Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

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.

Niacin receptor GPR109A inhibits insulin secretion and is down-regulated in type 2 diabetic islet beta-cells.

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.

Clodronate liposomes reduce excessive scar formation in a mouse model of burn injury by reducing collagen deposition and TGF-ß1 expression.

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.

SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB.

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.

SIRT1 interacts with metabolic transcriptional factors in the pancreas of insulin-resistant and calorie-restricted rats.

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.

Cellular and molecular effects of resveratrol in health and disease.

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.

The effects of caloric restriction and a high-fat diet on ovarian lifespan and the expression of SIRT1 and SIRT6 proteins in rats.

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.

SIRT1 and SIRT5 activity expression and behavioral responses to calorie restriction.

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.

Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1).

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.

[Effects of caloric restriction on the oxidative stress injury and the expression of SIRT3 in PC12 cell].

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.