MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.

AIM: Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored. METHODS: Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms. RESULTS: Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts. CONCLUSION: Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

MFN2-mediated mitochondrial fusion facilitates acute hypobaric hypoxia-induced cardiac dysfunction by increasing glucose catabolism and ROS production.

BACKGROUND: Rapid ascent to high-altitude environment which is characterized by acute hypobaric hypoxia (HH) may increase the risk of cardiac dysfunction. However, the potential regulatory mechanisms and prevention strategies for acute HH-induced cardiac dysfunction have not been fully clarified. Mitofusin 2 (MFN2) is highly expressed in the heart and is involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the significance of MFN2 in the heart under acute HH has not been investigated. METHODS AND RESULTS: Our study revealed that MFN2 upregulation in hearts of mice during acute HH led to cardiac dysfunction. In vitro experiments showed that the decrease in oxygen concentration induced upregulation of MFN2, impairing cardiomyocyte contractility and increasing the risk of QT prolongation. Additionally, acute HH-induced MFN2 upregulation promoted glucose catabolism and led to excessive mitochondrial reactive oxygen species (ROS) production in cardiomyocytes, ultimately resulting in decreased mitochondrial function. Furthermore, co-immunoprecipitation (co-IP) and mass spectrometry analyses indicated that MFN2 interacted with the NADH-ubiquinone oxidoreductase 23 kDa subunit (NDUFS8). Specifically, acute HH-induced MFN2 upregulation increased NDUFS8-dependent complex I activity. CONCLUSIONS: Taken together, our studies provide the first direct evidence that MFN2 upregulation exacerbates acute HH-induced cardiac dysfunction by increasing glucose catabolism and ROS production. GENERAL SIGNIFICANCE: Our studies indicate that MFN2 may be a promising therapeutic target for cardiac dysfunction under acute HH.

Outcome-based student assessment enhances academic performance in basic medical laboratory course.

Basic medical laboratory courses (BMLCs) play an important role in medical educational courses helping the student acquire three important skills of surgical operating, collaborative learning, and problem solving. The outcome-based student assessment (OBSA) is a learning evaluation method that establishes specific evaluation points based on performance of students in three aspects: surgical operating, collaborative learning, and problem solving in the BMLC curriculum practices. The purpose of the present randomized controlled trial study is to explore the efficiency of OBSA program in BMLCs. The 233 students attending BMLCs were randomly divided into 2 groups, 118 in the OBSA group and 115 in the control group. We conducted multiple-choice examination questions (MCQs) test and two questionnaires with the method of two-sample t test for statistics. The results of MCQs in total eight BMLC blocks showed that the academic performance of the OBSA group was significantly better than that of the control group (P < 0.05). In addition, the average scores of direct observation of procedural skills (DOPS) and mini-experimental evaluation exercise in OBSA group were significantly higher than those in control group (P < 0.05). The majority of the medical students preferred the OBSA and considered OBSA could effectively improve their surgical operating skills (83.9%), collaborative learning skills (92.1%), and problem-solving skills (91.1%). From the above, OBSA is an effective evaluation method for the implementation of the BMLC curriculum.

AKAP1 Deficiency Attenuates Diet-Induced Obesity and Insulin Resistance by Promoting Fatty Acid Oxidation and Thermogenesis in Brown Adipocytes.

Altering the balance between energy intake and expenditure is a major strategy for treating obesity. Nonetheless, despite the progression in antiobesity drugs on appetite suppression, therapies aimed at increasing energy expenditure are limited. Here, knockout ofAKAP1, a signaling hub on outer mitochondrial membrane, renders mice resistant to diet-induced obesity.AKAP1 knockout significantly enhances energy expenditure and thermogenesis in brown adipose tissues (BATs) of obese mice. Restoring AKAP1 expression in BAT clearly reverses the beneficial antiobesity effect in AKAP1-/- mice. Mechanistically, AKAP1 remarkably decreases fatty acid ß-oxidation (FAO) by phosphorylating ACSL1 to inhibit its activity in a protein-kinase-A-dependent manner and thus inhibits thermogenesis in brown adipocytes. Importantly, AKAP1 peptide inhibitor effectively alleviates diet-induced obesity and insulin resistance. Altogether, the findings demonstrate that AKAP1 functions as a brake of FAO to promote diet-induced obesity, which may be used as a potential therapeutic target for obesity.

Akap1 deficiency exacerbates diabetic cardiomyopathy in mice by NDUFS1-mediated mitochondrial dysfunction and apoptosis.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy, characterised by increased oxidative damage and mitochondrial dysfunction, contributes to the increased risk of heart failure in individuals with diabetes. Considering that A-kinase anchoring protein 121 (AKAP1) is localised in the mitochondrial outer membrane and plays key roles in the regulation of mitochondrial function, this study aimed to investigate the role of AKAP1 in diabetic cardiomyopathy and explore its underlying mechanisms. METHODS: Loss- and gain-of-function approaches were used to investigate the role of AKAP1 in diabetic cardiomyopathy. Streptozotocin (STZ) was injected into Akap1-knockout (Akap1-KO) mice and their wild-type (WT) littermates to induce diabetes. In addition, primary neonatal cardiomyocytes treated with high glucose were used as a cell model of diabetes. Cardiac function was assessed with echocardiography. Akap1 overexpression was conducted by injecting adeno-associated virus 9 carrying Akap1 (AAV9-Akap1). LC-MS/MS analysis and functional experiments were used to explore underlying molecular mechanisms. RESULTS: AKAP1 was downregulated in the hearts of STZ-induced diabetic mouse models. Akap1-KO significantly aggravated cardiac dysfunction in the STZ-treated diabetic mice when compared with WT diabetic littermates, as evidenced by the left ventricular ejection fraction (LVEF; STZ-treated WT mice [WT/STZ] vs STZ-treated Akap1-KO mice [KO/STZ], 51.6% vs 41.6%). Mechanistically, Akap1 deficiency impaired mitochondrial respiratory function characterised by reduced ATP production. Additionally, Akap1 deficiency increased cardiomyocyte apoptosis via enhanced mitochondrial reactive oxygen species (ROS) production. Furthermore, immunoprecipitation and mass spectrometry analysis indicated that AKAP1 interacted with the NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUFS1). Specifically, Akap1 deficiency inhibited complex I activity by preventing translocation of NDUFS1 from the cytosol to mitochondria. Akap1 deficiency was also related to decreased ATP production and enhanced mitochondrial ROS-related apoptosis. In contrast, restoration of AKAP1 expression in the hearts of STZ-treated diabetic mice promoted translocation of NDUFS1 to mitochondria and alleviated diabetic cardiomyopathy in the LVEF (WT/STZ injected with adeno-associated virus carrying gfp [AAV9-gfp] vs WT/STZ AAV9-Akap1, 52.4% vs 59.6%; KO/STZ AAV9-gfp vs KO/STZ AAV9-Akap1, 42.2% vs 57.6%). CONCLUSIONS/INTERPRETATION: Our study provides the first evidence that Akap1 deficiency exacerbates diabetic cardiomyopathy by impeding mitochondrial translocation of NDUFS1 to induce mitochondrial dysfunction and cardiomyocyte apoptosis. Our findings suggest that Akap1 upregulation has therapeutic potential for myocardial injury in individuals with diabetes.

MCUR1-Mediated Mitochondrial Calcium Signaling Facilitates Cell Survival of Hepatocellular Carcinoma via Reactive Oxygen Species-Dependent P53 Degradation.

AIMS: Levels of the mitochondrial calcium uniporter regulator 1 (MCUR1) increases during development of hepatocellular carcinoma (HCC). However, mechanisms of how mitochondrial Ca2+ homeostasis is modulated and its function remain limited in cancers. RESULTS: MCUR1 was frequently upregulated in HCC cells to enhance the Ca2+ uptake into mitochondria in an MCU-dependent manner, which significantly facilitated cell survival by inhibiting mitochondria-dependent intrinsic apoptosis and promoting proliferation of HCC cells, and thus led to poor prognosis. In vivo assay confirmed these results, indicating that overexpressed MCUR1 notably decreased the fraction of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and increased the positive Ki67 staining in xenograft tumors, while reduced MCUR1 expression was associated with impaired growth capacity of HCC cells in nude mice. The survival advantage conferred by MCUR1-mediated mitochondrial Ca2+ uptake was majorly caused by elevated production of mitochondrial reactive oxygen species and subsequent AKT/MDM2- induced P53 degradation, which regulated the expression level of apoptosis-related molecules and cell cycle-related molecules. Treatment of mitochondrial Ca2+-buffering protein parvalbumin remarkably inhibited the growth of HCC cells. Conclusions and Innovation: Our study provides evidence supporting a possible tumor-promoting role for MCUR1-mediated mitochondrial Ca2+ uptake and uncovers a mechanistic understanding that links change of mitochondrial Ca2+ homeostasis to cancer cell survival, which suggests a potential novel therapeutic target for HCC. Antioxid. Redox Signal. 28, 1120-1136.

MICU1 Alleviates Diabetic Cardiomyopathy Through Mitochondrial Ca2+-Dependent Antioxidant Response.

Diabetic cardiomyopathy is a major cause of mortality in patients with diabetes, but specific strategies for preventing or treating diabetic cardiomyopathy have not been clarified yet. MICU1 is a key regulator of mitochondrial Ca2+ uptake, which plays important roles in regulating mitochondrial oxidative phosphorylation and redox balance. To date, however, the significance of MICU1 in diabetic hearts has not been investigated. Here, we demonstrate that MICU1 was downregulated in db/db mouse hearts, which contributes to myocardial apoptosis in diabetes. Importantly, the reconstitution of MICU1 in diabetic hearts significantly inhibited the development of diabetic cardiomyopathy, as evidenced by enhanced cardiac function and reduced cardiac hypertrophy and myocardial fibrosis in db/db mice. Moreover, our in vitro data show that the reconstitution of MICU1 inhibited the apoptosis of cardiomyocytes, induced by high glucose and high fat, through increasing mitochondrial Ca2+ uptake and subsequently activating the antioxidant system. Finally, our results indicate that hyperglycemia and hyperlipidemia induced the downregulation of MICU1 by inhibiting Sp1 expression in diabetic cardiomyocytes. Collectively, our findings provide the first direct evidence that upregulated MICU1 preserves cardiac function in diabetic db/db mice, suggesting that increasing the expression or activity of MICU1 may be a pharmacological approach to ameliorate cardiomyopathy in diabetes.

Increased mitochondrial fission promotes autophagy and hepatocellular carcinoma cell survival through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways.

Mitochondrial morphology is dynamically remodeled by fusion and fission in cells, and dysregulation of this process is closely implicated in tumorigenesis. However, the mechanism by which mitochondrial dynamics influence cancer cell survival is considerably less clear, especially in hepatocellular carcinoma (HCC). In this study, we systematically investigated the alteration of mitochondrial dynamics and its functional role in the regulation of autophagy and HCC cell survival. Furthermore, the underlying molecular mechanisms and therapeutic application were explored in depth. Mitochondrial fission was frequently upregulated in HCC tissues mainly due to an elevated expression ratio of DNM1L to MFN1, which significantly contributed to poor prognosis of HCC patients. Increased mitochondrial fission by forced expression of DNM1L or knockdown of MFN1 promoted the survival of HCC cells both in vitro and in vivo mainly by facilitating autophagy and inhibiting mitochondria-dependent apoptosis. We further demonstrated that the survival-promoting role of increased mitochondrial fission was mediated via elevated ROS production and subsequent activation of AKT, which facilitated MDM2-mediated TP53 degradation, and NFKBIA- and IKK-mediated transcriptional activity of NFKB in HCC cells. Also, a crosstalk between TP53 and NFKB pathways was involved in the regulation of mitochondrial fission-mediated cell survival. Moreover, treatment with mitochondrial division inhibitor-1 significantly suppressed tumor growth in an in vivo xenograft nude mice model. Our findings demonstrate that increased mitochondrial fission plays a critical role in regulation of HCC cell survival, which provides a strong evidence for this process as drug target in HCC treatment.

Magnolol administration in normotensive young spontaneously hypertensive rats postpones the development of hypertension: role of increased PPAR gamma, reduced TRB3 and resultant alleviative vascular insulin resistance.

Patients with prehypertension are more likely to progress to manifest hypertension than those with optimal or normal blood pressure. However, the mechanisms underlying the development from prehypertension to hypertension still remain largely elusive and the drugs for antihypertensive treatment in prehypertension are absent. Here we determined the effects of magnolol (MAG) on blood pressure and aortic vasodilatation to insulin, and investigated the underlying mechanisms. Four-week-old male spontaneous hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto (WKY) control rats were used. Our results shown that treatment of young SHRs with MAG (100 mg/kg/day, o.g.) for 3 weeks decreased blood pressure, improved insulin-induced aorta vasodilation, restored Akt and eNOS activation stimulated by insulin, and increased PPARγ and decreased TRB3 expressions. In cultured human umbilical vein endothelial cells (HUVECs), MAG incubation increased PPARγ, decreased TRB3 expressions, and restored insulin-induced phosphorylated Akt and eNOS levels and NO production, which was blocked by both PPARγ antagonist and siRNA targeting PPARγ. Improved insulin signaling in HUVECs by MAG was abolished by upregulating TRB3 expression. In conclusion, treatment of young SHRs with MAG beginning at the prehypertensive stage decreases blood pressure via improving vascular insulin resistance that is at least partly attributable to upregulated PPARγ, downregulated TRB3 and consequently increased Akt and eNOS activations in blood vessels in SHRs.

Apocynum venetum leaf attenuates myocardial ischemia/reperfusion injury by inhibiting oxidative stress.

Apocynum venetum, a Chinese medicinal herb, is reported to be neuroprotective. However, whether Apocynum venetum leaf extract (AVLE) protects against ischemic myocardium remains elusive. Our present study was aimed to observe the effects of AVLE preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to investigate the possible mechanisms. Rats were treated with AVLE (500 mg/kg/d, o.g.) or distilled water once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. AVLE preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, AVLE reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, AVLE preconditioning significantly inhibited superoxide generation, gp91(phox) expression, malonaldialdehyde formation and enhanced superoxide dismutase (SOD) activity in I/R hearts. Furthermore, AVLE treatment increased Akt and extracellular regulated protein kinases 1/2 (ERK1/2) phosphorylations in I/R rat heart. Either the Phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin or the ERK1/2 inhibitor PD98059 blocked AVLE-stimulated anti-oxidative effects and cardioprotection. Our study demonstrated for the first time that AVLE reduces oxidative stress and exerts cardioprotection against MI/R injury in rats.

Vasonatrin peptide attenuates myocardial ischemia-reperfusion injury in diabetic rats and underlying mechanisms.

Diabetes mellitus increases morbidity/mortality of ischemic heart disease. Although atrial natriuretic peptide and C-type natriuretic peptide reduce the myocardial ischemia-reperfusion damage in nondiabetic rats, whether vasonatrin peptide (VNP), the artificial synthetic chimera of atrial natriuretic peptide and C-type natriuretic peptide, confers cardioprotective effects against ischemia-reperfusion injury, especially in diabetic patients, is still unclear. This study was designed to investigate the effects of VNP on ischemia-reperfusion injury in diabetic rats and to further elucidate its mechanisms. The high-fat diet-fed streptozotocin-induced diabetic Sprague-Dawley rats were subjected to ischemia-reperfusion operation. VNP treatment (100 µg/kg iv, 10 min before reperfusion) significantly improved the instantaneous first derivation of left ventricle pressure (±LV dP/dtmax) and LV systolic pressure and reduced LV end-diastolic pressure, apoptosis index, caspase-3 activity, plasma creatine kinase (CK), and lactate dehydrogenase (LDH) activities. Moreover, VNP inhibited endoplasmic reticulum (ER) stress by suppressing glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). These effects were mimicked by 8-bromine-cyclic guanosinemonophosphate (8-Br-cGMP), a cGMP analog, whereas they were inhibited by KT-5823, the selective inhibitor of PKG. In addition, pretreatment with tauroursodeoxycholic acid (TUDCA), a specific inhibitor of ER stress, could not further promote the VNP's cardioprotective effect in diabetic rats. In vitro H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation and incubated with or without VNP (10(-8) mol/l). Gene knockdown of PKG1α with siRNA blunted VNP inhibition of ER stress and apoptosis, while overexpression of PKG1α resulted in significant decreased ER stress and apoptosis. VNP protects the diabetic heart against ischemia-reperfusion injury by inhibiting ER stress via the cGMP-PKG signaling pathway. These results suggest that VNP may have potential therapeutic value for the diabetic patients with ischemic heart disease.

Acute insulin resistance mediated by advanced glycation endproducts in severely burned rats.

OBJECTIVE: Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. DESIGN: Prospective, randomized experimental study. SETTING: Animal research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. MEASUREMENTS AND MAIN RESULTS: We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn-induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. CONCLUSIONS: These findings suggest that severe burn-induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor for advanced glycation endproducts ameliorates postburn insulin resistance.

Semen cassiae attenuates myocardial ischemia and reperfusion injury in high-fat diet streptozotocin-induced type 2 diabetic rats.

Obese patients with type 2 diabetes mellitus (T2DM), which is characterized by hyperglycemia, are liable to more severe myocardial infarction. Semen Cassiae is proven to reduce serum lipid levels. This study investigated whether the Semen Cassiae extract (SCE) reduces myocardial ischemia and reperfusion (MI/R) injury with or without diabetes and the underlying mechanisms. The high-fat diet-fed streptozotocin (HFD-STZ) rat model was created as a T2DM model. Normal and DM rats received SCE treatment orally (10 mg/kg/day) for one week. Subsequently these animals were subjected to MI/R. Compared with the normal animals, DM rats showed increased plasma total cholesterol (TC) and triacylglycerol (TG), and more severe MI/R injury and cardiac functional impairment. SCE treatment significantly reduced the plasma TC and TG, improved the instantaneous first derivation of left ventricle pressure and reduced infarct size, decreased plasma creatine kinase and lactate dehydrogenase levels, and apoptosis index at the end of reperfusion in diabetic rats. Moreover, SCE treatment increased the antiapoptotic protein Akt and ERK1/2 phosphorylation levels. Pretreatment with a PI3K inhibitor wortmannin or an ERK1/2 inhibitor PD98059 not only blocked Akt and ERK1/2 phosphorylation respectively, but also inhibited the cardioprotective effects of SCE. However, SCE treatment did not show any effects on the MI/R injury in the normal rats. Our data suggest that SCE effectively improves myocardial function and reduces MI/R-induced injury in diabetic but not normal animals, which is possibly attributed to the reduced TC/TG levels and the triggered cell survival signaling Akt and ERK1/2.

Alpha-linolenic acid intake prevents endothelial dysfunction in high-fat diet-fed streptozotocin rats and underlying mechanisms.

BACKGROUND: Endothelial dysfunction is an important factor in the pathogenesis of diabetes related vascular complications, and acute alpha-linolenic acid (ALA) intake can increase flow-mediated dilation of the diabetic artery at 4 h postprandially. However, whether chronic ALA supplementation may prevent endothelial dysfunction in the process of diabetes and underlying mechanisms remains largely unknown. MATERIALS AND METHODS: The high-fat diet-fed streptozotocin (HFD-STZ) rats provided an animal model for T2DM. Age-matched normal and HFD-STZ rats randomly received normal diet or ALA (500 mg/kg per day). After 5 weeks of feeding, endothelial function was determined. RESULTS: Diabetes caused significant endothelial dysfunction (maximal vasorelaxation responses to ACh) in aortic segments, and ALA intake alleviated endothelial dysfunction. Superoxide production and peroxynitrite (ONOO-) formation were reduced with ALA supplement in diabetic vascular segments. Interestingly, ALA intake enhanced eNOS but inhibited iNOS activity in diabetic vessels. Moreover, ALA intake significantly increased eNOS phosphorylation. On the other hand, gp91phox and iNOS overexpression were reduced moderately with ALA intake in diabetic vessels. CONCLUSIONS: We concluded that ALA prevents diabetes-induced endothelial dysfunction by enhancing eNOS activity and attenuates oxidative/nitrative stress by inhibiting iNOS and NADPH oxidase expression and ONOO- production.

Achyranthes bidentata polypeptides reduces oxidative stress and exerts protective effects against myocardial ischemic/reperfusion injury in rats.

Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.

AMPK-regulated and Akt-dependent enhancement of glucose uptake is essential in ischemic preconditioning-alleviated reperfusion injury.

AIMS: Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury. METHODS: Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by (18)F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay. RESULTS: IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05). CONCLUSIONS: The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.

A novel mechanism for vascular insulin resistance in normotensive young SHRs: hypoadiponectinemia and resultant APPL1 downregulation.

Vascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. Clinical observation showed that lower plasma adiponectin concentration is significantly associated with hypertension. This study was aimed to determine whether hypoadiponectinemia induces vascular insulin resistance before systemic hypertension and the underlying mechanisms. Four-week-old young spontaneously hypertensive rats (ySHRs, normotensive) and adiponectin knockout (KO; APN(-/-)) mice were used to evaluate the role of hypoadiponectinemia in insulin-induced vasodilation of resistance vessels. ySHRs showed significant vascular insulin resistance as evidenced by the blunted vasorelaxation response to insulin in mesenteric arterioles compared with that of age-matched Wistar-Kyoto controls. Serum adiponectin and mesenteric arteriolar APPL1 (an adaptor protein that mediates adiponectin signaling) expression of ySHRs were significantly reduced. In addition, Akt and endothelial NO synthase phosphorylation and NO production in arterioles were markedly reduced, whereas extracellular signal-regulated protein kinases 1/2 (ERK1/2) phosphorylation and endothelin-1 secretion were augmented in ySHRs. APN(-/-) mice showed significantly decreased APPL1 expression and vasodilation evoked by insulin. More importantly, treatment of ySHRs in vivo with the globular domain of adiponectin for 1 week increased APPL1 expression and insulin-induced vasodilation, and restored the balance between insulin-stimulated endothelial vasodilator NO and vasoconstrictor endothelin-1. In cultured human umbilical vein endothelial cells, globular domain of adiponectin upregulated APPL1 expression. Suppression of APPL1 expression with small interfering RNA markedly blunted the globular domain of adiponectin-induced insulin sensitization as evidenced by reduced Akt/endothelial NO synthase and potentiated ERK1/2 phosphorylations. In conclusion, hypoadiponectinemia induces APPL1 downregulation in the resistance vessels, contributing to the development of vascular insulin resistance by differentially modulating the Akt/endothelial NO synthase/NO and ERK1/2/endothelin-1 pathways in vascular endothelium in normotensive ySHRs.

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin-interacting protein.

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin-interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG-induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG-induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG-treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.