardiac muscle with a prominent down-regulation of IRS-4 expression in a rat model of the metabolic syndrome. IRS-4 is not phosphorylated in response to insulin and IGF-I in primary muscle cells but appears to be involved in stress-induced cellular signaling.

	Materials and Methods

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Chemicals
Reagents for SDS-PAGE were supplied by Amersham Pharmacia Biotech (Braunschweig, Germany) and Sigma (München, Germany). BSA (fraction V, fatty acid free) was obtained from Boehringer (Mannheim, Germany), and protein A trisacryl beads were a product from Pierce Chemical Co. (Rockford, IL). Polyclonal anti-IRS-1 and anti-IRS-2 antiserum were gifts from Dr. J. A. Maassen (Leiden, The Netherlands). The polyclonal rabbit anti-IRS-4 antibody and the immunizing peptide (residues 1240–1257 of human IRS-4) were provided by Upstate Biotechnology (Lake Placid, NY). The antiphosphotyrosine antibody (RC-20) coupled to horseradish peroxidase (HRP) was from Transduction Laboratories, Inc. (Lexington, KY). HRP-conjugated goat-antirabbit IgG antibody as secondary antibody for enhanced chemiluminescence (ECL) detection was from Promega Corp. (Mannheim, Germany). Primary human skeletal muscle cells and supplement pack for growth medium were obtained from PromoCell (Heidelberg, Germany). Culture media were purchased from Life Technologies, Inc. (Berlin, Germany). All other chemicals were of the highest analytical grade commercially available and were purchased from Sigma.

Cell culture
HEK 293 cells were grown on 10-cm plates in DMEM supplemented with 10% fetal calf serum and streptomycin/penicillin. Cells were used upon reaching 80% confluence and were preincubated in serum-free medium for 2 h before stimulation with insulin.

Cardiomyocytes from adult rat heart of normal Wistar rats were isolated by perfusion of the heart with collagenase, as described in our earlier reports (18, 19). The final cell suspension was centrifuged and stored in liquid nitrogen until further use.

Primary human skeletal muscle cells obtained from satellite cells isolated from M. rectus abdominis of healthy Caucasian donors were supplied as proliferating myoblasts. Cells were cultured in -modified Eagle’s/Ham’s F-12 medium containing skeletal muscle cell growth medium supplement pack up to near confluence as recently described by us (20). For stimulation studies, 10⁶ cells/dish were plated in growth medium and were cultured for 5–6 d. Myoblasts were then washed with PBS and incubated for 24 h in the absence of serum before stimulation with insulin.

Preparation of tissues
Male Wistar rats weighing 280–320 g were used throughout the experiments. All animals had free access to food and drinking water, and all animal experimentation was conducted in accord with accepted standards of humane animal care. Animals were killed by decapitation and the heart and the gastrocnemius, quadriceps, and soleus muscle were removed (21). In parallel, the brain of the animal was rapidly excised and the hypothalamus was dissected, as detailed recently by us (22).

In some experiments, male obese Wistar Ottawa Karlsburg (WOKW) rats (23), at an age of 24–28 wk and weighing 400–450 g, and age-matched Wistar controls were used. Cardiac and soleus muscle of these animals was removed as outlined above.

Immunoprecipitation
Cells were washed twice with ice-cold PBS and lysis was performed by incubation in radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCL, pH 7.4; 1% Nonidet P-40; 0.25% sodium-deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM Na₃Vo₄; 1 mM NaF; and protease inhibitor cocktail) for 2 h at 4 C with gentle agitation. The suspension was centrifuged at 10,000 x g for 20 min and the supernatant (800 µl at 1 µg protein per microliter, if not otherwise indicated) was then incubated with antibodies (5 µl) against IRS-1, IRS-2, or IRS-4 at 4 C and gently rocked overnight. The immunocomplexes were adsorbed to protein A-Sepharose beads for 2 h at 4 C during gentle agitation and subsequently collected by centrifugation at 14,000 rpm for 30 sec at 4 C. Beads were then washed three times with ice-cold PBS, incubated for 10 min at 95 C with 20 µl electrophoresis buffer, and the complete supernatant was used for Western blot analysis.

Western blotting
Tissues were lysed in RIPA buffer except for brain, which was lysed in a buffer consisting of 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM sodium orthovanadate, 100 mM NaF, 1 mM EDTA, and protease inhibitor cocktail. The tissues (200 mg) were homogenized in 2 ml lysis buffer (10% wt/vol) using Ultra-Turrax tissue homogenizer (Jahnke Kunkel, Staufen, Germany). Lysates were cleared by centrifugation at 10,000 rpm for 20 min at 4 C. Protein determination of the supernatant was performed by the Bradford method using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Immunoprecipitates or total lysates were separated by SDS-PAGE using 7.5% horizontal gels and transferred to polyvinylidene fluoride filters in a semidry blotting apparatus (24). For phosphotyrosine detection, filters were blocked 60 min in Tris-buffered saline containing 0.05% Tween 20 and 1% BSA. Thereafter filters were incubated overnight with antiphosphotyrosine antibody (RC-20) coupled to HRP and subsequently processed for ECL detection using SuperSignal substrate (Pierce Chemical Co.). For detection of IRS-4, filters were blocked with Tris-buffered saline containing 0.05% Tween 20 and 10% nonfat dry milk and incubated overnight with 1 µg/ml antibody. Blocking of the IRS-4 antibody was achieved by preincubation of 1 µg/ml antibody with 10 µM immunizing peptide for 30 min at 4 C, followed by immediate use for immunodetection. After extensive washing, filters were incubated with goat-antirabbit HRP-coupled antibody and processed for ECL detection. Signals were visualized and evaluated on a LUMI Imager workstation using image analysis software (Roche Molecular Biochemicals, Mannheim, Germany).

Statistical analysis
All data analysis was performed using Prism (GraphPad Software, Inc., San Diego, CA) or t-ease (ISI, Philadelphia, PA) statistical software. Significance of reported differences was evaluated by using the null hypothesis and t statistics for paired data. A P value less than 0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IRS-4 is expressed in rat skeletal and cardiac muscle
In the present study, we used an antiserum raised against the carboxyl-terminal 18 amino acids of human IRS-4, somewhat different from the antisera used in earlier studies (11, 25). As shown in Fig. 1, the antiserum detects a prominent 160-kDa protein band in HEK 293 cell lysates that is completely absent when probing the blots with IRS-4 antiserum blocked with the immunizing peptide. Furthermore, immunoprecipitation of IRS-4 from HEK cell lysates revealed the presence of one protein band at 160 kDa (Fig. 1), most probably representing IRS-4. So far, rat IRS-4 has not been cloned; however, the peptide used for immunization has 44% homology with murine IRS-4 (15). Lysates of rat skeletal muscle with different fiber-type composition were probed with the IRS-4 antiserum using the blocked antiserum as a specificity control. As presented in Fig. 1, IRS-4 protein was specifically detected in red soleus muscle at a molecular mass of 150 kDa. In quadriceps and gastrocnemius, two muscles with mixed fiber-type composition, a faint band at 150 kDa was observed, potentially representing IRS-4. It should be noted that 20 µg skeletal muscle lysates were used for immunoblotting vs. only 1 µg HEK cell lysates.

View larger version (29K): [in this window] [in a new window]   Figure 1. Western blot analysis of IRS-4 expression in HEK 293 cells and rat skeletal muscle. A, HEK 293 cells were cultured as described in Materials and Methods and were lysed in RIPA buffer. One microgram cell lysate from three different cell batches (a–c) were resolved by SDS-PAGE and immunoblotted for IRS-4. IRS-4 antibody blocked with the immunizing peptide was used as a specificity control. IRS-4 was immunoprecipitated (IP) as described in Materials and Methods, and immunopellets were subjected to immunoblotting (ID) for IRS-4. Bands were detected using an ECL system and LUMI imager analyzer. Representative experiments of five replicate experiments are shown. B–D, Skeletal muscle samples from four individual rats (a–d) was lysed and subjected to immunoblotting for IRS-4 as outlined above, except using 20 µg tissue lysate per lane.

View larger version (25K): [in this window] [in a new window]   Figure 2. Protein expression of IRS-4 in cardiac and soleus muscle. A, Hearts were removed from individual rats (a–d), and tissue lysates (20 µg per lane) were subjected to immunoblotting for IRS-4, as outlined in Fig. 1. Cardiomyocytes were prepared from adult rat as described in Materials and Methods. Lysates were then immunoblotted for IRS-4. B, Heart and soleus muscle lysates were resolved by SDS-PAGE within the same gel and were immunoblotted for IRS-4. Signal intensities were quantified by LUMI imager software and are expressed as arbitrary units. Data are mean values of four different animals ± SD. *, Significantly different at P < 0.005.

View larger version (16K): [in this window] [in a new window]   Figure 3. Protein expression of IRS-4 in rat brain. Dissection of the hypothalamus was performed as described in Materials and Methods. Twenty micrograms hypothalamic lysates (a, b, d, e) or cerebellum (c) were then immunoblotted for IRS-4, as outlined in Fig. 1. A representative experiment of three is shown.

View larger version (42K): [in this window] [in a new window]   Figure 4. Expression of IRS proteins in heart and soleus muscle of WOKW rats. Heart and soleus muscle of WOKW and Wistar rats was removed, tissues were lysed, and immunoblotted (20 µg per lane) for IRS-4 (A), IRS-1 (B), and IRS-2 (C). Tissues from individual animals are marked a–d. Signal intensities were quantified by LUMI imager software and are expressed as arbitrary units. Data are mean values of four different animals ± SD. *, Significantly different at P < 0.05; **, significantly different at P < 0.005.

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of insulin on the tyrosine phosphorylation of IRS-4. A, Human skeletal muscle cells were cultured as detailed in Methods. After preincubation under serum-free conditions, the cells were stimulated with insulin (100 nM) for the indicated times followed by cell lysis and immunoprecipitation (IP) (800 µg protein) of IRS-4. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (ID) with anti-IRS-4 and antiphosphotyrosine antibodies, respectively. Signals were visualized using ECL detection. B, HEK cells were stimulated with the indicated concentrations of insulin for 10 min followed by cell lysis, immunoprecipitation (of 100 µg protein), and immunoblotting as outlined above. Representative blots of four replicate experiments are shown.

View larger version (18K): [in this window] [in a new window]   Figure 6. Tyrosine phosphorylation of IRS family members in human skeletal muscle cells in response to insulin. Human skeletal muscle cells were stimulated with insulin (100 nM) for 2.5 min followed by cell lysis and immunoprecipitation (of 800 µg protein) of IRS-1, IRS-2, and IRS-4. Immunoprecipitates were immunoblotted with antiphosphotyrosine and the corresponding IRS-antibodies, respectively. Representative blots of three replicate experiments are shown.

View larger version (38K): [in this window] [in a new window]   Figure 7. Tyrosine phosphorylation of IRS-4 in response to IGF-I and osmotic shock. Human skeletal muscle cells (left panel) or HEK 293 cells (right panel) were stimulated for 10 min with insulin (100 nM), IGF-I (70 nM), or osmotic shock (500 mM mannitol) followed by cell lysis and immunoprecipitation (800 µg protein) of IRS-4. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine and anti-IRS-4 antibodies, respectively. Representative blots of three replicate experiments are shown. Signal intensities were quantified by LUMI imager software and are expressed as arbitrary units. Data are mean values of three different experiments ± SD. *, Significantly different at P < 0.05; **, significantly different at P < 0.005.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IRS-1 and IRS-2 exhibit a widespread tissue distribution (17) and are expressed at the protein level in both red and white muscle (30, 31). We report here that IRS-4 protein is preferably detectable in oxidative muscle with the highest level in the heart. Oxidative red muscles are rich in type I fibers and have a higher content of GLUT4 (32) and a higher insulin sensitivity (33). Specific expression of IRS-4 in red soleus and cardiac muscle may suggest the potential involvement of IRS-4 in the regulation of GLUT4 translocation in these tissues, in agreement with the data by Zhou et al. (12) showing that overexpression of IRS-4 induces GLUT4 translocation. Unfortunately, we were not able to assess insulin signaling to IRS-4 in rat tissues. However, our results obtained in primary human skeletal muscle cells show that IRS-4 does not function as a substrate for the insulin and IGF-I receptor kinase. Interestingly, a prominent down-regulation of IRS-4 protein in cardiac and skeletal muscle was observed in WOKW rats, an animal model of obesity and insulin resistance (23, 27). On the other hand, IRS-1 was affected only in skeletal muscle in these animals with IRS-2 remaining unaltered, demonstrating a highly specific metabolic regulation of different IRS proteins. It must be kept in mind, however, that IRS-4-null mice have only mild defects in growth, reproduction, and glucose homeostasis (16). Future work will be needed to define the functional implications of normal and perturbed IRS-4 expression in heart and skeletal muscle.

A key finding of the present study consists of the observation that IRS-4 protein is expressed in primary human skeletal muscle cells at a considerable level without acting as a substrate for the insulin receptor kinase. This observation is consistent with the absence of insulin resistance in IRS-4-null mice (16). A potential explanation may be related to the relative abundance of IRS-1/2 vs. IRS-4 in muscle tissue. Thus, the insulin receptor may be saturated with IRS-1 and/or IRS-2 excluding IRS-4 from phosphorylation by the insulin receptor kinase. The inverse situation was reported for HEK 293 cells, in which IRS-4 is expressed at a 20- to 30-fold excess over IRS-1/2 (11). In these cells, as confirmed in the present study, IRS-4 is phosphorylated in response to insulin, whereas IRS-1 and IRS-2 do not participate in insulin signaling (11). We therefore concluded that IRS-4 is not a substrate for the insulin receptor kinase in muscle tissue under physiological conditions. Instead of the insulin receptor, the IGF-I receptor may represent an interesting candidate for using IRS-4 for downstream signaling. Qu et al. (25) reported that IRS-4 is implicated in IGF-I receptor mitogenic signaling in cell lines overexpressing IRS-4 and the IGF-I receptor. Furthermore, IRS-4-null mice exhibit a mild defect in growth (16). However, our data obtained with human myoblasts do not support a role for IRS-4 in IGF-I receptor signaling in primary muscle tissue.

In contrast to insulin and IGF-I, hyperosmotic shock was found to induce a prominent tyrosine phosphorylation of IRS-4 in human skeletal muscle cells. It is well established that protein tyrosine kinases play a pivotal role in the signaling of hyperosmotic stress (34, 35, 36), involving both members and nonmembers of the Src family of tyrosine kinases (36). Our results suggest that IRS-4 may be activated by nonreceptor tyrosine kinases and may be involved in stress-induced signaling in human skeletal muscle. Further work will be needed to identify the protein kinase mediating this effect. Interestingly, insulin-mimetic signaling to glucose transport involving tyrosine phosphorylation of IRS-1/2 by nonreceptor tyrosine kinases like pp59 (Lyn) has recently been reported (37), and this pathway may also include IRS-4.

In summary, we show here that IRS-4 protein is expressed in rat cardiac and soleus muscle and primary human skeletal muscle cells. However, IRS-4 does not function as a substrate for the insulin and IGF-I receptor, most likely because of competition with IRS-1 and/or IRS-2. It is suggested that IRS-4 may exert a physiological role in heart and skeletal muscle, potentially involving tyrosine kinases different from the insulin receptor.

	Acknowledgments

 
The secretarial assistance of Birgit Hurow is gratefully acknowledged.

	Footnotes

 
This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, EU COST Action B17, and the Jühling Foundation.

Abbreviations: ECL, Enhanced chemiluminescence; HEK, human embryonic kidney; HRP, horseradish peroxidase; IRS, insulin receptor substrate; RIPA, radioimmunoprecipitation assay; WOKW, Wistar Ottawa Karlsburg.

Received July 17, 2002.

Accepted for publication December 16, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kahn CR, White MF, Shoelson SE, Backer JM, Araki E, Cheatham B, Csermely P, Folli F, Goldstein BJ, Huertas P, Rothenburg PL, Saad MJ, Siddle K, Sun XJ, Wilden PA, Yamada K, Kahn SA 1993 The insulin receptor and its substrate: molecular determinants of early events in insulin action. Recent Prog Horm Res 48:291–339[Medline]
2. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[Abstract/Free Full Text]
3. Backer JM, Myers Jr MG, Shoelson SE, Chin DJ, Sun XJ, Miralpeix M, Hu P, Margolis B, Skolnik EY, Schlessinger J1992 Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J 11:3469–3479
4. Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly R, Myers Jr MJ, Backer JM, Ullrich A, White MF 1993 The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J 12:1929–1936[Medline]
5. Sun XJ, Crimmins DL, Myers Jr MG, Miralpeix M, White MF 1993 Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol 13:7418–7428[Abstract/Free Full Text]
6. White MF 1998 The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Recent Prog Horm Res 53:119–138[Medline]
7. Anai M, Ono H, Funaki M, Fukushima Y, Inukai K, Ogihara T, Sakoda H, Onishi Y, Yazaki Y, Kikuchi M, Oka Y, Asano T 1998 Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2. J Biol Chem 273:29686–29692[Abstract/Free Full Text]
8. Bruning JC, Winnay J, Cheatham B, Kahn CR 1997 Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells. Mol Cell Biol 17:1513–1521[Abstract]
9. He W, Craparo A, Zhu Y, O’Neill TJ, Wang LM, Pierce JH, Gustafson TA 1996 Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors. Evidence for two distinct phosphotyrosine-dependent interaction domains within IRS-2. J Biol Chem 271:11641–11645[Abstract/Free Full Text]
10. Lavan BE, Fantin VR, Chang ET, Lane WS, Keller SR, Lienhard GE 1997 A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family. J Biol Chem 272:21403–21407[Abstract/Free Full Text]
11. Fantin VR, Sparling JD, Slot JW, Keller SR, Lienhard GE, Lavan BE 1998 Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells. J Biol Chem 273:10726–10732[Abstract/Free Full Text]
12. Zhou L, Chen H, Xu P, Cong LN, Sciacchitano S, Li Y, Graham D, Jacobs AR, Taylor SI, Quon MJ 1999 Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells. Mol Endocrinol 13:505–514[Abstract/Free Full Text]
13. Uchida T, Myers Jr MG, White MF 2000 IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis. Mol Cell Biol 20:126–138[Abstract/Free Full Text]
14. Tsuruzoe K, Emkey R, Kriauciunas KM, Ueki K, Kahn CR 2001 Insulin receptor substrate 3 (IRS-3) and IRS-4 impair IRS-1- and IRS-2-mediated signaling. Mol Cell Biol 21:26–38[Abstract/Free Full Text]
15. Fantin VR, Lavan BE, Wang Q, Jenkins NA, Gilbert DJ, Copeland NG, Keller SR, Lienhard GE 1999 Cloning, tissue expression, and chromosomal location of the mouse insulin receptor substrate 4 gene. Endocrinology 140:1329–1337[Abstract/Free Full Text]
16. Fantin VR, Wang Q, Lienhard GE, Keller SR 2000 Mice lacking insulin receptor substrate 4 exhibit mild defects in growth, reproduction, and glucose homeostasis. Am J Physiol Endocrinol Metab 278:E127–E133
17. Giovannone B, Scaldaferri ML, Federici M, Porzio O, Lauro D, Fusco A, Sbraccia P, Borboni P, Lauro R, Sesti G 2000 Insulin receptor substrate (IRS) transduction system: distinct and overlapping signaling potential. Diabetes Metab Res Rev 16:434–441[CrossRef][Medline]
18. Dransfeld O, Rakatzi I, Sasson S, Gruzman A, Schmitt M, Haussinger D, Eckel J 2001 Eicosanoids participate in the regulation of cardiac glucose transport by contribution to a rearrangement of actin cytoskeletal elements. Biochem J 359:47–54[CrossRef][Medline]
19. Bähr M, Spelleken M, Bock M, von Holtey M, Kiehn R, Eckel J 1996 Acute and chronic effects of troglitazone (CS-045) on isolated rat ventricular cardiomyocytes. Diabetologia 39:766–774[CrossRef][Medline]
20. Dietze D, Koenen M, Röhrig K, Horikoshi H, Hauner H, Eckel J 2002 Impairment of insulin signaling in human skeletal muscle cells by co-culture with human adipocytes. Diabetes 51:2369–2376[Abstract/Free Full Text]
21. Ledwig D, Müller H, Bischoff H, Eckel J 2002 Early acarbose treatment ameliorates resistance of insulin-regulated GLUT4 trafficking in obese Zucker rats. Eur J Pharmacol 445:141–148[CrossRef][Medline]
22. Rizk NM, Stammsen D, Preibisch G, Eckel J 2001 Leptin and tumor necrosis factor- induce the tyrosine phosphorylation of signal transducer and activator of transcription proteins in the hypothalamus of normal rats in vivo. Endocrinology 142:3027–3032[Abstract/Free Full Text]
23. Van den Brandt J, Kovacs P, Kloting I 2000 Metabolic features in disease-resistant as well as in spontaneously hypertensive rats and newly established obese Wistar Ottawa Karlsburg inbred rats. Int J Obes Relat Metab Disord 24:1618–1622[CrossRef][Medline]
24. Wichelhaus A, Russ M, Petersen S, Eckel J 1994 G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats. Am J Physiol 267:H548–H555
25. Qu BH, Karas M, Koval A, LeRoith D 1999 Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation. J Biol Chem 274:31179–31184[Abstract/Free Full Text]
26. Numan S, Russell DS 1999 Discrete expression of insulin receptor substrate-4 mRNA in adult rat brain. Brain Res Mol Brain Res 72:97–102[Medline]
27. Van den Brandt J, Kovacs P, Klöting I 2002 Metabolic syndrome and aging in Wistar Ottawa Karlsburg W rats. Int J Obes 26:1–4[CrossRef][Medline]
28. Kovacs P, van den Brandt J, Klöting I 2000 Genetic dissection of the syndrome X in the rat. Biochem Biophys Res Commun 269:660–665[CrossRef][Medline]
29. Sesti G, Federici M, Hribal ML, Lauro D, Sbraccia P, Lauro R 2001 Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J 15:2099–2111[Abstract/Free Full Text]
30. Patti ME, Sun XJ, Bruening JC, Araki E, Lipes MA, White MF, Kahn CR 1995 4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice. J Biol Chem 270:24670–24673[Abstract/Free Full Text]
31. Zhou Q, Dohm GL 1997 Treadmill running increases phosphatidylinostol 3-kinase activity in rat skeletal muscle. Biochem Biophys Res Commun 236:647–650[CrossRef][Medline]
32. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, Holloszy JO 1990 Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol 259:E593–E598
33. James DE, Jenkins AB, Kraegen EW 1985 Heterogeneity of insulin action in individual muscles in vivo: euglycemic clamp studies in rats. Am J Physiol 248:E567–E574
34. Krump E, Nikitas K, Grinstein S 1997 Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils. J Biol Chem 272:17303–17311[Abstract/Free Full Text]
35. Junger WG, Hoyt DB, Davis RE, Herdon-Remelius C, Namiki S, Junger H, Loomis W, Altman A 1998 Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38. J Clin Invest 101:2768–2779[Medline]
36. Kapus A, Szaszi K, Sun J, Rizoli S, Rotstein OD 1999 Cell shrinkage regulates Src kinases and induces tyrosine phosphorylation of cortactin, independent of the osmotic regulation of Na+/H+ exchangers. J Biol Chem 274:8093–8102[Abstract/Free Full Text]
37. Muller G, Jung C, Wied S, Welte S, Frick W 2001 Insulin-mimetic signaling by the sulfonylurea glimepiride and phosphoinositolglycans involves distinct mechanisms for redistribution of lipid raft components. Biochemistry 40:14603–14620[CrossRef][Medline]

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