Chlorogenic acid prevents paraquat-induced apoptosis via Sirt1-mediated regulation of redox and mitochondrial function

Paraquat (PQ) is a widely used agro-chemical in agriculture and highly toxic to humans. Although the mechanism of PQ poisoning is not clear, it has been well documented that reactive oxygen species (ROS) generation and apoptosis play pivotal roles. Alternatively, chlorogenic acid (CA) is a biologically active dietary polyphenol, playing several therapeutic roles. However, it is not known whether CA has protective effect on PQ-induced apoptosis. Here, we investigated the effect of CA in preventing PQ-induced apoptosis and explored the underlying mechanisms. A549 cells were pretreated with 100 µM CA for 24?h and then exposed to 160 µM PQ for 24?h. We found that CA was effective in preventing PQ-induced apoptotic features, including the release of cytochrome c from the mitochondria to cytoplasm, the cleavages of caspase 3 and caspase 9, and the increases in levels of Bcl-2-associated X protein (Bax) and intracellular calcium ions. CA alleviated ROS production and prevented the reduction of antioxidant capacity in cells exposed to PQ by increasing NF-E2-related factor 2 (Nrf2), superoxide dismutase 2 (SOD2) and glutathione levels. In addition, CA also attenuated PQ-induced alterations of mitochondrial structure and function (such as the decreases in membrane potential and adenosine triphosphate level), and the impaired autophagic flux was improved by CA. Down-regulation of sirtuin 1 (Sirt1) by short hairpin RNA reversed the protective effects of CA. Thus, CA may be viewed as a potential drug to treat PQ-induced lung epithelial cell apoptosis and other disorders with similar pathologic mechanisms.     

急性脑缺血诱导的Sirt3蛋白表达下调通过破坏线粒体功能导致神经元损伤

本文旨在观察急性脑缺血对神经元沉默信息调节因子2相关酶类3(silent mating type information regulator 2 homolog 3,Sirt3)蛋白表达水平的影响,并阐明Sirt3在急性脑缺血中的病理意义.建立小鼠大脑中动脉栓塞(middle cerebral artery occlu...     

The possible role of intracellular polyamines in mitochondrial metabolic regulation.

Studies were made on the effects of very low concentrations of the polyamine, spermine, on rat liver mitochondrial metabolism associated with β-hydroxybutyrate. The respiratory control ratio and the rate of respiration during ADP-ATP conversion are significantly altered with shifts in spermine concentrations of as little as 15.7 nMoles/ml within the physiological Mg⁺⁺ concentration range. These spermine concentration changes are small compared to the estimated hepatic intracellular levels of spermine which have been reported to be between 200 and 1200 nMoles/gm wet weight under normal conditions. There is now evidence that exposure of an animal to certain environmental conditions induces changes of 164 nMoles/gm wet weight in intracellular levels of liver spermine in a few hours. Also there is evidence that the concentration of intracellular polyamines is influenced by endocrines since the levels of the enzymes responsible for their synthesis are markedly affected by hormonal changes. Therefore, alterations of polyamine levels may play a role in mitochondrial metabolic regulation $\text{in vivo}$.     

Intermediate filaments: structure, regulation of expression and possible function

New data are reviewed on intermediate filaments, i.e. on one of the cytoskeleton components. Structural proteins of intermediate filaments, their enzymatic modification, filament-associated proteins and the peculiarities of filament assembly are dealt with. The regularities of expression of intermediate filament proteins in normal tissues are analysed, as well as during differentiation and cultured cell growth. In the final part of the paper possible functions of intermediate filaments are discussed.     

Mitochondrial aldehyde dehydrogenase 2 accentuates aging-induced cardiac remodeling and contractile dysfunction: role of AMPK,Sirt1, and mitochondrial function

Cardiac aging is associated with compromised myocardial function and morphology although the underlying mechanism remains elusive. Aldehyde dehydrogenase 2 (ALDH2), an essential mitochondrial enzyme governing cardiac function, displays polymorphism in humans. This study was designed to examine the role of ALDH2 in aging-induced myocardial anomalies. Myocardial mechanical and intracellular Ca²⁺ properties were examined in young (4–5 months) and old (26–28 months) wild-type and ALDH2 transgenic mice. Cardiac histology, mitochondrial integrity, O₂⁻ generation, apoptosis, and signaling cascades, including AMPK activation and Sirt1 level were evaluated. Myocardial function and intracellular Ca²⁺ handling were compromised with advanced aging; the effects were accentuated by ALDH2. Hematoxylin and eosin and Masson trichrome staining revealed cardiac hypertrophy and interstitial fibrosis associated with greater left-ventricular mass and wall thickness in aged mice. ALDH2 accentuated aging-induced cardiac hypertrophy but not fibrosis. Aging promoted O₂⁻ release, apoptosis, and mitochondrial injury (mitochondrial membrane potential, levels of UCP-2 and PGC-1α), and the effects were also exacerbated by ALDH2. Aging dampened AMPK phosphorylation and Sirt1, the effects of which were exaggerated by ALDH2. Treatment with the ALDH2 activator Alda-1 accentuated aging-induced O₂⁻ generation and mechanical dysfunction in cardiomyocytes, the effects of which were mitigated by cotreatment with activators of AMPK and Sirt1, AICAR, resveratrol, and SRT1720. Examination of human longevity revealed a positive correlation between life span and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may accentuate myocardial remodeling and contractile dysfunction in aging, possibly through AMPK/Sirt1-mediated mitochondrial injury.     

Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

Sirtuins are a family of protein deacetylases that catalyze the nicotinamide adenine dinucleotide (NAD⁺)-dependent removal of acetyl groups from modified lysine side chains in various proteins. Sirtuins act as metabolic sensors and influence metabolic adaptation but also many other processes such as stress response mechanisms, gene expression, and organismal aging. Mammals have seven Sirtuin isoforms, three of them – Sirt3, Sirt4, and Sirt5 – located to mitochondria, our centers of energy metabolism and apoptosis initiation. In this review, we shortly introduce the mammalian Sirtuin family, with a focus on the mitochondrial isoforms. We then discuss in detail the current knowledge on the mitochondrial isoform Sirt5. Its physiological role in metabolic regulation has recently been confirmed, whereas an additional function in apoptosis regulation remains speculative. We will discuss the biochemical properties of Sirt5 and how they might contribute to its physiological function. Furthermore, we discuss the potential use of Sirt5 as a drug target, structural features of Sirt5 and of an Sirt5/inhibitor complex as well as their differences to other Sirtuins and the current status of modulating Sirt5 activity with pharmacological compounds.     

Glucosamine-induced alterations of mitochondrial function in pancreatic beta-cells: possible role of protein glycosylation

Chronic exposure of rat pancreatic islets and INS-1 insulinoma cells to glucosamine (GlcN) produced a reduction of glucose-induced (22.2 mM) insulin release that was associated with a reduction of ATP levels and ATP/ADP ratio compared with control groups. To further evaluate mitochondrial function and ATP metabolism, we then studied uncoupling protein-2 (UCP2), F1-F0-ATP-synthase, and mitochondrial membrane potential, a marker of F1-F0-ATP-synthase activity. UCP2 protein levels were unchanged after chronic exposure to GlcN on both pancreatic islets and INS-1 beta-cells. Due to the high number of cells required to measure mitochondrial F1-F0-ATP-synthase protein levels and mitochondrial membrane potential, we used INS-1 cells, and we found that chronic culture with GlcN increased F1-F0-ATP-synthase protein levels but decreased glucose-stimulated changes of mitochondrial membrane potential. Moreover, F1-F0-ATP-synthase was highly glycosylated, as demonstrated by experiments with N-glycosidase F and glycoprotein staining. Tunicamycin (an inhibitor of protein N-glycosylation), when added with GlcN in the culture medium, was able to partially prevent all these negative effects on insulin secretion, adenine nucleotide content, mitochondrial membrane potential, and protein glycosylation. Thus we suggest that GlcN-induced pancreatic beta-cell toxicity might be mediated by reduced cell energy production. An excessive protein N-glycosylation of mitochondrial F1-F0-ATP-synthase might lead to cell damage and secretory alterations in pancreatic beta-cells.     

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.Vascular endothelial cells (ECs) are fundamentally important in maintaining structural and functional homeostasis of blood vessels. Normal biological functions of ECs are highly sensitive to the biomechanical stimuli induced by blood flow, of which shear stress acting on the surface of EC has been recognized to be one of the most important vasoactive factors in EC.^{1, 2} A relatively high level of laminar shear stress is cytoprotective, whereas abnormal (low-magnitude or oscillatory) shear stress is a detrimental cellular stress to ECs.¹ Transduction of the mechanical signals involves multiple messenger molecules and signaling proteins, which collectively regulate important endothelial functions, such as gene expression, proliferation, migration, morphogenesis, permeability, thrombogenicity, and inflammation.²Autophagy (also known as macroautophagy) is an evolutionarily conserved cellular stress response.^{3, 4} Autophagy is a cellular self-digestion process, which is responsible for degradation of misfolded proteins and damaged organelles. Autophagic process is mainly mediated by the formation of autophagosome, a double-membrane vacuole structure containing engulfed cellular components. This process requires expression of a group of key genes involved in autophagy, including LC3A, beclin-1, Atg5, Atg7, and Atg12, for example.^{3, 5} Autophagosomes fuse with lysosomes, forming autolysosomes, where the cellular components are degraded by various hydrolases in an acidified environment.^{4, 5} In ECs, an autophagic response can be initiated by different stress stimuli.^{6, 7, 8} It is noted that the cellular outcome following autophagy induction in ECs varies depending on the nature of stimuli and specific experimental settings.^{6, 7, 9, 10} Moreover, there is evidence showing that autophagy may also be involved in modulating other EC functions such as angiogenesis and cellular senescence.^{11, 12} Therefore, understanding the regulatory mechanisms of autophagy in ECs will be important for discovery of strategies to protect normal endothelial functions. Recently, Guo et al. provided some evidence indicating that the autophagic process in EC might be affected by shear stress.¹³ This argument, however, was only based on observations of changed expression levels of LC3 and beclin-1; further experimental evidence is needed to confirm such an effect of shear stress on autophagy. More importantly, the mechanisms underlying this phenomenon are not understood. Different signaling pathways may be involved in modulating autophagy in ECs.^{14, 15, 16} For example, inhibition of the mTOR (mammalian target of rapamycin) pathway by rapamycin-induced endothelial autophagy and prevented energy stress-triggered cell damage.¹⁶ There is also evidence indicating a potential role of Sirt1.¹⁴ Moreover, accumulating evidence has suggested that reactive oxygen species (ROS) are closely implicated in modulating autophagic responses via complex interactions with other autophagy-related factors.¹⁵ Despite of these results, the signaling mechanisms of shear stress-regulated autophagy in EC remain to be defined. Hence, here we aim to delineate the impacts and underlying mechanisms of shear stress on autophagy in human vascular ECs.     

Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function

The positive role of PARP1 in regulation of various nuclear DNA transactions is well established. Although a mitochondrial localization of PARP1 has been suggested, its role in the maintenance of the mitochondrial DNA is currently unknown. Here we investigated the role of PARP1 in the repair of the mitochondrial DNA in the baseline and oxidative stress conditions. We used wild-type A549 cells or cells depleted of PARP1. Our data show that intra-mitochondrial PARP1 interacts with a key mitochondrial-specific DNA base excision repair (BER) enzymes, namely EXOG and DNA polymerase gamma (Polγ), which under oxidative stress become poly(ADP-ribose)lated (PARylated). Interaction between mitochondrial BER enzymes was significantly affected in the presence of PARP1. Moreover, the repair of the oxidative-induced damage to the mitochondrial DNA in PARP1-depleted cells was found to be more robust compared to control counterpart. In addition, mitochondrial biogenesis was enhanced in PARP1-depleted cells, including mitochondrial DNA copy number and mitochondrial membrane potential. This observation was further confirmed by analysis of lung tissue isolated from WT and PARP1 KO mice. In summary, we conclude that mitochondrial PARP1, in opposite to nuclear PARP1, exerts a negative effect on several mitochondrial-specific transactions including the repair of the mitochondrial DNA.     

Redox regulation of protein expression in Saccharomyces cerevisiae mitochondria: possible role of VDAC

Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage dependent anion selective channel) we studied the role of the cytosol and mitochondria redox states in regulation of the expression levels of some mitochondrial proteins. The studied proteins are MnSOD and subunits of the protein import machinery of the mitochondrial outer membrane, i.e. Tom70, Tom40 and Tob55 (Sam50). We have shown that both the cytosol and mitochondria redox states depend on the presence of a given VDAC isoform. The cytosol redox state is mediated by VDAC1, although VDAC2 has a quantitative effect, whereas the mitochondria redox state depends on the presence of both VDAC isoforms. Moreover, we have shown that the cytosol redox status but not the mitochondrial one is decisive for the expression levels of the studied mitochondrial proteins. Thus, expression levels of some mitochondrial proteins is influenced by VDAC and this regulatory process at least partially does not require its channel activity as VDAC2 does not form a channel. Thus, VDAC can be regarded as a participant of signaling pathways in S. cerevisiae cells.     

Downregulation of Sirt1 as aging change in advanced heart failure

Background

In congestive heart failure the balance between cell death and cell survival in cardiomyocytes is compromised. Sirtuin 1 (Sirt1) activates cell survival machinery and has been shown to be protective against ischemia/reperfusion injury in murine heart. The role of Sirt1 in heart failure, especially in human hearts is not clear.

Results

The expression of Sirt1 and other (associated) downstream molecules in human cardiomyocytes from patients with advanced heart failure was examined. Sirt1 was down-regulated (54.92% ± 7.80% in advanced heart failure samples compared with healthy control cardiomyocytes). The modulation of molecules involved in cardiomyocyte survival and death in advanced heart failure were also examined. The expression of Mn-superoxide dismutase and thioredoxin1, as well as an antiapoptotic molecule, Bcl-xL, were all significantly reduced in advanced heart failure cardiomyoctes (0.71 ± 0.02-fold, 0.61 ± 0.05-fold, and 0.53 ± 0.08-fold vs. control, respectively); whereas the expression of proapoptotic molecule Bax was significantly increased (1.62 ± 0.18-fold vs. control). Increased TUNEL-positive number of cardiomyocytes and oxidative stress, confirmed by 8-hydorxydeoxyguanosine staining, were associated with advanced heart failure. The AMPK-Nampt-Sirt1 axis also showed inhibition in advanced heart failure in addition to severely impaired AMPK activation. Increased p53 (acetyl form) and decreased FoxO1 translocation in the nucleus may be the mechanism of down-regulation of antioxidants and up-regulation of proapoptotic molecules due to low expression of Sirt1.

Conclusion

In advanced heart failure, low Sirt1 expression, like aging change may be a significant contributing factor in the downregulation of antioxidants and upregulation of proapoptotic molecules through the p53, FoxO1, and oxidative stress pathways.     

Sirt3 modulate renal ischemia-reperfusion injury through enhancing mitochondrial fusion and activating the ERK-OPA1 signaling pathway

Mitochondrial fusion is linked to heart and liver ischemia-reperfusion (IR) insult. Unfortunately, there is no report to elucidate the detailed influence of mitochondrial fusion in renal IR injury. This study principally investigated the mechanism by which mitochondrial fusion protected kidney against IR injury. Our results indicated that sirtuin 3 (Sirt3) was inhibited after renal IR injury in vivo and in vitro. Overexpression of Sirt3 improved kidney function, modulated oxidative injury, repressed inflammatory damage, and reduced tubular epithelial cell apoptosis. The molecular investigation found that Sirt3 overexpression attenuated IR-induced mitochondrial damage in renal tubular epithelial cells, as evidenced by decreased reactive oxygen species production, increased antioxidants sustained mitochondrial membrane potential, and inactivated mitochondria-initiated death signaling. In addition, our information also illuminated that Sirt3 maintained mitochondrial homeostasis against IR injury by enhancing optic atrophy 1 (OPA1)-triggered fusion of mitochondrion. Inhibition of OPA1-induced fusion repressed Sirt3 overexpression-induced kidney protection, leading to mitochondrial dysfunction. Further, our study illustrated that OPA1-induced fusion could be affected through ERK; inhibition of ERK abolished the regulatory impacts of Sirt3 on OPA1 expression and mitochondrial fusion, leading to mitochondrial damage and tubular epithelial cell apoptosis. Altogether, our results suggest that renal IR injury is closely associated with Sirt3 downregulation and mitochondrial fusion inhibition. Regaining Sirt3 and/or activating mitochondrial fission by modifying the ERK-OPA1 cascade may represent new therapeutic modalities for renal IR injury.     

Bakuchiol attenuates myocardial ischemia reperfusion injury by maintaining mitochondrial function: the role of silent information regulator 1

Ischemia reperfusion (IR) injury (IRI) is associated with poor prognoses in the settings of both cardiac surgery and ischemic heart disease and causes mitochondrial oxidative stress and cell death. Silent information regulator 1 (SIRT1), a member of the histone deacetylase family, exerts anti-IRI effects. Bakuchiol (BAK), an analog of resveratrol and a monoterpene phenol isolated from the seeds of Psoralea corylifolia (Leguminosae), protects tissues from injury. This study was designed to investigate the protective effects of BAK treatment in the setting of myocardial IRI and to elucidate the potential mechanism of those effects. Prior to induction of IR, isolated rat hearts or cardiomyocytes were exposed to BAK in either the absence or presence of the SIRT1 inhibitors Sirtinol and SIRT1 siRNA. BAK exerted cardioprotective effects, as evidenced by the improvements noted in cardiac function following ischemia, attenuated myocardial apoptosis, and changes in several biochemical parameters (including increases in the level of the anti-apoptotic protein Bcl2, decreases in the level of the pro-apoptotic protein Bax, and decreases in the cleaved Caspase 3 level). However, Sirtinol and SIRT1 siRNA each blocked BAK-induced cardioprotection by inhibiting SIRT1 signaling. Additionally, BAK significantly increased the activities of mitochondrial succinate dehydrogenase, cytochrome c oxidase, and mitochondrial superoxide dismutase and decreased the production of malondialdehyde. These findings suggested that BAK significantly attenuated IR-induced mitochondrial oxidative damage. However, Sirtinol and SIRT1 siRNA abolished BAK-dependent mitochondrial function. In summary, our results demonstrate that BAK treatment attenuates IRI by attenuating IR-induced mitochondrial oxidative damage via the activation of SIRT1/PGC-1α signaling.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease.

Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.