Zn-Mediated Fragmentation of N-Alkoxyphthalimides Enabling the Synthesis of gem-Difluoroalkenes.

Zn-mediated generation of alkoxyl radicals from N-alkoxyphthalimides emerged as an efficient approach for forming diverse and valuable alkyl radicals through ß-scission or a hydrogen atom transfer process. The alkyl radical species can be further trapped by α-trifluoromethyl alkenes to construct a series of gem-difluoroalkenes.

Physiological and Cognitive Determinants of Gait Variability of Asian Population: The Yishun Study.

INTRODUCTION: Gait variability is associated with frailty, dementia, and falls. Studies on the association of physiological and cognitive factors with gait variability have seldom included middle-aged adults, even though these adults already experienced loss of muscular strength and postural stability. This study aimed a) to examine and compare the trend of gait variability in men and women, across the adult age spectrum, and b) to identify and compare the contributions of physiological and cognitive factors to gait variability. METHODS: This was a population-based cross-sectional study at a single center. A random sample of 507 community-dwelling, well-functioning adults aged 21-90 years were studied. Cognition was measured using the Repeatable Battery for the Assessment of Neuropsychological Status. Physiological factors examined included visual contrast sensitivity (VCS), postural sway, hand reaction time, handgrip strength (HGS), knee extensor strength, and gait variability (coefficient of variation [CoV]). Multivariable regression models were used to examine the association between physiological and cognitive performance with gait CoV. RESULTS: Women walked with greater stride width CoV (p < 0.01) and double support time (DST) CoV (p < 0.01) than men. The stride width (p = 0.01) and DST variability (p = 0.03) were significantly higher in older men as compared to men in younger age-groups. Gait speed accounted for most of the gait CoV variances and attenuated the effects of physiological performance and/or attention cognition on most gait variability, except for CoV of DST and stride width. Adults with better VCS (ß = -0.19), faster hand reaction (ß = 0.12), and greater HGS (ß = -0.15) had lower variability in step length. CONCLUSION: The trends of stride width CoV and DST CoV across adult age spectrum were different between men and women. Greater stride width variability was partly attributed to greater HGS, possibly to better control lateral stability during walking. Physiological factors outweigh cognition in regulating most of the gait CoV in this study. They are modifiable and potential targets for healthy aging program.

Resveratrol improves estrus disorder induced by bisphenol A through attenuating oxidative stress, autophagy, and apoptosis.

Bisphenol A (BPA), as a widely used plasticizer, is easily absorbed by animals and humans. It has certain toxic effects on various tissues, including liver, heart, kidney, testis, and ovary. The toxic effects of BPA on animal reproduction have aroused widespread concern, but its regulatory mechanism and antidote in female animals estrus cycle remain unclear. In this study, the results displayed that BPA destroyed the normal estrus cycle of mice through decreasing the levels of progesterone and estradiol. Furthermore, BPA significantly increased the levels of oxidative stress, autophagy, and apoptosis in ovaries and granulosa cells. Interestingly, we found that the natural antioxidant resveratrol rescued estrus disorder and impaired estradiol secretion, reduced the abnormal reactive oxygen species accumulation, autophagy, and apoptosis in BPA exposed ovarian tissues. Moreover, transmission electron microscopy showed that resveratrol reduced BPA-induced autophagic vesicles formation and flow cytometry showed that resveratrol inhibited the increase of apoptotic cells induced by BPA on granulosa cells. Therefore, the supplement of resveratrol could restore BPA-induced estrus disorder by protecting ovarian granulosa cells. Overall, resveratrol is a potential drug to alleviate BPA-induced estrous cycle disorders and ovarian damage.

Physiological and Cognitive Determinants of Dual-Task Costs for Gait Parameters: The Yishun Study.

BACKGROUND: Studies indicate that physiological and cognitive aging are causally related and functionally interdependent. However, the relative contribution of physiological factors and cognition to dual-task costs (DTC) of gait parameters has not been well studied. In this cross-sectional study, we examined the trajectory of DTC of gait parameters across the adult age spectrum for both sexes and identified the contributions of physical and cognitive performance to DTC of gait. METHODS: A total of 492 community-dwelling adults, aged 21-90 years, were randomly recruited into the study. Participants were divided into 7 age groups, with 10-year age range for each group. Demographic data, height, body mass, education level, and information on comorbidities were recorded. Cognition was measured using the Repeatable Battery for the Assessment of Neuropsychological Status. Physical performance included visual contrast sensitivity, postural sway, hand reaction time, handgrip strength, knee extensor strength, and single-task and dual-task gait assessments. Stepwise multivariable regression was used to examine the association between physical and cognitive performance with DTC of gait parameters. RESULTS: Women were found to have significantly higher DTC of gait speed (p = 0.01), cadence (p < 0.01), and double support time (p < 0.01) than men. However, significant aging effect on DTC of gait speed (p = 0.01), step length (p = 0.01), and double support time (p = 0.01) was observed in men but not in women. Immediate memory was the primary determinant for the DTC of gait speed (ß = -0.25, p < 0.01), step length (ß = -0.22, p < 0.01), and cadence (ß = -0.15, p = 0.03) in men. Besides immediate memory, postural sway (ß = -0.13, p = 0.03) and hand reaction (ß = 0.14, p = 0.02) were also significantly associated with DTC of step length and cadence, respectively, in women. CONCLUSION: There were sex differences in the amplitude and trajectories of DTC of gait parameters. The DTC increased with age in men but not in women. Immediate memory was the primary determinant of DTC of gait parameters in men while immediate memory, postural sway, and reaction time were associated with DTC of gait in women. Future studies should investigate the clinical implications of the sex differences in the DTC with fall risks.

Reference Values of Gait Speed and Gait Spatiotemporal Parameters for a South East Asian Population: The Yishun Study.

BACKGROUND: Age-related slowing of gait has been reported to start as early as the fifth decade and accelerate beyond the seventh decade of life. A single cut-off for slow gait may not be appropriate for men and women of different ages. We aimed to report reference values for gait speed and spatiotemporal gait parameters of adult age groups in a South East Asian population. METHODS: A total of 507 community-dwelling adults, aged 21-90 years were recruited into the study through random sampling, filling quotas of 20-40 participants in each sex and age group (10-year age groups between 21 and 60 years; 5-year age groups beyond age 60 years). Demographic data, height, weight and information on comorbidities were recorded. Habitual gait speed and spatiotemporal parameters were measured, and the average of three trials was recorded using the GAITRite system. RESULTS: Gait speed peaked in their 40s for both men and women, but the trajectories differed slightly across age groups. Although similar for men in their 50s and 60s, gait speed was significantly slower among those aged 71 years and older. For women beyond 50 years old, gait slowed with age. After adjusting for height, women were found to walk significantly faster and with a longer step length than men. Women also walked with a significantly narrower stride width and less external rotation of the feet. The lowest quintile for gait speed in our study cohort was 0.9m/s, below the recommended cut-off of 1.0m/s. CONCLUSION: We established the reference values as well as the quintiles for gait speed and spatiotemporal gait parameters across adult age groups in a multi-ethnic Asian population. This contributes to a valuable database for gait assessment and evaluation of preventive or rehabilitative programs.

Urolithin A exerts antiobesity effects through enhancing adipose tissue thermogenesis in mice.

Obesity leads to multiple health problems, including diabetes, fatty liver, and even cancer. Here, we report that urolithin A (UA), a gut-microflora-derived metabolite of pomegranate ellagitannins (ETs), prevents diet-induced obesity and metabolic dysfunctions in mice without causing adverse effects. UA treatment increases energy expenditure (EE) by enhancing thermogenesis in brown adipose tissue (BAT) and inducing browning of white adipose tissue (WAT). Mechanistically, UA-mediated increased thermogenesis is caused by an elevation of triiodothyronine (T3) levels in BAT and inguinal fat depots. This is also confirmed in UA-treated white and brown adipocytes. Consistent with this mechanism, UA loses its beneficial effects on activation of BAT, browning of white fat, body weight control, and glucose homeostasis when thyroid hormone (TH) production is blocked by its inhibitor, propylthiouracil (PTU). Conversely, administration of exogenous tetraiodothyronine (T4) to PTU-treated mice restores UA-induced activation of BAT and browning of white fat and its preventive role on high-fat diet (HFD)-induced weight gain. Together, these results suggest that UA is a potent antiobesity agent with potential for human clinical applications.

Evaluation of ε-polylysine as antimicrobial alternative for liquid-stored boar semen.

ε-polylysine (ε-PL) has potent antibacterial effects and is often used in the food industry. However, no studies have clarified the antibacterial effects of ε-PL during storage of boar semen. In this study, boar semen samples were diluted with BTS buffer supplemented with different concentrations (0, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 g/L) of ε-PL and different combinations of ε-PL plus gentamicin during liquid storage at 17 °C for 5 days. Bacterial concentrations, bacterial community compositions, sperm quality parameters, and in vitro fertilization (IVF) were evaluated in order to analyze the antibacterial effects of these parameters during boar semen preservation. The results indicated that the optimum concentration of ε-PL was 0.16 g/L, which significantly improved sperm quality parameters, including sperm motility, plasma membrane integrity, mitochondrial membrane potential, and acrosome integrity, and changed bacterial proliferation and composition (P < 0.05). Moreover, compared with the control group, IVF parameters in the treatment groups also significantly improved (P < 0.05), although there were no significant differences among treatment groups. Interestingly, the antibacterial effect of 0.16 g/L ε-PL in combination with 0.125 g/L gentamycin was similar to that of 0.25 g/L gentamicin alone. In conclusion, our results showed that 0.16 g/L ε-PL is promising for the replacement of gentamicin to improve sperm quality parameters, sperm capacitation, and IVF by reducing bacterial concentrations and disrupting bacterial community composition.

Differences between porcine longissimus thoracis and semitendinosus intramuscular fat content and the regulation of their preadipocytes during adipogenic differentiation.

Intramuscular fat (IMF) plays an important role in pork quality. However, differences in the adipogenic regulation of IMF content between pig longissimus thoracis (LT) and semitendinosus (ST) remain unclear. Here, we found that IMF content of 180-day-old pig LT was greater than that of pig ST. Furthermore, lipid accumulation was earlier and greater in LT intramuscular preadipocytes (L-IMA) than in ST intramuscular preadipocytes (S-IMA) during differentiation. Interestingly, glucose consumption was lower in L-IMA than in S-IMA. Moreover, monounsaturated fatty acid content was greater in L-IMA than in S-IMA, whereas polyunsaturated fatty acid content was lower. Levels of the expression of key adipogenic genes were higher in L-IMA than S-IMA. Compared with S-IMA, adipogenic signals were more activated in L-IMA after adipogenic induction. In conclusion, IMF deposition differences between pig LT and ST were due to different glucose consumption, fatty acid composition, expression of key adipogenic genes and level of activating adipogenic signals between S-IMA and L-IMA during adipogenesis.

PDGFRα Regulated by miR-34a and FoxO1 Promotes Adipogenesis in Porcine Intramuscular Preadipocytes through Erk Signaling Pathway.

Suitable intramuscular fat (IMF) content improves porcine meat quality. The vital genes regulating IMF deposition are necessary for the selection and breeding of an IMF trait. However, the effect and mechanism of PDGFRα on IMF deposition are still unclear. Here, PDGFRα is moderately expressed in porcine longissimus dorsi muscle (LD), whereas it highly expressed in white adipose tissue (WAT). Moreover, PDGFRα-positive cells were located in the gaps of LD fibers which there were IMF adipocytes. Compared with 180-day-old and lean-type pigs, the levels of PDGFRα were much higher in one-day-old and fat-type pigs. Meanwhile the levels of PDGFRα gradually decreased during IMF preadipocyte differentiation. Furthermore, PDGFRα promoted adipogenic differentiation through activating Erk signaling pathway. Based on PDGFRα upstream regulation analysis, we found that the knockdown of FoxO1 repressed lipogenesis by downregulating PDGFRα, and miR-34a inhibited adipogenesis through targeting PDGFRα. Collectively, PDGFRα is a positive regulator of IMF deposition. Therefore, we suggest that PDGFRα is a possible target to improve meat quality.

miR-425-5p Inhibits Differentiation and Proliferation in Porcine Intramuscular Preadipocytes.

Intramuscular fat (IMF) content affects the tenderness, juiciness, and flavor of pork. An increasing number of studies are focusing on the functions of microRNAs (miRs) during porcine intramuscular preadipocyte development. Previous studies have proved that miR-425-5p was enriched in porcine skeletal muscles and played important roles in multiple physiological processes; however, its functions during intramuscular adipogenesis remain unclear. To explore the role of miR-425-5p in porcine intramuscular adipogenesis, miR-425-5p agomir and inhibitor were used to perform miR-425-5p overexpression and knockdown in intramuscular preadipocytes, respectively. Our results showed that the agomir of miR-425-5p dramatically inhibited intramuscular adipogenic differentiation and downregulated the expression levels of adipogenic marker genes PPARγ, FABP4, and FASN, whereas its inhibitor promoted adipogenesis. Interestingly, the agomir repressed proliferation of porcine intramuscular preadipocytes by downregulation of cyclin B and cyclin E. Furthermore, we demonstrated that miR-425-5p inhibited adipogenesis via targeting and repressing the translation of KLF13. Taken together, our findings identified that miR-425-5p is a novel inhibitor of porcine intramuscular adipogenesis possibly through targeting KLF13 and subsequently downregulating PPARγ.

Sirt1 AS lncRNA interacts with its mRNA to inhibit muscle formation by attenuating function of miR-34a.

Recent studies demonstrate the functions of long non-coding RNAs (lncRNAs) in mediating gene expression at the transcriptional or translational level. Our previous study identified a Sirt1 antisense (AS) lncRNA transcribed from the Sirt1 AS strand. However, its role and regulatory mechanism is still unknown in myogenesis. Here, functional analyses showed that Sirt1 AS lncRNA overexpression promoted myoblast proliferation, but inhibited differentiation. Mechanistically, Sirt1 AS lncRNA was found to activate its sense gene, Sirt1. The luciferase assay provided evidences that Sirt1 AS lncRNA interacted with Sirt1 3' UTR and rescued Sirt1 transcriptional suppression by competing with miR-34a. In addition, RNA stability assay showed that Sirt1 AS lncRNA prolonged Sirt1 mRNA half-life from 2 to 10 h. Ribonuclease protection assay further indicated that it fully bound to Sirt1 mRNA in the myoblast cytoplasm. Moreover, Sirt1 AS overexpression led to less mouse weight than the control because of less lean mass and greater levels of Sirt1, whereas the fat mass and levels of miR-34a were not altered. Based on the findings, a novel regulatory mechanism was found that Sirt1 AS lncRNA preferably interacted with Sirt1 mRNA forming RNA duplex to promote Sirt1 translation by competing with miR-34a, inhibiting muscle formation.

Mulberry 1-Deoxynojirimycin Inhibits Adipogenesis by Repression of the ERK/PPARγ Signaling Pathway in Porcine Intramuscular Adipocytes.

Intramuscular fat (IMF), which is modulated by adipogenensis of intramuscular adipocytes, plays a key role in pork quality associated with marbling, juiceness, and flavor. However, the regulatory mechanism of 1-deoxynojirimycin (DNJ) on adipogenesis is still unknown. Here, we found that both DNJ (2.0, 3.0, 4.0, 5.0, and 6.0 µM) and rosiglitazone (RSG; 0.1, 0.2, 0.3, 0.4, and 0.5 mM) had no effect on cell viability. Moreover, 4 µM DNJ significantly inhibited adipogenesis, whereas 0.4 mM RSG increased lipogenesis of porcine intramuscular adipocytes. Interestingly, DNJ sharply inhibited phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2), but did not change phosphorylation of AKT (protein kinase B) in intramuscular adipocytes. We further found that the inhibitory adipogenesis of DNJ was attenuated by RSG via up-regulation of PPARγ. On the basis of the above findings, we suggest that DNJ inhibited adipogenesis through the ERK/PPARγ signaling pathway in porcine intramuscular adipocytes.

Knockdown of CTRP6 inhibits adipogenesis via lipogenic marker genes and Erk1/2 signalling pathway.

C1q/tumor necrosis factor-related protein 6 (CTRP6), an adipose-tissue secretory factor, plays an important role in inflammatory reaction and carcinogenesis. However, the biological function of CTRP6 in adipogenesis remains unclear. In this study, we examined the effects of CTRP6 knockdown on lipogenesis of 3T3-L1 adipocytes. The results showed that after 3T3-L1 adipocytes transfected with anti-CTRP6 small interfering RNA (siRNA), not only levels of secreted CTRP6 protein in the culture medium but also the expression level of the CTRP6 protein in the 3T3-L1 adipocytes was significantly reduced (P < 0.01). In addition, the number of lipid droplets in the adipocytes was reduced, as well as the OD values reflecting the fat content being significantly decreased (P < 0.01). Meanwhile the levels of adipogenic markers, including peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), CCAAT/enhancer-binding protein ß (C/EBPß) and adipocyte fatty acid-binding protein 4 (aP2), were decreased after treatment with anti-CTRP6 siRNA, whereas the expression of adipose triglyceride lipase (ATGL) and triacylglycerol hydrolase (TGH) were increased. Furthermore, after transfection, activity of phosphorylated Erk1/2 (p-Erk1/2) was inhibited in the early stage of differentiation, but in terminal differentiation of adipocytes, its activity was activated. Taken together, the results indicate that knockdown of CTRP6 can inhibit adipogenesis of 3T3-L1 adipocytes through lipogenic marker genes and Erk1/2 signaling pathway.

3,3'-Diindolylmethane increases bone mass by suppressing osteoclastic bone resorption in mice.

3,3'-Diindolylmethane (DIM), a major acid-condensation product or metabolite of indole-3-carbinol which is found in cruciferous vegetables, has been shown to have anticancer, anti-inflammatory, and multiple immune stimulating effects. However, its function in bone metabolism is poorly understood. This study evaluated the effect of DIM on bone mass in mice under physiological and pathological conditions. Eight-week-old female mice received injections of a vehicle or 0.1mg/g of DIM, twice a week for four weeks. We found that DIM treatment significantly increased bone mass as assessed by dual-energy X-ray absorptiometry (DEXA) and micro-computed tomography (µCT). Further, Bone histomorphometric analyses showed that this treatment significantly reduced bone resorption parameters, but did not increase bone formation parameters. Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and explore function of DIM in skeletal pathological processes. Bone phenotype analyses revealed that the administration of DIM in this study effectively prevented OVX-induced bone loss resulting from increased bone resorption. Our results demonstrated that DIM increased bone mass by suppressing osteoclastic bone resorption in bone metabolism under both physiological and pathological conditions. Accordingly, DIM may be of value in the treatment and the possible prevention of bone diseases characterized by bone loss, such as postmenopausal osteoporosis.

Aryl hydrocarbon receptors in osteoclast lineage cells are a negative regulator of bone mass.

Aryl hydrocarbon receptors (AhRs) play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO) or transgenic mice, the cellular and molecular mechanism(s) in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR(RANKΔOc/ΔOc) (RANK(Cre/+);AhR(flox/flox)) mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR(RANKΔOc/ΔOc) mice. Control mice treated with 3-methylcholanthrene (3MC), an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR(CtskΔOc/ΔOc) (Ctsk(Cre/+);AhR(flox/flox)) mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs) from AhR(RANKΔOc/ΔOc) mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1), and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.

Association of Novel Polymorphisms in Lymphoid Enhancer Binding Factor 1 (LEF-1) Gene with Number of Teats in Different Breeds of Pig.

Lymphoid enhancer binding factor 1 (LEF-1) is a member of the T-cell specific factor (TCF) family, which plays a key role in the development of breast endothelial cells. Moreover, LEF-1 gene has been identified as a candidate gene for teat number trait. In the present study, we detected two novel mutations (NC_010450.3:g. 99514A>G, 119846C>T) by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism in exon 4 and intron 9 of LEF-1 in Guanzhong Black, Hanjiang Black, Bamei and Large White pigs. Furthermore, we analyzed the association between the genetic variations with teat number trait in these breeds. The 99514A>G mutation showed an extremely significant statistical relevance between different genotypes and teat number trait in Guanzhong (p<0.001) and Large White (p = 0.002), and significant relevance in Hanjiang (p = 0.017); the 119846C>T mutation suggested significant association in Guanzhong Black pigs (p = 0.042) and Large White pigs (p = 0.003). The individuals with "AG" or "GG" genotype displayed more teat numbers than those with "AA"; the individuals with "TC" or "CC" genotype showed more teat numbers than those with "TT". Our findings suggested that the 99514A>G and 119846C>T mutations of LEF-1 affected porcine teat number trait and could be used in breeding strategies to accelerate porcine teat number trait improvement of indigenous pigs breeds through molecular marker assisted selection.

Identification, stability and expression of Sirt1 antisense long non-coding RNA.

Natural antisense transcripts (NATs) exist ubiquitously as pivotal molecules to regulate coding gene expression. Sirtuin 1 (Sirt1) is a NAD-dependent deacetylase which is involved in myogenesis. However, whether Sirt1 transcribes NAT during C2C12 differentiation is still unknown. In this study, we identified a Sirt1 NAT which was designated as Sirt1 antisense long non-coding RNA (AS lncRNA) by sequencing and bioinformatic analysis. The level of Sirt1 AS lncRNA was greater in spleen but less in muscle tissue. The expression of both Sirt1 mRNA and Sirt1 AS lncRNA decreased during C2C12 myogenic differentiation, whereas the levels of miR-34a, which targets Sirt1, increased gradually. We further found that the half-life of Sirt1 AS lncRNA was 10h, but that of Sirt1 mRNA was 6h in C2C12 cells treated with 2 µg/ml Actinomycin D. Therefore, compared with Sirt1 mRNA, Sirt1 AS lncRNA was more stable. Overexpression of Sirt1 AS lncRNA increased the levels of Sirt1 protein, whereas overexpression of Sirt1 AS lncRNA mutant did not affect the level of Sirt1 protein in C2C12 cells. Moreover, downregulation of Sirt1 mRNA caused by miR-34a was counteracted by Sirt1 AS lncRNA in C2C12 cells. Taken together, we identified a novel NAT of Sirt1 which implicated in myogenesis through regulating Sirt1 expression.

Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing-thawing.

Low-density lipoproteins (LDL) is known to protect boar sperm during freezing-thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing-thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen-thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen-thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen-thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation.

PU.1 is an Ets family transcription factor involved in the myelo-lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR-155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR-155 regulates adipogenesis not through PU.1, but via C/EBPß which is another target of miR-155. We also checked the expression levels of PU.1 mRNA and antisense long non-coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral-mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes.

Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes.

Adipose tissue is an important energy reservoir, and its over-development results in obesity in humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and regulatory mechanism of Sirt1 and camptothecin on porcine preadipocyte apoptosis are still largely unknown. Here, we evaluated whether Sirt1 had any role in the basal cellular and camptothecin-induced conditions in porcine preadipocytes. Flow cytometric analysis shows that viable cells decrease as well as early apoptotic and late apoptotic cells increase after knockdown of Sirt1 in porcine preadipocytes. Camptothecin induces porcine preadipocyte apoptosis in a dose-dependent manner, assessed with the Hoechst staining and western blot analysis. Interestingly, silencing of Sirt1 significantly enhances sensitivity of porcine preadipocytes to camptothecin, which may be due to upregulation of p53, acetylated p53, Bax, cleaved caspase-3 and downregulation of Bcl-2. On the contrary, under the Sirt1 overexpression condition viable cells' number significantly increases when challenging with camptothecin, and the protein levels of cleaved caspase-3, p53, acetylated p53 and Bax are downregulated. We also find that hyperacetylated p53 is the major effect of Sirt1 knockdown by overexpression of a mutated p53, whereas Sirt1 overexpression prevents camptothecin-induced apoptosis through p53 deacetylation in preadipocytes. Furthermore, repressing preadipocyte apoptosis of Sirt1 is mediated by direct interaction with cleaved caspase-3 using immunoprecipitation and inhibition of caspase-3 transcriptional activity using luciferase reporter assays.