胰岛素血糖依赖性和非依赖性心肌保护作用及抗肾上腺素能心肌保护新机制
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摘要
研究背景
“葡萄糖-钾-胰岛素”(GIK)极化液作为辅助治疗措施用于急性心肌缺血(AMI)的治疗已有近半个世纪的历史,基于动物及体外实验的基础研究均多次证实GIK及其关键成分胰岛素在保护缺血心肌方面的有益作用。然而,GIK应用于AMI治疗的临床试验结果不一,围绕GIK临床有效性及其作用机制的诸多争议性问题,至今在基础和临床研究领域尚未有确凿的实验依据给予明确回答。例如,有学者撰文分析GIK临床试验阴性结果的可能原因是GIK使用过程中血糖控制不当,但这一观点尚未得到实验确证,胰岛素降糖作用(即血糖依赖性作用)在胰岛素心肌保护效应中充当着怎样的角色,有待进一步探讨,且对于这一问题的研究在GIK/胰岛素的临床应用具有重要价值。
我们在前期的研究工作中发现,胰岛素除代谢调节作用外,可以直接激活心血管内源性Akt-eNOS系统,激活细胞“生存信号”保护缺血心脏。我们和国际上多个实验室也相继报道了胰岛素糖代谢作用以外的直接心血管保护效应,如抑制心肌细胞凋亡、保护血管内皮、抗炎、抗氧化应激、正性肌力作用等。然而,以往对于上述胰岛素非代谢性效应的观察往往同时与胰岛素降糖过程相伴随,胰岛素降糖以外的心肌保护作用与其代谢调节作用的相互关系及各自的作用尚不明确。
针对这一问题,最近危重症治疗研究领域根据临床和动物实验结果率先提出,危重症治疗中胰岛素带来的治疗收益完全取决于并得益于其降糖作用,而胰岛素降糖以外的作用甚微。然而遗憾的是,上述对于胰岛素应用于危重症的临床和实验观察并未评价心脏损伤和心血管终点事件。因此,胰岛素在AMI中的心肌保护作用是否应完全归功于其降糖作用,抑或存在血糖非依赖性的直接保护效应?阐明胰岛素对缺血心肌的保护效应与血糖的关系,并寻找血糖非依赖性心肌保护效应存在的直接证据和作用机制,将对胰岛素在AMI的临床应用提供重要的实验依据和指导价值。
实验目的
利用接近于临床的大动物模型,探讨心肌缺血再灌注过程中,胰岛素血糖依赖性和非依赖性作用的相互关系及各自对胰岛素心肌保护效应的意义和相关机制。
实验方法
1.将犬随机分为4组:正常胰岛素、正常血糖组(Normal Insulin/Euglycemia, NI/NG组);正常胰岛素、高血糖组(Normal Insulin/Hyperglycemia, NI/HG组);高胰岛素、正常血糖组(HighInsulin/Euglycemia, HI/NG组) ;高胰岛素、高血糖组(High Insulin/Hyperglycemia, HI/HG组)。为阻断因血糖波动引起的内源性胰岛素释放,通过结扎犬胰周血管阻断胰岛素分泌入血。建立犬急性心肌缺血再灌注(MI/R)模型:利用电磁流量计和血管狭窄器制造50分钟的局部定量缺血,使左前降支(LAD)血流量降低80%,此后释放狭窄器恢复血流,再灌注4小时。于再灌注前5分钟微量泵静脉输注50%葡萄糖和GIK极化液用于对血糖和血浆胰岛素水平的干预。
2.实时测量血糖,血浆胰岛素、C肽水平、游离脂肪酸含量,以进行血糖和胰岛素水平控制和代谢评价。于缺血前、缺血后50 min及再灌注4 h分别同时从前室间隔静脉和左股动脉取血,立即进行血气分析,检测PO2、PCO2、pH、血红蛋白(Hb)及血样饱和度(SO2),用于计算LAD灌注区心肌组织氧耗率和心肌呼吸指数。
3.通过多道生理记录和分析处理系统持续记录犬心电、冠脉LAD血流量和血流动力学指标。于再灌注结束后用伊文氏蓝/TTC双染色法测定心肌梗死面积。血浆肌钙蛋白T作为心肌损伤标志物,用于细胞坏死程度的评价。采用免疫组织化学方法分别检测Caspase-3活化及TUNEL凋亡指数用于定性和定量检测细胞凋亡。
4.采用石蜡包埋切片的苏木素-伊红染色方法,评价再灌注后心肌坏死周边区白细胞浸润程度,同时测量组织中髓过氧化物酶(MPO)含量以定量评估组织炎症反应水平。检测组织丙二醛(MDA)含量用于评估脂质过氧化水平,测量超氧化物歧化酶(SOD)以评价内源性抗氧化能力。
5.在数据采集和统计过程中采用严格排除标准以保证各实验组纳入的动物样本具有可比性。
实验结果
1.本实验成功建立犬急性心肌缺血再灌注损伤模型。再灌注后心肌出现明显梗死区,细胞凋亡和坏死,及组织炎症反应和氧化损伤。
2.胰腺手术造成的胰腺缺血和局限性炎症在实验观察时程内对全身性炎症反应和心脏功能影响甚微。通过平行实验,在建立心肌缺血再灌注模型前分别实施胰周血管结扎手术和假手术以对比观察。两组间血清胰淀粉酶、胰脂肪酶、心肌肌钙蛋白T(cTnT)和肌酸激酶同工酶(CK-MB)的基础值和术后5小时测量值均无显著性差异,且血流动力学的同步观测显示心脏功能亦无明显变化。
3.高血糖存在情况下心肌I/R损伤程度进一步加重。表现为高血糖组较正常血糖组心肌收缩功能恢复障碍进一步加重,梗死面积扩大,细胞凋亡和坏死增多,炎症和氧化损伤加重(NI/HG vs. NI/NG, HI/HG vs. HI/NG, n=8, P<0.05)。
4.胰岛素干预可显著增加冠脉流量,提高代谢效率,增强心肌收缩功能,且这些有益作用不受血糖高低的影响。高胰岛素组较正常胰岛素组再灌注后收缩功能(心率压力指数、最大收缩速率)明显提高,LAD灌流区血流量增加,心肌组织葡糖糖摄取率明显升高,游离脂肪酸(FFA)摄取率轻微下降,心脏呼吸指数明显上升(HI/NG vs. NI/NG, HI/HG vs. NI/HG, n=8, P<0.05),提示胰岛素诱导心肌氧化代谢底物由FFA向葡萄糖转变。且在心功能明显改善的前提下,心肌氧耗并未显著增高,表明心肌氧利用率的提高。
5.当血糖钳夹在生理水平时,胰岛素水平升高可进一步缩小梗死面积,抑制细胞坏死和凋亡,降低炎症反应和氧化应激(HI/NG vs. NI/NG, n=8, P<0.05)。但此作用需以降糖为前提,高血糖存在时,上述保护作用消失(HI/HG vs. NI/HG, n=8, P>0.05)。
结论
1.高血糖可显著加重心肌损伤并掩盖胰岛素保护作用,因而胰岛素降糖作用在其抗I/R损伤保护缺血心肌的效应中扮演着关键角色。
2.胰岛素具有独立于降糖效应、直接的心血管保护作用,其中包括不依赖于血糖控制水平的直接正性肌力作用、冠脉扩张作用及对心肌氧化代谢效率的促进作用。更为重要的是,胰岛素减轻心肌损伤,抑制细胞凋亡,降低组织炎症反应和氧化应激的有益作用,虽然依赖于正常血糖的维持,但独立于降糖作用而存在。
Part II
胰岛素抗肾上腺素能心肌保护新机制的研究
研究背景
急性心肌缺血发生时普遍存在着心脏交感-肾上腺素能系统的激活,一系列证据提示,交感-肾上腺素能活性增强及其介导的儿茶酚胺分泌释放是加重缺血心肌损伤、诱导致命性心律失常发生的重要原因之一,且与心脏重构、心力衰竭的发生发展密切相关。其中,儿茶酚胺对心脏的直接作用主要由β-肾上腺素受体(adrenergic receptor, AR)所介导,因此β受体阻滞剂被广泛应用于临床AMI的治疗。
越来越多的证据表明,胰岛素除了在维持心肌代谢稳态中发挥重要作用外,还在多种生理、病理过程中发挥关键调控作用。通过前述实验一的研究,我们在大动物MI/R模型上首次证实了胰岛素独立于降糖作用之外直接的心肌保护作用。例如,我们在前期研究中发现,胰岛素可激活“PI3K-Akt-eNOS”细胞生存信号抑制心肌细胞凋亡,保护冠脉内皮,抑制炎性反应和氧化应激,促进再灌注后心脏功能的恢复。此外,我们和国际上多个实验室也发现了胰岛素的直接心脏正性变力作用,表现为在整体动物模型中增强心肌收缩功能,在细胞水平增强收缩幅度和钙瞬变。
心肌细胞膜上同时存在着大量的胰岛素受体和β受体。已有研究报道,在人脂肪细胞和平滑肌细胞中,胰岛素可直接对抗儿茶酚胺的脂解作用;相关分子水平的研究也支持胰岛素对β受体信号调控作用的存在。最近有细胞实验显示,心肌细胞中PI3K信号可抵消cAMP信号系统介导的Ca2+内流和正性变力作用。然而,在心肌细胞中,胰岛素对β受体激动效应可能的调控作用所知甚少,目前仅有的少数文献报道尚存争议。
儿茶酚胺的正性肌力作用主要通过β受体内传,经cAMP的生成激活蛋白激酶A (PKA),进而磷酸化一系列下游分子,参与对兴奋-收缩偶联的调控。其中PKA的靶分子之一肌浆网受磷蛋白(PLB)的磷酸化水平可直接调控SERCA2a活性,而后者是与心肌细胞Ca~(2+)释放、摄取、贮存有关的肌浆网内主要钙转运蛋白之一,也是推动心肌收缩舒张过程的关键蛋白酶。此外,MI/R伴发的一系列细胞内环境稳态的改变,如儿茶酚胺的大量释放,细胞内钙超载,氧化应激等,均可通过改变PLB磷酸化状态及SERCA2活性,加重MI/R心肌收缩功能的损伤。然而,MI/R时βAR激活加重心肌损伤,以及胰岛素对βAR系统的拮抗作用是否与肌浆网钙调控蛋白的功能调节有关,目前尚不明确。
实验目的
本研究旨在探讨急性MI/R过程中,胰岛素是否对βAR系统具有调控作用;在回答第一个问题的基础上,我们将进一步围绕细胞内肌浆网钙调控蛋白的调节,寻求胰岛素调控βAR系统作用的分子机制。
实验方法
1.采用Langendorff离体灌流系统制备大鼠离体心脏缺血再灌注模型。离体灌流心脏随机分为以下5组:⑴sham MI/R组;⑵MI/R组:30min局部缺血/2h再灌注;⑶MI/R+Ins组:再灌注前5min开始给予10-7mol/L胰岛素;⑷MI/R+ISO组:缺血前10min开始给予10~(-9)mol/L异丙肾上腺素(ISO);⑸MI/R+Ins+ISO组:再灌注前5min给予10~(-7) mol/L胰岛素并于再灌注前10min给予10~(-9) mol/L ISO直至再灌注结束。
2.于左心室插入充水球囊,由多道生理记录系统连续采集左室压力指标,实时测量心脏收缩功能。于再灌注结束后用伊文氏蓝/TTC双染色法测定心肌梗死面积。于心肌缺血前15min、缺血30min末、再灌注1h、再灌注2h分别收集冠脉流出液,用分光光度法测定肌酸激酶(CK)、乳酸脱氢酶(LDH)活性。DNA ladder方法和TUNEL原位法分别定性和定量测量心肌细胞凋亡。
3.取再灌注后心肌组织,制备心肌细胞肌浆网膜蛋白以测定Ca~(2+)-ATPase活性。Western Blot检测SERCA2a蛋白表达水平、PLB及磷酸化PLB水平。PepTag非放射性蛋白激酶检测试剂盒测定组织PKA活性。
4.酶解法分离成年大鼠钙耐受性心室肌细胞,并建立化学模拟缺血再灌注(SI/R)模型。随机分组,缺血15 min后按以下溶液进行再灌注30 min:⑴对照组:台式液;⑵胰岛素(10~-8 -10~-5 mol/L)组;⑶ISO(10~(-9) mol/L)组;⑷ISO (10~(-9) mol/L) + Insulin (10(~-8) -10~(-5) mol/L)组。采用IonOptix可视化动缘探测系统同步检测心肌细胞收缩/舒张功能和钙瞬变。与SI/R相对应,正常灌流情况下同期记录上述四种不同药物干预措施引起的细胞收缩和钙瞬变的变化。
实验结果
1.与对照组相比,ISO干预组于缺血前和缺血早期出现短暂的心率(HR)加快、左室收缩压(LVSP)增高;然而再灌注1 h后,HR、LVSP、±LVdP/dtmax开始显著下降,左室舒张末压(LVEDP)明显升高。表明β受体激动加重再灌注后心肌收缩功能的障碍。胰岛素的给予可明显对抗ISO的负向作用,表现为HR、LVSP、±LVdP/dtmax降低程度减轻,LVEDP升高减缓(n=8, P<0.05)。
2.与对照组相比,单独ISO干预使再灌注后梗死范围进一步扩大,冠脉流出液中CK和LDH活性显著增加,DNA梯状条带的染色强度和TUNEL凋亡阳性染色细胞数(n=8, P<0.05)增加。而同时给予胰岛素梗死面积缩小33.1%,降低CK和LDH水平分别为35.1%和22.5% ,同时抑制ISO增加凋亡的作用(n=8, P<0.05)。
3.在正常灌流的成年大鼠心室肌细胞,胰岛素(10~(-7 )mol/L)可显著增加细胞收缩幅度和钙瞬变幅度,ISO (10~(-9) mol/L)则显示出更为明显的正性肌力作用,收缩峰值和钙瞬变幅度的增幅分别达19.1%和85% (n=20, P<0.05)。同时给予10~(-8)-10~(-6) mol/L不同浓度梯度的胰岛素,对ISO的正性肌力反应并不产生显著影响。
4.在模拟I/R的心肌细胞,胰岛素(10~(-7) mol/L)和ISO(10~(-9) mol/L)使细胞收缩峰值各增加17%和81%,钙瞬变增幅15%和109%。然而两者合用时,细胞收缩峰值只增加47%,钙瞬变增幅降至76% (n=20, P<0.05 vs. MI/R+ISO组),提示I/R心肌细胞中,胰岛素可拮抗ISO的正性肌力作用。此外,此拮抗作用也呈现出剂量依赖性特点(10~(-8)-10~(-5) mol/L)。
5.与sham组相比,I/R心肌组织内SERCA2a蛋白表达水平和活性均明显降低,且在ISO处理组进一步下降。单纯胰岛素干预或胰岛素与ISO联用,SERCA2a蛋白表达水平不变,而活性均较相应对照组增高。I/R心肌中PLB磷酸化(PKA磷酸化位点)水平明显降低,胰岛素和ISO可分别升高PLB磷酸化4.2倍和3.1倍,二者联用可进一步增加PLB磷酸化1.7倍。与sham组对比,MI/R本身可诱导2.5倍PKA活性的增高,而ISO可进一步使PKA活性大幅增高约30.7倍(P < 0.01)。胰岛素自身对MI/R心肌PKA活性并无明显影响,然而对ISO诱导的PKA激活产生显著的抑制效应(P < 0.01)。
结论
1.胰岛素可显著抑制MI/R过程中异丙肾上腺素(ISO)诱发β受体激动所带来的心肌损伤,减轻心肌收缩功能障碍、缩小梗死面积,减少细胞坏死和凋亡;
2.胰岛素可浓度依赖性减弱ISO对I/R心肌细胞的正性变力作用;
3.胰岛素对β受体信号系统中的PKA活性及其下游钙转运蛋白的调节,可能是MI/R过程中胰岛素拮抗β受体激活效应的机制之一。上述结果提示胰岛素心血管保护作用的抗肾上腺素能新机制。
Background
Experimental evidence has provided strong supports for the cardioprotective effects of“Glucose-insulin-potassium (GIK)”solution since its introduction as an adjunct to the contemporary management of acute myocardial ischemia (AMI). GIK solution, however, has not been uniformly successful in clinical practice over the last few decades and few trials and experiments to date have been adequately powered to explore the mechanism underlying the mixed results. Although it has been proposed that insulin therapy alone without achieving euglycemia may not improve outcomes in AMI, it remains completely unevidenced whether insulin-titrated euglycemia maintenance is requisite for the cardioprotective effects of insulin. The definite relationship between glycemic control, cardioprotection, and insulin therapy itself requires further elucidation.
Meanwhile, our recent studies as well as others’have proposed direct cell survival signalings favorably modulated by insulin against myocardial ischemia/reperfusion (MI/R) injury. In addition to its anti-apoptotic action, anti-inflammatory, anti-oxidative stress and positive inotropic effects have also been well documented, and are believed to further enhance cardiac performanc after reperfusion. However, latest reports from the research on critically ill patients and animal models suggested that the survival benefits from intensive insulin therapy were due to successful glycemic control, rather than other glycemia-independent effects of insulin. However, cardiac outcome was not addressed, thus it remains unclear whether the well-documented benefits of insulin in the ischemic/reperfused heart should be attributed exclusively to glucose normalization, or combined with other mechanisms inherently originating from insulin per se. The clarification of the existence and contribution of glycemic-control-independent actions of insulin may greatly advance the understanding and application of GIK in AMI management.
Objectives
The present study was designed to investigate, in a clinically relevant large animal model of myocardial ischemia/reperfusion (MI/R), the relationship between glycemia-dependent and -independent effects of insulin and their contributions to insulin’s cardioprotection.
Methods
1. Endogenous insulin production in canines was abolished by peripancreatic vessel ligation. Aninals were then randomly assigned to four groups: Normal plasma insulin/euglycemia (NI/NG), normal insulin/hyperglycemia (NI/HG), high insulin/euglycemia (HI/NG), and high insulin/hyperglycemia (HI/HG), which were achieved by controlled intravenous infusion of glucose/insulin.
2. Anesthetized open-chest dogs were subjected to MI/R (50 min/4 h) by partially occluding the left anterior descending coronary artery (LAD) (80% reduction in its blood flow). Gluose/insulin administration started at 5 min before reperfusion and continued thoughout the experiment.
3. Blood glucose and plasma concentrations of insulin, C-peptide and free fatty acid (FFA) were measured for metabolic control and evaluations. Blood samples from the left femoral artery and the anterior interventricular vein were analyzed for PO2, PCO2, pH, hemoglobin (Hb) concentration and oxygen saturation (SO2) in order to calculate oxygen consumption rate (MVO2) and respiratory quotient (RQ) of the LAD perfused region.
4. Left ventricular (LV) pressure was continuously monitored by a hemodynamic analyzing system. Myocardial infarction was measured using the Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) double staining. Plasma level of cardiac troponin-T (cTnT) was assayed as a biomarker for myocardial ischemic injury. Cell apoptosis was analyzed by immunohistochemical detection of caspase-3 activation and TUNEL assay.
5. Myocardial inflammation was evaluated by histochemical detection of leukocyte infiltration as well as measurement of tissue myeloperoxidase (MPO) activity. Oxidative stress was estimated by measuring the tissue content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD).
6. Strict criteria were established to guarantee that all animals included in the final data analysis were healthy and exposed to comparable degrees of regional myocardial ischemia.
Results
1. Canine acute MI/R model was successfully established. Significant myocardial infarction, cell apoptosis and necrosis, inflammatory reaction and oxidative stress were observed as expected.
2. The ischemic insult due to pancreas surgery resulted in limited pancreatic inflammation and minimal influence on systemic inflammation and cardiac function within 5 hours’protocol. Plasma levels of amylase, lipase, cTnT and creatine kinase-MB, as well as hemodynamic indices at baseline and 5 hours after the pancreas surgery showed no statistical differences between pancreatic-ligated versus non-operated dogs subjected to MI/R.
3. Hyperglycemia alone significantly aggravated MI/R injury, as evidenced by worse functional recovery, larger infarct size, increased cell apoptosis and necrosis, and enhanced inflammation and oxidative injury in HG groups compared to NG groups.
4. Insulin increased conoray perfusion and metabolic efficiency as well as promoted contractile function of the I/R myocardium, which were minimally affected by glucose levels. Compared to NI groups, both HI groups showed higher rate-pressure product and +LVdp/dtmax with increased LAD blood flow during reperfusion. In addition, a robust elevation in anterior LV glucose uptake was also observed following insulin infusion, accompanied by a slight decline in FFA uptake and increase in respiratory quotient, suggesting insulin-mediatd switch of myocardial substrate uptake from fat to carbohydrate. Interestingly, MVO2 of the anterior LV was not elevated in HI groups though contractile function increased, indicating an apparent increase in O2 utilization efficiency.
5. When blood glucose was clamped at physiologic level, insulin elevation (HI/NG vs. NI/NG) further exerted protection against MI/R injury, as evidenced by improved functional recovery, decreased myocardial infarct size, reduced cell necrosis and apoptosis, and alleviated inflammatory and oxidative stress. However, maintaining high glucose concentration in high insulin animals (HI/HG) markedly blunted or abolished the above protective effects of insulin.
Conclusions
1. Insulin-titrated maintenance of euglycemia protects heart from MI/R injury, whereas hyperglycemia oppositely affects MI/R injury and masks the insulin-induced cardiac benefits. Thus insulin-mediated hyperglycemia prevention is of critical importance to ensure insulin’s full efficacy in protecting heart against MI/R.
2. Insulin has direct cardioprotective effects independent of its glucose-lowering ability. Thus the cardiac benefits of insulin against I/R injury should not be attributed exclusively to glucose normalization.
Part II
Background
Myocardial ischemia involves a large and progressive release of catecholamines from adrenergic nerve terminals, and overshooting of myocardialβ- adrenergic receptors (?-AR) by catecholamines may further aggravate MI/R injury, induce lethal arrhythmias, and contribute to post-infarction remodeling and heart failure.
Increasing evidence showed that insulin, apart from its role in the metabolic regulation, participates in a variety of physiological and pathological cardiovascular process. In the above Part I study, we have demonstrated that insulin has direct cardioprotective effects independent of its glucose-lowering ability. For example, through the activation of a cell survival signaling“PI3K-Akt-eNOS”, insulin exerts protection for the I/R heart by inhibiting cell apoptosis, preserving endothelial function, and suppressing myocardial inflammation and oxidative stress. Moreover, a direct positive inotropic effect of insulin has been reported as evidenced by increased contractile function in in vivo models and elevated twitch amplitude and calcium transient in single ventricular myocyetes.
Previous investigations have revealed a counter-regulatory effect of insulin onβ-adrenergic catecholamine action in isolated human fat cells and cultured smooth muscle cells. Recent studies also provided important evidence for the existence of interaction between myocardial insulin signaling and ?-AR signaling. However, existing data concerning the functional effects of insulin on the myocardial response to ?-AR activation are quite limited and inconsistent. Moreover, the major cardiac effects of catecholamines are mediated by ?-AR through the activation of cAMP-dependent protein kinase A (PKA), which in turn phosphorylates a series of target moleculars responsible for the regulation of excitation-contraction coupling. Phospholamban is one of the PKA target proteins, and at the same time a major regulator of SERCA2, which modulates the rate of SR Ca~(2+) uptake, leads to the regulation of relaxation velocity, SR Ca~(2+) load and myocardial contractility. Several events associated with MI/R, such as catecholamines release, intracellular Ca~(2+) overload and oxidative stress, participate in the modulation of PLB phosphorylation and SERCA2 activity. However, it remains unclear howβ-AR stimulation influences these SR Ca~(2+) handling proteins in the I/R heart and whether these influences could be modified by the application of insulin.
Objectives
This study aimed to investigate the ability of insulin to modulateβ-adrenergic actions on post-ischemic injury and myocardial contractility in acute MI/R and the underlying mechanism.
Methods
1. Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h). Hearts (n=16-20 per group) were randomized to receive one of the following treatments: (1) sham MI/R; (2) MI/R vehicle; (3) MI/R receiving insulin (10-7 mol/L) 5 min before and throughout reperfusion; (4) MI/R receiving ISO (10~(-9) mol/L) 10 min before ischemia and throughout reperfusion; (5) MI/R receiving insulin 5 min before reperfusion plus ISO (10~(-9) mol/L) 10 min before ischemia, both throughout reperfusion.
2. A water-filled latex balloon was inserted into the left ventricle; cardiac function was continuously recorded by a hemodynamic analyzing system via a pressure transducer connected to the balloon. Myocardial infarction was measured using the Evans blue/TTC double staining. Coronary effluent was collected and creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically. Cardiomyocyte apoptosis was analyzed by detection of DNA fragmentation (DNA ladders) and TUNEL in situ assay.
3. Ca~(2+)-ATPase activity of the isolated SR membrane was measured in the reperfused hearts. Expressions of SERCA2a, PLB and phosphorylated PLB in the SR were determined by Western blot. Tissue homogenates were prepared for non-radioactive determination of PKA activity using PepTag Assay.
4. Calcium-tolerant ventricular myocytes were isolated from adult male rats by a standard enzymatic technique. Isolated myocytes were exposed to simulated ischemia for 15 min using chemical anoxia solution, and then reperfused with one of the following agents for another 30 min: (1) vehicle; (2) insulin (10~(-8) -10~(-5) mol/L); (3) isoproterenol (ISO, 10~(-9) mol/L); (4) ISO (10~(-9) mol/L) +insulin (10~(-8)-10~(-5) mol/L). The mechanical contraction and Ca2+ transients of ventricular myocytes were assessed by a video-based motion edge-detection system. Responses to the above agents were also observed in normal myocytes.
Results
1. ISO administration produced a transient increase in HR and LVSP, followed by a sharp decline after 1 h reperfusion and end up with a greatly dampened HR and LVDP and elevated LVEDP compared with the untreated I/R hearts, suggesting thatβ-AR activation during MI/R aggravates post-ischemic cardiac dysfunction. More importantly, insulin largely reversed the ISO-induced functional impairment, inhibiting ISO-induced declines in HR and LVSP by 34.0% and 23.0% respectively, and preventing ISO-induced elevation in LVEDP by 28.7% (n=8, all P<0.05).
2. ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were all inhibited by treatment of insulin (n=8, all P<0.05).
3. In normal ventricular myocytes, insulin (10~(-7) mol/L) exerted significant inotropic effect as evidenced by increased peak twitch amplitude (PTA) and calcium transient amplitude (ΔFFI). ISO with a submaximal concentration of 10~(-9) mol/L, showed an even greater inotropic effect with a 19.1% increase of PTA and 85% increase ofΔFFI. Exposure to insulin (10~(-8)-10~(-6) mol/L), either before or after the administration of ISO, displayed no significant differences with ISO alone.
4. In simulated I/R cardiomyocytes, insulin (10~(-7)mol/L) and ISO(10~(-9) mol/L)both mediated distinct positive inotropic responses, with 17% and 81% increases of PTA along with 15% and 109% increases ofΔFFI, respectively. When applied together, however, insulin attenuated the inotropic response to ISO as evidenced by an 18.7% reduction in PTA and a 23.9% reduction inΔFFI (n=20 myocytes from 8 hearts, both P<0.05). This inhibitory effect also acts in a concentration-dependent manner (10~(-8)-10~(-5) mol/L).
5. Both protein expression and activity of SERCA2a in untreated I/R hearts were significantly reduced compared with sham hearts, and were further depressed when ISO was applied. Reperfusion with insulin produced a 13.9% increase in SERCA2a activity compared with vehicle, and a 15.2% increase compared with ISO alone (both P<0.05). Phosphorylation of PLB at Ser16 was significantly decresed in I/R hearts. Insulin and ISO triggered a 4.2- and 3.1-fold increase in PLB phosphorylation, and insulin further facilitated ISO-induced PLB phosphorylation, a 1.7-fold increase relative to ISO alone (P<0.01). As for PKA activity, MI/R resulted in 2.5-fold increase in PKA activation compared with sham group. Application of ISO produced an overt elevation in PKA activity as evidenced by a 30.7-fold increase (P<0.01 vs. MI/R group). Interestingly, insulin itself exerted no significant influence on PKA activity, but markedly inhibited ISO-mediated PKA activation in I/R hearts (P<0.01).
1. Insulin significantly suppresses ISO-elicited cardiac injury including myocardial contractile dysfunction, cell apoptosis and necrosis in isolated I/R hearts, which may contribute to insulin-induced cardioprotection in MI/R.
2. Insulin attenuates ISO-induced positive inotropic effect on simulated I/R cardiomyocytes in a concentration-dependent manner.
3. The regulation of ?-adrenergic postreceptorial signaling such as PKA and its downstream calcium handling proteins is a likely mechanism that accounted for the insulin-induced counterbalance of -adrenergic activation in MI/R. Conclusions
“葡萄糖-钾-胰岛素”(GIK)极化液作为辅助治疗措施用于急性心肌缺血(AMI)的治疗已有近半个世纪的历史,基于动物及体外实验的基础研究均多次证实GIK及其关键成分胰岛素在保护缺血心肌方面的有益作用。然而,GIK应用于AMI治疗的临床试验结果不一,围绕GIK临床有效性及其作用机制的诸多争议性问题,至今在基础和临床研究领域尚未有确凿的实验依据给予明确回答。例如,有学者撰文分析GIK临床试验阴性结果的可能原因是GIK使用过程中血糖控制不当,但这一观点尚未得到实验确证,胰岛素降糖作用(即血糖依赖性作用)在胰岛素心肌保护效应中充当着怎样的角色,有待进一步探讨,且对于这一问题的研究在GIK/胰岛素的临床应用具有重要价值。
我们在前期的研究工作中发现,胰岛素除代谢调节作用外,可以直接激活心血管内源性Akt-eNOS系统,激活细胞“生存信号”保护缺血心脏。我们和国际上多个实验室也相继报道了胰岛素糖代谢作用以外的直接心血管保护效应,如抑制心肌细胞凋亡、保护血管内皮、抗炎、抗氧化应激、正性肌力作用等。然而,以往对于上述胰岛素非代谢性效应的观察往往同时与胰岛素降糖过程相伴随,胰岛素降糖以外的心肌保护作用与其代谢调节作用的相互关系及各自的作用尚不明确。
针对这一问题,最近危重症治疗研究领域根据临床和动物实验结果率先提出,危重症治疗中胰岛素带来的治疗收益完全取决于并得益于其降糖作用,而胰岛素降糖以外的作用甚微。然而遗憾的是,上述对于胰岛素应用于危重症的临床和实验观察并未评价心脏损伤和心血管终点事件。因此,胰岛素在AMI中的心肌保护作用是否应完全归功于其降糖作用,抑或存在血糖非依赖性的直接保护效应?阐明胰岛素对缺血心肌的保护效应与血糖的关系,并寻找血糖非依赖性心肌保护效应存在的直接证据和作用机制,将对胰岛素在AMI的临床应用提供重要的实验依据和指导价值。
实验目的
利用接近于临床的大动物模型,探讨心肌缺血再灌注过程中,胰岛素血糖依赖性和非依赖性作用的相互关系及各自对胰岛素心肌保护效应的意义和相关机制。
实验方法
1.将犬随机分为4组:正常胰岛素、正常血糖组(Normal Insulin/Euglycemia, NI/NG组);正常胰岛素、高血糖组(Normal Insulin/Hyperglycemia, NI/HG组);高胰岛素、正常血糖组(HighInsulin/Euglycemia, HI/NG组) ;高胰岛素、高血糖组(High Insulin/Hyperglycemia, HI/HG组)。为阻断因血糖波动引起的内源性胰岛素释放,通过结扎犬胰周血管阻断胰岛素分泌入血。建立犬急性心肌缺血再灌注(MI/R)模型:利用电磁流量计和血管狭窄器制造50分钟的局部定量缺血,使左前降支(LAD)血流量降低80%,此后释放狭窄器恢复血流,再灌注4小时。于再灌注前5分钟微量泵静脉输注50%葡萄糖和GIK极化液用于对血糖和血浆胰岛素水平的干预。
2.实时测量血糖,血浆胰岛素、C肽水平、游离脂肪酸含量,以进行血糖和胰岛素水平控制和代谢评价。于缺血前、缺血后50 min及再灌注4 h分别同时从前室间隔静脉和左股动脉取血,立即进行血气分析,检测PO2、PCO2、pH、血红蛋白(Hb)及血样饱和度(SO2),用于计算LAD灌注区心肌组织氧耗率和心肌呼吸指数。
3.通过多道生理记录和分析处理系统持续记录犬心电、冠脉LAD血流量和血流动力学指标。于再灌注结束后用伊文氏蓝/TTC双染色法测定心肌梗死面积。血浆肌钙蛋白T作为心肌损伤标志物,用于细胞坏死程度的评价。采用免疫组织化学方法分别检测Caspase-3活化及TUNEL凋亡指数用于定性和定量检测细胞凋亡。
4.采用石蜡包埋切片的苏木素-伊红染色方法,评价再灌注后心肌坏死周边区白细胞浸润程度,同时测量组织中髓过氧化物酶(MPO)含量以定量评估组织炎症反应水平。检测组织丙二醛(MDA)含量用于评估脂质过氧化水平,测量超氧化物歧化酶(SOD)以评价内源性抗氧化能力。
5.在数据采集和统计过程中采用严格排除标准以保证各实验组纳入的动物样本具有可比性。
实验结果
1.本实验成功建立犬急性心肌缺血再灌注损伤模型。再灌注后心肌出现明显梗死区,细胞凋亡和坏死,及组织炎症反应和氧化损伤。
2.胰腺手术造成的胰腺缺血和局限性炎症在实验观察时程内对全身性炎症反应和心脏功能影响甚微。通过平行实验,在建立心肌缺血再灌注模型前分别实施胰周血管结扎手术和假手术以对比观察。两组间血清胰淀粉酶、胰脂肪酶、心肌肌钙蛋白T(cTnT)和肌酸激酶同工酶(CK-MB)的基础值和术后5小时测量值均无显著性差异,且血流动力学的同步观测显示心脏功能亦无明显变化。
3.高血糖存在情况下心肌I/R损伤程度进一步加重。表现为高血糖组较正常血糖组心肌收缩功能恢复障碍进一步加重,梗死面积扩大,细胞凋亡和坏死增多,炎症和氧化损伤加重(NI/HG vs. NI/NG, HI/HG vs. HI/NG, n=8, P<0.05)。
4.胰岛素干预可显著增加冠脉流量,提高代谢效率,增强心肌收缩功能,且这些有益作用不受血糖高低的影响。高胰岛素组较正常胰岛素组再灌注后收缩功能(心率压力指数、最大收缩速率)明显提高,LAD灌流区血流量增加,心肌组织葡糖糖摄取率明显升高,游离脂肪酸(FFA)摄取率轻微下降,心脏呼吸指数明显上升(HI/NG vs. NI/NG, HI/HG vs. NI/HG, n=8, P<0.05),提示胰岛素诱导心肌氧化代谢底物由FFA向葡萄糖转变。且在心功能明显改善的前提下,心肌氧耗并未显著增高,表明心肌氧利用率的提高。
5.当血糖钳夹在生理水平时,胰岛素水平升高可进一步缩小梗死面积,抑制细胞坏死和凋亡,降低炎症反应和氧化应激(HI/NG vs. NI/NG, n=8, P<0.05)。但此作用需以降糖为前提,高血糖存在时,上述保护作用消失(HI/HG vs. NI/HG, n=8, P>0.05)。
结论
1.高血糖可显著加重心肌损伤并掩盖胰岛素保护作用,因而胰岛素降糖作用在其抗I/R损伤保护缺血心肌的效应中扮演着关键角色。
2.胰岛素具有独立于降糖效应、直接的心血管保护作用,其中包括不依赖于血糖控制水平的直接正性肌力作用、冠脉扩张作用及对心肌氧化代谢效率的促进作用。更为重要的是,胰岛素减轻心肌损伤,抑制细胞凋亡,降低组织炎症反应和氧化应激的有益作用,虽然依赖于正常血糖的维持,但独立于降糖作用而存在。
Part II
胰岛素抗肾上腺素能心肌保护新机制的研究
研究背景
急性心肌缺血发生时普遍存在着心脏交感-肾上腺素能系统的激活,一系列证据提示,交感-肾上腺素能活性增强及其介导的儿茶酚胺分泌释放是加重缺血心肌损伤、诱导致命性心律失常发生的重要原因之一,且与心脏重构、心力衰竭的发生发展密切相关。其中,儿茶酚胺对心脏的直接作用主要由β-肾上腺素受体(adrenergic receptor, AR)所介导,因此β受体阻滞剂被广泛应用于临床AMI的治疗。
越来越多的证据表明,胰岛素除了在维持心肌代谢稳态中发挥重要作用外,还在多种生理、病理过程中发挥关键调控作用。通过前述实验一的研究,我们在大动物MI/R模型上首次证实了胰岛素独立于降糖作用之外直接的心肌保护作用。例如,我们在前期研究中发现,胰岛素可激活“PI3K-Akt-eNOS”细胞生存信号抑制心肌细胞凋亡,保护冠脉内皮,抑制炎性反应和氧化应激,促进再灌注后心脏功能的恢复。此外,我们和国际上多个实验室也发现了胰岛素的直接心脏正性变力作用,表现为在整体动物模型中增强心肌收缩功能,在细胞水平增强收缩幅度和钙瞬变。
心肌细胞膜上同时存在着大量的胰岛素受体和β受体。已有研究报道,在人脂肪细胞和平滑肌细胞中,胰岛素可直接对抗儿茶酚胺的脂解作用;相关分子水平的研究也支持胰岛素对β受体信号调控作用的存在。最近有细胞实验显示,心肌细胞中PI3K信号可抵消cAMP信号系统介导的Ca2+内流和正性变力作用。然而,在心肌细胞中,胰岛素对β受体激动效应可能的调控作用所知甚少,目前仅有的少数文献报道尚存争议。
儿茶酚胺的正性肌力作用主要通过β受体内传,经cAMP的生成激活蛋白激酶A (PKA),进而磷酸化一系列下游分子,参与对兴奋-收缩偶联的调控。其中PKA的靶分子之一肌浆网受磷蛋白(PLB)的磷酸化水平可直接调控SERCA2a活性,而后者是与心肌细胞Ca~(2+)释放、摄取、贮存有关的肌浆网内主要钙转运蛋白之一,也是推动心肌收缩舒张过程的关键蛋白酶。此外,MI/R伴发的一系列细胞内环境稳态的改变,如儿茶酚胺的大量释放,细胞内钙超载,氧化应激等,均可通过改变PLB磷酸化状态及SERCA2活性,加重MI/R心肌收缩功能的损伤。然而,MI/R时βAR激活加重心肌损伤,以及胰岛素对βAR系统的拮抗作用是否与肌浆网钙调控蛋白的功能调节有关,目前尚不明确。
实验目的
本研究旨在探讨急性MI/R过程中,胰岛素是否对βAR系统具有调控作用;在回答第一个问题的基础上,我们将进一步围绕细胞内肌浆网钙调控蛋白的调节,寻求胰岛素调控βAR系统作用的分子机制。
实验方法
1.采用Langendorff离体灌流系统制备大鼠离体心脏缺血再灌注模型。离体灌流心脏随机分为以下5组:⑴sham MI/R组;⑵MI/R组:30min局部缺血/2h再灌注;⑶MI/R+Ins组:再灌注前5min开始给予10-7mol/L胰岛素;⑷MI/R+ISO组:缺血前10min开始给予10~(-9)mol/L异丙肾上腺素(ISO);⑸MI/R+Ins+ISO组:再灌注前5min给予10~(-7) mol/L胰岛素并于再灌注前10min给予10~(-9) mol/L ISO直至再灌注结束。
2.于左心室插入充水球囊,由多道生理记录系统连续采集左室压力指标,实时测量心脏收缩功能。于再灌注结束后用伊文氏蓝/TTC双染色法测定心肌梗死面积。于心肌缺血前15min、缺血30min末、再灌注1h、再灌注2h分别收集冠脉流出液,用分光光度法测定肌酸激酶(CK)、乳酸脱氢酶(LDH)活性。DNA ladder方法和TUNEL原位法分别定性和定量测量心肌细胞凋亡。
3.取再灌注后心肌组织,制备心肌细胞肌浆网膜蛋白以测定Ca~(2+)-ATPase活性。Western Blot检测SERCA2a蛋白表达水平、PLB及磷酸化PLB水平。PepTag非放射性蛋白激酶检测试剂盒测定组织PKA活性。
4.酶解法分离成年大鼠钙耐受性心室肌细胞,并建立化学模拟缺血再灌注(SI/R)模型。随机分组,缺血15 min后按以下溶液进行再灌注30 min:⑴对照组:台式液;⑵胰岛素(10~-8 -10~-5 mol/L)组;⑶ISO(10~(-9) mol/L)组;⑷ISO (10~(-9) mol/L) + Insulin (10(~-8) -10~(-5) mol/L)组。采用IonOptix可视化动缘探测系统同步检测心肌细胞收缩/舒张功能和钙瞬变。与SI/R相对应,正常灌流情况下同期记录上述四种不同药物干预措施引起的细胞收缩和钙瞬变的变化。
实验结果
1.与对照组相比,ISO干预组于缺血前和缺血早期出现短暂的心率(HR)加快、左室收缩压(LVSP)增高;然而再灌注1 h后,HR、LVSP、±LVdP/dtmax开始显著下降,左室舒张末压(LVEDP)明显升高。表明β受体激动加重再灌注后心肌收缩功能的障碍。胰岛素的给予可明显对抗ISO的负向作用,表现为HR、LVSP、±LVdP/dtmax降低程度减轻,LVEDP升高减缓(n=8, P<0.05)。
2.与对照组相比,单独ISO干预使再灌注后梗死范围进一步扩大,冠脉流出液中CK和LDH活性显著增加,DNA梯状条带的染色强度和TUNEL凋亡阳性染色细胞数(n=8, P<0.05)增加。而同时给予胰岛素梗死面积缩小33.1%,降低CK和LDH水平分别为35.1%和22.5% ,同时抑制ISO增加凋亡的作用(n=8, P<0.05)。
3.在正常灌流的成年大鼠心室肌细胞,胰岛素(10~(-7 )mol/L)可显著增加细胞收缩幅度和钙瞬变幅度,ISO (10~(-9) mol/L)则显示出更为明显的正性肌力作用,收缩峰值和钙瞬变幅度的增幅分别达19.1%和85% (n=20, P<0.05)。同时给予10~(-8)-10~(-6) mol/L不同浓度梯度的胰岛素,对ISO的正性肌力反应并不产生显著影响。
4.在模拟I/R的心肌细胞,胰岛素(10~(-7) mol/L)和ISO(10~(-9) mol/L)使细胞收缩峰值各增加17%和81%,钙瞬变增幅15%和109%。然而两者合用时,细胞收缩峰值只增加47%,钙瞬变增幅降至76% (n=20, P<0.05 vs. MI/R+ISO组),提示I/R心肌细胞中,胰岛素可拮抗ISO的正性肌力作用。此外,此拮抗作用也呈现出剂量依赖性特点(10~(-8)-10~(-5) mol/L)。
5.与sham组相比,I/R心肌组织内SERCA2a蛋白表达水平和活性均明显降低,且在ISO处理组进一步下降。单纯胰岛素干预或胰岛素与ISO联用,SERCA2a蛋白表达水平不变,而活性均较相应对照组增高。I/R心肌中PLB磷酸化(PKA磷酸化位点)水平明显降低,胰岛素和ISO可分别升高PLB磷酸化4.2倍和3.1倍,二者联用可进一步增加PLB磷酸化1.7倍。与sham组对比,MI/R本身可诱导2.5倍PKA活性的增高,而ISO可进一步使PKA活性大幅增高约30.7倍(P < 0.01)。胰岛素自身对MI/R心肌PKA活性并无明显影响,然而对ISO诱导的PKA激活产生显著的抑制效应(P < 0.01)。
结论
1.胰岛素可显著抑制MI/R过程中异丙肾上腺素(ISO)诱发β受体激动所带来的心肌损伤,减轻心肌收缩功能障碍、缩小梗死面积,减少细胞坏死和凋亡;
2.胰岛素可浓度依赖性减弱ISO对I/R心肌细胞的正性变力作用;
3.胰岛素对β受体信号系统中的PKA活性及其下游钙转运蛋白的调节,可能是MI/R过程中胰岛素拮抗β受体激活效应的机制之一。上述结果提示胰岛素心血管保护作用的抗肾上腺素能新机制。
Background
Experimental evidence has provided strong supports for the cardioprotective effects of“Glucose-insulin-potassium (GIK)”solution since its introduction as an adjunct to the contemporary management of acute myocardial ischemia (AMI). GIK solution, however, has not been uniformly successful in clinical practice over the last few decades and few trials and experiments to date have been adequately powered to explore the mechanism underlying the mixed results. Although it has been proposed that insulin therapy alone without achieving euglycemia may not improve outcomes in AMI, it remains completely unevidenced whether insulin-titrated euglycemia maintenance is requisite for the cardioprotective effects of insulin. The definite relationship between glycemic control, cardioprotection, and insulin therapy itself requires further elucidation.
Meanwhile, our recent studies as well as others’have proposed direct cell survival signalings favorably modulated by insulin against myocardial ischemia/reperfusion (MI/R) injury. In addition to its anti-apoptotic action, anti-inflammatory, anti-oxidative stress and positive inotropic effects have also been well documented, and are believed to further enhance cardiac performanc after reperfusion. However, latest reports from the research on critically ill patients and animal models suggested that the survival benefits from intensive insulin therapy were due to successful glycemic control, rather than other glycemia-independent effects of insulin. However, cardiac outcome was not addressed, thus it remains unclear whether the well-documented benefits of insulin in the ischemic/reperfused heart should be attributed exclusively to glucose normalization, or combined with other mechanisms inherently originating from insulin per se. The clarification of the existence and contribution of glycemic-control-independent actions of insulin may greatly advance the understanding and application of GIK in AMI management.
Objectives
The present study was designed to investigate, in a clinically relevant large animal model of myocardial ischemia/reperfusion (MI/R), the relationship between glycemia-dependent and -independent effects of insulin and their contributions to insulin’s cardioprotection.
Methods
1. Endogenous insulin production in canines was abolished by peripancreatic vessel ligation. Aninals were then randomly assigned to four groups: Normal plasma insulin/euglycemia (NI/NG), normal insulin/hyperglycemia (NI/HG), high insulin/euglycemia (HI/NG), and high insulin/hyperglycemia (HI/HG), which were achieved by controlled intravenous infusion of glucose/insulin.
2. Anesthetized open-chest dogs were subjected to MI/R (50 min/4 h) by partially occluding the left anterior descending coronary artery (LAD) (80% reduction in its blood flow). Gluose/insulin administration started at 5 min before reperfusion and continued thoughout the experiment.
3. Blood glucose and plasma concentrations of insulin, C-peptide and free fatty acid (FFA) were measured for metabolic control and evaluations. Blood samples from the left femoral artery and the anterior interventricular vein were analyzed for PO2, PCO2, pH, hemoglobin (Hb) concentration and oxygen saturation (SO2) in order to calculate oxygen consumption rate (MVO2) and respiratory quotient (RQ) of the LAD perfused region.
4. Left ventricular (LV) pressure was continuously monitored by a hemodynamic analyzing system. Myocardial infarction was measured using the Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) double staining. Plasma level of cardiac troponin-T (cTnT) was assayed as a biomarker for myocardial ischemic injury. Cell apoptosis was analyzed by immunohistochemical detection of caspase-3 activation and TUNEL assay.
5. Myocardial inflammation was evaluated by histochemical detection of leukocyte infiltration as well as measurement of tissue myeloperoxidase (MPO) activity. Oxidative stress was estimated by measuring the tissue content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD).
6. Strict criteria were established to guarantee that all animals included in the final data analysis were healthy and exposed to comparable degrees of regional myocardial ischemia.
Results
1. Canine acute MI/R model was successfully established. Significant myocardial infarction, cell apoptosis and necrosis, inflammatory reaction and oxidative stress were observed as expected.
2. The ischemic insult due to pancreas surgery resulted in limited pancreatic inflammation and minimal influence on systemic inflammation and cardiac function within 5 hours’protocol. Plasma levels of amylase, lipase, cTnT and creatine kinase-MB, as well as hemodynamic indices at baseline and 5 hours after the pancreas surgery showed no statistical differences between pancreatic-ligated versus non-operated dogs subjected to MI/R.
3. Hyperglycemia alone significantly aggravated MI/R injury, as evidenced by worse functional recovery, larger infarct size, increased cell apoptosis and necrosis, and enhanced inflammation and oxidative injury in HG groups compared to NG groups.
4. Insulin increased conoray perfusion and metabolic efficiency as well as promoted contractile function of the I/R myocardium, which were minimally affected by glucose levels. Compared to NI groups, both HI groups showed higher rate-pressure product and +LVdp/dtmax with increased LAD blood flow during reperfusion. In addition, a robust elevation in anterior LV glucose uptake was also observed following insulin infusion, accompanied by a slight decline in FFA uptake and increase in respiratory quotient, suggesting insulin-mediatd switch of myocardial substrate uptake from fat to carbohydrate. Interestingly, MVO2 of the anterior LV was not elevated in HI groups though contractile function increased, indicating an apparent increase in O2 utilization efficiency.
5. When blood glucose was clamped at physiologic level, insulin elevation (HI/NG vs. NI/NG) further exerted protection against MI/R injury, as evidenced by improved functional recovery, decreased myocardial infarct size, reduced cell necrosis and apoptosis, and alleviated inflammatory and oxidative stress. However, maintaining high glucose concentration in high insulin animals (HI/HG) markedly blunted or abolished the above protective effects of insulin.
Conclusions
1. Insulin-titrated maintenance of euglycemia protects heart from MI/R injury, whereas hyperglycemia oppositely affects MI/R injury and masks the insulin-induced cardiac benefits. Thus insulin-mediated hyperglycemia prevention is of critical importance to ensure insulin’s full efficacy in protecting heart against MI/R.
2. Insulin has direct cardioprotective effects independent of its glucose-lowering ability. Thus the cardiac benefits of insulin against I/R injury should not be attributed exclusively to glucose normalization.
Part II
Background
Myocardial ischemia involves a large and progressive release of catecholamines from adrenergic nerve terminals, and overshooting of myocardialβ- adrenergic receptors (?-AR) by catecholamines may further aggravate MI/R injury, induce lethal arrhythmias, and contribute to post-infarction remodeling and heart failure.
Increasing evidence showed that insulin, apart from its role in the metabolic regulation, participates in a variety of physiological and pathological cardiovascular process. In the above Part I study, we have demonstrated that insulin has direct cardioprotective effects independent of its glucose-lowering ability. For example, through the activation of a cell survival signaling“PI3K-Akt-eNOS”, insulin exerts protection for the I/R heart by inhibiting cell apoptosis, preserving endothelial function, and suppressing myocardial inflammation and oxidative stress. Moreover, a direct positive inotropic effect of insulin has been reported as evidenced by increased contractile function in in vivo models and elevated twitch amplitude and calcium transient in single ventricular myocyetes.
Previous investigations have revealed a counter-regulatory effect of insulin onβ-adrenergic catecholamine action in isolated human fat cells and cultured smooth muscle cells. Recent studies also provided important evidence for the existence of interaction between myocardial insulin signaling and ?-AR signaling. However, existing data concerning the functional effects of insulin on the myocardial response to ?-AR activation are quite limited and inconsistent. Moreover, the major cardiac effects of catecholamines are mediated by ?-AR through the activation of cAMP-dependent protein kinase A (PKA), which in turn phosphorylates a series of target moleculars responsible for the regulation of excitation-contraction coupling. Phospholamban is one of the PKA target proteins, and at the same time a major regulator of SERCA2, which modulates the rate of SR Ca~(2+) uptake, leads to the regulation of relaxation velocity, SR Ca~(2+) load and myocardial contractility. Several events associated with MI/R, such as catecholamines release, intracellular Ca~(2+) overload and oxidative stress, participate in the modulation of PLB phosphorylation and SERCA2 activity. However, it remains unclear howβ-AR stimulation influences these SR Ca~(2+) handling proteins in the I/R heart and whether these influences could be modified by the application of insulin.
Objectives
This study aimed to investigate the ability of insulin to modulateβ-adrenergic actions on post-ischemic injury and myocardial contractility in acute MI/R and the underlying mechanism.
Methods
1. Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h). Hearts (n=16-20 per group) were randomized to receive one of the following treatments: (1) sham MI/R; (2) MI/R vehicle; (3) MI/R receiving insulin (10-7 mol/L) 5 min before and throughout reperfusion; (4) MI/R receiving ISO (10~(-9) mol/L) 10 min before ischemia and throughout reperfusion; (5) MI/R receiving insulin 5 min before reperfusion plus ISO (10~(-9) mol/L) 10 min before ischemia, both throughout reperfusion.
2. A water-filled latex balloon was inserted into the left ventricle; cardiac function was continuously recorded by a hemodynamic analyzing system via a pressure transducer connected to the balloon. Myocardial infarction was measured using the Evans blue/TTC double staining. Coronary effluent was collected and creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically. Cardiomyocyte apoptosis was analyzed by detection of DNA fragmentation (DNA ladders) and TUNEL in situ assay.
3. Ca~(2+)-ATPase activity of the isolated SR membrane was measured in the reperfused hearts. Expressions of SERCA2a, PLB and phosphorylated PLB in the SR were determined by Western blot. Tissue homogenates were prepared for non-radioactive determination of PKA activity using PepTag Assay.
4. Calcium-tolerant ventricular myocytes were isolated from adult male rats by a standard enzymatic technique. Isolated myocytes were exposed to simulated ischemia for 15 min using chemical anoxia solution, and then reperfused with one of the following agents for another 30 min: (1) vehicle; (2) insulin (10~(-8) -10~(-5) mol/L); (3) isoproterenol (ISO, 10~(-9) mol/L); (4) ISO (10~(-9) mol/L) +insulin (10~(-8)-10~(-5) mol/L). The mechanical contraction and Ca2+ transients of ventricular myocytes were assessed by a video-based motion edge-detection system. Responses to the above agents were also observed in normal myocytes.
Results
1. ISO administration produced a transient increase in HR and LVSP, followed by a sharp decline after 1 h reperfusion and end up with a greatly dampened HR and LVDP and elevated LVEDP compared with the untreated I/R hearts, suggesting thatβ-AR activation during MI/R aggravates post-ischemic cardiac dysfunction. More importantly, insulin largely reversed the ISO-induced functional impairment, inhibiting ISO-induced declines in HR and LVSP by 34.0% and 23.0% respectively, and preventing ISO-induced elevation in LVEDP by 28.7% (n=8, all P<0.05).
2. ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were all inhibited by treatment of insulin (n=8, all P<0.05).
3. In normal ventricular myocytes, insulin (10~(-7) mol/L) exerted significant inotropic effect as evidenced by increased peak twitch amplitude (PTA) and calcium transient amplitude (ΔFFI). ISO with a submaximal concentration of 10~(-9) mol/L, showed an even greater inotropic effect with a 19.1% increase of PTA and 85% increase ofΔFFI. Exposure to insulin (10~(-8)-10~(-6) mol/L), either before or after the administration of ISO, displayed no significant differences with ISO alone.
4. In simulated I/R cardiomyocytes, insulin (10~(-7)mol/L) and ISO(10~(-9) mol/L)both mediated distinct positive inotropic responses, with 17% and 81% increases of PTA along with 15% and 109% increases ofΔFFI, respectively. When applied together, however, insulin attenuated the inotropic response to ISO as evidenced by an 18.7% reduction in PTA and a 23.9% reduction inΔFFI (n=20 myocytes from 8 hearts, both P<0.05). This inhibitory effect also acts in a concentration-dependent manner (10~(-8)-10~(-5) mol/L).
5. Both protein expression and activity of SERCA2a in untreated I/R hearts were significantly reduced compared with sham hearts, and were further depressed when ISO was applied. Reperfusion with insulin produced a 13.9% increase in SERCA2a activity compared with vehicle, and a 15.2% increase compared with ISO alone (both P<0.05). Phosphorylation of PLB at Ser16 was significantly decresed in I/R hearts. Insulin and ISO triggered a 4.2- and 3.1-fold increase in PLB phosphorylation, and insulin further facilitated ISO-induced PLB phosphorylation, a 1.7-fold increase relative to ISO alone (P<0.01). As for PKA activity, MI/R resulted in 2.5-fold increase in PKA activation compared with sham group. Application of ISO produced an overt elevation in PKA activity as evidenced by a 30.7-fold increase (P<0.01 vs. MI/R group). Interestingly, insulin itself exerted no significant influence on PKA activity, but markedly inhibited ISO-mediated PKA activation in I/R hearts (P<0.01).
1. Insulin significantly suppresses ISO-elicited cardiac injury including myocardial contractile dysfunction, cell apoptosis and necrosis in isolated I/R hearts, which may contribute to insulin-induced cardioprotection in MI/R.
2. Insulin attenuates ISO-induced positive inotropic effect on simulated I/R cardiomyocytes in a concentration-dependent manner.
3. The regulation of ?-adrenergic postreceptorial signaling such as PKA and its downstream calcium handling proteins is a likely mechanism that accounted for the insulin-induced counterbalance of -adrenergic activation in MI/R. Conclusions
引文
1. Sodi-Pallares D, Testelli MR, Fishleder BL, Bisteni A, Medrano GA, Friedland C, De Micheli A: Effects of an intravenous infusion of a potassium-glucose-insulin solution on the electrocardiographic signs of myocardial infarction. A preliminary clinical report. Am J Cardiol 1962;9:166-181.
2. Das UN: Is insulin an endogenous cardioprotector? Crit Care 2002;6:389-393.
3. 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.
4. Opie LH: The glucose hypothesis: relation to acute myocardial ischaemia. J Mol Cell Cardiol 1970;1:107-114.
5. Cooney GJ, Denyer GS, Jenkins AB, Storlien LH, Kraegen EW, Caterson ID: In vivo insulin sensitivity of the pyruvate dehydrogenase complex in tissues of the rat. Am J Physiol 1993;265:E102-107.
6. Czech MP, Klarlund JK, Yagaloff KA, Bradford AP, Lewis RE: Insulin receptor signaling. Activation of multiple serine kinases. J Biol Chem 1988;263:11017-11020.
7. Sabia PJ, Powers ER, Ragosta M, Sarembock IJ, Burwell LR, Kaul S: An association between collateral blood flow and myocardial viability in patients with recent myocardial infarction. N Engl J Med 1992;327:1825-1831.
8. Taegtmeyer H: Energy metabolism of the heart: from basic concepts to clinical applications. Curr Probl Cardiol 1994;19:59-113.
9. Tune JD, Mallet RT, Downey HF: Insulin improves cardiac contractile function and oxygen utilization efficiency during moderate ischemia without compromising myocardial energetics. J Mol Cell Cardiol 1998;30:2025-2035.
10. Schipke JD: Cardiac efficiency. Basic Res Cardiol 1994;89:207-240.
11. Jonassen AK, Sack MN, Mjos OD, Yellon DM: Myocardial protection byinsulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191-1198.
12. Gao 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 phosphorylation. Circulation 2002;105:1497-1502.
13. Sack 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;41:1404-1407.
14. Yu Q, Gao F, Ma XL: Insulin says NO to cardiovascular disease. Cardiovasc Res 2011;89:516-524.
15. Zhang 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;9:777-783.
16. Ma 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;69:57-65.
17. Li J, Zhang H, Wu F, Nan Y, Ma H, Guo W, Wang H, Ren J, Das UN, Gao F: Insulin inhibits tumor necrosis factor-alpha induction in myocardial ischemia/reperfusion: role of Akt and endothelial nitric oxide synthase phosphorylation. Crit Care Med 2008;36:1551-1558.
18. Li J, Wu F, Zhang H, Fu F, Ji L, Dong L, Li Q, Liu W, Zhang Y, Lv A, Wang H, Ren J, Gao F: Insulin inhibits leukocyte-endothelium adherence via an Akt-NO-dependent mechanism in myocardial ischemia/reperfusion. J Mol Cell Cardiol 2009;47:512-519.
19. Diaz R, Paolasso EA, Piegas LS, Tajer CD, Moreno MG, Corvalan R, Isea JE, Romero G: Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) Collaborative Group. Circulation 1998;98:2227-2234.
20. Ceremuzynski L, Budaj A, Czepiel A, Burzykowski T, Achremczyk P,Smielak-Korombel W, Maciejewicz J, Dziubinska J, Nartowicz E, Kawka-Urbanek T, Piotrowski W, Hanzlik J, Cieslinski A, Kawecka-Jaszcz K, Gessek J, Wrabec K: Low-dose glucose-insulin-potassium is ineffective in acute myocardial infarction: results of a randomized multicenter Pol-GIK trial. Cardiovasc Drugs Ther 1999;13:191-200.
21. Stanley AW, Jr., Moraski RE, Russell RO, Rogers WJ, Mantle JA, Kreisberg RA, McDaniel HG, Rackley CE: Effects of glucose-insulin-potassium on myocardial substrate availability and utilization in stable coronary artery disease. Studies on myocardial carbohydrate, lipid and oxygen arterial-coronary sinus differences in patients with coronary artery disease. Am J Cardiol 1975;36:929-937.
22. Malmberg K, Ryden L, Efendic S, Herlitz J, Nicol P, Waldenstrom A, Wedel H, Welin L: Randomized trial of insulin-glucose infusion followed by subcutaneous insulin treatment in diabetic patients with acute myocardial infarction (DIGAMI study): effects on mortality at 1 year. J Am Coll Cardiol. 1995:57-65.
23. Mellbin LG, Malmberg K, Norhammar A, Wedel H, Ryden L: The impact of glucose lowering treatment on long-term prognosis in patients with type 2 diabetes and myocardial infarction: a report from the DIGAMI 2 trial. Eur Heart J 2008;29:166-176.
24. Mehta SR, Yusuf S, Diaz R, Zhu J, Pais P, Xavier D, Paolasso E, Ahmed R, Xie C, Kazmi K, Tai J, Orlandini A, Pogue J, Liu L: Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial. JAMA 2005;293:437-446.
25. Lell WA, Nielsen VG, McGiffin DC, Schmidt FE, Jr., Kirklin JK, Stanley AW, Jr.: Glucose-insulin-potassium infusion for myocardial protection during off-pump coronary artery surgery. Ann Thorac Surg 2002;73:1246-1251; discussion 1251-1242.
26. Deedwania P, Kosiborod M, Barrett E, Ceriello A, Isley W, Mazzone T,Raskin P: Hyperglycemia and acute coronary syndrome: a scientific statement from the American Heart Association Diabetes Committee of the Council on Nutrition, Physical Activity, and Metabolism. Circulation. 2008:1610-1619.
27. Kosuge M, Kimura K, Ishikawa T, Shimizi T, Hibi K, Toda N, Tahara Y, Kanna M, Tsukahara K, Okuda J, Nozawa N, Umemura S: Persistent hyperglycemia is associated with left ventricular dysfunction in patients with acute myocardial infarction. Circ J 2005;69:23-28.
28. Chaudhuri A, Janicke D, Wilson MF, Tripathy D, Garg R, Bandyopadhyay A, Calieri J, Hoffmeyer D, Syed T, Ghanim H, Aljada A, Dandona P: Anti-Inflammatory and Profibrinolytic Effect of Insulin in Acute ST-Segment-Elevation Myocardial Infarction. In, 2004:849-854.
29. Vincent AM, McLean LL, Backus C, Feldman EL: Short-term hyperglycemia produces oxidative damage and apoptosis in neurons. Faseb J 2005;19:638-640.
30. Creagh-Brown BC, Quinlan GJ, Evans TW, Burke-Gaffney A: The RAGE axis in systemic inflammation, acute lung injury and myocardial dysfunction: an important therapeutic target? Intensive Care Med 2010;36:1644-1656.
31. Van den Berghe G, Wouters PJ, Bouillon R, Weekers F, Verwaest C, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P: Outcome benefit of intensive insulin therapy in the critically ill: Insulin dose versus glycemic control. Crit Care Med 2003;31:359-366.
32. Ellger B, Debaveye Y, Vanhorebeek I, Langouche L, Giulietti A, Van Etten E, Herijgers P, Mathieu C, Van den Berghe G: Survival benefits of intensive insulin therapy in critical illness: Impact of maintaining normoglycemia versus glycemia-independent actions of insulin. In Diabetes. 2006:1096-1105.
33. Kamishima T, Quayle JM: Ca2+-induced Ca2+ release in cardiac and smooth muscle cells. Biochem Soc Trans 2003;31:943-946.
34. Janczewski AM, Spurgeon HA, Stern MD, Lakatta EG: Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells. Am J Physiol 1995;268:H916-920.
35. Shen AC, Jennings RB: Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 1972;67:441-452.
36. Diehl AM: Cytokine regulation of liver injury and repair. Immunol Rev 2000;174:160-171.
37. Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ: The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction. Mol. Cell. Biol. 2004;24:1844-1854.
38. Zarain-Herzberg A, MacLennan DH, Periasamy M: Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene. J Biol Chem 1990;265:4670-4677.
39. Bers DM, Bassani JW, Bassani RA: Na-Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle. Ann N Y Acad Sci 1996;779:430-442.
40. Mattiazzi A, Mundina-Weilenmann C, Guoxiang C, Vittone L, Kranias E: Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions. Cardiovascular Research 2005;68:366-375.
41. Phillips RM, Narayan P, Gomez AM, Dilly K, Jones LR, Lederer WJ, Altschuld RA: Sarcoplasmic reticulum in heart failure: central player or bystander? Cardiovascular Research 1998;37:346-351.
42. Seehase M, Quentin T, Wiludda E, Hellige G, Paul T, Schiffmann H: Gene expression of the Na-Ca2+ exchanger, SERCA2a and calsequestrin after myocardial ischemia in the neonatal rabbit heart. Biol Neonate 2006;90:174-84.
43. Su H, Sun X, Ma H, Zhang H-F, Yu Q-J, Huang C, Wang X-M, Luan R-H, Jia G-L, Wang H-C, Gao F: Acute hyperglycemia exacerbates myocardial ischemia/reperfusion injury and blunts cardioprotective effect of GIK. In, 2007:E629-635.
44. Mundina-Weilenmann C, Said M, Vittone L, Ferrero P, Mattiazzi A: Phospholamban phosphorylation in ischemia-reperfused heart. Effect ofpacing during ischemia and response to a beta-adrenergic challenge. Mol Cell Biochem 2003;252:239-246.
45. Netticadan T, Temsah R, Osada M, Dhalla NS: Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion. Am J Physiol 1999;277:C384-391.
46. Sande JB, Sjaastad I, Hoen IB, Bokenes J, Tonnessen T, Holt E, Lunde PK, Christensen G: Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity. Cardiovascular Research 2002;53:382-391.
47. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P: Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 1999;5:1135-1142.
48. Temsah RM, Dyck C, Netticadan T, Chapman D, Elimban V, Dhalla NS: Effect of beta-adrenoceptor blockers on sarcoplasmic reticular function and gene expression in the ischemic-reperfused heart. J Pharmacol Exp Ther 2000;293:15-23.
49. De Leiris J, Opie LH, Lubbe WF: Effects of free fatty acid and enzyme release in experimental glucose on myocardial infarction. Nature 1975;253:746-747.
50. Komatsu E, Fukuyama H, Misawa T, Kaminishi T, Yamaguchi I, Miyazawa K: Ventricular wall motion and NE release in post-ischemic reperfused myocardium. Jpn Circ J 1991;55:900-903.
51. Lameris TW, de Zeeuw S, Alberts G, Boomsma F, Duncker DJ, Verdouw PD, Veld AJ, van Den Meiracker AH: Time course and mechanism of myocardial catecholamine release during transient ischemia in vivo. Circulation 2000;101:2645-2650.
52. Brodde O-E, Michel MC: Adrenergic and Muscarinic Receptors in the HumanHeart. In, 1999:651-690.
53. Engelhardt S, Hein L, Wiesmann F, Lohse MJ: Progressive hypertrophy and heart failure in beta 1-adrenergic receptor transgenic mice. In, 1999:7059-7064.
54. Vetter U, Kupferschmid C, Lang D, Pentz S: Insulin-like growth factors and insulin increase the contractility of neonatal rat cardiocytes in vitro. Basic Res Cardiol 1988;83:647-654.
55. Ren J, Walsh MF, Hamaty M, Sowers JR, Brown RA: Augmentation of the inotropic response to insulin in diabetic rat hearts. Life Sci 1999;65:369-380.
56. Aulbach F, Simm A, Maier S, Langenfeld H, Walter U, Kersting U, Kirstein M: Insulin stimulates the L-type Ca2+ current in rat cardiac myocytes. Cardiovasc Res 1999;42:113-120.
57. Heyliger CE, Pierce GN, Singal PK, Beamish RE, Dhalla NS: Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy. Basic Res Cardiol 1982;77:610-618.
58. Vadlamudi RV, McNeill JH: Effect of experimental diabetes on isolated rat heart responsiveness to isoproterenol. Can J Physiol Pharmacol 1984;62:124-131.
59. Nishio Y, Kashiwagi A, Kida Y, Kodama M, Abe N, Saeki Y, Shigeta Y: Deficiency of cardiac beta-adrenergic receptor in streptozocin-induced diabetic rats. Diabetes 1988;37:1181-1187.
60. Wichelhaus A, Russ M, Petersen S, Eckel J: G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats. Am J Physiol 1994;267:H548-555.
61. Baltensperger K, Karoor V, Paul H, Ruoho A, Czech MP, Malbon CC: The beta-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase. In, 1996:1061-1064.
62. Shumay E, Song X, Wang HY, Malbon CC: pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation. Mol Biol Cell 2002;13:3943-3954.
63. Karoor V, Wang L, Wang H-y, Malbon CC: Insulin stimulates sequestration of beta -adrenergic receptors and enhanced association of beta -adrenergic receptors with Grb2 via Tyrosine 350. J. Biol. Chem. 1998;273:33035-33041.
64. Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM: Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating beta-arrestin-1. Mol Cell Biol 2002;22:6272-6285.
65. Gavi S, Yin D, Shumay E, Wang H-y, Malbon CC: The 15-Amino Acid Motif of the C Terminus of the {beta}2-Adrenergic Receptor Is Sufficient to Confer Insulin-Stimulated Counterregulation to the {beta}1-Adrenergic Receptor. Endocrinology 2005;146:450-457.
66. Leblais V, Jo S-H, Chakir K, Maltsev V, Zheng M, Crow MT, Wang W, Lakatta EG, Xiao R-P: Phosphatidylinositol 3-Kinase Offsets cAMP-Mediated Positive Inotropic Effect via Inhibiting Ca2+ Influx in Cardiomyocytes. In, 2004:1183-1190.
67. Shumay E, Gavi S, Wang H-y, Malbon CC: Trafficking of {beta}2-adrenergic receptors: insulin and {beta}-agonists regulate internalization by distinct cytoskeletal pathways. J. Cell Sci. 2004;117:593-600.
68. Mulder AH, Tack CJ, Olthaar AJ, Smits P, Sweep FC, Bosch RR: Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation. Am J Physiol Endocrinol Metab 2005;289:E627-633.
69. Morisco C, Marrone C, Trimarco V, Crispo S, Monti MG, Sadoshima J, Trimarco B: Insulin resistance affects the cytoprotective effect of insulin in cardiomyocytes through an impairment of MAPK phosphatase-1 expression. Cardiovasc Res 2007;76:453-464.
70. Egert S, Nguyen N, Schwaiger M: Contribution of alpha-adrenergic and beta-adrenergic stimulation to ischemia-induced glucose transporter (GLUT) 4 and GLUT1 translocation in the isolated perfused rat heart. Circ Res 1999;84:1407-1415.
71. Wang H-y, Doronin S, Malbon CC: Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta -adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor. J. Biol. Chem. 2000;275:36086-36093.
72. Zhang HX, Zang YM, Huo JH, Liang SJ, Zhang HF, Wang YM, Fan Q, Guo WY, Wang HC, Gao F: Physiologically tolerable insulin reduces myocardial injury and improves cardiac functional recovery in myocardial ischemic/reperfused dogs. J Cardiovasc Pharmacol 2006;48:306-313.
73. Das UN: Glucose, insulin, and coronary heart disease. In, 2008:1075-a-1076.
74. Dandona P, Chaudhuri A, Ghanim H, Mohanty P: Insulin as an anti-inflammatory and antiatherogenic modulator. J Am Coll Cardiol 2009;53:S14-20.
75. Grund F, Sommerschild HT, Kirkeboen KA, Ilebekk A: Preconditioning with ischaemia reduces both myocardial oxygen consumption and infarct size in a graded pattern. J Mol Cell Cardiol 1997;29:3067-3079.
76. Recchia FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, Hintze TH: Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 1998;83:969-979.
77. Goyal A, Mehta SR, Diaz R, Gerstein HC, Afzal R, Xavier D, Liu L, Pais P, Yusuf S: Differential clinical outcomes associated with hypoglycemia and hyperglycemia in acute myocardial infarction. Circulation 2009;120:2429-2437.
78. Webster KA: Stress hyperglycemia and enhanced sensitivity to myocardial infarction. Curr Hypertens Rep 2008;10:78-84.
79. Smit JW, Romijn JA: Acute insulin resistance in myocardial ischemia: causes and consequences. Semin Cardiothorac Vasc Anesth 2006;10:215-219.
80. Opie LH: Metabolic management of acute myocardial infarction comes to the fore and extends beyond control of hyperglycemia. Circulation 2008;117:2172-2177.
81. Zarich SW, Nesto RW: Implications and treatment of acute hyperglycemia in the setting of acute myocardial infarction. Circulation 2007;115:e436-439.
82. Lazar HL MM, Chipkin S: Insulin is the protective component of glucose-insulin-potassium solutionsduring coronary artery bypass graft surgery in patients with diabetes. Circulation 2008; 118:S705.
83. Muniyappa R, Montagnani M, Koh KK, Quon MJ: Cardiovascular actions of insulin. Endocr Rev 2007;28:463-491.
84. Howell NJ, Ashrafian H, Drury NE, Ranasinghe AM, Contractor H, Isackson H, Calvert M, Williams LK, Freemantle N, Quinn DW, Green D, Frenneaux M, Bonser RS, Mascaro JG, Graham TR, Rooney SJ, Wilson IC, Pagano D: Glucose-insulin-potassium reduces the incidence of low cardiac output episodes after aortic valve replacement for aortic stenosis in patients with left ventricular hypertrophy: results from the Hypertrophy, Insulin, Glucose, and Electrolytes (HINGE) trial. Circulation;123:170-177.
85. Vlasselaers D: Glucose-insulin-potassium: much more than enriched myocardial fuel. Circulation;123:129-130.
86. Ellger B, Langouche L, Richir M, Debaveye Y, Vanhorebeek I, Teerlink T, Van Leeuwen PA, Van den Berghe G: Modulation of regional nitric oxide metabolism: blood glucose control or insulin? Intensive Care Med 2008;34:1525-1533.
87. van den Heuvel I, Vanhorebeek I, Van den Berghe G: The importance of strict blood glucose control with insulin therapy in the intensive care unit. Curr Diabetes Rev 2008;4:227-233.
88. Vasilyev N, Williams T, Brennan M-L, Unzek S, Zhou X, Heinecke JW, Spitz DR, Topol EJ, Hazen SL, Penn MS: Myeloperoxidase-Generated Oxidants Modulate Left Ventricular Remodeling but Not Infarct Size After Myocardial Infarction. In, 2005:2812-2820.
89. Ling PR, Smith RJ, Bistrian BR: Acute effects of hyperglycemia and hyperinsulinemia on hepatic oxidative stress and the systemic inflammatory response in rats. Crit Care Med 2007;35:555-560.
90. Esumi K, Nishida M, Shaw D, Smith TW, Marsh JD: NADH measurements in adult rat myocytes during simulated ischemia. Am J Physiol 1991;260:H1743-1752.
91. Lindemann JP, Jones LR, Hathaway DR, Henry BG, Watanabe AM: beta-Adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles. J Biol Chem 1983;258:464-471.
92. Hopkins TA, Dyck JR, Lopaschuk GD: AMP-activated protein kinase regulation of fatty acid oxidation in the ischaemic heart. Biochem Soc Trans 2003;31:207-212.
93. Stanley WC, Lopaschuk GD, Hall JL, McCormack JG: Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Cardiovascular Research 1997;33:243-257.
94. Remondino A, Kwon SH, Communal C, Pimentel DR, Sawyer DB, Singh K, Colucci WS: Beta-adrenergic receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of the mitochondrial pathway. Circ Res 2003;92:136-138.
95. Engfeldt P, Hellmer J, Wahrenberg H, Arner P: Effects of insulin on adrenoceptor binding and the rate of catecholamine-induced lipolysis in isolated human fat cells. J Biol Chem 1988;263:15553-15560.
96. Karoor V, Baltensperger K, Paul H, Czech MP, Malbon CC: Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin. J Biol Chem 1995;270:25305-25308.
97. Austin C, Chess-Williams R: The in-vitro effects of insulin and the effects of acute fasting on cardiac beta-adrenoceptor responses in the short-term streptozotocin-diabetic rat. J Pharm Pharmacol 1994;46:326-331.
98. Ferrara N, Abete P, Corbi G, Paolisso G, Longobardi G, Calabrese C, Cacciatore F, Scarpa D, Iaccarino G, Trimarco B, Leosco D, Rengo F: Insulin-induced changes in beta-adrenergic response: an experimental study in the isolated rat papillary muscle. Am J Hypertens 2005;18:348-353.
99. Lohse MJ: Molecular mechanisms of membrane receptor desensitization.Biochim Biophys Acta 1993;1179:171-188.
100. Strasser RH, Marquetant R, Kubler W: Independent sensitization of beta-adrenoceptors and adenylate cyclase in acute myocardial ischaemia. Br J Clin Pharmacol 1990;30 Suppl 1:27S-35S.
101. Sichelschmidt OJ, Hahnefeld C, Hohlfeld T, Herberg FW, Schror K: Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity. Cardiovasc Res 2003;58:602-610.
102. Gavi S, Yin D, Shumay E, Wang HY, Malbon CC: Insulin-like growth factor-I provokes functional antagonism and internalization of beta1-adrenergic receptors. Endocrinology 2007;148:2653-2662.
103. Krause SM, Jacobus WE, Becker LC: Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic "stunned" myocardium. Circ Res 1989;65:526-530.
104. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD: PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med 2004;10:248-254.
105. Vittone L, Mundina-Weilenmann C, Said M, Mattiazzi A: Mechanisms involved in the acidosis enhancement of the isoproterenol-induced phosphorylation of phospholamban in the intact heart. J Biol Chem 1998;273:9804-9811.
2. Das UN: Is insulin an endogenous cardioprotector? Crit Care 2002;6:389-393.
3. 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.
4. Opie LH: The glucose hypothesis: relation to acute myocardial ischaemia. J Mol Cell Cardiol 1970;1:107-114.
5. Cooney GJ, Denyer GS, Jenkins AB, Storlien LH, Kraegen EW, Caterson ID: In vivo insulin sensitivity of the pyruvate dehydrogenase complex in tissues of the rat. Am J Physiol 1993;265:E102-107.
6. Czech MP, Klarlund JK, Yagaloff KA, Bradford AP, Lewis RE: Insulin receptor signaling. Activation of multiple serine kinases. J Biol Chem 1988;263:11017-11020.
7. Sabia PJ, Powers ER, Ragosta M, Sarembock IJ, Burwell LR, Kaul S: An association between collateral blood flow and myocardial viability in patients with recent myocardial infarction. N Engl J Med 1992;327:1825-1831.
8. Taegtmeyer H: Energy metabolism of the heart: from basic concepts to clinical applications. Curr Probl Cardiol 1994;19:59-113.
9. Tune JD, Mallet RT, Downey HF: Insulin improves cardiac contractile function and oxygen utilization efficiency during moderate ischemia without compromising myocardial energetics. J Mol Cell Cardiol 1998;30:2025-2035.
10. Schipke JD: Cardiac efficiency. Basic Res Cardiol 1994;89:207-240.
11. Jonassen AK, Sack MN, Mjos OD, Yellon DM: Myocardial protection byinsulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191-1198.
12. Gao 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 phosphorylation. Circulation 2002;105:1497-1502.
13. Sack 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;41:1404-1407.
14. Yu Q, Gao F, Ma XL: Insulin says NO to cardiovascular disease. Cardiovasc Res 2011;89:516-524.
15. Zhang 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;9:777-783.
16. Ma 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;69:57-65.
17. Li J, Zhang H, Wu F, Nan Y, Ma H, Guo W, Wang H, Ren J, Das UN, Gao F: Insulin inhibits tumor necrosis factor-alpha induction in myocardial ischemia/reperfusion: role of Akt and endothelial nitric oxide synthase phosphorylation. Crit Care Med 2008;36:1551-1558.
18. Li J, Wu F, Zhang H, Fu F, Ji L, Dong L, Li Q, Liu W, Zhang Y, Lv A, Wang H, Ren J, Gao F: Insulin inhibits leukocyte-endothelium adherence via an Akt-NO-dependent mechanism in myocardial ischemia/reperfusion. J Mol Cell Cardiol 2009;47:512-519.
19. Diaz R, Paolasso EA, Piegas LS, Tajer CD, Moreno MG, Corvalan R, Isea JE, Romero G: Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiologicos Latinoamerica) Collaborative Group. Circulation 1998;98:2227-2234.
20. Ceremuzynski L, Budaj A, Czepiel A, Burzykowski T, Achremczyk P,Smielak-Korombel W, Maciejewicz J, Dziubinska J, Nartowicz E, Kawka-Urbanek T, Piotrowski W, Hanzlik J, Cieslinski A, Kawecka-Jaszcz K, Gessek J, Wrabec K: Low-dose glucose-insulin-potassium is ineffective in acute myocardial infarction: results of a randomized multicenter Pol-GIK trial. Cardiovasc Drugs Ther 1999;13:191-200.
21. Stanley AW, Jr., Moraski RE, Russell RO, Rogers WJ, Mantle JA, Kreisberg RA, McDaniel HG, Rackley CE: Effects of glucose-insulin-potassium on myocardial substrate availability and utilization in stable coronary artery disease. Studies on myocardial carbohydrate, lipid and oxygen arterial-coronary sinus differences in patients with coronary artery disease. Am J Cardiol 1975;36:929-937.
22. Malmberg K, Ryden L, Efendic S, Herlitz J, Nicol P, Waldenstrom A, Wedel H, Welin L: Randomized trial of insulin-glucose infusion followed by subcutaneous insulin treatment in diabetic patients with acute myocardial infarction (DIGAMI study): effects on mortality at 1 year. J Am Coll Cardiol. 1995:57-65.
23. Mellbin LG, Malmberg K, Norhammar A, Wedel H, Ryden L: The impact of glucose lowering treatment on long-term prognosis in patients with type 2 diabetes and myocardial infarction: a report from the DIGAMI 2 trial. Eur Heart J 2008;29:166-176.
24. Mehta SR, Yusuf S, Diaz R, Zhu J, Pais P, Xavier D, Paolasso E, Ahmed R, Xie C, Kazmi K, Tai J, Orlandini A, Pogue J, Liu L: Effect of glucose-insulin-potassium infusion on mortality in patients with acute ST-segment elevation myocardial infarction: the CREATE-ECLA randomized controlled trial. JAMA 2005;293:437-446.
25. Lell WA, Nielsen VG, McGiffin DC, Schmidt FE, Jr., Kirklin JK, Stanley AW, Jr.: Glucose-insulin-potassium infusion for myocardial protection during off-pump coronary artery surgery. Ann Thorac Surg 2002;73:1246-1251; discussion 1251-1242.
26. Deedwania P, Kosiborod M, Barrett E, Ceriello A, Isley W, Mazzone T,Raskin P: Hyperglycemia and acute coronary syndrome: a scientific statement from the American Heart Association Diabetes Committee of the Council on Nutrition, Physical Activity, and Metabolism. Circulation. 2008:1610-1619.
27. Kosuge M, Kimura K, Ishikawa T, Shimizi T, Hibi K, Toda N, Tahara Y, Kanna M, Tsukahara K, Okuda J, Nozawa N, Umemura S: Persistent hyperglycemia is associated with left ventricular dysfunction in patients with acute myocardial infarction. Circ J 2005;69:23-28.
28. Chaudhuri A, Janicke D, Wilson MF, Tripathy D, Garg R, Bandyopadhyay A, Calieri J, Hoffmeyer D, Syed T, Ghanim H, Aljada A, Dandona P: Anti-Inflammatory and Profibrinolytic Effect of Insulin in Acute ST-Segment-Elevation Myocardial Infarction. In, 2004:849-854.
29. Vincent AM, McLean LL, Backus C, Feldman EL: Short-term hyperglycemia produces oxidative damage and apoptosis in neurons. Faseb J 2005;19:638-640.
30. Creagh-Brown BC, Quinlan GJ, Evans TW, Burke-Gaffney A: The RAGE axis in systemic inflammation, acute lung injury and myocardial dysfunction: an important therapeutic target? Intensive Care Med 2010;36:1644-1656.
31. Van den Berghe G, Wouters PJ, Bouillon R, Weekers F, Verwaest C, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P: Outcome benefit of intensive insulin therapy in the critically ill: Insulin dose versus glycemic control. Crit Care Med 2003;31:359-366.
32. Ellger B, Debaveye Y, Vanhorebeek I, Langouche L, Giulietti A, Van Etten E, Herijgers P, Mathieu C, Van den Berghe G: Survival benefits of intensive insulin therapy in critical illness: Impact of maintaining normoglycemia versus glycemia-independent actions of insulin. In Diabetes. 2006:1096-1105.
33. Kamishima T, Quayle JM: Ca2+-induced Ca2+ release in cardiac and smooth muscle cells. Biochem Soc Trans 2003;31:943-946.
34. Janczewski AM, Spurgeon HA, Stern MD, Lakatta EG: Effects of sarcoplasmic reticulum Ca2+ load on the gain function of Ca2+ release by Ca2+ current in cardiac cells. Am J Physiol 1995;268:H916-920.
35. Shen AC, Jennings RB: Kinetics of calcium accumulation in acute myocardial ischemic injury. Am J Pathol 1972;67:441-452.
36. Diehl AM: Cytokine regulation of liver injury and repair. Immunol Rev 2000;174:160-171.
37. Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ: The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction. Mol. Cell. Biol. 2004;24:1844-1854.
38. Zarain-Herzberg A, MacLennan DH, Periasamy M: Characterization of rabbit cardiac sarco(endo)plasmic reticulum Ca2(+)-ATPase gene. J Biol Chem 1990;265:4670-4677.
39. Bers DM, Bassani JW, Bassani RA: Na-Ca exchange and Ca fluxes during contraction and relaxation in mammalian ventricular muscle. Ann N Y Acad Sci 1996;779:430-442.
40. Mattiazzi A, Mundina-Weilenmann C, Guoxiang C, Vittone L, Kranias E: Role of phospholamban phosphorylation on Thr17 in cardiac physiological and pathological conditions. Cardiovascular Research 2005;68:366-375.
41. Phillips RM, Narayan P, Gomez AM, Dilly K, Jones LR, Lederer WJ, Altschuld RA: Sarcoplasmic reticulum in heart failure: central player or bystander? Cardiovascular Research 1998;37:346-351.
42. Seehase M, Quentin T, Wiludda E, Hellige G, Paul T, Schiffmann H: Gene expression of the Na-Ca2+ exchanger, SERCA2a and calsequestrin after myocardial ischemia in the neonatal rabbit heart. Biol Neonate 2006;90:174-84.
43. Su H, Sun X, Ma H, Zhang H-F, Yu Q-J, Huang C, Wang X-M, Luan R-H, Jia G-L, Wang H-C, Gao F: Acute hyperglycemia exacerbates myocardial ischemia/reperfusion injury and blunts cardioprotective effect of GIK. In, 2007:E629-635.
44. Mundina-Weilenmann C, Said M, Vittone L, Ferrero P, Mattiazzi A: Phospholamban phosphorylation in ischemia-reperfused heart. Effect ofpacing during ischemia and response to a beta-adrenergic challenge. Mol Cell Biochem 2003;252:239-246.
45. Netticadan T, Temsah R, Osada M, Dhalla NS: Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion. Am J Physiol 1999;277:C384-391.
46. Sande JB, Sjaastad I, Hoen IB, Bokenes J, Tonnessen T, Holt E, Lunde PK, Christensen G: Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity. Cardiovascular Research 2002;53:382-391.
47. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P: Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 1999;5:1135-1142.
48. Temsah RM, Dyck C, Netticadan T, Chapman D, Elimban V, Dhalla NS: Effect of beta-adrenoceptor blockers on sarcoplasmic reticular function and gene expression in the ischemic-reperfused heart. J Pharmacol Exp Ther 2000;293:15-23.
49. De Leiris J, Opie LH, Lubbe WF: Effects of free fatty acid and enzyme release in experimental glucose on myocardial infarction. Nature 1975;253:746-747.
50. Komatsu E, Fukuyama H, Misawa T, Kaminishi T, Yamaguchi I, Miyazawa K: Ventricular wall motion and NE release in post-ischemic reperfused myocardium. Jpn Circ J 1991;55:900-903.
51. Lameris TW, de Zeeuw S, Alberts G, Boomsma F, Duncker DJ, Verdouw PD, Veld AJ, van Den Meiracker AH: Time course and mechanism of myocardial catecholamine release during transient ischemia in vivo. Circulation 2000;101:2645-2650.
52. Brodde O-E, Michel MC: Adrenergic and Muscarinic Receptors in the HumanHeart. In, 1999:651-690.
53. Engelhardt S, Hein L, Wiesmann F, Lohse MJ: Progressive hypertrophy and heart failure in beta 1-adrenergic receptor transgenic mice. In, 1999:7059-7064.
54. Vetter U, Kupferschmid C, Lang D, Pentz S: Insulin-like growth factors and insulin increase the contractility of neonatal rat cardiocytes in vitro. Basic Res Cardiol 1988;83:647-654.
55. Ren J, Walsh MF, Hamaty M, Sowers JR, Brown RA: Augmentation of the inotropic response to insulin in diabetic rat hearts. Life Sci 1999;65:369-380.
56. Aulbach F, Simm A, Maier S, Langenfeld H, Walter U, Kersting U, Kirstein M: Insulin stimulates the L-type Ca2+ current in rat cardiac myocytes. Cardiovasc Res 1999;42:113-120.
57. Heyliger CE, Pierce GN, Singal PK, Beamish RE, Dhalla NS: Cardiac alpha- and beta-adrenergic receptor alterations in diabetic cardiomyopathy. Basic Res Cardiol 1982;77:610-618.
58. Vadlamudi RV, McNeill JH: Effect of experimental diabetes on isolated rat heart responsiveness to isoproterenol. Can J Physiol Pharmacol 1984;62:124-131.
59. Nishio Y, Kashiwagi A, Kida Y, Kodama M, Abe N, Saeki Y, Shigeta Y: Deficiency of cardiac beta-adrenergic receptor in streptozocin-induced diabetic rats. Diabetes 1988;37:1181-1187.
60. Wichelhaus A, Russ M, Petersen S, Eckel J: G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats. Am J Physiol 1994;267:H548-555.
61. Baltensperger K, Karoor V, Paul H, Ruoho A, Czech MP, Malbon CC: The beta-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase. In, 1996:1061-1064.
62. Shumay E, Song X, Wang HY, Malbon CC: pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation. Mol Biol Cell 2002;13:3943-3954.
63. Karoor V, Wang L, Wang H-y, Malbon CC: Insulin stimulates sequestration of beta -adrenergic receptors and enhanced association of beta -adrenergic receptors with Grb2 via Tyrosine 350. J. Biol. Chem. 1998;273:33035-33041.
64. Dalle S, Imamura T, Rose DW, Worrall DS, Ugi S, Hupfeld CJ, Olefsky JM: Insulin induces heterologous desensitization of G-protein-coupled receptor and insulin-like growth factor I signaling by downregulating beta-arrestin-1. Mol Cell Biol 2002;22:6272-6285.
65. Gavi S, Yin D, Shumay E, Wang H-y, Malbon CC: The 15-Amino Acid Motif of the C Terminus of the {beta}2-Adrenergic Receptor Is Sufficient to Confer Insulin-Stimulated Counterregulation to the {beta}1-Adrenergic Receptor. Endocrinology 2005;146:450-457.
66. Leblais V, Jo S-H, Chakir K, Maltsev V, Zheng M, Crow MT, Wang W, Lakatta EG, Xiao R-P: Phosphatidylinositol 3-Kinase Offsets cAMP-Mediated Positive Inotropic Effect via Inhibiting Ca2+ Influx in Cardiomyocytes. In, 2004:1183-1190.
67. Shumay E, Gavi S, Wang H-y, Malbon CC: Trafficking of {beta}2-adrenergic receptors: insulin and {beta}-agonists regulate internalization by distinct cytoskeletal pathways. J. Cell Sci. 2004;117:593-600.
68. Mulder AH, Tack CJ, Olthaar AJ, Smits P, Sweep FC, Bosch RR: Adrenergic receptor stimulation attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes by inhibiting GLUT4 translocation. Am J Physiol Endocrinol Metab 2005;289:E627-633.
69. Morisco C, Marrone C, Trimarco V, Crispo S, Monti MG, Sadoshima J, Trimarco B: Insulin resistance affects the cytoprotective effect of insulin in cardiomyocytes through an impairment of MAPK phosphatase-1 expression. Cardiovasc Res 2007;76:453-464.
70. Egert S, Nguyen N, Schwaiger M: Contribution of alpha-adrenergic and beta-adrenergic stimulation to ischemia-induced glucose transporter (GLUT) 4 and GLUT1 translocation in the isolated perfused rat heart. Circ Res 1999;84:1407-1415.
71. Wang H-y, Doronin S, Malbon CC: Insulin activation of mitogen-activated protein kinases Erk1,2 is amplified via beta -adrenergic receptor expression and requires the integrity of the Tyr350 of the receptor. J. Biol. Chem. 2000;275:36086-36093.
72. Zhang HX, Zang YM, Huo JH, Liang SJ, Zhang HF, Wang YM, Fan Q, Guo WY, Wang HC, Gao F: Physiologically tolerable insulin reduces myocardial injury and improves cardiac functional recovery in myocardial ischemic/reperfused dogs. J Cardiovasc Pharmacol 2006;48:306-313.
73. Das UN: Glucose, insulin, and coronary heart disease. In, 2008:1075-a-1076.
74. Dandona P, Chaudhuri A, Ghanim H, Mohanty P: Insulin as an anti-inflammatory and antiatherogenic modulator. J Am Coll Cardiol 2009;53:S14-20.
75. Grund F, Sommerschild HT, Kirkeboen KA, Ilebekk A: Preconditioning with ischaemia reduces both myocardial oxygen consumption and infarct size in a graded pattern. J Mol Cell Cardiol 1997;29:3067-3079.
76. Recchia FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, Hintze TH: Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 1998;83:969-979.
77. Goyal A, Mehta SR, Diaz R, Gerstein HC, Afzal R, Xavier D, Liu L, Pais P, Yusuf S: Differential clinical outcomes associated with hypoglycemia and hyperglycemia in acute myocardial infarction. Circulation 2009;120:2429-2437.
78. Webster KA: Stress hyperglycemia and enhanced sensitivity to myocardial infarction. Curr Hypertens Rep 2008;10:78-84.
79. Smit JW, Romijn JA: Acute insulin resistance in myocardial ischemia: causes and consequences. Semin Cardiothorac Vasc Anesth 2006;10:215-219.
80. Opie LH: Metabolic management of acute myocardial infarction comes to the fore and extends beyond control of hyperglycemia. Circulation 2008;117:2172-2177.
81. Zarich SW, Nesto RW: Implications and treatment of acute hyperglycemia in the setting of acute myocardial infarction. Circulation 2007;115:e436-439.
82. Lazar HL MM, Chipkin S: Insulin is the protective component of glucose-insulin-potassium solutionsduring coronary artery bypass graft surgery in patients with diabetes. Circulation 2008; 118:S705.
83. Muniyappa R, Montagnani M, Koh KK, Quon MJ: Cardiovascular actions of insulin. Endocr Rev 2007;28:463-491.
84. Howell NJ, Ashrafian H, Drury NE, Ranasinghe AM, Contractor H, Isackson H, Calvert M, Williams LK, Freemantle N, Quinn DW, Green D, Frenneaux M, Bonser RS, Mascaro JG, Graham TR, Rooney SJ, Wilson IC, Pagano D: Glucose-insulin-potassium reduces the incidence of low cardiac output episodes after aortic valve replacement for aortic stenosis in patients with left ventricular hypertrophy: results from the Hypertrophy, Insulin, Glucose, and Electrolytes (HINGE) trial. Circulation;123:170-177.
85. Vlasselaers D: Glucose-insulin-potassium: much more than enriched myocardial fuel. Circulation;123:129-130.
86. Ellger B, Langouche L, Richir M, Debaveye Y, Vanhorebeek I, Teerlink T, Van Leeuwen PA, Van den Berghe G: Modulation of regional nitric oxide metabolism: blood glucose control or insulin? Intensive Care Med 2008;34:1525-1533.
87. van den Heuvel I, Vanhorebeek I, Van den Berghe G: The importance of strict blood glucose control with insulin therapy in the intensive care unit. Curr Diabetes Rev 2008;4:227-233.
88. Vasilyev N, Williams T, Brennan M-L, Unzek S, Zhou X, Heinecke JW, Spitz DR, Topol EJ, Hazen SL, Penn MS: Myeloperoxidase-Generated Oxidants Modulate Left Ventricular Remodeling but Not Infarct Size After Myocardial Infarction. In, 2005:2812-2820.
89. Ling PR, Smith RJ, Bistrian BR: Acute effects of hyperglycemia and hyperinsulinemia on hepatic oxidative stress and the systemic inflammatory response in rats. Crit Care Med 2007;35:555-560.
90. Esumi K, Nishida M, Shaw D, Smith TW, Marsh JD: NADH measurements in adult rat myocytes during simulated ischemia. Am J Physiol 1991;260:H1743-1752.
91. Lindemann JP, Jones LR, Hathaway DR, Henry BG, Watanabe AM: beta-Adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles. J Biol Chem 1983;258:464-471.
92. Hopkins TA, Dyck JR, Lopaschuk GD: AMP-activated protein kinase regulation of fatty acid oxidation in the ischaemic heart. Biochem Soc Trans 2003;31:207-212.
93. Stanley WC, Lopaschuk GD, Hall JL, McCormack JG: Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Cardiovascular Research 1997;33:243-257.
94. Remondino A, Kwon SH, Communal C, Pimentel DR, Sawyer DB, Singh K, Colucci WS: Beta-adrenergic receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of the mitochondrial pathway. Circ Res 2003;92:136-138.
95. Engfeldt P, Hellmer J, Wahrenberg H, Arner P: Effects of insulin on adrenoceptor binding and the rate of catecholamine-induced lipolysis in isolated human fat cells. J Biol Chem 1988;263:15553-15560.
96. Karoor V, Baltensperger K, Paul H, Czech MP, Malbon CC: Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin. J Biol Chem 1995;270:25305-25308.
97. Austin C, Chess-Williams R: The in-vitro effects of insulin and the effects of acute fasting on cardiac beta-adrenoceptor responses in the short-term streptozotocin-diabetic rat. J Pharm Pharmacol 1994;46:326-331.
98. Ferrara N, Abete P, Corbi G, Paolisso G, Longobardi G, Calabrese C, Cacciatore F, Scarpa D, Iaccarino G, Trimarco B, Leosco D, Rengo F: Insulin-induced changes in beta-adrenergic response: an experimental study in the isolated rat papillary muscle. Am J Hypertens 2005;18:348-353.
99. Lohse MJ: Molecular mechanisms of membrane receptor desensitization.Biochim Biophys Acta 1993;1179:171-188.
100. Strasser RH, Marquetant R, Kubler W: Independent sensitization of beta-adrenoceptors and adenylate cyclase in acute myocardial ischaemia. Br J Clin Pharmacol 1990;30 Suppl 1:27S-35S.
101. Sichelschmidt OJ, Hahnefeld C, Hohlfeld T, Herberg FW, Schror K: Trapidil protects ischemic hearts from reperfusion injury by stimulating PKAII activity. Cardiovasc Res 2003;58:602-610.
102. Gavi S, Yin D, Shumay E, Wang HY, Malbon CC: Insulin-like growth factor-I provokes functional antagonism and internalization of beta1-adrenergic receptors. Endocrinology 2007;148:2653-2662.
103. Krause SM, Jacobus WE, Becker LC: Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic "stunned" myocardium. Circ Res 1989;65:526-530.
104. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, DePaoli-Roach AA, Robbins J, Hewett TE, Bibb JA, Westfall MV, Kranias EG, Molkentin JD: PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat Med 2004;10:248-254.
105. Vittone L, Mundina-Weilenmann C, Said M, Mattiazzi A: Mechanisms involved in the acidosis enhancement of the isoproterenol-induced phosphorylation of phospholamban in the intact heart. J Biol Chem 1998;273:9804-9811.