RESUMO
The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 µm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Núcleo Celular , DNA Mitocondrial/genética , Genômica , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , MicroRNAs/genética , Cimento de Policarboxilato/química , RNA Mensageiro/genética , Análise de Sequência de DNA , Vírus da Estomatite Vesicular IndianaRESUMO
OBJECTIVES: We previously found niacin receptor GPR109A was expressed in murine islet beta-cells, and signaling through GPR109A inhibited glucose stimulated insulin secretion (GSIS). However, the expression of GPR109A in human islets and its functional relevance is still not known. METHODS: The expression of GPR109A was examined by antibody staining and in situ hybridization on pancreatic paraffin sections. GPR109A was cloned and expressed in INS-1 islet beta-cells. Intracellular cAMP and GSIS were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of GPR109A was confirmed in murine islet beta-cells and further detected in human counterparts by using commercially available polyclonal antibodies. In situ hybridization study detected the transcripts of GPR109A, but not that of closely related GPR109B. Furthermore, GPR109A was significantly reduced in islets from diabetic individuals and animal model of db/db mice as compared to their respective controls. Further, GPR109A levels in insulinoma were also reduced dramatically as compared to islets found in corresponding non-tumor normal tissues. Quantitative RT-PCR analysis demonstrated that GPR109A transcripts were severely down-regulated in rodent insulinoma cell lines as compared to that of freshly isolated islets from mice. Finally, human and murine GPR109A expression cassettes were transfected into INS-1 cells, which resulted in reduced accumulation of cAMP and insulin secretion after incubation with niacin. The effect could be completely abrogated by pretreatment with pertussis toxin. CONCLUSIONS: These results demonstrate that GPR109A is functionally expressed in both human and murine islet beta-cells. However, the role of GPR109A in the prevention of diabetes or insulinoma needs further study.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Idoso , Animais , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Imunofluorescência , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Insulinoma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Clodronate liposome injection is an effective approach to selectively and specifically depleting macrophages. Macrophages play a crucial role in cutaneous wound healing and are associated with excessive scar formation. Use of clodronate liposomes to enhance cutaneous wound healing and reduce scar formation could represent a major advance in wound therapy and hypertrophic scar treatment. This study aimed to investigate the effects of subcutaneous or intraperitoneal injection of clodronate liposomes on cutaneous wound healing and scar formation. A burn injury mouse model was used. Mice were treated with subcutaneous or intraperitoneal injection of clodronate liposomes. Wound healing time was analyzed and scar tissues were harvested for hematoxylin and eosin (HE) staining, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses. Wound healing time in treated mice was extended. HE showed that the basal layer of the epidermis in treated scars was flattened, the dermis layer was not significantly thickened, and collagen fibers were well arranged, with few cells and micro vessels. RT-PCR and Western blot analyses showed that the levels of TGF-ß1 and collagen I-α2 were decreased in treated mice. Clodronate liposomes reduce excessive scar formation and delay cutaneous wound healing possibly by reducing collagen deposition and macrophage-derived TGF-ß1 expression.
Assuntos
Queimaduras/metabolismo , Queimaduras/patologia , Cicatriz/metabolismo , Ácido Clodrônico/administração & dosagem , Colágeno/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Queimaduras/tratamento farmacológico , Cicatriz/tratamento farmacológico , Cicatriz/patologia , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Lipossomos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Fatores de Tempo , Cicatrização/efeitos dos fármacos , Cicatrização/imunologiaRESUMO
Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50 µM H(2)O(2) for 6h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H(2)O(2)-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H(2)O(2)-induced NF-κB activation, whereas nicotinamide enhanced H(2)O(2)-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.
Assuntos
Apoptose/fisiologia , Miócitos Cardíacos/fisiologia , NF-kappa B/metabolismo , Estresse Oxidativo , Sirtuína 3/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Regulação da Expressão Gênica , Genes bcl-2/genética , Peróxido de Hidrogênio/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Niacinamida/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/antagonistas & inibidores , Sirtuína 3/genética , Superóxido Dismutase/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Sirtuin 1 (SIRT1) is one member of the silent information regulator 2 (Sir2)-like family of proteins involved in glucose homeostasis in mammals. It has been reported that SIRT1 modulates endocrine signaling of glucose and fat homeostasis by regulating transcription factors such as forkhead transcription factor 3a (FOXO3a), glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor gamma (PPARγ) and PPARγ coactivator (PGC-1α). However, it is still not clear how SIRT1 is involved in the development of insulin resistance. To determine the location and expression of SIRT1 and its target proteins in rats and analyze the interactions and functions of these proteins in insulin resistance. Forty-eight male Sprague-Dawley rats were randomly divided into four regimen groups: normal control (NC), calorie restriction (CR), high-fat (HFa), and high-fructose (HFr). Animals were fed for 12 weeks and blood samples collected from tail veins at weeks 2, 4, 6, 8 and 12 after fasting for 16 h. Baseline metabolic parameters such as fasting blood sugar, insulin, cholesterol and triglycerides were analyzed. A glucose tolerance test was carried out at the end of the study. Visceral fat, consisting of epididymis and perirenal fat, was isolated and weighed. The pancreas from each animal was also immediately removed. Immunohistochemical staining was performed to detect the locations of SIRT1, FOXO3a, GLUT4, PPARγ and PGC-1α in the ß-cell of the rat pancreas. Expression in the pancreas was analyzed by western blotting. Blood biochemical analysis indicated that the HFa and HFr groups were insulin-resistant. Immunohistochemical staining showed that GLUT4 was a nuclear protein. SIRT1, FOXO3a, PPARγ and PGC-1α were present in both the nucleus and the cytoplasm of ß-cells of pancreatic islets. The expression of SIRT1, GLUT4 and PGC-1α increased significantly in response to CR, but decreased in the HFr and HFa groups. FOXO3a was similar in the CR and the NC groups, whereas it declined in the HFa and HFr groups. PPARγ was elevated in the HFa group, but dropped in the CR and HFr groups. These data suggest that SIRT1 and its regulators are involved in the development of insulin resistance.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina/genética , PPAR gama/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Restrição Calórica , Dieta Hiperlipídica , Proteína Forkhead Box O3 , Frutose/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Masculino , Pâncreas/citologia , Pâncreas/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses diverse biochemical and physiological actions, including anti-inflammatory, anti-oxidation, anti-proliferation and promotion of differentiation, and chemopreventive effects. Recently, it is attracting increased attention due to its health benefits, especially in common age-related diseases such as cardiovascular disease, cancer, type 2 diabetes, and neurological conditions. In this review, we discuss the latest cellular and molecular findings that account for the beneficial actions of resveratrol.
Assuntos
Estilbenos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Doenças Cardiovasculares/prevenção & controle , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Inibidores da Agregação Plaquetária/farmacologia , Resveratrol , Estilbenos/farmacologiaRESUMO
BACKGROUND AND AIMS: Caloric restriction (CR) extends mammals' lifespans and suppresses ovary development. Sirtuins are involved in these mechanisms. If, and to what extent CR affects ovarian lifespan and follicle development is largely unknown. We investigated the effects of moderate and severe caloric restriction compared with a high-fat dietary regimen on ovarian follicle reserves in rats. METHODS: Female Sprague-Dawley rats (n=48) randomly divided into four groups including normal control (NC), 25% caloric restriction (MCR), 45% CR (SCR) and high-fat diet (HF) were maintained on these regimens for 2 months. RESULTS: Histological analysis showed that both the 25 and 45% CR rats had a significantly higher percentage of primordial follicles and a larger number of healthy follicles than the NC rats, whereas the HF rats did not differ significantly from the NC rats. Immunohistochemical analysis revealed that SIRT1 and SIRT6 proteins were present in the nucleus and cytoplasm of the oocytes. The 25% CR diet increased the expression of both SIRT1 and SIRT6 in the ovary, whereas the 45% CR and HF diets caused a decrease in SIRT1 expression. The level of SIRT6 protein did not change with the 45% CR diet, and it appeared slightly lower in the HF than in the NC groups. CONCLUSIONS: Caloric restriction may inhibit the transition from primordial to developing follicles and extend the entire growth phase of a follicle to preserve the reserve of germ cells. SIRT1 and SIRT6 are both associated with these effects.
Assuntos
Restrição Calórica , Dieta Hiperlipídica , Folículo Ovariano/metabolismo , Sirtuína 1/biossíntese , Sirtuínas/biossíntese , Animais , Peso Corporal/fisiologia , Colesterol/sangue , Ingestão de Energia/fisiologia , Feminino , Folículo Ovariano/patologia , Ratos , Ratos Sprague-Dawley , Sirtuína 1/metabolismo , Sirtuínas/metabolismo , Triglicerídeos/sangueRESUMO
To investigate the effects of calorie restriction (CR) on behavioral performance and expression of SIRT1 and SIRT5 in rat cerebral tissues. Beginning at 18 months of age, 60 rats were randomly divided into a CR group (n = 30) and a group that remained fed ad libitum (AL; n = 30). CR rats were restricted to a diet of 60% of their daily food consumption. After 6 months of CR, CR rats displayed a maximum 50% reduction in escape latency (AL 20 ± 0.3 s vs. CR 10 ± 0.2 s) and a 3.2 s decrease in time and distance to target when evaluated in Morris water maze tests. The levels of SIRT1 and SIRT5 protein in cerebral tissues of CR rats were elevated compared to AL rats (P < 0.05). CR retarded declines in cognitive ability and enhanced the expression of both SIRT1 and SIRT5 proteins in the cerebral tissue of CR rats compared with AL rats.
Assuntos
Comportamento Animal , Restrição Calórica , Sirtuína 1/metabolismo , Sirtuínas/metabolismo , Animais , Western Blotting , Imunofluorescência , Masculino , Aprendizagem em Labirinto , Células PC12 , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The insulin-like growth factor-1 (IGF-1) plays an important role in the regulation of reproductive function. In the present study, we examined the effects of caloric restriction (CR) on the reproductive lifespan in rats and investigated the potential role of IGF-1. After 10 weeks of treatment, we determined the distribution of the ovarian follicles at various stages and measured the plasma level of IGF-1, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG). Our results show that IGF-1 level was decreased after CR and correlated with the decrease in the levels of LH, FSH and ESG. Moreover, a higher percentage of primordial follicles and surviving follicles was observed in CR rats than in control rats (P<0.05). Immunohistochemical analysis showed that IGF-1 was extensively expressed in the cytoplasm of granulosa cells in the surviving follicles at different stages but not in the atretic follicles. Taken together, these results suggest that caloric restriction promotes the reproductive capacity of female rats via modulating the level of IGF-1, which then regulate pituitary gonadotrope cells to reduce the release of LH, FSH and ESG, and modulate follicular development.
Assuntos
Restrição Calórica , Fator de Crescimento Insulin-Like I/metabolismo , Animais , Estrogênios/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Células da Granulosa/metabolismo , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Folículo Ovariano/fisiologia , Ratos , Reprodução/fisiologiaRESUMO
OBJECTIVE: To study the regulation effects of CR (caloric restriction) and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell. METHODS: The cells were divided into four groups: H2O2, H2O2 + CR, CR and control (high glucose). For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose; for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 µmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 (from 10 to 120 µmol/L). The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot methods detected the expression of SIRT3, Caspase-3. RESULTS: After pretreating with 60 µmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group (74.01 ± 2.21)% retained above 70%, and have statistical significance contrasted with control group (P < 0.05); After pretreating with 120 µmol/L H2O2, the viability of PC12 cells declined significantly (38.22 ± 3.34)%. So 60 µmol/L H2O2 is our experiment concentration. The viability of H2O2 group (74.01 ± 2.21)% was much lower than CR + H2O2 group (97.26 ± 1.92)% (P < 0.05). After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR + H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3 was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group (6857 ± 157) (P < 0.05) increased and decreased in H2O2 group (3786 ± 160) (P < 0.05) contrasted with control group (5256 ± 143). The expression of SIRT3 in CR + H2O2 group (5056 ± 121) (P < 0.05) increased contrasted with H2O2 group (3786 ± 160). We also detected that Caspase-3 in H2O2 group (8499 ± 426) (P < 0.001) was much higher than control group than (5342 ± 420), but in the CR + H2O2 group (5750 ± 438) the expression of Caspase-3 was much lower than H2O2 group (8499 ± 426) (P < 0.001). RT-PCR also showed that the expression of SIRT3 in CR group (7214 ± 148) increased and decreased in H2O2 group (4807 ± 143) (P < 0.05) contrasted with control group (6204 ± 134). The expression of SIRT3 in CR + H2O2 group (6195 ± 166) increased contrasted with H2O2 group (4807 ± 143) (P < 0.05). CONCLUSION: CR causes anti-oxidative injury and has apoptotic effects in PC12 cell. It up-regulates the expression of SIRT3 and the effects of CR-SIRT3 can prevent PC12 cell from H2O2-induced apoptosis. And SIRT3 may be a novel molecule of regulating target in the delay of neuronal senescence.
Assuntos
Restrição Calórica , Estresse Oxidativo , Sirtuína 3/metabolismo , Animais , Peróxido de Hidrogênio/farmacologia , Células PC12 , RatosRESUMO
Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a.
Assuntos
Apoptose , Fatores de Transcrição Forkhead/fisiologia , Oócitos/fisiologia , Ovário/fisiologia , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Ligante Fas/metabolismo , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Crescimento e Desenvolvimento/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The sirtuin proteins are nicotinamide adenine dinucleotide dependent deacetylases and adenosine diphosphate (ADP)-ribosyl transferases associated with metabolic balance and lifespan extension. Sirtuin 1 (SIRT1) and sirtuin 4 (SIRT4) have been reported to regulate insulin secretion, but their association with the development of insulin resistance and nonalcoholic fatty liver disease remain undefined. The aim of this study was to determine the expression of SIRT1 and SIRT4 in the liver and pancreas of rats fed with different diets and analyze the association of these proteins with insulin resistance and nonalcoholic fatty liver disease. Male Sprague-Dawley rats were randomly divided into the following 4 diet treatment groups: normal control (NC), calorie restriction (CR), high-fat (HFa), and high-fructose (HFr), and these groups were maintained for 12 weeks. Blood biochemical analysis and histopathology indicated that HFa and HFr groups were insulin resistant and developed nonalcoholic fatty livers. SIRT1 was present in the nucleus and cytoplasm of the pancreatic beta-cells, while SIRT4 was located in the cytoplasm. Treatment with the CR diet increased the expression of SIRT1 in both the pancreas and liver, while treatment with the HFa and HFr diets caused a decrease. SIRT4 was upregulated in the liver of rats treated with the HFa diet, but did not change with the CR diet treatment. These data suggest that SIRT1 and SIRT4 were both involved in the development of insulin resistance and nonalcoholic fatty liver disease.
Assuntos
Restrição Calórica , Resistência à Insulina/fisiologia , Sirtuína 1/metabolismo , Sirtuínas/metabolismo , Animais , Glicemia/metabolismo , Glicemia/fisiologia , Peso Corporal/fisiologia , Colesterol/sangue , Jejum/sangue , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Insulina/metabolismo , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/fisiologia , Masculino , Hepatopatia Gordurosa não Alcoólica , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
The pool of ovarian primordial follicles is established during embryonic development or at birth. During the development from primordial to primary, secondary, and antral follicles, only a small portion of follicles can mature and successfully ovulate; the others are destined to degenerate through apoptotic or atretic loss. As aging advances, females ultimately enter the cessation phase of the estrous cycle and are no longer capable of fertilization. The presumption is that if we can slow down the process of folliculogenesis or decrease follicle loss, females may have a larger ovarian follicular reserve and a longer reproductive lifespan. In our study, rats underwent intragastric administration with tea polyphenols, quercetin (meletin), genistein, or resveratrol, once a day for 4 months (from age 12 to 15 months), to test whether they have positive effects on follicular reserve or ovarian functions. The results showed that rats treated with tea polyphenols (27.8 +/- 3.2) and quercetin (36.5 +/- 4.1) had a comparable number of healthy follicles to those of controls (26.9 +/- 3.8), although significantly fewer atretic follicles were observed in the tea polyphenol group (43.4 +/- 5.9 vs 79.7 +/- 7.5; p < 0.001). Remarkably, both genistein- and resveratrol-treated rats had more healthy follicles (respectively, 42.8 +/- 3.9, p < 0.05; and 51.9 +/- 6.4, p < 0.001) and fewer atretic follicles (respectively, 58.4 +/- 8.0, p < 0.05; and 51.0 +/- 6.2, p < 0.01) than controls. These results indicate that genistein and resveratrol can increase the ovarian follicular reserve and prolong the ovarian lifespan in rats, and their positive effects may be not only due to their intervention in the transition from primordial to primary follicle, but also due to the inhibiting effect on follicular atresia.
Assuntos
Envelhecimento/fisiologia , Flavonoides/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Fenóis/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Contagem de Células , Avaliação Pré-Clínica de Medicamentos , Ciclo Estral/efeitos dos fármacos , Feminino , Genisteína/farmacologia , Folículo Ovariano/fisiologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Polifenóis , Quercetina/farmacologia , Ratos , Ratos Sprague-Dawley , Resveratrol , Estilbenos/farmacologia , Chá/químicaRESUMO
To investigate the activity of senescence-associated beta-galactosidase (SA-beta-GAL) in the hippocampus of aging rats. Hippocampi of 6-, 18-, and 24-month-old rats were observed by histochemical staining for SA-beta-GAL and cytochemical staining for SA-beta-GAL in cultured hippocampal neurons. The activity of SA-beta-GAL doubled in hippocampal pyramidal cells of the CA3 region in rats between 6 and 18 months (14.57+/-2.74% vs. 31.66+/-14.12% SA-beta-GAL-positive, respectively), and reached 50.76+/-14.41% positive at 24 months. The activity of SA-beta-GAL also increased as a function of time upon prolonged culture of cultured hippocampal neurons with 95% of cells being SA-beta-GAL-positive at 20 days in vitro. Interestingly, no SA-beta-GAL-positive cells were found in neurons of the hippocampal dentate gyrus, a neurogenic region of the brain, at any age examined. SA-beta-GAL can be used as a senescence biomarker in determining senescent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging.
Assuntos
Região CA3 Hipocampal/fisiologia , Senescência Celular , Células Piramidais/fisiologia , beta-Galactosidase/biossíntese , Animais , Biomarcadores , Região CA3 Hipocampal/enzimologia , Células Cultivadas , Masculino , Neurônios/enzimologia , Neurônios/fisiologia , Células Piramidais/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
Spliceosomal introns play a key role in eukaryotic genome evolution and protein diversity. A large Rab GTPase family has been identified in a unicellular eukaryote Trichomonas vaginalis. However, the characteristics of introns in Rab genes of T. vaginalis have not been investigated previously. In this study, we identified a 25-bp spliceosomal intron in the T. vaginalis Rab1a (TvRab1a) gene, the smallest intron in T. vaginalis to be characterized to date. This intron contains a canonical splice site at both 5' (GT) and 3' (AG) ends, and a putative branch-point sequence (TCTAAC) that matches the Trichomonad consensus sequence of ACTAAC except for the first nucleotide. The position and phase of the TvRab1a intron are evolutionarily conserved in Rab1 homologous genes across at least five eukaryotic supergroups, including Opisthokonta, Amoebozoa, Excavata, Chromalveolata, and Plantae. These results strongly suggest that the TvRab1a intron is likely to be an ancient spliceosomal intron, and it can therefore be used as a phylogenetic marker to evaluate particular eukaryotic groupings. Identification and characterization of the TvRabla intron may provide an insight into the evolution of the large Rab repertoire in T. vaginalis.
Assuntos
Íntrons , Spliceossomos/genética , Trichomonas vaginalis/genética , Proteínas rab1 de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Evolução Molecular , Genoma de Protozoário , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spliceossomos/enzimologia , Trichomonas vaginalis/enzimologiaRESUMO
Atherosclerosis is a chronic immuno-inflammatory disease associated with blood lipids disorder. Many studies have demonstrated that caloric restriction (CR) can prevent atherosclerosis and extend lifespan. Sir2 protein, mammal's SIRT1, has been reported to at least partly contribute to the protective effect of CR. Hence, we hypothesize that SIRT1 is a key regulator in the pathogenesis of atherosclerosis and that upregulation of SIRT1 in endothelial cells may mimic CR's beneficial effect on vascular health. The recent studies have demonstrated that endothelial SIRT1 is an anti-atherosclerosis factor and the possible mechanism may be related to inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis, upregulate endothelial nitric oxide synthase (eNOS) expression, and improve endothelium relaxation function. We infer that SIRT1 may be a novel target for atherosclerosis prevention and treatment.
Assuntos
Aterosclerose/prevenção & controle , Sirtuína 1/metabolismo , Animais , Aterosclerose/etiologia , Restrição Calórica , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Humanos , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2-7) are unknown. The present study shows that exposure of H9c2 cells to 50 microM H(2)O(2) for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 microM) eliminated H(2)O(2)-induced apoptosis. Resveratrol also prevented H(2)O(2)-induced caspase-3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase-3 activity. These results suggest that resveratrol protects cardiomyocytes from H(2)O(2)-induced apoptosis by activating SIRT1,3,4,7.
Assuntos
Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Sirtuínas/genética , Estilbenos/farmacologia , Animais , Caspase 3/metabolismo , Linhagem Celular , Miócitos Cardíacos/citologia , Ratos , Resveratrol , Sirtuína 1RESUMO
Recent studies in mice have shown that resveratrol can protect the liver from fat accumulation induced by high fat diet. However, the exact mechanism is largely unknown. To explore the possible mechanism, we investigated the anti-lipogenic effect of resveratrol in vitro model. Oil Red O staining revealed that resveratrol could significantly ameliorate the excessive triglyceride accumulation in HepG2 cells induced by palmitate. The results of RT-PCR and Western blotting showed that resveratrol upregulated the expression of Sirt1 and forkhead box O1 (FOXO1), whereas downregulated the expression of sterol regulatory element binding protein1 (SREBP1). Moreover, resveratrol was shown to inhibit the activity of SREBP1, as evaluated by immunofluorescence assay. Our results suggest that resveratrol may attenuate fat deposition by inhibiting SREBP1 expression via Sirt1-FOXO1 pathway and thus may have application for the treatment of NAFLD.
Assuntos
Fígado Gorduroso/metabolismo , Hipolipemiantes/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Estilbenos/farmacologia , Triglicerídeos/metabolismo , Animais , Restrição Calórica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Modelos Biológicos , Ácido Palmítico/farmacologia , Resveratrol , Transdução de Sinais , Sirtuína 1 , Sirtuínas/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/biossínteseRESUMO
Loss of cardiomyocytes through apoptosis has been proposed as a cause of ventricular remodeling and heart failure. Ischemia- and hypoxia-induced apoptosis of cardiomyocytes reportedly plays an important role in many cardiac pathologies. We investigated whether resveratrol (Res) has direct cytoprotective effects against ischemia/hypoxia for cardiomyocytes. Exposure of H9c2 embryonic rat heart-derived cells to hypoxia for 24h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, while treatment with 20 microM Res greatly decreased hypoxia-induced apoptosis in these cells. Exposure of the cells to Res (20 microM) caused rapid activation of SIRT1, which had a dual effect on FoxO1 function: SIRT1 increased FoxO1's ability to induce cell cycle arrest, but inhibited FoxO1's ability to induce cell death. This effect could be reversed by SIRT1 inhibition. Results of our study indicate that Res inhibits hypoxia-induced apoptosis via the SIRT1-FoxO1 pathway in H9c2 cells. This polyphenol may have potential in preventing cardiovascular disease, especially in coronary artery disease (CAD) patients.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Citoproteção , Miócitos Cardíacos/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Isquemia Miocárdica/patologia , Proteínas do Tecido Nervoso/metabolismo , Ratos , Resveratrol , Sirtuína 1 , Sirtuínas/agonistas , Sirtuínas/metabolismoRESUMO
Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.