Advanced Maternal Age‐associated SIRT1 Deficiency Compromises Trophoblast Epithelial−Mesenchymal Transition through an Increase in Vimentin Acetylation

Advanced maternal age (AMA) pregnancies are rapidly increasing and are associated with aberrant trophoblast cell function, poor placentation, and unfavorable pregnancy outcomes, presumably due to premature placental senescence. SIRT1 is an NAD⁺‐dependent deacetylase with well‐known antiaging effects, but its connection with placental senescence is unreported. In this study, human term placentas and first‐trimester villi were collected from AMA and normal pregnancies, and a mouse AMA model was established by cross breeding young and aged male and female C57 mice. SIRT1 expression and activity in HTR8/SVneo cells were genetically or pharmacologically manipulated. Trophoblast‐specific Sirt1‐knockout (KO) mouse placentas were generated by mating Elf5‐Cre and Sirt1 ^fl/fl mice. Trophoblast cell mobility was assessed with transwell invasion and wound‐healing assays. SIRT1‐binding proteins in HTR8/SVneo cells and human placental tissue were identified by mass spectrometry. We identified SIRT1 as the only differentially expressed sirtuin between AMA and normal placentas. It is downregulated in AMA placentas early in the placental life cycle and is barely impacted by paternal age. SIRT1 loss upregulates P53 acetylation and P21 expression and impairs trophoblast invasion and migration. Sirt1‐KO mouse placentas exhibit senescence markers and morphological disruption, along with decreased fetal weight. In trophoblasts, SIRT1 interacts with vimentin, regulating its acetylation. In conclusion, SIRT1 promotes trophoblast epithelial−mesenchymal transition (EMT) to enhance invasiveness by modulating vimentin acetylation. AMA placentas are associated with premature senescence during placentation due to SIRT1 loss. Therefore, SIRT1 may be an antiaging therapeutic target for improving placental development and perinatal outcomes in AMA pregnancies.     

SIRT1 but not its increased expression is essential for lifespan extension in caloric‐restricted mice

The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1^+/− mice was identical (51%) to that observed in wild-type mice, but SIRT1^+/− mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.     

Drosophila Sirtuin 6 mediates developmental diet‐dependent programming of adult physiology and survival

Organisms in the wild experience unpredictable and diverse food availability throughout their lifespan. Over‐/under‐nutrition during development and in adulthood is known to dictate organismal survival and fitness. Studies using model systems have also established long‐term effects of developmental dietary alterations on life‐history traits. However, the underlining genetic/molecular factors, which differentially couple nutrient inputs during development with fitness later in life are far less understood. Using Drosophila and loss/gain of function perturbations, our serendipitous findings demonstrate an essential role of Sirtuin 6 in regulating larval developmental kinetics, in a nutrient‐dependent manner. The absence of Sirt6 affected ecdysone and insulin signalling and led to accelerated larval development. Moreover, varying dietary glucose and yeast during larval stages resulted in enhanced susceptibility to metabolic and oxidative stress in adults. We also demonstrate an evolutionarily conserved role for Sirt6 in regulating physiological homeostasis, physical activity and organismal lifespan, known only in mammals until now. Our results highlight gene‐diet interactions that dictate thresholding of nutrient inputs and physiological plasticity, operative across development and adulthood. In summary, besides showing its role in invertebrate ageing, our study also identifies Sirt6 as a key factor that programs macronutrient‐dependent life‐history traits.     

Progression of Chronic Liver Inflammation and Fibrosis Driven by Activation of c-JUN Signaling in Sirt6 Mutant Mice

The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6^−/−) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7–8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6^−/− mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6^−/− mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.     

SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin

Cadmium is one of the most toxic metal compounds found in the environment. It is well established that Cd induces hepatotoxicity in humans and multiple animal models. Melatonin, a major secretory product of the pineal gland, has been reported to protect against Cd-induced hepatotoxicity. However, the mechanism behind this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd induced mitochondrial-derived superoxide anion-dependent autophagic cell death. Specifically, Cd decreased SIRT3 protein expression and activity and promoted the acetylation of SOD2, superoxide dismutase 2, mitochondrial, thus decreasing its activity, a key enzyme involved in mitochondrial ROS production, although Cd did not disrupt the interaction between SIRT3 and SOD2. These effects were ameliorated by overexpression of SIRT3. However, a catalytic mutant of SIRT3 (SIRT3^H248Y) lacking deacetylase activity lost the capacity to suppress Cd-induced autophagy. Notably, melatonin treatment enhanced the activity but not the expression of SIRT3, decreased the acetylation of SOD2, inhibited mitochondrial-derived O₂^•− production and suppressed the autophagy induced by 10 μM Cd. Moreover, 3-(1H-1,2,3-triazol-4-yl)pyridine, a confirmed selective SIRT3 inhibitor, blocked the melatonin-mediated suppression of autophagy by inhibiting SIRT3-SOD2 signaling. Importantly, melatonin suppressed Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin exerts a hepatoprotective effect on mitochondrial-derived O₂^•−-stimulated autophagic cell death that is dependent on the SIRT3/SOD2 pathway.     

Prohibitin depletion extends lifespan of a TORC2/SGK‐1 mutant through autophagy and the mitochondrial UPR

Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar effect on lifespan. While PHB depletion shortens lifespan of wild‐type animals, it enhances longevity of a plethora of metabolically compromised mutants, including target of rapamycin complex 2 (TORC2) mutants sgk‐1 and rict‐1. Here, we show that sgk‐1 mutants have impaired mitochondrial homeostasis, lipogenesis and yolk formation, plausibly due to alterations in membrane lipid and sterol homeostasis. Remarkably, all these features are suppressed by PHB depletion. Our analysis shows the requirement of SRBP1/SBP‐1 for the lifespan extension of sgk‐1 mutants and the further extension conferred by PHB depletion. Moreover, although the mitochondrial unfolded protein response (UPR^mt) and autophagy are induced in sgk‐1 mutants and upon PHB depletion, they are dispensable for lifespan. However, the enhanced longevity caused by PHB depletion in sgk‐1 mutants requires both, the UPR^mt and autophagy, but not mitophagy. We hypothesize that UPR^mt induction upon PHB depletion extends lifespan of sgk‐1 mutants through autophagy and probably modulation of lipid metabolism.     

Metabolic and Behavioral Compensations in Response to Caloric Restriction: Implications for the Maintenance of Weight Loss

Background

Metabolic and behavioral adaptations to caloric restriction (CR) in free-living conditions have not yet been objectively measured.

Methodology and Principal Findings

Forty-eight (36.8±1.0 y), overweight (BMI 27.8±0.7 kg/m²) participants were randomized to four groups for 6-months; Control: energy intake at 100% of energy requirements; CR: 25% calorie restriction; CR+EX: 12.5% CR plus 12.5% increase in energy expenditure by structured exercise; LCD: low calorie diet (890 kcal/d) until 15% weight reduction followed by weight maintenance. Body composition (DXA) and total daily energy expenditure (TDEE) over 14-days by doubly labeled water (DLW) and activity related energy activity (AREE) were measured after 3 (M3) and 6 (M6) months of intervention. Weight changes at M6 were −1.0±1.1% (Control), −10.4±0.9% (CR), −10.0±0.8% (CR+EX) and −13.9±0.8% (LCD). At M3, absolute TDEE was significantly reduced in CR (−454±76 kcal/d) and LCD (−633±66 kcal/d) but not in CR+EX or controls. At M6 the reduction in TDEE remained lower than baseline in CR (−316±118 kcal/d) and LCD (−389±124 kcal/d) but reached significance only when CR and LCD were combined (−351±83 kcal/d). In response to caloric restriction (CR/LCD combined), TDEE adjusted for body composition, was significantly lower by −431±51 and −240±83 kcal/d at M3 and M6, respectively, indicating a metabolic adaptation. Likewise, physical activity (TDEE adjusted for sleeping metabolic rate) was significantly reduced from baseline at both time points. For control and CR+EX, adjusted TDEE (body composition or sleeping metabolic rate) was not changed at either M3 or M6.

Conclusions

For the first time we show that in free-living conditions, CR results in a metabolic adaptation and a behavioral adaptation with decreased physical activity levels. These data also suggest potential mechanisms by which CR causes large inter-individual variability in the rates of weight loss and how exercise may influence weight loss and weight loss maintenance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00099151","term_id":"NCT00099151"}}NCT00099151     

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.     

Sirt6 deficiency contributes to mitochondrial fission and oxidative damage in podocytes via ROCK1‐Drp1 signalling pathway

ObjectivesIncreasing evidence suggests that mitochondrial dysfunction is the key driver of angiotensin II (Ang II)‐induced kidney injury. This study was designed to investigate whether Sirtuin 6 (Sirt6) could affect Ang II‐induced mitochondrial damage and the potential mechanisms.Materials and MethodsPodocyte‐specific Sirt6 knockout mice were infused with Ang II and cultured podocytes were stimulated with Ang II to evaluate the effects of Sirt6 on mitochondrial structure and function in podocytes. Immunofluorescence staining was used to detect protein expression and mitochondrial morphology in vitro. Electron microscopy was used to assess mitochondrial morphology in mice. Western blotting was used to quantify protein expression.ResultsMitochondrial fission and decreased Sirt6 expression were observed in podocytes from Ang II‐infused mice. In Sirt6‐deficient mice, Ang II infusion induced increased apoptosis and mitochondrial fragmentation in podocytes than that in Ang II‐infused wild‐type mice. In cultured human podocytes, Sirt6 knockdown exacerbated Ang II‐induced mitochondrial fission, whereas Sirt6 overexpression ameliorated the Ang II‐induced changes in the balance between mitochondrial fusion and fission. Functional studies revealed that Sirt6 deficiency exacerbated mitochondrial fission by promoting dynamin‐related protein 1 (Drp1) phosphorylation. Furthermore, Sirt6 mediated Drp1 phosphorylation by promoting Rho‐associated coiled coil‐containing protein kinase 1 (ROCK1) expression.ConclusionOur study has identified Sirt6 as a vital factor that protects against Ang II‐induced mitochondrial fission and apoptosis in podocytes via the ROCK1‐Drp1 signalling pathway.

Schematic of the molecular action proposed in this study. Schematic depicting that Ang II‐induced sirt6 expression decline promotes mitochondrial fission and podocyte injury through ROCK1‐Drp1 signalling. SIRT6 reduction leads to increased levels of ROCK1, thereby enhancing Drp1 phosphorylation at the Ser637 site. The Drp1 phosphorylation finally results in podocyte injury by inducing mitochondrial fission and apoptosis.     

CNS SIRT3 Expression Is Altered by Reactive Oxygen Species and in Alzheimer’s Disease

Progressive mitochondrial dysfunction contributes to neuronal degeneration in age-mediated disease. An essential regulator of mitochondrial function is the deacetylase, sirtuin 3 (SIRT3). Here we investigate a role for CNS Sirt3 in mitochondrial responses to reactive oxygen species (ROS)- and Alzheimer’s disease (AD)-mediated stress. Pharmacological augmentation of mitochondrial ROS increases Sirt3 expression in primary hippocampal culture with SIRT3 over-expression being neuroprotective. Furthermore, Sirt3 expression mirrors spatiotemporal deposition of β-amyloid in an AD mouse model and is also upregulated in AD patient temporal neocortex. Thus, our data suggest a role for SIRT3 in mechanisms sensing and tackling ROS- and AD-mediated mitochondrial stress.     

Sirtuin在热量限制延长寿命机制中的研究进展

热量限制(caloric restriction, CR)可以引起细胞、生物体寿命延长和降低衰老相关疾病的发生,其中Sirtuin起着关键作用．Sirtuin将机体能量代谢和基因表达调控相偶联,通过赖氨酸去乙酰化改变蛋白质的活性和稳定性,从而调节衰老进程．酵母中度CR影响其复制寿命和时序寿命,主要依赖于激活Sir2,增加细胞内NAD+/NADH的比例和调节尼克酰胺浓度来实现．类似的机制也存在于秀丽线虫和果蝇中．哺乳动物在CR条件下SIRT1蛋白表达应答性上升,细胞中NAM磷酸基转移酶能够直接影响NAM和NAD+浓度,并影响SIRT1活性．NO表达增加能导致SIRT1上调和线粒体合成增加．SIRT1可能通过改变组蛋白、p53、NES1、FOXO等底物蛋白的乙酰化影响到细胞和个体的衰老．表明不同生物体中的Sirtuin及其同源类似物在CR条件下对衰老进程和寿命都起着非常重要的作用．     

Loss of Sirt1 Function Improves Intestinal Anti-Bacterial Defense and Protects from Colitis-Induced Colorectal Cancer

Dysfunction of Paneth and goblet cells in the intestine contributes to inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Here, we report a role for the NAD⁺-dependent histone deacetylase SIRT1 in the control of anti-bacterial defense. Mice with an intestinal specific Sirt1 deficiency (Sirt1^int−/−) have more Paneth and goblet cells with a consequent rearrangement of the gut microbiota. From a mechanistic point of view, the effects on mouse intestinal cell maturation are mediated by SIRT1-dependent changes in the acetylation status of SPDEF, a master regulator of Paneth and goblet cells. Our results suggest that targeting SIRT1 may be of interest in the management of IBD and CAC.     

Sirtuin 3 regulates mitochondrial protein acetylation and metabolism in tubular epithelial cells during renal fibrosis

Proximal tubular epithelial cells (TECs) demand high energy and rely on mitochondrial oxidative phosphorylation as the main energy source. However, this is disturbed in renal fibrosis. Acetylation is an important post-translational modification for mitochondrial metabolism. The mitochondrial protein NAD⁺-dependent deacetylase sirtuin 3 (SIRT3) regulates mitochondrial metabolic function. Therefore, we aimed to identify the changes in the acetylome in tubules from fibrotic kidneys and determine their association with mitochondria. We found that decreased SIRT3 expression was accompanied by increased acetylation in mitochondria that have separated from TECs during the early phase of renal fibrosis. Sirt3 knockout mice were susceptible to hyper-acetylated mitochondrial proteins and to severe renal fibrosis. The activation of SIRT3 by honokiol ameliorated acetylation and prevented renal fibrosis. Analysis of the acetylome in separated tubules using LC–MS/MS showed that most kidney proteins were hyper-acetylated after unilateral ureteral obstruction. The increased acetylated proteins with 26.76% were mitochondrial proteins which were mapped to a broad range of mitochondrial pathways including fatty acid β-oxidation, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation. Pyruvate dehydrogenase E1α (PDHE1α), which is the primary link between glycolysis and the TCA cycle, was hyper-acetylated at lysine 385 in TECs after TGF-β1 stimulation and was regulated by SIRT3. Our findings showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in TECs. The deacetylation of PDHE1α by SIRT3 at lysine 385 plays a key role in metabolic reprogramming associated with renal fibrosis.Subject terms: Protein-protein interaction networks, End-stage renal disease     

SIRT2‐knockdown rescues GARS‐induced Charcot‐Marie‐Tooth neuropathy

Charcot‐Marie‐Tooth disease is the most common inherited peripheral neuropathy. Dominant mutations in the glycyl‐tRNA synthetase (GARS) gene cause peripheral nerve degeneration and lead to CMT disease type 2D. The underlying mechanisms of mutations in GARS (GARS^CMT2D) in disease pathogenesis are not fully understood. In this study, we report that wild‐type GARS binds the NAD⁺‐dependent deacetylase SIRT2 and inhibits its deacetylation activity, resulting in the acetylated α‐tubulin, the major substrate of SIRT2. The catalytic domain of GARS tightly interacts with SIRT2, which is the most CMT2D mutation localization. However, CMT2D mutations in GARS cannot inhibit SIRT2 deacetylation, which leads to a decrease of acetylated α‐tubulin. Genetic reduction of SIRT2 in the Drosophila model rescues the GARS‐induced axonal CMT neuropathy and extends the life span. Our findings demonstrate the pathogenic role of SIRT2‐dependent α‐tubulin deacetylation in mutant GARS‐induced neuropathies and provide new perspectives for targeting SIRT2 as a potential therapy against hereditary axonopathies.     

NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10

A member of the sirtuin family of NAD⁺-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD⁺-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3^−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD⁺-dependent deacetylase, SIRT3.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.