Insulin Prevents Cardiomyocytes From Oxidative Stress–Induced Apoptosis Through Activation of PI3 Kinase/Akt
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.
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 hypertrophy1 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.
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 1×105 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.
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 β-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 p85α.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 [γ-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 [γ-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 (4×105) 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
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.
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.
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.
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.
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⇓).
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⇑).
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.
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.
- Copyright © 2000 by American Heart Association
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