Resveratrol, a SIRT1 activator, attenuates aging-associated alterations in skeletal muscle and heart in mice.

Aging is associated with impairment of multiple organs, including skeletal muscle and heart. In this study, we investigated whether resveratrol, an activator of an NAD+-dependent protein deacetylase Sirtuin-1 (SIRT1), attenuates age-related sarcopenia and cardiomyocyte hypertrophy in mice. Treatment of mice with resveratrol (0.4 g/kg diet) from 28 weeks of age for 32 weeks prevented aging-associated shortening of rotarod riding time. In the tibialis anterior (TA) muscle, histogram analysis showed that the atrophic muscle was increased in 60-week-old (wo) mice compared with 20-wo mice, which was attenuated by resveratrol. In the heart, resveratrol attenuated an aging-associated increase in the cardiomyocyte diameter. Acetylated proteins were increased and autophagic activity was reduced in the TA muscle of 60-wo mice compared with those of 20-wo mice. Resveratrol treatment reduced levels of acetylated proteins and restored autophagic activity in the TA muscle. Aging-related reduction in myocardial autophagy was also suppressed by resveratrol. Skeletal muscle-specific SIRT1 knockout mice showed increases in acetylated proteins and atrophic muscle fibers and reduced autophagic activity in the TA muscle. These results suggest that activation of SIRT1 by treatment with resveratrol suppresses sarcopenia and cardiomyocyte hypertrophy by restoration of autophagy in mice.

SIRT1 in the cardiomyocyte counteracts doxorubicin-induced cardiotoxicity via regulating histone H2AX.

AIMS: Cardiotoxicity by doxorubicin predicts worse prognosis of patients. Accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. SIRT1, an NAD+-dependent histone/protein deacetylase, protects cells by deacetylating target proteins. We investigated whether SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating Ser139 phosphorylation of histone H2AX, a critical signal of the DNA damage response. METHODS AND RESULTS: Doxorubicin (5 mg/kg per week, x4) was administered to mice with intact SIRT1 (Sirt1f/f) and mice that lack SIRT1 activity in cardiomyocytes (Sirt1f/f;MHCcre/+). Reductions in left ventricular fractional shortening and ejection fraction by doxorubicin treatment were more severe in Sirt1f/f;MHCcre/+ than in Sirt1f/f. Myocardial expression level of type-B natriuretic peptide was 2.5-fold higher in Sirt1f/f;MHCcre/+ than in Sirt1f/f after doxorubicin treatment. Sirt1f/f;MHCcre/+ showed larger fibrotic areas and higher nitrotyrosine levels in the heart after doxorubicin treatment. Although doxorubicin-induced DNA damage evaluated by TUNEL staining was enhanced in Sirt1f/f;MHCcre/+, the myocardium from Sirt1f/f;MHCcre/+ showed blunted Ser139 phosphorylation of H2AX by doxorubicin treatment. In H9c2 cardiomyocytes, SIRT1 knockdown attenuated Ser139 phosphorylation of H2AX, increased DNA damage, and enhanced caspase-3 activation under doxorubicin treatment. Immunostaining revealed that acetylation level of H2AX at Lys5 was higher in hearts from Sirt1f/f;MHCcre/+. In H9c2 cells, acetyl-Lys5-H2AX level was increased by SIRT1 knockdown and reduced by SIRT1 overexpression. Ser139 phosphorylation in response to doxorubicin treatment was blunted in a mutant H2AX with substitution of Lys5 to Gln (K5Q) that mimics acetylated lysine compared with that in wild-type H2AX. Expression of K5Q-H2AX as well as S139A-H2AX, which cannot be phosphorylated at Ser139, augmented doxorubicin-induced caspase-3 activation. Treatment of mice with resveratrol, a SIRT1 activator, attenuated doxorubicin-induced cardiac dysfunction, which was associated with a reduction in acetyl-Lys5-H2AX level and a preserved phospho-Ser139-H2AX level. CONCLUSION: These findings suggest that SIRT1 counteracts doxorubicin-induced cardiotoxicity by mediating H2AX phosphorylation through its deacetylation in cardiomyocytes.

Downregulation of IGFBP5 contributes to replicative senescence via ERK2 activation in mouse embryonic fibroblasts.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) are secretory proteins that regulate IGF signaling. In this study, we investigated the role of IGFBP5 in replicative senescence in embryonic mouse fibroblasts (MEFs). During passages according to the 3T3 method, MEFs underwent senescence after the 5th passage (P5) based on cell growth arrest, an increase in the number of cells positive for senescence-associated ß-galactosidase (SA-ß-GAL) staining, and upregulation of p16 and p19. In P8 MEFs, IGFBP5 mRNA level was markedly reduced compared with that in P2 MEFs. Downregulation of IGFBP5 via siRNA in P2 MEFs increased the number of SA-ß-GAL-positive cells, upregulated p16 and p19, and inhibited cell growth. Incubation of MEFs with IGFBP5 during serial passage increased the cumulative population doubling and decreased SA-ß-GAL positivity compared with those in vehicle-treated cells. IGFBP5 knockdown in P2 MEFs increased phosphorylation levels of ERK1 and ERK2. Silencing of ERK2, but not that of ERK1, blocked the increase in the number of SA-ß-GAL-positive cells in IGFBP5-knockdown cells. The reduction in the cell number and upregulation of p16 and p21 in IGFBP5-knockdown cells were attenuated by ERK2 knockdown. Our results suggest that downregulation of IGFBP5 during serial passage contributes to replicative senescence via ERK2 in MEFs.

Ubiquitin-dependent rapid degradation conceals a cell-protective function of cytoplasmic SIRT3 against oxidative stress.

SIRT3 is an NAD+-dependent protein deacetylase localized in mitochondria. Several studies reported localization of SIRT3 in the cytoplasm or nucleus, but data of these studies were not consistent. We detected expression of mitochondrial (SIRT3mt) and cytoplasmic (SIRT3ct) Sirt3 mRNAs in the mouse brain, and we also found SIRT3 immunostaining of mitochondria and cytoplasm in the brain and cultured neural cells. However, expression levels of SIRT3ct in COS cells transfected with SIRT3ct cDNA were much lower than those of SIRT3mt. We found that SIRT3ct but not SIRT3mt was promptly degraded by ubiquitin-dependent degradation, in which SIRT3ct degradation was mediated mainly by ubiquitination of NH2-terminal methionine and partly by that of lysine residues of SIRT3ct. SIRT3ct expression level was significantly enhanced by the treatment of cells with staurosporine or H2O2. H2O2 treatment promoted nuclear translocation of SIRT3ct and induced histone H3 deacetylation and superoxide dismutase 2 expression. Overexpression of SIRT3ct decreased cell death caused by H2O2 at levels similar to those achieved by overexpression of SIRT3mt. Knockdown of Sirt3 mRNA increased cell death caused by amyloid-ß (Aß), and overexpression of SIRT3ct suppressed the toxic function of Aß in PC12 cells. These results indicate that SIRT3ct promotes cell survival under physiological and pathological conditions.

Aging-associated inflammation and fibrosis in arachnoid membrane.

BACKGROUND: The physiological and pathological significance of the arachnoid membrane (AM) is still unknown. In this study, we investigated various characteristics of the AM, focusing on the influence of inflammation and fibrosis. METHODS: Small pieces of AM sample were obtained during neurosurgical procedures from 74 cases. The clinical and pathological characteristics of the hyperplastic AM group (≥ 50 µm) and the non-hyperplastic AM group (< 50 µm) were compared. Then, potential correlations between AM thickness and clinical characteristics were analyzed. Moreover, VEGFα, TGFß, and TGFα levels were quantitated by real time PCR. Then, the potential correlations between AM thickness and these inflammatory or anti-inflammatory markers, and the influence of the original disease were calculated. RESULTS: The median age of the patients in hyperplastic AM group was significantly older than that of the non-hyperplastic AM group. Moreover, the number of fibroblasts, CD68+ cells, CD86+ cells, and CD206+ cells in the hyperplastic AM group was significantly higher than that in the non-hyperplastic AM group. The AM thickness was significantly correlated to age and number of fibroblasts, CD68+ cells, CD86+ cells, and CD206+ cells. The thickness of the AM was significantly correlated to the messenger RNA expression levels of VEGFα (ρ = 0.337), and the VEGFα expression levels were significantly correlated with TGFß and TNFα. CONCLUSIONS: The AM hyperplasia was influenced by aging and could be a result of inflammation and fibrosis through cytokine secretion from the inflammatory cells and fibroblasts in the AM.

SIRT1 decelerates morphological processing of oligodendrocyte cell lines and regulates the expression of cytoskeleton-related oligodendrocyte proteins.

SIRT1 is involved in the regulation of a variety of biological processes such as metabolism, stress response, autophagy and differentiation. Although progenitor cells of oligodendrocytes (OPCs) express high level of SIRT1, its function on differentiation is unknown. Because we have shown that SIRT1 plays a pivotal role in differentiation of neural precursor cells, we hypothesized that SIRT1 may also participate in the differentiation of oligodendrocytes (OLGs). We examined whether SIRT1 was expressed in two human oligodendrocyte cell lines: KG-1-C and MO 3.13 OLG. Transfection of cell lines with SIRT1-siRNA and SIRT2-siRNA promoted the extension of cellular processes. SIRT1-siRNA and SIRT2-siRNA increased acetyl-α-tubulin level, conversely, over expression of SIRTs resulted in decreased the ratio of acetyl-α-tubulin to α-tubulin. We also found knockdown of SIRT1 and SIRT2 induced overexpression of ßIV-tubulin and tubulin polymerization promoting protein (TPPP) (OLG-specific cytoskeleton-related molecules) that distributed widely in cell bodies. Taken together, SIRT1 may play a role in oligodenroglial differentiation and myelinogenesis.

Different Antioxidative and Antiapoptotic Effects of Piceatannol and Resveratrol.

Resveratrol affords protection against reactive oxygen species (ROS)-related diseases via activation of SIRT1, an NAD+-dependent deacetylase. However, the low bioavailability of resveratrol limits its therapeutic applications. Since piceatannol is a hydroxyl analog of resveratrol with higher bioavailability, it could be an alternative to resveratrol. In this study, we compared the cytotoxicity, antioxidative activity, and mechanisms of cytoprotection of piceatannol with those of resveratrol. In C2C12 cells incubated with piceatannol, electrospray ionization mass spectrometry analysis showed that piceatannol was present in the intracellular fraction. A high concentration (50 µM) of piceatannol, but not resveratrol, induced mitochondrial depolarization and apoptosis. However, piceatannol at 10 µM inhibited the increase in mitochondrial ROS level induced by antimycin A, and this ROS reduction was greater than that by resveratrol. The reduction in hydrogen peroxide-induced ROS by piceatannol was also greater than that by resveratrol or vitamin C. Piceatannol reduced antimycin A-induced apoptosis more than did resveratrol. SIRT1 knockdown abolished the antiapoptotic activity of resveratrol, whereas it blocked only half of the antiapoptotic activity of piceatannol. Piceatannol, but not resveratrol, induced heme oxygenase-1 (HO1) expression, which was blocked by knockdown of the transcription factor NRF2, but not by SIRT1 knockdown. HO1 knockdown partially blocked the reduction of ROS by piceatannol. Furthermore, the antiapoptotic action of piceatannol was abolished by HO1 knockdown. Our results suggest that the therapeutic dose of piceatannol protects cells against mitochondrial ROS more than does resveratrol via SIRT1- and NRF2/HO1-dependent mechanisms. The activation of NRF2/HO1 could be an advantage of piceatannol compared with resveratrol for cytoprotection. SIGNIFICANCE STATEMENT: This study showed that piceatannol and resveratrol were different in cytotoxicity, oxidant-scavenging activities, and mechanisms of cytoprotection. Protection by piceatannol against apoptosis induced by reactive oxygen species was superior to that by resveratrol. In addition to the sirtuin 1-dependent pathway, piceatannol exerted nuclear factor erythroid 2-related factor 2/heme oxygenase-1-mediated antioxidative and antiapoptotic effects, which could be an advantage of piceatannol compared with resveratrol.

Resveratrol improves motor function in patients with muscular dystrophies: an open-label, single-arm, phase IIa study.

Muscular dystrophies (MDs) are inherited disorders characterized by progressive muscle weakness. Previously, we have shown that resveratrol (3,5,4'-trihydroxy-trans-stilbene), an antioxidant and an activator of the protein deacetylase SIRT1, decreases muscular and cardiac oxidative damage and improves pathophysiological conditions in animal MD models. To determine whether resveratrol provides therapeutic benefits to patients with MDs, an open-label, single-arm, phase IIa trial of resveratrol was conducted in 11 patients with Duchenne, Becker or Fukuyama MD. The daily dose of resveratrol was 500 mg/day, which was increased every 8 weeks to 1000 and then 1500 mg/day. Primary outcomes were motor function, evaluated by a motor function measure (MFM) scale, muscular strength, monitored with quantitative muscle testing (QMT), and serum creatine kinase (CK) levels. Adverse effects and tolerability were evaluated as secondary outcomes. Despite the advanced medical conditions of the patients, the mean MFM scores increased significantly from 34.6 to 38.4 after 24 weeks of medication. A twofold increase was found in the mean QMT scores of scapula elevation and shoulder abduction. Mean CK levels decreased considerably by 34%. Diarrhoea and abdominal pain was noted in six and three patients, respectively. Resveratrol may provide some benefit to MD patients.

SIRT1 deficiency interferes with membrane resealing after cell membrane injury.

Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000-3001 µm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms.

Resveratrol Decreases Oxidative Stress by Restoring Mitophagy and Improves the Pathophysiology of Dystrophin-Deficient mdx Mice.

We previously showed that treatment with resveratrol (3,5,4'-trihydroxy-trans-stilbene), an activator of the NAD+-dependent deacetylase SIRT1 at 4 g/kg food for 32 weeks, significantly decreased the muscular reactive oxygen species (ROS) levels and ameliorated the pathology of mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Here, we treated mdx mice with various doses of resveratrol (0.04, 0.4, and 4 g/kg food) for 56 weeks and examined the effects on serum creatine kinase levels and physical activities. Because resveratrol promotes autophagy, we also investigated whether autophagy including mitochondrial autophagy (mitophagy) is involved in resveratrol's effects. Autophagy/mitophagy-related genes and autophagic flux were downregulated in the muscle of mdx mice, and these phenomena were reversed by resveratrol with significant ROS reduction. Resveratrol at 4 g/kg food reduced the number of immature myofibers containing central nuclei and fine fibers < 400 µm2 and increased that of thicker myofibers in the quadriceps, suggesting that resveratrol decreased myofiber wasting and promoted muscular maturation. Accordingly, resveratrol at 0.4 g/kg food reduced the creatine kinase levels to one-third of those in untreated mdx mice and significantly increased the animals' physical activities. In C2C12 myoblast cells, resveratrol promoted mitophagy and eliminated mitochondria containing high superoxide levels. The clearance of damaged mitochondria and ROS reduction by resveratrol was completely suppressed by an autophagy inhibitor (chloroquine) and by knocking down Atg5 or Pink1, essential genes for autophagy and mitophagy, respectively. Thus, resveratrol is a potential therapeutic agent for DMD, and the clearance of damaged mitochondria probably contributes to its action.

Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice.

Autophagy activation improves the phenotype in mdx mice, a Duchenne muscular dystrophy (DMD) model, although the underlying mechanisms are obscure. We previously found that resveratrol, a strong inducer of autophagy, ameliorates the cardiac pathology of mdx mice. Autophagy could eliminate damaged mitochondria, a major source of intracellular reactive oxygen species (ROS), although there is no evidence for mitochondriopathy in dystrophic cardiomyopathy. To elucidate resveratrol's function, we investigated the deletion of mitochondrial DNA (mtDNA), autophagy of damaged mitochondria (mitophagy), and ROS accumulation in the mdx mouse heart. Low levels of normal mtDNA and abnormal accumulations of mitochondria-containing autophagosomes were found in the mdx mouse heart. Administering resveratrol to mdx mice for 56 weeks ameliorated the cardiomyopathy, with significant reductions in the amount of mtDNA deletion, the number of mitochondria-containing autophagosomes, and the ROS levels. Resveratrol induced nuclear FoxO3a accumulation and the expression of autophagy-related genes, which are targets of FoxOs. The most effective dose in mdx mice was 0.4 g resveratrol/kg food. In conclusion, resveratrol improved cardiomyopathy by promoting mitophagy in the mdx mouse heart. We propose that acquired mitochondriopathy worsens the pathology of DMD and is a potential therapeutic target for the cardiomyopathy in DMD patients.

Pterostilbene and Its Glucoside Induce Type XVII Collagen Expression.

The glycosylation of pterostilbene by cultured plant cells of Phytolacca americana gave pterostilbene 4'-O-ß-D-glucoside. Both pterostilbene and its 4'-0-ß-D- glucoside induced type XVII collagen expression in the EpiDermFT EFT-400 human skin cell model. Pterostilbene 4'-O-ß-D-glucoside strongly induced type XVII collagen expression rather than pterostilbene.

SIRT1: A Novel Target for the Treatment of Muscular Dystrophies.

Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and is caused by mutations in the gene that encodes the cytoskeletal protein dystrophin. The treatment for DMD is limited to glucocorticoids, which are associated with multiple side effects. Thus, the identification of novel therapeutic targets is urgently needed. SIRT1 is an NAD(+)-dependent histone/protein deacetylase that plays roles in diverse cellular processes, including stress resistance and cell survival. Studies have shown that SIRT1 activation provides beneficial effects in the dystrophin-deficient mdx mouse, a model of DMD. SIRT1 activation leads to the attenuation of oxidative stress and inflammation, a shift from the fast to slow myofiber phenotype, and the suppression of tissue fibrosis. Although further research is needed to clarify the molecular mechanisms underlying the protective role of SIRT1 in mdx mice, we propose SIRT1 as a novel therapeutic target for patients with muscular dystrophies.

Biochemical effects of the flavanol-rich lychee fruit extract on the melanin biosynthesis and reactive oxygen species.

An ingredient of fruit polyphenol, resveratrol, is known to have an inhibitory effect on melanogenesis. In order to examine the functional differences between resveratrol and other fruit polyphenols, we compared biochemical effects of a resveratrol-free polyphenol, flavanol-rich lychee fruit extract (FRLFE), with other phenolic compounds including resveratrol. FRLFE as well as hydroquinone and resveratrol suppressed growth of B16F1 melanoma cells more significantly than rhododendrol or arbutin. Resveratrol suppressed mushroom tyrosinase at the lowest concentration (23.0 µmol/L) among the compounds tested. FRLFE and hydroquinone suppressed tyrosinase at almost the same concentration (half maximal inhibitory concentration [IC50 ], 83.5 and 94.6 µmol/L, respectively), which was higher than rhododendrol, ascorbic acid and arbutin (IC50 , 245, 345 and 421 µmol/L, respectively). Western blot analysis revealed that although resveratrol decreased expressions of tyrosinase and tyrosinase-related protein 1, FRLFE did not affect their expressions. Both FRLFE and resveratrol suppressed antimycin A-mediated reactive oxygen species (ROS) production in melanocytic cells. Resveratrol-mediated ROS suppression was inhibited by nicotinamide, a SIRT1 inhibitor. However, FRLFE-mediated suppression was not affected by nicotinamide. Moreover, FRLFE directly decreased superoxide in vitro, as detected by superoxide dismutase-like scavenging activity assay. These results suggest that FRLFE can protect melanocytes from cytotoxicity caused by an excess amount of melanin and ROS in a different manner from resveratrol.

The effects of resveratrol and SIRT1 activation on dystrophic cardiomyopathy.

The muscular dystrophies, which cause progressive weakening of the skeletal muscles, are frequently associated with cardiomyopathy. In fact, the leading cause of mortality in patients with Duchenne muscular dystrophy, the most common and most severe type of muscular dystrophy, is heart failure due to cardiomyopathy. Therefore, more effective methods for treating cardiomyopathy are expected to improve long-term outcomes for patients with Duchenne muscular dystrophy. Our recent preclinical data show that treatment with the SIRT1 activator resveratrol is beneficial for dystrophic cardiomyopathy. We examined the effects of resveratrol treatment in two different models of muscular dystrophy: dystrophin-deficient mdx mice and δ-sarcoglycan-deficient TO-2 hamsters. In both models, resveratrol suppressed cardiac hypertrophy, preserved cardiac function, and reduced tissue fibrosis in the diseased heart. Importantly, resveratrol significantly improved survival in TO-2 hamsters. Resveratrol also attenuated skeletal muscle pathology in mdx mice. These promising results indicate resveratrol's potential for clinical translation to treat cardiomyopathy in patients with muscular dystrophies.

Chloroquine potentiates temozolomide cytotoxicity by inhibiting mitochondrial autophagy in glioma cells.

Mitochondrial autophagy eliminates damaged mitochondria and decreases reactive oxygen species (ROS). The autophagy inhibitor chloroquine (CQ) potentiates temozolomide (TMZ) cytotoxicity in glioma cells, but it is not known whether CQ does this by inhibiting mitochondrial autophagy. The effects of CQ and TMZ on MitoSOX Red fluorescence, a mitochondrial ROS indicator, and cell death were examined in rat C6 glioma cells. Mitochondrial autophagy was monitored by the colocalization of MitoTracker Red fluorescence and EGFP-LC3 dots. Mitochondrial content was measured by MitoTracker Green fluorescence and immunoblotting for a mitochondrial protein. Finally, CQ's effects on tumor cells derived from a glioblastoma patient and human U87-MG glioblastoma cells were assessed. TMZ (100-1,000 µM) alone did not affect mitochondrial ROS or cell death in C6 cells, but when administered with CQ (10 µM), it increased mitochondrial ROS and cell death. Antioxidants significantly suppressed the CQ-augmented cell death in TMZ-treated cells, indicating that mitochondrial ROS were involved in this cell death. TMZ treatment reduced MitoTracker Green fluorescence and mitochondrial protein levels, and these effects were inhibited by CQ. TMZ also increased the colocalization of EGFP-LC3 dots with mitochondria, and CQ enhanced this effect. CQ potentiated TMZ-induced cytotoxicity in patient-derived glioblastoma cells as well as human U87-MG glioblastoma cells. These results suggest that CQ increases cellular ROS and augments TMZ cytotoxicity in glioma cells by inhibiting mitochondrial autophagy.

Sirt1-deficient mice have hypogonadotropic hypogonadism due to defective GnRH neuronal migration.

Hypogonadatropic hypogonadism (HH) can be acquired through energy restriction or may be inherited as congenital hypogonadotropic hypogonadism and its anosmia-associated form, Kallmann's syndrome. Congenital hypogonadotropic hypogonadism is associated with mutations in a group of genes that impact fibroblast growth factor 8 (FGF8) function. The Sirt1 gene encodes a nicotinamide adenine dinucleotide-dependent histone deacetylase that links intracellular metabolic stress to gene expression. Herein Sirt1(-/-) mice are shown to have HH due to failed GnRH neuronal migration. Sirtuin-1 (Sirt1) catalytic function induces GnRH neuronal migration via binding and deacetylating cortactin. Sirt1 colocalized with cortactin in GnRH neurons in vitro. Sirt1 colocalization with cortactin was regulated in an FGF8/fibroblast growth factor receptor-1 dependent manner. The profound effect of Sirt1 on the hormonal status of Sirt1(-/-) mice, mediated via defective GnRH neuronal migration, links energy metabolism directly to the hypogonadal state. Sirt1-cortactin may serve as the distal transducer of neuronal migration mediated by the FGF8 synexpression group of genes that govern HH.

Translocation of glycogen synthase kinase-3ß (GSK-3ß), a trigger of permeability transition, is kinase activity-dependent and mediated by interaction with voltage-dependent anion channel 2 (VDAC2).

Glycogen synthase kinase-3ß (GSK-3ß) is a major positive regulator of the mitochondrial permeability transition pore (mPTP), a principle trigger of cell death, under the condition of oxidative stress. However, the mechanism by which cytosolic GSK-3ß translocates to mitochondria, promoting mPTP opening, remains unclear. Here we addressed this issue by analyses of the effect of site-directed mutations in GSK-3ß on mitochondrial translocation and protein/protein interactions upon oxidative stress. H9c2 cardiomyoblasts were transfected with GFP-tagged GSK-3ß (WT), a mutant GSK-3ß insensitive to inhibitory phosphorylation (S9A), or kinase-deficient GSK-3ß (K85R). Time lapse observation revealed that WT and S9A translocated from the cytosol to the mitochondria more promptly than did K85R after exposure to oxidative stress. H2O2 increased the density of nine spots on two-dimensional gel electrophoresis of anti-GSK-3ß-immunoprecipitates by more than 3-fold. MALDI-TOF/MS analysis revealed that one of the spots contained voltage-dependent anion channel 2 (VDAC2). Knockdown of VDAC2, but not VDAC1 or VDAC3, by siRNA attenuated both the mitochondrial translocation of GSK-3ß and mPTP opening under stress conditions. The mitochondrial translocation of GSK-3ß was attenuated also when Lys-15, but not Arg-4 or Arg-6, in the N-terminal domain of GSK-3ß was replaced with alanine. The oxidative stress-induced mitochondrial translocation of GSK-3ß was associated with an increase in cell death, which was suppressed by lithium chloride (LiCl), a GSK-3ß inhibitor. These results demonstrate that GSK-3ß translocates from the cytosol to mitochondria in a kinase activity- and VDAC2-dependent manner in which an N-terminal domain of GSK-3ß may function as a mitochondrial targeting sequence.

SIRT1 regulates lamellipodium extension and migration of melanoma cells.

Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells.

Regulation of FOXOs and p53 by SIRT1 modulators under oxidative stress.

Excessive reactive oxygen species (ROS) induce apoptosis and are associated with various diseases and with aging. SIRT1 (sirtuin-1), an NAD+-dependent protein deacetylase, decreases ROS levels and participates in cell survival under oxidative stress conditions. SIRT1 modulates the transcription factors p53, a tumor suppressor and inducer of apoptosis, and the forkhead O (FOXO) family, both of which play roles for cell survival and cell death. In this study, we aimed to know which is working greatly among p53 and FOXOs transcription factors in SIRT1's cell protective functions under oxidative stress conditions. The antimycin A-induced increase in ROS levels and apoptosis was enhanced by SIRT1 inhibitors nicotinamide and splitomicin, whereas it was suppressed by a SIRT1 activator, resveratrol, and a SIRT1 cofactor, NAD+. SIRT1-siRNA abolished the effects of splitomicin and resveratrol. p53-knockdown experiment in C2C12 cells and experiment using p53-deficient HCT116 cells showed that splitomicin and resveratrol modulated apoptosis by p53-dependent and p53-independent pathways. In p53-independent cell protective pathway, we found that FOXO1, FOXO3a, and FOXO4 were involved in SOD2's upregulation by resveratrol. The knockdown of these three FOXOs by siRNAs completely abolished the SOD2 induction, ROS reduction, and anti-apoptotic function of resveratrol. Our results indicate that FOXO1, FOXO3a and FOXO4, are indispensable for SIRT1-dependent cell survival against oxidative stress, although deacetylation of p53 has also some role for cell protective function of SIRT1.