胰岛素保护缺血再灌注心脏：PI3-KAkt和JNKs信号通路间的交互作用

https://www.wendangku.net/doc/447607781.html,

Research Paper

Insulin protects isolated hearts from ischemia/reperfusion injury:

cross-talk between PI3-K/Akt and JNKs

LIU Hai-Tao1, ZHANG Hai-Feng2, SI Rui1, ZHANG Quan-Jiang2, ZHANG Kun-Ru2, GUO Wen-Yi1,

WANG Hai-Chang1,*, GAO Feng2,*

1Department of Cardiology, Xijing Hospital; 2Department of Physiology, the Fourth Military Medical University, Xi’an 710032, China

Abstract: Our previous results have demonstrated that insulin reduces myocardial ischemia/reperfusion (MI/R) injury and increases the postischemic myocardial functions via activating the cellular survival signaling, i.e., phosphatidylinositol 3-kinase (PI3-K)-Akt-endothelial nitric oxide synthase (eNOS)-nitric oxide (NO) cascade. However, it remains largely controversial whether c-Jun NH2-terminal kinase (JNK) is involved in the effects of insulin on MI/R injury. Therefore, the aims of the present study were to investigate the role of JNK, especially the cross-talk between JNK and previously expatiated Akt signaling, in the protective effect of insulin on I/R myocardium. Isolated hearts from adult Sprague-Dawley rats were subjected to 30 min of regional ischemia and followed by 2 or 4 h of reperfusion (n=6). The hearts were pretreated with PI3-K inhibitor LY294002, or phosphorylated-JNK inhibitor SP600125, respectively, then perfused retrogradely with insulin, and the mechanical functions of hearts, including the heart rate (HR), left ventricular developed pressure (LVDP) and instantaneous first derivation of left ventricular pressure (±LVd p/d t max) were measured. At the end of reperfusion, the infarct size (IS) and apoptotic index (AI) were examined. MI/R caused significant cardiac dysfunction and myocardial apoptosis (strong TUNEL-positive staining). Compared with the control group, insulin treatment in MI/R rats exerted protective effects as evidenced by reduced myocardial IS [(28.9±2.0)% vs (45.0±4.0)%, n=6, P<0.01], inhibited cardiomyocyte apoptosis [decreased AI: (16.0±0.7)% vs (27.6±1.3)%, n=6, P<0.01] and improved recovery of cardiac systolic/diastolic function (including LVDP and ±LVd p/d t max) at the end of reperfusion. Moreover, insulin resulted in 1.7-fold and 1.5-fold increases in Akt and JNK phosphorylation in I/R myocardium, respectively (n=6, P<0.05). Inhibition of Akt activation with LY294002 abolished, and inhibition of JNK activation with SP600125 enhanced the cardioprotection by insulin, respectively. And the abolishment by L Y294002 could be partly converted by SP600125 pretreatment. In addition, SP600125 also decreased the Akt phosphorylation (n=6, P<0.05). These results demonstrate that insulin simultaneously activates both Akt and JNK, and the latter further increases the phosphorylation of Akt which attenuates MI/R injury and improves heart function; this cross-talk between Akt and JNK in the insulin signaling is involved in insulin-induced cardioprotective effect.

Key words: ischemia/reperfusion injury; insulin; apoptosis; Akt; JNK; cross-talk

胰岛素保护缺血/再灌注心脏：PI3-K/Akt和JNKs信号通路间的交互作用

刘海涛1，张海锋2，司瑞 1，张全江2，张昆茹 2，郭文仪1，王海昌1,*，高峰2,*

第四军医大学1西京医院心血管内科；2生理学教研室，西安 710032

摘 要：我们前期研究表明胰岛素可激活细胞内信号转导机制如磷脂酰肌醇3-激酶-蛋白激酶B-内皮型一氧化氮合酶-一氧化氮(PI3-K-Akt-eNOS-NO)信号通路，减轻心肌缺血/再灌注(ischemia/reperfusion, I/R)损伤，改善缺血后心肌功能恢复。然而c-Jun氨基末端激酶(c-Jun NH2-terminal kinase, JNK)信号通路在胰岛素保护I/R心肌中的作用尚不清楚，本研究旨在探讨JNK

Received 2007-07-20 Accepted 2007-08-29

This work was supported by the National Science Fundation of China for Outstanding Young Investigators (No. 30625033), the National Natural Science Foundation of China (No. 30471923, 30500577), and the National Basic Research Development Program of China (No. 2007CB512106).

*Corresponding author. GAO Feng: Tel: +86-29-84776423; Fax: +86-29-84776423; E-mail: fgao@https://www.wendangku.net/doc/447607781.html,; WANG Hai-Chang: Tel: +86-29-84773469; Fax: +86-29-84773469; E-mail: wanghc@https://www.wendangku.net/doc/447607781.html,

信号通路在胰岛素保护I/R心肌中的作用及其与PI3-K/Akt信号通路间的相互关系。离体Sprague-Dawley大鼠心脏缺血30 min 后施行2 h或4 h的再灌注，缺血前用LY294002 (15 mmol/L)和SP600125 (10 mmol/L)灌注15 min，分别阻断PI3-K/Akt和磷酸化JNK (phosphorylated-JNK, p-JNK)活化，观测心脏功能、心肌梗死、细胞凋亡和蛋白磷酸化水平。与对照组相比，胰岛素再灌注2 h后，心率、左心室发展压和左心室收缩/舒张最大速率均明显增加，梗死面积减少约16.1% [(28.9±2.0)% vs (45.0±4.0)%, n=6, P<0.01]，细胞凋亡指数从(27.6±1.3)%减少到(16.0±0.7)% (n=6, P<0.01)，Akt的活性增加1.7倍(n=6, P< 0.05)，同时JNK活性增加1.5倍(n=6, P<0.05)。用LY294002处理后，胰岛素对I/R心肌的保护作用消失；而用SP600125处理可增强胰岛素的保护作用，且可部分逆转LY294002的抑制作用。进一步观察发现SP600125减弱了Akt的磷酸化(n=6, P< 0.05)。上述结果表明，在I/R心肌中，胰岛素可同时激活PI3-K/Akt及JNK信号通路，且通过后者进一步增加Akt活化，从而减轻I/R损伤，改善心肌功能。这种PI3-K/Akt与JNK信号通路交互机制对胰岛素保护I/R心肌有重要意义。

关键词：缺血/再灌注损伤；胰岛素；凋亡；蛋白激酶B；c-J u n氨基末端激酶；交互机制

中图分类号：R331.3

Reperfusion therapy has become a practical and effective strategy in the salvage of ischemic myocardium. The di-rect enhancement of cardiac cellular tolerance against myocardial ischemia/reperfusion (MI/R) injury should fur-ther improve patient outcome in acute coronary syndromes (ACS). Our previous studies demonstrated that insulin might play an important role in attenuating MI/R injury in vivo when it was administered during reperfusion and activa-tion of the cellular survival signaling, that is, phosphatidyl-inositol 3-kinase (PI3-K)-Akt-endothelial nitric oxide syn-thase (eNOS)-nitric oxide (NO) cascade, is one of the cen-tral mechanisms underlying the protective effect of insulin against MI/R injury[1-5]. Besides the PI3-K/Akt pathway, the mitogen-activated protein kinase (MAPK) cascades also are the downstream molecules of insulin in the heart[6,7], which have been shown to play an important regulatory role in a variety of cellular processes[8,9]. MAPKs phospho-rylate selected intracellular proteins, including transcrip-tion factors, which subsequently regulate gene expression by transcriptional and post-transcriptional mechanisms[10]. MAPKs are, in turn, activated by phosphorylation at con-served threonine and tyrosine residues by upstream dual-specific MAPK kinases (MAPKKs), which themselves are activated by MAPKK kinases (MAPKKKs)[11]. In particular, the c-Jun NH

2

-terminal kinases (JNKs) are a group of MAPKs that play a role in apoptosis, proliferation, and embryonic morphogenesis in both transcription-dependent and -independent mechanisms[10,12,13]. However, regarding the MAPK signaling cascades are one of the main signaling pathways of insulin, it remains largely elusive whether JNK is also involved in insulin-induced anti-apoptotic effects on I/R myocardium.

In addition, JNK activities were shown to be antago-nized by Akt kinase activity in numerous cell systems, and this cross-talk may underlie many of the prosurvival effects of Akt[14]. It has been demonstrated that Akt regu-lates several proteins to suppress the JNK pathway, such as extracellular regulated kinases (ERKs), JNK-interacting protein (JIP) 1[15-18], and MAPK/ERK kinase (MEK)[15,19]. Indeed, the role of Akt-JNK cross-talk in the cardiopro-tective effects of insulin against MI/R is unclear so far. Therefore, the aims of the present study were: (1) to investigate whether the JNK signaling pathway participates in the protective effect of insulin, and if so, (2) to further explore the possible role of Akt-JNK cross-talk in insulin-induced anti-apoptosis and subsequent attenuation of MI/R injury.

1 MATERIALS AND METHODS

1.1 Experimental protocols

The experiments performed were in adherence with Guide-lines on the Use of Laboratory Animals in the National Institutes of Health and were approved by the Fourth Mili-tary Medical University Committee on Animal Care.

The experimental protocol was shown in Fig.1. Isolated hearts from adult male Sprague-Dawley rats [(200±50) g] were randomly assigned to the following groups. (1) Sham group: the hearts were subjected to the MI/R procedure (30 min of regional ischemia and followed by 2 or 4 h of reperfusion) except that the suture that was passed under the left coronary artery was left untied (n=6); (2) Control (V ehicle) group: the hearts were subjected to 30 min of regional ischemia and followed by 2 or 4 h of reperfusion with vehicle (n=6); (3) Insulin group: insulin (Novo Nordisk, 60 U/L) was administered 15 min before MI/R procedure (n=6); (4) Insulin + L Y294002 (a specific inhibitor for PI3-K) group: the hearts were pretreated with LY294002 (15 mmol/L)[19] for 15 min before insulin administration (n=6);

(5) Insulin + SP600125 (a specific inhibitor for JNK) group:

LIU Hai-Tao et al : Cross-talk between Akt and JNK 653

the hearts were pretreated with SP600125 (10 mmol /L)[12,20,21]for 15 min before insulin administration (n =6); (6) Insulin +LY294002 + SP600125 group: the hearts were pretreated with SP600125 and LY294002 for 15 min before insulin administration (n =6). The doses of the reagents were based on our previous experiments in rat hearts.

1.2 Isolated heart preparation and MI/R

After exteriorized through a left thoracic incision, the hearts were placed rapidly in ice-cold Krebs-Henseleit buffer to arrest and prevent them from ischemic preconditioning.The hearts were then perfused retrogradely through the ascending aorta by a peristaltic pump at a constant flow rate of 10 mL/min, with perfusion medium containing (in mmol/L): NaCl 11.9, NaHCO 3 24.9, KCl 4.74, KH 2PO 4 1.19,

MgSO 4 1.2, CaCl 2 1.8 and glucose 11, oxygenated and kept at pH 7.4 by gassing with the mixture of 95% O 2 and 5%CO 2[21,22]. The heart was kept throughout at constant tem-perature of 36.5 o C by being placed in a recirculation glass jacket. The initial perfusion pressure was 60 mmHg. When the heart began contracting spontaneously, a latex balloon,connected to a pressure transducer, was inserted into the left ventricle via the left atrium. The balloon was then filled with saline buffer at a steady diastolic pressure of 4-8 mmHg to measure left ventricular pressure. Two silver electrodes were fixed, one at the apex and another at the atria, for electrocardiogram recording of a bipolar derivative. The efficiency of the peristaltic pump in maintaining a stable coronary flow was determined by collecting the effluent

Fig. 1. Protocol chart. Isolated hearts from adult male Sprague-Dawley rats [(200±50) g] were randomly assigned to the following groups. (1) Sham group: the hearts were subjected to the MI/R procedure (30 min of regional ischemia and followed by 2 or 4 h of reperfusion) except that the suture that was passed under the left coronary artery was left untied (n =6); (2) Control (Vehicle) group:the hearts were subjected to 30 min of regional ischemia and followed by 2 or 4 h of reperfusion with vehicle (n =6); (3) Insulin group:insulin (Novo Nordisk, 60 U/L) was administered 15 min before MI/R procedure (n =6); (4) Insulin + LY294002 (a specific inhibitor for PI3-K) group: the hearts were pretreated with LY294002 (15 mmol/L) for 15 min before insulin administration (n =6); (5) Insulin +SP600125 (a specific inhibitor for JNK) group: the hearts were pretreated with SP600125 (10 mmol /L) for 15 min before insulin administration (n =6); (6) Insulin + L Y294002 + SP600125 group: the hearts were pretreated with SP600125 and LY294002 for 15 min before insulin administration (

n

=6).

Acta Physiologica Sinica, October 25, 2007, 59 (5): 651-659 654

from the heart in a graduated glass container at fixed intervals. All variables were recorded using a computer acquisition data system[23].

Making a slipknot with a 4-0 silk around the left anterior descending (LAD) coronary artery produced myocardial ischemia. After 30 min of ischemia, the slipknot was released and the heart was reperfused for 2 h to measure infarct size (IS) or 4 h for terminal deoxynucleotidyl nick-end labeling (TUNEL) and Western blot assays[24].

1.3 Determination of myocardial infarction

To determine myocardial infarct, the LAD coronary artery was ligated again after 2 h of reperfusion, and perfused with 3 mL/kg of 1% Evans blue to stain the area at risk (AAR). The heart was then removed and divided into 4-5 slices of 1-2 mm width transversally. The Evans blue so-lution stained the perfused myocardium leaving the oc-cluded vascular bed uncolored. All the colored non-ischemic tissue and the non-colored AAR were weighed to calculate the percentage of AAR with respect to the whole left ventricle. To distinguish between viable ischemic and in-farcted tissue, the slices were incubated with TTC (10 mg/mL, 20 min, at 37 o C). In the presence of intact dehy-drogenase enzyme systems (normal myocardium), TTC formed dark red formation, while areas of necrosis lack-ing dehydrogenase activity were not stained, and TTC-stained tissue was weighed. IS was then calculated and expressed as percentage of AAR[25].

1.4 Western blot analysis

Heart tissue samples were lysed with lysis buffer. After sonication, the lysates were centrifuged; proteins were sepa-rated by electrophoresis on SDS-PAGE and transferred onto a polyvinylidene difluoride-plus membrane. After be-ing blocked with 5% fat-free milk, the immunoblots were probed with anti-p-JNK and anti-p-Akt antibodies over-night at 4 o C followed by incubation with the correspond-ing secondary antibodies at room temperature for 1 h. The blots were visualized with ECL-plus reagent. p-JNK and p-Akt immunoblots were then stripped with strip buffer at 50 o C for 30 min and reblotted for total JNK and Akt[26].

1.5 TUNEL assay

Hearts were washed by perfusion with ice-cold PBS for 1 min followed by a fixation step with ice-cold 4% parafor-maldehyde in phosphate buffer (peristaltic pump flow: 50 mL/min for 10 min). After 24 h in fixation solution, hearts were cryosectioned at a thickness of 10 μm and serial sections were stained with TUNEL reagents (Roche Co., USA), according to the manufacturer’s instructions. The digoxigenin-conjugated dUTP was incorporated to the ends of DNA fragments by terminal deoxynucleotidyl transferase

(TdT). The signal of TdT-mediated dUTP nick-end label-ing was then detected by an anti-fluorescein antibody con-jugation with alkaline phosphatase, a reporter enzyme that catalytically generates a red-colored product from vector red substrate. For each slide, 10 fields were randomly chosen and a defined rectangular field area (40 × objective) was used, a total of 200 cells per field were counted. The apoptotic index (AI) was determined (i.e., number of posi-tively stained apoptotic myocytes/total number of myocytes counted ×100%) from a total of 80 fields per heart. The assays were performed in a blinded manner[26].

1.6Statistical analysis

All values were presented as means±SEM. Differences were compared by ANOV A or Student’s t-test, when appropriate. P<0.05 was considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA).

2 RESUL TS

2.1 Cardiac hemodynamics in isolated hearts sub-jected to MI/R

Baseline values for functional parameters were obtained after stabilization. In sham group, the left ventricular deve-loped pressure (L VDP), heart rate (HR) and instantaneous first derivation of left ventricular pressure (±LVd p/d t

max

) remained at a relatively stable level throughout the whole MI/R procedure. In the control group, the left ventricular pressure showed a large scatter, which was frequently found despite a large sample. Coronary occlusion caused a

significant decline in L VDP, HR and ±L Vd p/d t

max

. However, compared with the control, administration of insulin at 60 U/L

induced a significant increase in L VDP, HR and ±L Vd p/d t

max by 26%, 25%, 29% and 28% (n=6, all P<0.05) (Fig.2), respectively. In insulin + LY294002 group, the values of

LVDP, HR, ±LVd p/d t

max

decreased by 34%, 23%, 43% and 42% (n=6, P<0.05 vs insulin group), respectively. All values between the control and insulin + L Y294002 groups had no statistical significance. Nevertheless, SP600125 con-verted the abolishment caused by LY294002 when it was administered together with LY294002, that is, compared

with those in insulin + L Y294002 group, L VDP, +L Vd p/d t

max and -L Vd p/d t

max

in insulin + L Y294002 + SP600125 group increased by 30%, 24% and 33%, respectively (n=6, P< 0.05) at 1 h after reperfusion, while HR was not signifi-cantly altered (P>0.05) (Fig.2). These results indicate that

LIU Hai-Tao et al: Cross-talk between Akt and JNK655

both Akt and JNK play roles in the effects of insulin on MI/R hearts.

2.2 Myocardial IS in isolated hearts subjected to MI/R

To determine myocardial injury, the effects of insulin, insulin + LY294002, insulin + SP600125, and insulin + L Y294002 + SP600125 on myocardial IS were determined. There were no significant differences in AAR/LV among all groups (data not shown). Occlusion of the LAD coro-nary artery for 30 min followed by reperfusion with ve-hicle for 2 h resulted in an IS of (45.0±4.0)%. Compared with the control, administration of insulin at 60 U/L caused a significant reduction in IS by approximately 16.1% (n=6, P<0.01) (Fig.3). Pretreatment of the isolated heart with the PI3-K inhibitor LY294002 abolished the reduction of IS by insulin [(43.5±5.0)% vs (28.9±2.0)%, n=6, P< 0.05]. While hearts in insulin + SP600125 group showed a smaller IS compared with that in insulin group(P<0.05). Interestingly, cotreatment with L Y294002 and SP600125 attenuated the inhibition of LY294002 on insulin-induced reduction of myocardial infarction (n=6, P<0.05) (Fig.3). These data suggest that insulin exerts cardioprotection via a PI3-K/Akt-dependent mechanism and that JNK activa-tion plays a role in this action of insulin.

2.3 Cardiomyocyte apoptosis in isolated hearts sub-jected to MI/R

In sham I/R hearts, few nuclei were stained TUNEL positively. In contrast, TUNEL-positive nuclei were preva-lent in the control I/R hearts (Fig.4). Administration of insulin reduced the percentage of TUNEL-positive nuclei from (27.6±1.3)% to (16.0±0.7)% (n=6, P<0.01 vs control) (Fig.4). However, after the hearts were treated with insu-lin + L Y294002, TUNEL-positive cells increased to (21.9±0.8)% (n=6, P<0.01 vs insulin) (Fig.4). These results

Fig. 2. Cardiac hemodynamics in isolated hearts subjected to ischemia/reperfusion and receiving different treatments. A: Left ventricular developed pressure (LVDP). B: Heart rate (HR). C and D: The instantaneous first derivation of left ventricular pressure (±LVd p/d t

max

). I, ischemia; R, reperfusion; LY, LY294002; SP, SP600125. means±SEM, n=6. *P<0.05 vs control, #P<0.05 vs insulin, +P<0.05 vs insulin + LY.

Acta Physiologica Sinica , October 25, 2007, 59 (5): 651-659

656indicate that insulin exerts an anti-apoptotic effect on I/R heart via a PI3-K/Akt-dependent pathway.

Insulin plus SP600125 reduced the apoptotic cells com-pared with insulin alone [(11.6±0.5)% vs (16.0±0.7)%, n =6,P <0.01]. In another group, in which hearts were pretreated

with SP600125 plus LY294002 15 min before ischemia and reperfusion with insulin during reperfusion, it was found that the number of TUNEL-positive nuclei in I/R hearts declined to (15.0±1.6)%, less than that in insulin +LY294002 group (n =6, P <0.05) (Fig.4). These data de-monstrate that administration of SP600125 plus L Y294002could abate the effect caused by LY294002, suggesting

Fig. 3. Infarct size in isolated hearts subjected to ischemia/reperfusion and receiving different treatments. TTC-negative stain-ing portions (infarct) and TTC-positive staining portions within ischemic/reperfused regions (area at risk, AAR) were weighed and infarct size was expressed as percent of infarct area relative to AAR. LY, LY294002; SP, SP600125. means±SEM, n =6.

**

P<0.01 vs control, #P<0.05 vs insulin, +P<0.05 vs

insulin + LY

.

Fig. 4. Apoptosis in isolated hearts subjected to ischemia/reperfusion and receiving different treatments. Data were ex-pressed as percentage of nuclei staining positive for TUNEL. LY ,L Y294002; SP, SP600125. means±SEM, n =6. **P<0.01 vs sham,

##

P<0.01 vs control, ++P <0.01 vs insulin, ?P <0.05 vs insulin + LY

.

Fig. 5. Total and phosphorylated Akt and JNK in isolated hearts subjected to ischemia/reperfusion and receiving different treatments.A : Phosphorylated Akt. Insets: Representative blots of phosphorylated and total Akt. **P <0.01 vs control, ##P <0.01 vs insulin. B :Phosphorylated JNK. Insets: Representative blots of phosphorylated and total JNK. **P <0.01 vs control, ##P <0.01 vs insulin. Data obtained from quantitative densitometry were presented as means±SEM of at least six independent experiments. LY, LY294002; SP,

SP600125.

LIU Hai-Tao et al: Cross-talk between Akt and JNK657

that JNK may act as a proapoptotic factor in MI/R hearts.

2.4 Akt and JNK activation in isolated hearts sub-jected to MI/R

Using Western blot analysis, the levels of JNK and Akt phosphorylation were examined after 4 h of reperfusion (Fig.5). It was observed that after 4 h of reperfusion with vehicle, Akt and JNK phosphorylation levels slightly in-creased in I/R myocardium and there was no statistical significance in the expressions. However, treatment with insulin markedly increased both Akt and JNK phosphory-lation in I/R hearts (n=3, P<0.01 vs control), suggesting the activation of insulin signaling transduction pathway in MI/R (Fig.5).

Previously we have demonstrated that LY294002 treat-ment inhibited the anti-apoptotic effects of insulin on MI/R rat hearts[1]. Here we examined the possibility that JNK inhibition leads to convert the action of LY294002 on cell apoptosis. Rat hearts were pretreated with a selective JNK inhibitor, SP600125, for 15 min, before LY294002 treat-ment, then Akt phosphorylation was assessed after 4-hour reperfusion. As anticipated, L Y294002 and SP600125 did inhibit insulin-induced Akt and JNK phosphorylation, re-spectively (n=6, P<0.01). Cotreatment with LY294002 and SP600125 almost completely abolished both Akt and JNK activation induced by insulin (n=6, P<0.01). Interestingly, it was found that inhibition of JNK activity by SP600125 simultaneously decreased phosphorylation of Akt (n=6, P< 0.01), which seemed different from that by LY294002. The tight temporal correlation between these two events supported the notion that PI3-K/Akt activation in isolated hearts might be related to JNK activation.

3 DISCUSSION

In the present study, it was observed that, consistent with our previous results, administration of insulin at the onset of reperfusion exerted cardioprotective effects on MI/R hearts as evidenced by improved cardiac functions, reduced IS and cell apoptosis via activation of the PI3-K/ Akt pathway, i.e., the survival signaling. In addition, along with Akt activation, there was also increased phosphoryla-tion of JNK when insulin was administered, which aggra-vated MI/R injury. The main findings of this study are that insulin-induced JNK activation could further increase Akt phosphorylation and this cross-talk between the Akt and JNK signaling seems to play an important role in the cardio-protection exerted by insulin.

The “decision” of a cell to live or to die is derived from the dominance of both prodeath or prosurvival proteins and cellular factors. Our previous study showed that in vivo treatment with insulin reduced postischemic apoptotic death in both cardiomyocytes and coronary endothelial cells by activating the PI3-K-Akt-eNOS pathway (the survival sig-naling pathway), which might further contribute to the prolonged improvement of cardiac performance following MI/R[1-5]. Consistent with those results, inhibition of Akt activation by L Y294002 in the present study almost com-pletely abolished the protective effects of insulin on iso-lated I/R hearts, indicating that the prosurvival factor Akt plays a key role in this beneficial action of insulin.

In a variety of cultured cells, JNK has been implicated in the cell death pathway, and several mechanisms have been proposed[11-13]. Previous studies have shown that JNK activation could be detected in myocardium within minutes after the onset of reperfusion[12]. Here we also found JNK activation was accompanied with reperfusion in the iso-lated rat hearts. These data suggest a possible role for JNK in the death of cardiomyocytes following MI/R, but do not provide the causality. It was reported that insulin induced stimulation of JNK[27], and in rat hippocampus, the neuroprotection of insulin against ischemic brain injury was via negative regulation of the JNK signaling pathway[28]. In the present study, we treated the isolated MI/R hearts with a JNK inhibitor, SP600125, and found that there was not only an inhibition of JNK phosphorylation, but also a reduction in IS and TUNEL-positive nuclei, and an im-provement in the heart functions. Interestingly, although LY294002 abolished the reduction of MI/R injury and the improvement of heart function caused by insulin, this abo-lishment could be partly converted by SP600125 pretreat-ment possibly due to the inhibition of the proapoptotic ef-fects of JNK. In addition, previous studies showed that SP600125 could not only inhibit JNK itself, but also sup-press the activation of JNK downstream members, for example, C-JUN, A TF-2, ELK-1, p53, and so on, thus in-duces anti-apoptotic effect. These data suggest that acti-vation of JNK induced by insulin aggravates myocardial injury and decreases the mechanical work of isolated hearts after regional MI/R.

Although insulin treatment protects the hearts from MI/R injury and improves the prolonged myocardial performance, whether there exists a cross-talk between insulin-induced Akt and JNK remains largely unclear. In the present study, we found that the activity of Akt could be regulated by JNK. Specifically, reperfusion with insulin resulted in both increased Akt and JNK phosphorylation. Pretreatment with LY294002, the PI3-K inhibitor, inhibited phosphorylation of Akt but not JNK. SP600125 pretreatment inhibited the

Acta Physiologica Sinica, October 25, 2007, 59 (5): 651-659 658

activation of JNK, and the Akt activation induced by insu-lin was simultaneously decreased, although SP600125 itself had no effect on Akt activation[15]. These data sug-gest that JNK activation induced by insulin increases Akt phosphorylation. The attenuation of Akt phosphorylation by the inhibition of JNK by SP600125 might be due to direct JNK-mediated phosphorylation of Akt, or change of upstream of PI3-K/Akt due to the regulation of PTEN (3’-phosphoinositide phosphatase) by JNK[29,30]. Further stu-dies are expected to elucidate this issue. It was reported that the dissociation of Akt from its negative regulator JIP1 is regulated by JNK[17]. Moreover, Kilter et al.[31] reported that JNKs are necessary for the reactivation of Akt after ischemic injury. Inhibition of JNK phosphorylation induced reduction in Akt activity. They further identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia. Taken together, it seems that more phos-phorylated JNK induced by insulin, more phosphorylated Akt is produced, which is involved in insulin-induced cardioprotective effects on MI/R hearts, although JNK it-self to some extent increases MI/R injury after activation. Future investigations are proposed to observe whether Akt activation activated by JNK could reversely inhibit phos-phorylation of JNK and exert impelling anti-apoptotic effect, and whether the balance between Akt and JNK may play a role in the beneficial effects of insulin on I/R myocardium. In summary, the present study demonstrates for the first time that insulin simultaneously actives both Akt and JNK, and the latter further increases the phosphorylation of Akt which attenuates MI/R injury and improves heart function. This cross-talk between Akt and JNK in the insulin signal-ing is involved in insulin-induced cardioprotective effect.

REFERENCES

1Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphoryla-tion. Circulation 2002; 105(12): 1497-1502.

2Ma H, Zhang HF, Yu L, Zhang QJ, Li J, Huo JH, Li X, Guo WY, Wang HC, Gao F. Vasculoprotective effect of insulin in the ischemic/reperfused canine heart: role of Akt-stimulated NO production. Cardiovasc Res 2006; 69(1): 57-65.

3Zhang HF, Fan Q, Qian XX, Lopez BL, Christopher TA, Ma XL, Gao F. Role of insulin in the anti-apoptotic effect of glucose-insulin-potassium in rabbits with acute myocardial ischemia and reperfusion. Apoptosis 2004; 9(6): 777-783.

4Zhang B, Zhang HF, Fan Q, Ma XL, Gao F. Insulin improves

cardiac myocytes contractile function recovery in simulated ischemia-reperfusion: key role of Akt. Chin Sci Bull 2003; 48: 1364-1369.

5Yu J, Zhang HF, Wu F, Li QX, Ma H, Guo WY, Wang HC, Gao

F. Insulin improves cardiomyocyte contractile function through

enhancement of SERCA2a activity in simulated ischemia/ reperfusion. Acta Pharmacol Sin 2006; 27(7): 919-926.

6Blakesley VA, Scrimgeour A, Esposito D, Le Roith D. Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev 1996; 7

(2): 153-159.

7Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature 2001; 414(6865): 799-806. 8Hausenloy DJ, Mocanu MM, Yellon DM. Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning. Cardiovasc Res 2004; 63(2): 305-312. 9Schaeffer HJ, Weber MJ. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 1999; 19(4): 2435-2444.

10Paraskevas S, Aikin R, Maysinger D, Lakey JR, Cavanagh TJ, Hering B, Wang R, Rosenberg L. Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival. FEBS Lett 1999; 455(3): 203-208.

11Fleming Y, Armstrong CG, Morrice N, Paterson A, Goedert M, Cohen P. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.

Biochem J 2000; 352 (Pt 1): 145-154.

12Ferrandi C, Ballerio R, Gaillard P, Giachetti C, Carboni S, Vitte PA, Gotteland JP, Cirillo R. Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol 2004; 142(6): 953-960.

13Kogel D, Schomburg R, Copanaki E, Prehn JH. Regulation of gene expression by the amyloid precursor protein: inhibition of the JNK/c-Jun pathway. Cell Death Differ 2005; 12(1): 1-9. 14Cerezo A, Martinez-A C, Lanzarot D, Fischer S, Franke TF, Rebollo A. Role of Akt and c-Jun N-terminal kinase 2 in apoptosis induced by interleukin-4 deprivation. Mol Biol Cell 1998; 9(11): 3107-3118.

15Heo YS, Kim SK, Seo CI, Kim YK, Sung BJ, Lee HS, Lee JI, Park SY, Kim JH, Hwang KY, Hyun YL, Jeon YH, Ro S, Cho JM, Lee TG, Yang CH. Structural basis for the selective inhibi-tion of JNK1 by the scaffolding protein JIP1 and SP600125.

EMBO J 2004; 23(11): 2185-2195.

16Kim AH, Yano H, Cho H, Meyer D, Monks B, Margolis B, Birnbaum MJ, Chao MV. Akt1 regulates a JNK scaffold during excitotoxic apoptosis. Neuron 2002; 35(4): 697-709.

LIU Hai-Tao et al: Cross-talk between Akt and JNK659

17Song JJ, Lee YJ. Dissociation of Akt1 from its negative regulator JIP1 is mediated through the ASK1-MEK-JNK signal transduc-tion pathway during metabolic oxidative stress: a negative feed-back loop. J Cell Biol 2005; 170(1): 61-72.

18Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma M, Davis RJ. The JIP group of mitogen-activated protein kinase scaffold proteins.

Mol Cell Biol 1999; 19(10): 7245-7254.

19Park HS, Kim MS, Huh SH, Park J, Chung J, Kang SS, Choi EJ.

Akt (protein kinase B) negatively regulates SEK1 by means of protein phosphorylation. J Biol Chem 2002; 277(4): 2573-2578. 20Engelbrecht AM, Niesler C, Page C, Lochner A. p38 and JNK have distinct regulatory functions on the development of apoptosis during simulated ischaemia and reperfusion in neona-tal cardiomyocytes. Basic Res Cardiol 2004; 99(5): 338-350. 21Lochner A, Genade S, Hattingh S, Marais E, Huisamen B, Moolman JA. Comparison between ischaemic and anisomycin-induced preconditioning: role of p38 MAPK. Cardiovasc Drugs Ther 2003; 17(3): 217-230.

22Cardenas G, Torres JC, Zamora J, Banos G. Isolated heart func-tion during ischemia and reperfusion in sucrose-fed rats: effect of insulin infusion. Cardiovasc Pathol 2005; 14(5): 256-264.

23Zaha V, Francischetti I, Doenst T. Insulin improves postischemic recovery of function through PI3K in isolated working rat heart.

Mol Cell Biochem 2003; 247(1-2): 229-232.

24Sack MN, Yellon DM. Insulin therapy as an adjunct to reperfusion after acute coronary ischemia: a proposed direct myocardial cell survival effect independent of metabolic modulation. J Am Coll Cardiol 2003; 41(8): 1404-1407.

25Zacharowski K, Olbrich A, Otto M, Hafner G, Thiemermann C.

Effects of the prostanoid EP3-receptor agonists M&B 28767 and

GR 63799X on infarct size caused by regional myocardial ischaemia in the anaesthetized rat. Br J Pharmacol 1999; 126(4): 849-858.

26Gao F, Gong B, Christopher TA, Lopez BL, Karasawa A, Ma XL. Anti-apoptotic effect of benidipine, a long-lasting vasodilat-ing calcium antagonist, in ischaemic/reperfused myocardial cells.

Br J Pharmacol 2001; 132(4): 869-878.

27Müssig K, Fiedler H, Staiger H, W eigert C, Lehmann R, Schleicher ED, H?ring HU. Insulin-induced stimulation of JNK and the PI3-kinase/mTOR pathway leads to phosphorylation of serine 318 of IRS-1 in C2C12 myotubes. Biochem Biophys Res Commun 2005; 335(3): 819-825.

28Hui L, Pei DS, Zhang QG, Guan QH, Zhang GY. The neuro-protection of insulin on ischemic brain injury in rat hippocam-pus through negative regulation of JNK signaling pathway by PI3K/Akt activation. Brain Res 2005; 1052(1): 1-9.

29Vivanco I, Palaskas N, Tran C, Finn SP, Getz G, Kennedy NJ, Jiao J, Rose J, Xie W, Loda M, Golub T, Mellinghoff IK, Davis RJ, Wu H, Sawyers CL. Identification of the JNK signaling pathway as a functional target of the tumor suppressor PTEN.

Cancer Cell 2007; 11(6): 555-569.

30Zhang QG, Wu DN, Han D, Zhang GY. Critical role of PTEN in the coupling between PI3K/Akt and JNK1/2 signaling in is-chemic brain injury. FEBS Lett 2007; 581(3): 495-505.

31Shao Z, Bhattacharya K, Hsich E, Park L, Walters B, Germann U, Wang YM, Kyriakis J, Mohanlal R, Kuida K, Namchuk M, Salituro F, Yao YM, Hou WM, Chen X, Aronovitz M, Tsichlis PN, Bhattacharya S, Force T, Kilter H. c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo. Circ Res 2006; 98(1): 111-118.