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(Circulation. 2000;102:2873.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Insulin Prevents Cardiomyocytes From Oxidative Stress–Induced Apoptosis Through Activation of PI3 Kinase/Akt

Ryuichi Aikawa; Masao Nawano, MD, PhD, PhD; Yaping Gu, MD; Hideki Katagiri, MD, PhD; Tomoichiro Asano, MD, PhD; Weidong Zhu, MD, PhD; Ryozo Nagai, MD, PhD; Issei Komuro, MD, PhD

From the Department of Cardiovascular Medicine (R.A., Y.G., W.Z., R.N., I.K.) and the Department of Metabolic Diseases, University of Tokyo Graduate School of Medicine (M.N., H.K., T.A.), Tokyo, Japan.

Correspondence to Issei Komuro, MD, PhD, Department of Medicine III, School of Medicine, Chiba University, 1-9-1 Inohara, Chuo-ku, Chiba 260-8670, Japan. E-mail komuro-tky{at}umin.ac.jp

	Abstract

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Background—Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Reactive oxygen species induce apoptosis in many types of cells including cardiomyocytes. Because insulin has been reported to have protective effects, we examined whether insulin prevents cardiomyocytes from oxidative stress–induced apoptotic death.

Methods and Results—Cultured cardiomyocytes of neonatal rats were stimulated by hydrogen peroxide (H₂O₂). Apoptosis was evaluated by means of the TUNEL method and DNA laddering. Incubation with 100 µmol/L H₂O₂ for 24 hours increased the number of TUNEL-positive cardiac myocytes (control, 4% versus H₂O₂, 23%). Pretreatment with 10^-6 mol/L insulin significantly decreased the number of H₂O₂-induced TUNEL-positive cardiac myocytes (12%) and DNA fragmentation induced by H₂O₂. Pretreatment with a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin, and overexpression of dominant negative mutant of PI3K abolished the cytoprotective effect of insulin. Insulin strongly activated both PI3K and the putative downstream effector Akt. Moreover, a proapoptotic protein, Bad, was significantly phosphorylated and inactivated by insulin through PI3K.

Conclusions—These results suggest that insulin protects cardiomyocytes from oxidative stress–induced apoptosis through the PI3K pathway.

Key Words: apoptosis • insulin • myocytes • cardiomyopathy • hypertrophy • heart diseases

	Introduction

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Many studies have shown that apoptosis plays an important role in cardiac development and diseases. It has been recently reported that apoptosis is observed in the hearts of patients with myocardial infarction, cardiomyopathy, and cardiac hypertrophy^{1 2 3} and that loss of cardiomyocytes by apoptosis causes heart failure.² Therefore, it is very important to find molecules that inhibit cardiomyocyte apoptosis to prevent the development of heart failure.^{4 5} Apoptosis occurs after ischemia and ischemia followed by reperfusion in several organs including the hearts.¹ Recent studies have indicated that insulin-like growth factor 1 (IGF-1) effectively protects myocardium from reperfusion injury and reduces cell death after acute myocardial infarction.^{4 5} IGF-1 has been reported to inhibit serum withdrawal–induced apoptosis in some cell types, including cardiomyocytes in vitro.^{6 7 8} We have demonstrated that reactive oxygen species generated during ischemia and reperfusion induce apoptosis in cardiac myocytes.⁹ Although insulin protects some cell types from apoptosis,^{6 7} it remains unclear whether insulin inhibits reactive oxygen species–induced apoptosis in cardiac myocytes.

In many cell types, insulin evokes its effects by activating a signaling cascade of protein tyrosine kinases and lipid kinases.¹⁰ Insulin activates 2 main signaling pathways: the phosphatidylinositol 3 kinase (PI3K) pathway and the Ras/mitogen-activated the protein kinase (MAPK) pathway.¹⁰ PI3K exhibits both lipid and protein kinase activities and plays critical roles in a variety of cell functions, including proliferation and survival in many cell types.^{6 11} The presumable downstream targets of PI3K are p70 S6 kinase,¹² some isoforms of protein kinase C, and a newly identified class of protein kinase C–like serine/threonine kinase Akt.¹³ The PI3K-specific inhibitor wortmannin inhibited the protective action of many growth factors,⁶ and activation of PI3K and its putative effector Akt was sufficient to protect fibroblasts against UV-induced apoptosis,¹¹ suggesting that PI3K plays an important role in preventing apoptosis through Akt. Moreover, it has recently been reported that the proapoptotic factor Bad is phosphorylated and inactivated by IGF-1 through PI3K and Akt.¹⁴ One of the MAPK family, extracellular signal-regulated kinase (ERK), is activated by a variety of growth factors, cytokines, and phorbol esters and plays pivotal roles in proliferation and differentiation in many types of cells.¹⁵ We and others have reported that oxidative stress activates ERKs through Ras small G protein and that the activation of ERKs protects cardiomyocytes from apoptosis.^{9 16} In the present study, we examined whether insulin protects cardiomyocytes from oxidative stress–induced apoptosis.

	Methods

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Cell Culture
Primary cultures of cardiac myocytes were prepared from ventricles of 1-day old Wistar rats, as described previously.¹⁷ In brief, cardiomyocytes were plated at a field density of 1x10⁵ cells/cm² on 35-mm culture dishes with 2 mL of culture medium (DMEM with 10% FBS). Twenty-four hours after seeding, the culture medium was changed to serum-free DMEM, and cells were cultured for 48 hours before stimulation.

Adenovirus
Cardiomyocytes were infected by adenovirus for 12 hours at 24 hours after plating. Infections were performed at a multiplicity of infection of 20. Infection with an adenovirus encoding ß-galactosidase showed that >95% of cardiomyocytes expressed the transgene (data not shown). An adenovirus encoding the HA-epitope tagged dominant negative PI3K cDNA contains a deletion at amino acid residues 479 to 512 of p85.¹⁸

In Vitro Measurement of PI3K Activity
The PI3K activity was measured as described previously.¹⁹ Briefly, PI3K was immunoprecipitated with antibody against phosphotyrosine (4G10), and the immunoprecipitates were suspended in kinase buffer (10 mmol/L Tris pH 7.5, 1 mmol/L EDTA, 100 mmol/L MgCl₂) containing phosphatidylinositol substrate (0.3 µg/µL) and 5 µCi [-³²P]ATP. The reactions were quenched after 10 minutes with 20 mL of 8 mol/L HCl and 160 mL of CHCl₃/MeOH (1:1 vol/vol ratio) and separated on thin-layer chromatography plates precoated with ammonium oxalate. The migration solution contained CHCl₃/MeOH/H₂O/NH₄OH (120:94:23.2:4 vol/vol ratio). The phosphorylated lipids were revealed by autoradiography.

Western Blot Analysis of Phosphorylated Akt and Bad
We used a polyclonal phosphorylated Akt-specific antibody, which recognizes only activated Akt with phosphorylation at the Ser 473 site (New England BioLabs Inc). To analyze Bad phosphorylation, we used a polyclonal phospho-Bad antibody (New England BioLabs Inc), which detects phophorylated Ser-136 of Bad. The anti-rabbit IgG antibody conjugated with horseradish peroxidase was used as the second antibody, and immunocomplexes were visualized with an enhanced chemiluminescence (ECL) detection kit (Boehringer Mannheim) according to the manufacturer’s directions.

Assay of Endogenous ERK Activity
Endogenous ERK activity was measured by myelin basic protein (MBP) assay.⁹ In brief, ERKs were immunoprecipitated with anti-ERK polyclonal antibody,²⁰ and the immune complex was incubated with [-³²P]ATP and MBP as a substrate. The samples were subjected to SDS-PAGE, and the gel was dried and subjected to autoradiography.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling
Cardiomyocytes cultured on a cover glass were fixed with 4% paraformaldehyde solution for 30 minutes at room temperature. Cells were first incubated with a monoclonal antibody against myosin heavy chain (MF-20) for 1 hour at 37°C and next with an anti-mouse IgG conjugated with rhodamine for 1 hour at room temperature. Next, 50 µL TUNEL reaction mixture containing both terminal deoxynucleotidyl transferase and FITC-dUTP was added on each sample for 1 hour at 37°C. These samples were analyzed by fluorescence microscopy as described previously.⁹

Agarose Gel Electrophoresis for DNA Fragmentation
Only fragmented DNA was extracted as described previously.²¹ Cells (4x10⁵) lysed in 0.2 mL of lysis buffer (10 mmol/L Tris-HCl pH 7.4, 10 mmol/L EDTA pH 8.0, 0.5% Triton X-100) were first incubated with 40 µg RNase (Boehringer Mannheim) for 1 hour at 37°C and next with 100 mg proteinase K (Boehringer Mannheim) for 1 hour at 37°C. The fragmented DNA was electrophoretically fractionated on 1.5% agarose gel and stained with ethidium bromide as described previously.⁹

Statistics
Statistical comparison within groups was carried out with 1-way ANOVA and Dunnett’s t test. The accepted level of significance was P<0.05.

	Results

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Insulin Prevents Cardiomyocytes From Oxidative Stress–Induced Apoptosis
We first examined whether insulin prevents cardiomyocytes from oxidative stress–induced apoptosis. When cardiac myocytes cultured in serum-free media for 24 hours were stained by TUNEL method, 4% of cardiac myocytes were positive, as reported before (Figure 1, A and B, and Figure 2).⁹ When cardiac myocytes were incubated with 100 µmol/L H₂O₂ for 24 hours, the number of TUNEL-positive cardiac myocytes was significantly increased (23%) (Figure 1, C and D, and Figure 2). Some nuclei of these TUNEL-positive cells were condensed and contracted, suggesting that H₂O₂ induced apoptosis in cardiac myocytes. When cardiomyocytes were pretreated with insulin for 30 minutes before addition of H₂O₂, the number of TUNEL-positive cardiomyocytes was significantly decreased (10^-6 mol/L insulin 11%, 10^-7 mol/L insulin 17%) (Figure 1, E and F, and Figure 2). To confirm that H₂O₂ induces apoptosis in cardiac myocytes, we examined DNA ladder formation by agarose gel electrophoresis.⁹ Cardiac myocytes that were cultured in serum-free medium for 48 hours showed a faint DNA ladder (Figure 3, lane A). When cardiac myocytes were exposed to 100 µmol/L H₂O₂ for 24 hours, extracted genomic DNA showed a prominent DNA ladder characteristic of apoptosis (Figure 3, lane B). DNA ladder formation was significantly decreased when cardiac myocytes were incubated with 10^-6 mol/L insulin for 30 minutes before exposure to H₂O₂ (Figure 3, lane C). These results suggest that insulin prevents cardiomyocytes from oxidative stress–induced apoptosis.

View larger version (87K): [in this window] [in a new window]   Figure 1. Insulin protects cardiomyocytes from H2O2-induced apoptosis. TUNEL analysis was performed as described in Methods. Cardiomyocytes were marked by staining with monoclonal anti-myosin heavy chain antibody (MF-20) followed by incubation with anti-mouse IgG antibody conjugated with TRITC (A, C, E, and G). TUNEL staining was performed with FITC-conjugated dUTP (B, D, F, and H). A and B, Unstimulated cardiomyocytes. C and D, Cardiomyocytes incubated with 100 µmol/L H2O2 for 24 hours. E and F, Cardiomyocytes incubated with 100 µmol/L H2O2 for 24 hours after pretreatment with 10-6 mol/L insulin for 30 minutes. G and H, Cardiomyocytes were infected with adenovirus encoding D.N.PI3K before treatment with insulin and H2O2. Magnification x200 (bar=20 µm).

View larger version (35K): [in this window] [in a new window]   Figure 2. Number of cardiac myocytes undergoing apoptosis after exposure to H2O2. One hundred MF-20–positive cardiac myocytes were counted; number of TUNEL-positive cells was presented as percentage from 4 independent experiments (mean±SEM). *P<0.05 vs control. Ins indicates insulin; Wo, wortmannin; and LY, LY294002.

View larger version (106K): [in this window] [in a new window]   Figure 3. H2O2-induced DNA fragmentation in cardiomyocytes with or without insulin. Cardiomyocyte lysates were first incubated with RNase and then with proteinase K. By this method, only fragmented DNA was extracted. DNA was separated by electrophoresis in 1.5% agarose gels and stained by ethidium bromide. A, Untreated cardiomyocytes; B and C, cardiac myocytes incubated with 100 µmol/L H2O2 for 48 hours with (C) or without (B) pretreatment with 10-6 mol/L insulin for 30 minutes. D, C+ overexpression of D.N.PI3K. Molecular weight is shown at left.

Specific PI3K Inhibitors or Overexpression of D.N.PI3K Abolishes the Protective Effect of Insulin
Recently, accumulating evidence has suggested that PI3K plays a pivotal role in protecting cells from apoptosis.^{6 7} We thus examined whether the protective effect of insulin is mediated by PI3K. When cardiomyocytes were preincubated with a specific PI3K inhibitor, wortmannin (10^-7 mol/L), for 6 hours,⁶ the amount of H₂O₂-induced apoptosis was increased (42%) (Figure 2). The cardioprotective effect of insulin was attenuated by the pretreatment with wortmannin and another specific PI3K inhibitor, LY294002, and the number of TUNEL-positive cells was increased to the level of H₂O₂ treatment without any pretreatments (26%) (Figure 2). To confirm that insulin inhibits H₂O₂-induced apoptosis through activation of PI3K, we overexpressed dominant negative mutant of PI3K (D.N.PI3K) by using recombinant adenovirus. The number of TUNEL-positive cells was increased to the almost same level of H₂O₂ treatment without any pretreatments by overexpression of D.N.PI3K (23%) (Figure 1, G and H, and Figure 2). DNA fragmentation also became more prominent by overexpression of D.N.PI3K compared with insulin treatment without D.N.PI3K (Figure 3, lane C and D). These results suggest that activation of PI3K plays a critical role in insulin-induced prevention of H₂O₂-induced apoptosis in cardiac myocytes.

Insulin Activates PI3K in Cardiac Myocytes
We next examined whether insulin actually activates PI3K in cardiac myocytes. When 10^-6 mol/L of insulin was added to the culture media, PI3K was activated from 5 minutes in cardiac myocytes (Figure 4A). The PI3K activity peaked at 15 minutes and returned to the basal levels at 60 minutes. Insulin activated PI3K in a concentration-dependent manner, and PI3K was maximally activated by 10^-6 mol/L of insulin (Figure 4B). Insulin-induced activation of PI3K was significantly suppressed by pretreatment with 10^-8 mol/L wortmannin and was abolished with 10^-7 mol/L wortmannin (Figure 4C). These results suggest that insulin strongly activates PI3K in cardiac myocytes.

View larger version (24K): [in this window] [in a new window]   Figure 4. Activation of PI3K by insulin. A, Cardiac myocytes were cultured in serum-free media for 48 hours and then incubated with 10-6 mol/L insulin for indicated periods of time. B, Cardiac myocytes were exposed to indicated concentrations of insulin (10-8 to 10-5 mol/L) for 15 minutes. C, Cardiac myocytes were preincubated with wortmannin (Wo, 10-9 to 10-7 mol/L) for 6 hours before addition of 10-6 mol/L insulin (Ins). After immunoprecipitation with anti-phosphotyrosine antibody (4G10), in vitro kinase assay was performed with phosphatidylinositol used as substrate. Lipids were separated by thin-layer chromatography; phosphoinositide monophosphate was revealed by autoradiography. Amount of PIP was quantified by densitometric scanning. These data represent average percentage of controls (100%) from 4 independent experiments (mean±SEM). *P<0.05, **P<0.01 vs zero-time control.

Insulin Activates Akt Through PI3K in Cardiac Myocytes
The serine/threonine protein kinase Akt is one of the downstream effectors of the PI3K signaling pathway, and the activation of Akt has been reported to play a critical role in protecting cells from apoptotic cell death.^{11 14 22} Therefore, we examined whether insulin activates Akt in cultured cardiac myocytes by using activated Akt-specific antibody. When cardiac myocytes were exposed to 10^-6 mol/L insulin, Akt was strongly activated (Figure 5A). The phosphorylation of Akt was significantly enhanced from 15 minutes and peaked at 60 to 120 minutes after addition of insulin (Figure 5A). Phosphorylated levels of Akt were thereafter decreased, but the levels were still significantly higher than basal levels at 240 minutes. Insulin-induced activation of Akt was almost completely suppressed by the pretreatment with 10^-7 mol/L wortmannin for 6 hours or significantly attenuated by the pretreatment with 10^-5 mol/L LY294002 for 6 hours (Figure 5B), suggesting that insulin activates Akt through the PI3K pathway in cardiac myocytes.

View larger version (27K): [in this window] [in a new window]   Figure 5. Insulin-induced phosphorylation of Akt. A, Cardiomyocytes were cultured in serum-free media for 48 hours and then incubated with 10-6 mol/L insulin for indicated periods of time. B, Cardiac myocytes were preincubated with either 10-5 mol/L LY294002 (LY) for 6 hours, 10-7 mol/L wortmannin (Wo) for 6 hours, or 20 µmol/L PD98059 (PD) for 60 minutes before addition of 10-6 mol/L insulin. Western blot analysis was performed with phosphorylated Akt-specific antibody. Phosphorylated Akt was detected by ECL system; phosphorylated levels of Akt were quantified by densitometric scanning. Data represent average percentage of controls (100%) from 3 independent experiments (mean±SEM). *P<0.05, **P<0.01 vs zero-time control.

Insulin Phosphorylates Bad Through PI3K in Cardiac Myocytes
Akt has been reported to phosphorylate and inactivate a proapoptotic protein, Bad, and thereby inhibit cell death.¹⁴ We thus examined whether insulin phosphorylates Bad in cultured cardiac myocytes by using the antibody that specifically recognizes phosphorylated Bad at Ser-136. Insulin significantly phosphorylated Bad in cardiac myocytes (Figure 6A). The phosphorylated levels of Bad increased from 30 minutes and peaked at 60 minutes after exposure to 10^-6 mol/L insulin. Insulin-induced activation of Bad was strongly suppressed by the pretreatment with 10^-7 mol/L wortmannin or 10^-5 mol/L LY294002 for 6 hours (Figure 6B).

View larger version (33K): [in this window] [in a new window]   Figure 6. Insulin-induced phosphorylation of Bad. A, Cardiac myocytes were maintained in serum-free media for 48 hours and then incubated with 10-6 mol/L insulin for indicated periods of time. B, Cardiac myocytes were preincubated with either 10-7 mol/L wortmannin (Wo) for 6 hours or 10-5 mol/L LY294002 (LY) for 6 hours before addition of 10-6 mol/L insulin. Western blot analysis was performed with phosphorylated Bad -specific antibodies. Phosphorylated Bad was detected by ECL detection system; phosphorylated levels of Bad were quantified by densitometric scanning. Data represent average percentage of controls (100%) from 4 independent experiments (mean±SEM). *P<0.05 vs zero-time control.

ERKs Are Activated by Insulin Independent of PI3K Pathway in Cardiac Myocytes
We have recently reported that insulin activates ERKs through a small G protein, Ras,²³ and the Src family tyrosine kinases,²⁴ and that ERKs play an important role in protecting cardiomyocytes from H₂O₂-induced apoptosis.⁹ We thus questioned the relation between PI3K and ERKs in cardiac myocytes. Although activation of ERKs by insulin was completely suppressed by a specific MEK inhibitor, PD98059 (20 µmol/L), insulin-induced ERK activation was not affected by the pretreatment with 10^-7 mol/L wortmannin for 6 hours (Figure 7). In addition, insulin-induced activation of Akt was not inhibited by PD98059 (Figure 5B).

View larger version (30K): [in this window] [in a new window]   Figure 7. Effects of wortmannin on insulin-induced activation of ERKs. Cardiac myocytes were preincubated with either 10-7 mol/L wortmannin (Wo) for 6 hours or 20 µmol/L PD98059 (PD) for 60 minutes before addition of 10-6 mol/L insulin. ERKs were immunoprecipitated from cell lysates with polyclonal antibody against ERKs (Y91); kinase activity was assayed with MBP used as substrate. After electrophoresis, gel was dried and subjected to autoradiography. Representative autoradiogram from 3 independent experiments is shown.

	Discussion

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Insulin and IGF-1 can protect cells from apoptosis that is induced under a wide variety of circumstances such as growth factor withdrawal, UV irradiation, exposure to anticancer drugs, and ischemia/reperfusion.^{5 6 7 11 25} Therefore, we examined whether insulin protects cardiomyocytes from oxidative stress–induced apoptosis. The pretreatment of cultured cardiomyocytes with insulin inhibited H₂O₂-induced apoptosis (Figure 1, 2, and 3). To determine the mechanism of antiapoptotic action of insulin, we examined the role of PI3K because insulin has been reported to have antiapoptotic effects through PI3K in many cell types.^{6 7 11} The antiapoptotic effect of insulin was abolished both by the preincubation with wortmannin and by overexpression of D.N.PI3K (Figure 2). Because <10^-7 mol/L wortmannin is highly specific to PI3K,^{6 7} these results suggest that insulin exhibits antiapoptotic effects on cardiomyocytes through PI3K. In addition, the number of H₂O₂-induced TUNEL-positive cardiac myocytes was increased by the pretreatment with 10^-7 mol/L wortmannin (Figure 2). H₂O₂ itself slightly activated PI3K in cardiac myocytes (data not shown). Although it is unknown at present how H₂O₂ activates PI3K in cardiomyocytes, these results suggest that PI3K is an important survival factor for cardiac myocytes, as in other types of cells.

Insulin rapidly and dose-dependently activated PI3K in cardiac myocytes, which was completely suppressed by 10^-7 mol/L wortmannin (Figure 4C). PI3K has been reported to phosphorylate and activate many lipids and proteins, including the serine/threonine kinase Akt. Akt was initially described as an oncogene and is activated by a variety of growth factors through the PI3K-dependent pathway. Activation of Akt has been reported to inhibit apoptosis, which is induced by serum depletion, UV irradiation, and chemical agents in many cell types such as neurons, fibroblasts, and lymphoid cells.^{6 7 11 22 25} In this study, insulin markedly activated Akt in cultured cardiomyocytes, and this activation was strongly suppressed by specific PI3K inhibitors such as wortmannin and LY 294002 (Figure 5). Moreover, overexpression of D.N.PI3K strongly suppressed insulin-induced Akt activation (data not shown). Taken together, PI3K may mediate an antiapoptotic effect of insulin in cardiac myocytes through activating Akt.

Several members of the Bcl-2 family such as Bcl-2, Bcl-xL, Mcl-1, A1, and Bag-1 promote survival, whereas other members such as Bcl-xS, Bad, Bax, and Bak induce cell death.^{2 14} The Bcl-2 family proteins form homodimers and/or heterodimers, and it depends on the balance between homodimers and heterodimers whether cells undergo apoptosis or not.^{8 14} Unphosphorylated Bad is thought to induce cell death, possibly by forming heterodimers with Bcl-2 and by concomitantly generating Bax homodimers.¹⁴ It has recently been reported that PI3K phosphorylates Bad through Akt and that phosphorylated Bad dissociates from Bcl-2 and shows the antiapoptotic effects.^{14 26} In cardiomyocytes, insulin induced phosphorylation of Bad at Ser-136, which was strongly suppressed by wortmannin and LY294002 (Figure 6). Taken together, insulin may protect cardiomyocytes from H₂O₂-induced apoptosis at least part by phosphorylating Bad through the PI3K/ Akt pathway.

Insulin shows its metabolic and mitogenic effects by activating a complex signaling cascade of serine/threonine kinases including MAPK/ERKs.^{10 20} We have reported that when ERKs were inhibited by PD98059, the number of H₂O₂-induced TUNEL-positive cardiac myocytes was increased.⁹ In the present study, blockade of PI3K by wortmannin also increased the number of apoptotic cells. Moreover, pretreatment with both PD98059 and wortmannin more strongly augmented H₂O₂-induced apoptosis in cardiac myocytes as compared with single treatment (data not shown). Because PD98059 inhibited activation of ERKs but not of PI3K and wortmannin did not affect the activity of ERKs, both PI3K and ERKs may be independently involved in the protection of cardiac myocytes from apoptosis. Recent studies have indicated that the Ras-MAPK pathway phosphorylates Bad at Ser-112 in mammalian cells,²⁷ and therefore, it is possible that ERKs may cooperate with PI3K to protect cardiac myocytes from H₂O₂-induced apoptosis by phosphorylating different serine residues.

Many studies have indicated that glucose-insulin-potassium (GIK) therapy is effective for cardiomyocyte protection.^{28 29} Quite recently, Diaz et al²⁹ have reported that GIK infusion is very useful to treat patients with acute myocardial infarction. In particular, GIK therapy significantly improved nonfatal severe heart failure caused by ischemic myocardial injuries.²⁹ It has been reported that IGF-1 protects myocardium from reperfusion injury and reduces cell death after myocardial infarction.^{4 5} In the present study, we demonstrated one possible mechanism by which insulin exhibits protective effects on cardiac myocytes. Further studies are necessary to determine whether this mechanism works in in vivo situations.

	Acknowledgments

 
This work was supported by a Grant-in-Aid for Scientific Research, Developmental Scientific Research, and Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan; by Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief; R&D Promotion and Product Review of Japan; and by a Grant-in-Aid from The Tokyo Biochemical Research Foundation (to I. Komuro). We wish to thank Dr T. Kadowaki for advice and K. Kuwabara and C. Masuo for the excellent technical assistance.

Received March 13, 2000; revision received June 26, 2000; accepted June 30, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest. 1994;94:1621–1628.
   
2. Olivetti G, Abbi R, Quaini F, et al. Apoptosisin the failing human heart. N Engl J Med. 1997;336:1131–1141.[Abstract/Free Full Text]
   
3. Teiger E, Than VD, Richard L, et al. Apoptosis in pressure overload-induced heart hypertrophy in the rat. J Clin Invest. 1996;97:2891–2897.[Medline] [Order article via Infotrieve]
   
4. Li Q, Li B, Wang X, et al. Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J Clin Invest. 1997;100:1991–1999.[Medline] [Order article via Infotrieve]
   
5. Buerke M, Murohara T, Skurk C, et al. Cardioprotective effect of insulin-like growth factor I in myocardial ischemia followed by reperfusion. Proc Natl Acad Sci U S A. 1995;92:8031–8035.[Abstract/Free Full Text]
   
6. Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995;267:2003–2006.[Abstract/Free Full Text]
   
7. Parrizas M, Saltiel AR, LeRoith D. Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. J Biol Chem. 1997;272:154–161.[Abstract/Free Full Text]
   
8. Wang L, Ma W, Markovich R, et al. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res. 1998;83:516–522.[Abstract/Free Full Text]
   
9. Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activates extracellular signal-regulated kinases through src and ras in cultured cardiac myocytes of neonatal rats. J Clin Invest. 1997;100:1813–1821.[Medline] [Order article via Infotrieve]
   
10. White MF, Kahn CR. The insulin signaling system. J Biol Chem. 1994;269:1–4.[Free Full Text]
    
11. Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol. 1997;17:1595–1606.[Abstract]
    
12. Downward J. Signal transduction. Regulating S6 kinase. Nature. 1994;371:378–379.[Medline] [Order article via Infotrieve]
    
13. Franke TF, Yang SI, Chan TO, et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995;81:727–736.[Medline] [Order article via Infotrieve]
    
14. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997;91:231–241.[Medline] [Order article via Infotrieve]
    
15. Boulton TG, Nye SH, Robbins DJ, et al. ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell. 1991;65:663–675.[Medline] [Order article via Infotrieve]
    
16. von Harsdorf R, Li PF, Dietz R. Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis. Circulation. 1999;99:2934–2941.[Abstract/Free Full Text]
    
17. Komuro I, Kaida T, Shibazaki Y, et al. Stretching cardiac myocytes stimulates proto-oncogene expression. J Biol Chem. 1990;265:3595–3598.[Abstract/Free Full Text]
    
18. Katagiri H, Asano T, Inukai K, et al. Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3–L1 adipocytes. Am J Physiol. 1997;272:E326–E331.[Abstract/Free Full Text]
    
19. Pons S, Asano T, Glasheen E, et al. The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase. Mol Cell Biol. 1995;15:4453–4465.[Abstract]
    
20. Tobe K, Kadowaki T, Hara K, et al. Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection. J Biol Chem. 1992;267:21089–21097.[Abstract/Free Full Text]
    
21. Sellins KS, Cohen JJ. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol. 1987;139:3199–3206.[Abstract]
    
22. Dudek H, Datta SR, Franke TF, et al. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science. 1997;275:661–665.[Abstract/Free Full Text]
    
23. Zou Y, Komuro I, Yamazaki T, et al. Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes. J Biol Chem. 1996;271:33592–33597.[Abstract/Free Full Text]
    
24. Aikawa R, Komuro I, Yamazaki T, et al. Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes. Circ Res. 1999;84:458–466.[Abstract/Free Full Text]
    
25. Sell C, Baserga R, Rubin R. Insulin-like growth factor I (IGF-I) and the IGF-I receptor prevent etoposide-induced apoptosis. Cancer Res. 1995;55:303–306.[Abstract/Free Full Text]
    
26. del Peso L, Gonzalez-Garcia M, Page C, et al. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997;278:687–689.[Abstract/Free Full Text]
    
27. Scheid MP, Schubert KM, Duronio V. Regulation of bad phosphorylation and association with Bcl-x(L) by the MAPK/Erk kinase. J Biol Chem. 1999;274:31108–31113.[Abstract/Free Full Text]
    
28. Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction: an overview of randomized placebo-controlled trials. Circulation. 1997;96:1152–1156.[Abstract/Free Full Text]
    
29. Diaz R, Paolasso EA, Piegas LS, et al. Metabolic modulation of acute myocardial infarction: the ECLA (Estudios Cardiologicos Latinoamerica) Collaborative Group. Circulation. 1998;98:2227–2234.[Abstract/Free Full Text]
    

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