Doxycycline in Extremely Low Dose Improves Glycemic Control and Islet Morphology in Mice Fed a High-Fat Diet.

PURPOSE: Chronic low-grade inflammation is detected in obese and diabetic individuals. Tetracyclines, used as antibiotics for years, have been demonstrated to have diverse non-bactericidal effects, including anti-tumor and anti-inflammatory activities. This study aimed to investigate whether doxycycline at sub-antimicrobial concentrations could improve glycemic control in mice fed a high-fat diet, through its anti-inflammatory activities. METHODS: C57BL/6J mice were fed with a high-fat diet to induce diabetic and obese conditions. Three sub-antimicrobial dosages of doxycycline (200, 20, and 2 µg/mL) were added to drinking water for 23 weeks during the housing phase. RESULTS: Doxycycline at 200 µg/mL tended to increase body weight, islet mass, and the percentage of large islets (diameter >350 µm). At 20 µg/mL, doxycycline significantly improved glucose tolerance and decreased fasting blood glucose. At 2 µg/mL, doxycycline increased the percentage of small islets (diameter <80 µm). Serum C-reactive protein and lipopolysaccharide levels significantly decreased while the beta-cell ratio increased in all doxycycline-administered mice. CONCLUSION: Our results suggest that doxycycline, even at an extremely low dose, could improve glycemic control and islet morphology via its anti-inflammatory activities.

SIRT1 knock-in mice preserve ovarian reserve resembling caloric restriction.

Previous studies have proposed that caloric restriction (CR) regulates many cell functions and prolongs the lifespan of an organism. Our previous studies proposed that CR also prevents follicular activation and preserves the ovarian reserve in mice by activating SIRT1. To test if SIRT1 preserves the ovarian reserve and prolongs the ovarian longevity, we generated SIRT1 knock-in mice that can overexpress SIRT1 in oocytes of the mouse. Ovaries of the mice at ages 35 days and 15 months were collected, and the follicular development and follicular reserve were examined. The vaginal opening and onset of estrus of transgenic female mice (both the homozygous and heterozygous for SIRT1 overexpression) were later than that of wild-type mice. Both the homozygous and heterozygous SIRT1-overexpressing mice had a larger and stronger reproductive capacity than wild-type mice. Moreover, 35-day-old and 15-month-old homozygous and heterozygous SIRT1-overexpressing mice also had a higher mean number and percentage of healthy follicles, fewer atretic follicles than wild-type mice, and the mean number and percentage of primordial follicles in both the homozygous and heterozygous SIRT1-overexpressing mice were higher than wild-type mice at the same age. However, the phenotypes of heterozygous and homozygous transgenic mice came no difference. Immunohistochemistry showed increased expression of SIRT1 and FOXO3a, and decreased expression of mTOR in both the homozygous and heterozygous SIRT1-overexpressing mice compared with wild-type mice. Thus, oocyte-specific SIRT1-overexpressing mice continuously activate FOXO3a and suppress mTOR and have a larger reproductive capacity, larger follicle reserve and longer ovarian lifespan.

Silent Information Regulator 1 Negatively Regulates Atherosclerotic Angiogenesis via Mammalian Target of Rapamycin Complex 1 Signaling Pathway.

BACKGROUND: This study aimed to investigate the interactions between silent information regulator 1 (SIRT1) and mammalian target of rapamycin (mTOR) in intraplaque angiogenesis and their potential mechanisms through in vivo and in vitro studies. METHODS: An atherosclerosis model was established in 12 rabbits on a high-cholesterol diet. The rabbits were equally divided into 3 groups: a control group (high-lipid diet), RAP group (high-lipid diet supplemented with rapamycin) and RAP + NAM group (high-lipid diet supplemented with rapamycin and nicotinamide). At the end of 4 weeks, the area of plaques in the aorta was determined and the protein expression of CD31 and vascular endothelial growth factor (VEGF) was detected through hematoxylin and eosin staining and immunohistochemical staining, respectively. For in vitro study, a hypoxia model was established in human umbilical vein endothelial cells (HUVECs) by using the chemical method (CoCl2). The MTT assay, scratch assay and tube formation assay were performed to evaluate the proliferation and angiogenesis abilities of HUVECs. Reverse transcription polymerase chain reaction was used to examine the mRNA levels of SIRT1, hypoxia-inducible factor-1α (HIF-1α), mTOR and p70 ribosomal S6 kinase (p70S6K). Western blotting was used to examine the protein levels of SIRT1, HIF-1α, mTOR, p-mTOR, p-raptor and p-p70S6K. RESULTS: The results of the in vivo study indicated a significant inhibitory effect of rapamycin on plaque size and intraplaque angiogenesis (0.05 ± 0.02mm2 versus 5.44 ± 0.50mm2, P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm2 versus 0.05 ± 0.02mm2, P < 0.05). Compared with the RAP group, CD31- and VEGF-positive vessels were abundant in the RAP + NAM group. The RAP group showed lower expression of p-mTOR, p-p70S6K and HIF-1α than did the control group (P < 0.05), whereas the RAP + NAM group showed slightly higher expression of these factors than did the RAP group (P < 0.05). Furthermore, in vitro studies revealed that the inhibitory effect of rapamycin on the angiogenic ability of HUVECs and its significant inhibitory effects on the protein level of HIF-1α and the phosphorylation of proteins involved in the mTORC1 pathway, including mTOR, raptor and p70S6K (P < 0.05), were enhanced by cotreatment with SRT1720 and rapamycin (P < 0.05). In contrast to mTOR and SIRT1, the mRNA levels of p70S6K and HIF-1α were reduced by rapamycin (P < 0.05) and further reduced by cotreatment with SRT1720 and rapamycin. CONCLUSIONS: The study results indicate that SIRT1 might negatively regulate atherosclerotic angiogenesis via mTORC1 and HIF-1α signaling pathway and cointervention of SIRT1 and mTOR may serve as a crucial therapeutic strategy in cardiovascular medicine.

Moderate calorie restriction attenuates age­associated alterations and improves cardiac function by increasing SIRT1 and SIRT3 expression.

Calorie restriction (CR) extends the lifespan of mammals and improves cardiac function by attenuation of age­associated alterations. Sirtuins (SIRT) are involved in these mechanisms, however, the extent to which CR affects cardiac function and sirtuin expression remains unknown. Therefore, the present study aimed to determine to what extent CR affects cardiac function and sirtuin expression. A total of 60 female Sprague­Dawley rats were randomly divided into four groups, including normal control (NC), 25% calorie restriction (25% CR), 45% calorie restriction (45% CR) and high­fat diet (HF). The groups were maintained on these specific regimens for 2 months. CR rats were observed to have significantly lower body weight, heart weight, and left ventricle mass index compared with NC and HF rats. Visceral fat, triglyceride, and low density lipoprotein levels were significantly decreased in CR rats. Compared with the 25% CR group, the 45% CR group heart function decreased. The heart rate, left ventricular systolic pressure, +dp/dt and ­dp/dt of the 45% CR rats decreased, whereas the left ventricular end­diastolic pressure increased. To explore the molecular mechanism of CR on cardiac function, immunoblotting was used to detect the protein expression of SIRT1 and SIRT3. The 25% CR diet increased the expression of SIRT1 and SIRT3 in myocardium, whereas the 45% CR and HF diets resulted in a decrease in SIRT1 and SIRT3 expression. Moderate calorie restriction (25% CR) improves cardiac function by attenuation of age­associated alterations in rats. SIRT1 and SIRT3 are associated with these effects.

Transforming growth factor-ß1 and lysophosphatidic acid activate integrin ß6 gene promoter in Hep-3B cells.

Although it is difficult to detect αvß6 integrin (αvß6) in normal epithelia cells, its expression is upregulated during wound healing and carcinogenesis. Overexpression of αvß6 has been demonstrated in epithelial cell carcinomas, such as adenocarcinoma of the colon and ovary. However, the expression of αvß6 has not been reported in hepatocellular carcinoma (HCC). We previously indicated that LPA may induce αvß6-mediated TGF-ß1 signaling mechanisms during the pathogenesis of lung injury and fibrosis. In addition, transforming growth factor-ß1 (TGF-ß1) and lysophosphatidic acid (LPA) have been demonstrated to participate in the progression of HCC. In the present study, we hypothesized that TGF-ß1 and LPA would serve a key role in the subunit integrin ß6 (Itgß6) transcriptional regulatory mechanism in HCC. It was identified that human HCC tissues and Hep-3B cells expressed Itgß6. Treatment of Hep-3B with TGF-ß1 or LPA increased the expression of Itgß6. Furthermore, truncation experiments indicated a positive regulatory region at -326 to -157 bp of the Itgß6 promoter. TGF-ß1 and LPA increased transcriptional activation at this regulatory region. To the best of our knowledge, the present study was the first to demonstrate Itgß6 expression in HCC, and the data indicate that TGF-ß1 and LPA regulate Itgß6 expression through the Itgß6 gene promoter, which is an important factor in the development of HCC.

Nicotinic Acid Receptor GPR109A Exerts Anti-Inflammatory Effects Through Inhibiting the Akt/mTOR Signaling Pathway in MIN6 Pancreatic ß cells.

OBJECTIVES: We found that activation of the nicotinic acid receptor GPR109A, expressed by the MIN6 murine pancreatic ß cell line, inhibits nitric oxide accumulation induced by IFN-γ and TNF-α, implicating an anti-inflammatory effect of GPR109A in MIN6 cells. Nevertheless, the mechanism of its anti-inflammatory effect is still unknown. In this study, we used palmitic acid to stimulate MIN6 cells to induce inflammatory cytokine production and explored the mechanism by which GPR109A exerts anti-inflammatory effects. MATERIALS AND METHODS: RT-PCR and immunocytochemical staining were used to detect the expression of GPR109A in MIN6 cells. Western blotting was used to detect the activation of the Akt/mTOR signaling pathway and expression of the inflammatory cytokine INF-γ, in MIN6 cells, following treatments with palmitic acid and palmitic acid+nicotinic acid, or with different concentrations of nicotinic acid and 3-hydroxybutyrate. RESULTS: In MIN6 cells, GPR109A transcripts and protein are expressed and GPR109A protein is mainly located in the cell membrane and cytoplasm. Palmitic acid enhanced the phosphorylation of Akt and p70S6K and elevated the expression of IFN-γ. Co-treatment with nicotinic acid, which is an agonist of GPR109A, inhibited the palmitic acid-induced phosphorylation of Akt, mTOR, and p70S6K, as well as the expression of IFN-γ. CONCLUSIONS: GPR109A may inhibit inflammatory cytokine production, induced by palmitic acid, by MIN6 cells possibly via inhibiting the Akt/mTOR signaling pathway.

Low dose doxycycline decreases systemic inflammation and improves glycemic control, lipid profiles, and islet morphology and function in db/db mice.

The aim of this study was to determine whether low dose doxycycline as an anti-inflammatory agent could improve glucose metabolism in diabetic animals. Therefore, doxycycline was supplemented in drinking water to 6-week-old male db/db mice for 10 weeks. Doxycycline reduced perirenal/epididymal fat, Lee's index, and liver cholesterol. Blood HDL-cholesterol increased, but total cholesterol and aspartate transaminase decreased. Glucose and insulin tolerances were improved, accompanying with reduced fasting blood glucose, insulin, HOMA-IR and advanced glycation end products. Islet number, ß-cell percentage and mass increased, while islet size decreased. Consistently, less apoptosis but more ß-cell proliferation were found in islets of treated mice. Freshly isolated islets from treated mice showed higher insulin content and enhanced glucose stimulated insulin secretion (GSIS). In addition, purified islets of Balb/c mice showed increased GSIS after cultivation in vitro with doxycycline, but not with chloramphenicol and levofloxacin. Inflammation markers, including lipopolysaccharides (LPS) and C-reactive protein (CRP) in serum as well as CD68-positive cells in treated islets, decreased significantly. Finally, LPS stimulated the production of inflammatory factors but inhibited GSIS of MIN6 cells; however, the effects were completely reversed by doxycycline. The results support further study of possible long-term usage of sub-antimicrobial doxycycline in diabetic patients.

The Effect of Metformin on the Expression of GPR109A, NF-κB and IL-1ß in Peripheral Blood Leukocytes from Patients with Type 2 Diabetes Mellitus.

OBJECTIVES: Type 2 Diabetes Mellitus (T2DM), which often accompanies dyslipidemia, is considered an inflammatory disease. GPR109A, as a niacin receptor, is up-regulated under high glucose concentration. Activation of GPR109A reduces GSIS and exerts anti-inflammatory effects by regulating NF-κB/IL-1ß signaling. Metformin improves hyperglycemia, increases insulin sensitivity and attenuates the activation of the NF-κB pathway in T2DM. We aimed to examine whether metformin plays beneficial effects in T2DM by regulating the GPR109A signaling. METHODS: 117 T2DM patients were involved in this study and divided into two groups, the control group (without metformin) and the Metformin (Met) group (orally given metformin, 500mg-2000mg/d). Peripheral blood samples were collected from all the patients for testing PBL counts, biochemical data, and C peptide. Total RNA was isolated from PBLs. RT-PCR and immunocytochemistry were used to examine the expression of GPR109A, NF-κB and IL-1ß in PBLs. RESULTS: FPG, HbA1c and LDL levels were lower and 2hr C peptide was higher in the Met group than in the control group (P<0.05). RT-PCR showed that mRNA levels of GPR109A, NF-κB and IL-1ß were lower in the Met group than in the control group (P<0.05). Correlation analysis showed that there was a positive correlation between GPR109A and IL-1ß (p<0.01, r=0.425) in the control group, GPR109A and IL-1ß (p<0.05, r=0.256), GPR109A, and NF-κB (p<0.05,r=0.295) in the Met group. Immunocytochemistry showed that the GPR109A and NF-κB proteins were localized in the nucleus and cytoplasm of PBLs; however, there were no significant differences in the protein expression between the two groups. CONCLUSIONS: The results suggest that Met may reduce GPR109A expression in PBLs of T2DM patients by suppressing NF-κB/IL-1ß signaling. Up-regulated expression of GPR109A may be an inflammatory consequence and the improvement of inflammation may down-regulate the expression of GPR109A in T2DM.

mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway.

Atherosclerosis (AS) is a chronic immuno­inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C­C chemokine receptor type 7/nuclear factor­κB (LXR­CCR7/NF­κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1­LXR­CCR7/NF­κB signaling (SIRT1 signaling) in AS pathogenesis. To induce foam cell formation, U937 cells were differentiated into macrophages by exposure to phorbol 12­myristate 13­acetate (PMA) for 24 h, followed by treatment with palmitate and oxidized low density lipoprotein for a further 24 h. Oil red O staining revealed a large accumulation of lipid droplets present in foam cells. Western blot analysis demonstrated increased protein levels of phosphorylated (p)­mTOR and its downstream factor p­ribosomal protein S6 kinase (p70S6K). Reverse transcription­quantitative polymerase chain reaction and western blot analyses additionally revealed decreased expression of SIRT1, LXRα and CCR7 and increased expression of NF­κB and its downstream factor tumor necrosis factor­α (TNF­α) in an atherogenetic condition induced by lysophosphatidic acid (LPA). In addition, abundant lipid droplets accumulated in U937­LPA­treated foam cells. Rapamycin, an mTOR inhibitor, suppressed the expression and activity of mTOR and p70S6K, however enhanced expression of SIRT1, LXRα, and CCR7. Conversely, rapamycin deceased TNF­α and NF­κB activity, the latter of which was further confirmed by immunofluorescence analysis demonstrating increased levels of NF­κB present in the cytoplasm compared with the nucleus. The findings of the present study suggest that mTOR signaling promotes foam cell formation and inhibits foam cell egress via suppression of SIRT1 signaling.

VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells.

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Muts) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

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.

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.

GPR109A Expression in the Murine Min6 Pancreatic Beta Cell Line, and Its Relation with Glucose Metabolism and Inflammation.

BACKGROUND: Nicotinic acid has been used clinically to manage dyslipidemia for many years, and its receptor, GPR109A, is expressed in various tissues or cells. It is not known if GPR109A is also expressed in pancreatic beta cells or if it takes part in maintaining homeostasis of glucose metabolism. AIMS: In this study, the expression of GPR109A was investigated in the murine Min6 pancreatic beta cell line. The anti-inflammatory role of GPR109A in MIN6 cells was also explored. METHODS: RT-PCR, western blotting, and immunocytochemical staining were used to detect the expression of GPR109A in MIN6 cells. Real-Time RT-PCR was used to investigate GPR109A mRNA levels influenced by IFN-γ and glucose. Cell viability and cytoplasmic nitrite levels were measured colorimetrically. RESULTS: We have identified that MIN6 cell, a mouse pancreatic beta cell line, expresses GPR109A transcripts and protein. GPR109A protein is mainly located in the cell membrane and cytoplasm. GPR109A mRNA increased more than 9-fold in MIN6 cells incubated with IFN-γ. High glucose inhibited GPR109A mRNA expression. Nitric oxide accumulation, induced by IFN-γ/TNF-α, was inhibited by nicotinic acid and 3-hydroxy-butyrate. CONCLUSIONS: Our results suggest that the expression of GPR109A in pancreatic beta cells is not only influenced by inflammation and glucose, but also plays a protective role under inflammatory conditions. Moreover, the MIN6 cell line may serve as a cellular model for further investigation of GPR109A-mediated signal transduction in modulating metabolism and diabetes.

Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

Calorie restriction inhibits ovarian follicle development and follicle loss through activating SIRT1 signaling in mice.

BACKGROUND: Silent information regulator 2 related enzyme 1 (SIRT1) is one of the key factors in the mechanism of calorie restriction (CR) extending lifespan of animals. The aim of the study is to investigate if CR prolongs ovarian lifespan in mice through activating SIRT1 signaling. METHODS: In the present study, 21 female C57BL/6 mice were divided into three groups: the control (n = 7), CR (n = 7), and SRT1720 (n = 7) groups. After the 26-week treatment, the number of ovarian follicles at each stage was counted, and Western blot was performed. RESULTS: The number of surviving follicles in ovaries of the SRT1720 group was less than that of the CR group but more than that of the normal control (NC) group. The number of atretic follicles in the ovaries of the SRT1720 group was similar to that of the CR group but less than that of the NC group. The number of primordial follicles in the ovaries of the SRT1720 group was less than that of the CR group but more than that of the NC group. The numbers of primary follicles, secondary follicles, antral follicles, and corpora lutea in the SRT1720 group were similar to those in the CR group. Western blot analysis showed that the expression of SIRT1, SIRT6, FOXO3a, and NRF1 proteins was upregulated, and p53 was downregulated in both the CR group and the SRT1720 group compared to the control group. CONCLUSIONS: Our results indicate that CR inhibits the activation of primordial follicles and development of follicles at different stages, thus preserving the reserve of follicle pool (at least partly) through activating SIRT1 signaling.

Cloning and characterization of the human integrin ß6 gene promoter.

The integrin ß6 (ITGB6) gene, which encodes the limiting subunit of the integrin αvß6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5'-RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region -24 to -18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located -289 to -150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

Caloric restriction promotes the reserve of follicle pool in adult female rats by inhibiting the activation of mammalian target of rapamycin signaling.

Caloric restriction (CR) is known to increase the number of primordial follicles and prolong the reproductive life span. However, how CR modulates follicular development is not well understood. In the present study, we examined the effects of CR on follicular development in rats and investigated the underlying mechanism. After 10 weeks of CR or high-fat diet, ovarian follicles at different developmental stages were examined by histological analysis. Plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estrogen (ESG) were measured, and the levels of mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and phosphorylated p70S6K in the ovary were detected by Western blot. The results showed that the reserve of follicle pool in CR rats was increased, accompanied by decreased level of phosphorylated p70S6K in the ovary, and decreased serum LH, FSH, and ESG levels. Taken together, these results suggest that CR may suppress ovarian follicular development and enhance the follicle pool reserve by inhibiting mTOR signaling.

Direct evidence of sirtuin downregulation in the liver of non-alcoholic fatty liver disease patients.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) has become the most common form of chronic liver disease in the world. Recent studies in cultured cells and mice have shown that sirtuin, especially sirtuin 1 (SIRT1), is a key metabolic sensor for regulating metabolic homeostasis and thus has the potential to ameliorate NAFLD. For the purposes of this study, we hypothesized that the inhibition of sirtuin signaling might contribute to the development of NAFLD. METHODS: Tissue was obtained from hepatectomy specimens (10 samples), and medicolegal autopsies (10 samples). Liver tissue sections were stained with H&E. Expression of sirtuin in liver tissues in NAFLD and control group was investigated by RT-PCR and Western blotting. RESULTS: RT-PCR and Western blotting demonstrated decreased expression of SIRT1, SIRT3, SIRT5, and SIRT6 in the NAFLD group in comparison with the control group. Increased expression of lipogenic genes including sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC) was noted within the NAFLD group. In contrast to the other SIRT genes, the expression of SIRT4 was upregulated. CONCLUSION: Our study provides direct evidence of the downregulation of sirtuin signaling that suppresses lipid synthesis in the liver of NAFLD patients, which may promote NAFLD development.