Dietary Arachidonic Acid Has a Time-Dependent Differential Impact on Adipogenesis Modulated via COX and LOX Pathways in Grass Carp Ctenopharyngodon idellus

In this study, we explored the function of arachidonic acid (ARA) in adipogenesis in the grass carp (Ctenopharyngodon idellus) using in vivo and in vitro models. An 8‐week feeding trial was performed using three isonitrogenous and isoenergetic purified diets: ARA‐free, ARA, and ARA + acetylsalicylic acid [ASA, a cyclooxygenase (COX) inhibitor]. Fish were sampled after 4 and 8 weeks of feeding. Results showed that ARA‐fed fish had a significantly lower intraperitoneal fat index (IPFI) and smaller adipocytes; these decreases were reversed by ASA after 8 weeks of feeding. Nevertheless, at week 4, the IPFI and adipocyte size were higher in the ARA group, and they were comparable to those of fish fed ARA + ASA. To further investigate the influence of ARA on adipocyte differentiation, confluent pre‐adipocytes of grass carp were incubated with ARA for 3 days. This in vitro experiment demonstrated that ARA promoted adipogenesis in a dose‐dependent manner. Pre‐treatment with the lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid attenuated the pro‐adipogenic function of ARA. However, after treatment with ARA for 8 days, adipocytes had a lower lipid content than cells treated with oleic acid, and ASA could suppress this effect. We thus revealed the dual function of ARA in adipogenesis in grass carp. The LOX pathway may play a key role in pro‐adipogenesis after short‐term treatment with ARA, whereas the COX pathway is possibly responsible for the inhibition of adipogenesis after long‐term treatment.     

Adipogenic and Myogenic Potentials of Chicken Embryonic Fibroblasts in vitro: Combination of Fatty Acids and Insulin Induces Adipogenesis

The regulation of adipocyte differentiation is an important factor for production efficiency and meat quality in the poultry industry. The purpose of this study was to develop a new in vitro model of adipogenic differentiation of chicken embryonic fibroblasts (CEF). In this study, CEF were isolated at embryonic day (E) 5, and adipogenic differentiation was induced with supplementation of fatty acids (FA) and/or insulin (Ins) for 48 h. Oil-Red-O staining showed that lipid accumulation in E5 CEF was greater when supplemented with a combination of FA and Ins (FI) than other treatment groups (p < 0.05). Genes involved in differentiation of preadipocytes, fatty acid transport, and triacylglycerol synthesis were upregulated in the FI group compared to all other treatment groups (p < 0.01). Under myogenic media, the E5 CEF formed myotubes and expressed myogenic markers, myosin heavy chain (MHC), and myogenin (MyoG), suggesting myogenic potential of E5 CEF. To determine the permissive age window for adipogenic differentiation of CEF, E5, E6, and E7 CEF were induced for adipogenesis with FI treatment in 1%, 5%, or 10% chicken serum (CS). Among all embryonic ages, E5 with 10% CS showed the most lipid accumulation and the least myotube formation with the lowest expression of MHC and MyoG. These data indicate both adipogenic and myogenic potentials of E5 CEF, providing a new in vitro model for a better understanding of the processes of adipogenic and myogenic differentiation in chickens.     

Dietary Omega-3 Fatty Acids Prevented Adipocyte Hypertrophy by Downregulating DGAT-2 and FABP-4 in a Sex-Dependent Fashion

Obesity is characterized by an increase in fat mass primarily as a result of adipocyte hypertrophy. Diets enriched in omega (n)‐3 polyunsaturated fatty acids (PUFA) are suggested to reduce obesity, however, the mechanisms are not well understood. We investigated the effect of n‐3 PUFA on adipocyte hypertrophy and the key genes involved in adipocyte hypertrophy. Female C57BL/6 mice were fed semi‐purified diets (20 % w/w fat) containing high n‐3 PUFA before mating, during pregnancy, and until weaning. Male and female offspring were continued on high n‐3 PUFA (10 % w/w), medium n‐3 PUFA (4 % w/w), or low n‐3 PUFA (2 % w/w) diet for 16 weeks postweaning. Adipocyte area was quantified using microscopy, and gonadal mRNA expression of acyl CoA:diacylglycerol acyltransferase‐2 (DGAT‐2), fatty acid binding protein‐4 (FABP‐4) and leptin were measured. The high n‐3 PUFA group showed higher levels of total n‐3 PUFA in gonadal TAG compared to the medium and low n‐3 PUFA groups (P < 0.001). The high n‐3 PUFA male group had a lower adipocyte area compared to the medium and low n‐3 PUFA group (P < 0.001); however, no difference was observed in females. The high n‐3 PUFA male group showed lower mRNA expression of FABP‐4, DGAT‐2 and leptin compared to the low n‐3 PUFA group, with no difference in females. Plasma lipid levels were lower in the high n‐3 PUFA group compared to the other groups. Our findings show for the first time that n‐3 PUFA prevents adipocyte hypertrophy by downregulating FABP‐4, DGAT‐2 and leptin; the effects are however sex‐specific.     

Suppression of breast cancer cells in vitro by polyamidoamine‐dendrimer‐mediated 5‐fluorouracil chemotherapy combined with antisense micro‐RNA 21 gene therapy

A specific micro‐RNA (miRNA), micro‐RNA 21 (miR‐21), is strongly overexpressed in breast cancer cells. Antisense inhibition of miRNA function, an important tool for uncovering miRNA biology, which is often used to knockdown miRNA, can cause a notable inhibition of cell growth. In this study, 5‐fluorouracil (5‐FU) was conjugated to polyamidoamine dendrimers via direct encapsulation; this method was then combined with antisense micro‐RNA 21 (as‐miR‐21) strategies to evaluate the effects of the growth suppression of breast cancer cells. Our results show that as‐miR‐21 strategies significantly improved the chemosensitivity of free 5‐FU on breast cancer cells (MCF‐7). In addition, not only could as‐miR‐21 effectively increase the apoptotic cell numbers but it could also bring down the migration ability of MCF‐7 cells. Our results provide invaluable information for the future design of drug–polymer complexes for multimodal cancer treatments. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Preparation and characterization of bamboo fiber‐graft‐lauryl methacrylate and its composites with polypropylene

In this study, lauryl methacrylate grafted onto bamboo fibers (BF‐g‐LM) was prepared to improve the interfacial compatibility between hydrophilic bamboo fibers (BFs) and hydrophobic plastic. The lauryl methacrylate (LM) was initiated by benzoxyl peroxide (BPO) and grafted onto BFs via a free‐radical chain‐transfer reaction. LM was grafted onto BFs with ether bonds, and differential scanning calorimetry indicated that the ether pyrolysis of BF‐g‐LM occurred at 280°C. The optimum preparation conditions were obtained as follows: 0.30 g of pretreated bamboo flour was immersed in 0.225 mol/L LM. The reaction was then initiated by 0.025 mol/L BPO, and this reaction was sustained for 4 h at 80°C. BF‐g‐LM served as coupling agent between the BFs and the polypropylene (PP) matrix, as shown by scanning electron microscopy analysis. The elongation at break of the BF/PP composites with BF‐g‐LM increased to two times compared to that of the BF/PP composites without BF‐g‐LM. The impact strength and maximum deflection also increased to 75 and 580%, respectively. In conclusion, BF‐g‐LM is a promising coupling agent that can be used in BF‐reinforced thermoplastic composites. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2377–2382, 2013     

BMP7 Drives Human Adipogenic Stem Cells into Metabolically Active Beige Adipocytes

Adult humans have a substantial amount of inducible‐brown (or beige) fat, which is associated with increased energy expenditure and reduced weight gain via thermogenesis. Despite the identification of key regulators of beige adipogenesis, impacts of dietary factors on adaptive thermogenesis are largely unknown, partly due to a lack of validated human cell models. Bone morphogenetic protein 7 (BMP7) is known to promote brown adipogenesis in rodent and human progenitor cells. However, controversy still surrounds the cellular identity in BMP7‐mediated transition of white to brown adipocytes. The aim of this study was to confirm BMP7‐derived human adipocytes as a relevant in vitro model of human beige adipocyte by verifying the cellular lineage and metabolic activity. In this study, we hypothesized that pre‐exposure of the stromal vascular (SV) fraction of primary human adipogenic precursor cells (hASC) to BMP7 would convert metabolically active brown adipocytes. Our results showed that exposure of hASC to human BMP7 was associated with significant escalation of (1) UCP1 gene expression, a signature gene of brown adipocytes, (2) beige specific marker gene expression (i.e., CD137 and TMEM26), (3) glucose and fatty acid uptake, and (4) basal and cAMP‐stimulated oxygen consumption rate compared to white adipocyte control. Taken together, we demonstrated that BMP7 mediates conversion of hASC into metabolically active beige adipocytes. By confirming the cellular identity and metabolic activity, this BMP7‐induced human beige adipocytes from hASC should aid in the discovery and assessment of bioactive molecules to promote adaptive thermogenesis.     

Bioavailability and pharmacokinetics of bufadienolides‐loaded lipid microspheres after different administrations to rats

The degradation kinetics in vitro and pharmacokinetics of bufadienolides loaded in lipid microsphere (BU‐LM) after intravenous (IV) administration were investigated and compared with bufadienolides solution (BU‐S). The bioavailabilities of BU‐LM after intraportal (F_IP), intraduodenal (F_ID), and intragastric (F_IG) administrations to rats were also evaluated. The degradation kinetics showed that BU‐LM could protect bufadienolides from rapid metabolism in the plasma and gastrointestinal tract. The pharmacokinetic study revealed that BU‐LM after IV administration exhibited longer half‐life, longer mean residence time and higher apparent volume of distribution than intravenous BU‐S. After different routes of administration of BU‐LM, cinobufagin (C), and resibufogenin (R) were metabolized completely and could not be detected at an oral dose of 6 mg/kg. Furthermore, C exhibited a complete hepatic first‐pass effect after IP administration of 0.5 mg/kg BU‐LM. Bufalin (B), the lowest component of the BU‐LM could be detected at any administration and the F_IG, F_ID, and F_IP for B were 43.05, 88.93, and 93.31%, respectively. R had a F_ID and F_IP of only 1.51 and 2.77%, respectively. The results demonstrated that the stomach appeared to be the dominating site of metabolism for B and the liver for C and R. Practical applications: Bufadienolides, consisting of bufalin (B), binobufagin (C), and resibufogenin (R) are easily hydrolyzed (catalyzed by acids, bases, and enzymes) and are metabolized rapidly in rats because of their chemical structures, especially C. However, the three bufadienolides could be protected from degradation effectively and the degradation half‐lives could be extended to different degrees by using lipid microspheres (LM) as a delivery system. Also, after intravenous administration to rats, bufadienolides‐loaded lipid microspheres (BU‐LM) could slow down the metabolism and elimination of bufadienolides, delay the release and increase the targeted effect of the drug compared with bufadienolides aqueous solution (BU‐S_iv). Consequently, LM is a promising alternative vehicle for bufadienolides.     

Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)₂D₃ in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)₂D₃ enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)₂D₃ were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)₂D₃ induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts.     

N‐3 fatty acids and conjugated linoleic acids of longissimus muscle in beef cattle

The objective of the experiment with cattle was to produce high quality beef under different feeding conditions and to increase the concentration of essential fatty acids in muscle. In total 10 German Simmental (GS) bulls and 9 German Holstein (GH) steers were kept either on pasture (grass feeding) or in stable (concentrate feeding). Despite biohydrogenation in the rumen, linolenic acid (C18:3n‐3) contained in grass was absorbed and deposited into the lipids of muscle. This led to a significantly (p ≤ 0.05) higher content of n‐3 fatty acids in the muscle lipids of grazing cattle. The relative amount of total n‐3 fatty acids increased from 1.4 g/100 g fatty acid methyl ester (%FAME) in the intensively fed Simmental bulls to 5.5 %FAME in grass fed cattle. The n‐6/n‐3 ratio of pasture grazing GS bulls was 1.3 in contrast to 13.7 of the animals kept in the byre. The total n‐3 fatty acid concentration in beef muscle increased from 24.6 mg (concentrate) to 108.6 mg/100 g wet weight (grazing). In GH steers the total n‐3 fatty acid concentration was significantly (p ≤ 0.05) increased up to 86.3 mg/100 g wet weight in pasture grazing steers compared to 28.8 mg/100 g wet weight in animals fed the concentrate. The relative content (%FAME) of CLAcis‐9, trans‐11 (0.6 vs 0.56 %FAME in GS; 0.55 vs 0.52 %FAME in GH) in muscle was not significantly increased by grazing on pasture in comparison to concentrate feeding neither in GS bulls nor in GH steers, respectively.     

Structure,Activity and Stereoselectivity of NADPH‐Dependent Oxidoreductases Catalysing the S‐Selective Reduction of the Imine Substrate 2‐Methylpyrroline

Oxidoreductases from Streptomyces sp. GF3546 [3546‐IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2‐methylpyrroline (2MPN) to (S)‐2‐methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue—Tyr169—in 3546‐IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.     

Synergistic inhibition of human glioma cell line by temozolomide and PAMAM‐mediated miR‐21i

Temozolomide (TMZ) is a promising chemotherapeutic agent for treating glioblastomas. However, resistance develops quickly and with a high frequency. Efforts to overcome chemoresistance are, therefore, critically needed. In present study, a poly(amidoamine; PAMAM) dendrimer was used as a vector to deliver microRNA‐21 inhibitor (miR‐21i) into U87 cells and the chemosensitivity of the combination effect of miR‐21i and TMZ for glioma therapy was investigated. Flow cytometry analysis showed the uptake efficiency of microRNA‐21 inhibitor after complexation with PAMAM. Real‐time PCR and in situ hybridization indicated that, compared with TMZ or miR‐21i treated cells, cells simultaneously treated with miR‐21i and TMZ showed a remarkable decrease in the microRNA‐21 (miR‐21) level. The transfection of miR‐21i enhanced the chemosensitivity by significantly decreasing the IC50 value of TMZ to glioma cells. Knockdown of miR‐21 promoted the cells' apoptosis, and at the same time, inhibited cell invasion. In conclusion, the combination treatment of glioma cells with TMZ and miR‐21i could yield a synergistic effect in inhibition of human glioma cell line. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Fish Oil and Olive Oil Supplementation in Late Pregnancy and Lactation Differentially Affect Oxidative Stress and Inflammation in Sows and Piglets

This study was conducted to compare the effects of fish oil and olive oil supplementation in late pregnancy and during lactation on oxidative stress and inflammation in sows and their piglets. A total of 24 sows were fed a basal diet supplemented with additional corn starch (CON), fish oil (FO) or olive oil (OO). Sows fed an OO diet during late gestation had a higher piglet birth weight compared with CON‐fed and FO‐fed sows (P < 0.05). Furthermore, sows from the OO group had a higher milk fat content than sows from CON and FO groups, and a lower pre‐weaning mortality of piglets was observed in the OO group (P < 0.05). Maternal FO supplementation resulted in increased malondialdehyde concentration in sow plasma, colostrum, milk and piglet plasma than in CON and OO groups (P < 0.05). However, an increased total antioxidant capacity (T‐ACC) and activities of glutathione peroxidase (GSH‐Px) and total superoxide dismutase (T‐SOD) were also observed in the FO group (P < 0.05). Sows fed an OO diet had significantly decreased interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α) concentrations in milk compared with CON and FO fed sows (P < 0.05). Moreover, lower plasma IL‐1β and TNF‐α levels were observed in piglets from the OO group compared with the CON group (P < 0.05). Collectively, these results suggest that an OO diet is most beneficial in late gestation and during lactation in sows. However, FO increases the susceptibility to oxidative stress in sows and piglets.     

Cellular uptakes, biostabilities and anti-miR-210 activities of chiral arginine-PNAs in leukaemic K562 cells

A series of 18‐mer peptide nucleic acids (PNAs) targeted against micro‐RNA miR‐210 was synthesised and tested in a cellular system. Unmodified PNAs, R₈‐conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2‐modified (R) or C5‐modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT‐PCR on K562 chronic myelogenous leukaemic cells indicated that arginine‐conjugated and backbone‐modified PNAs display good cellular uptake, with best performances for the C2‐modified series. Resistance to enzymatic degradation was found to be higher for the backbone‐modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR‐210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin‐treated cells. Interestingly, the anti‐miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone‐modified PNAs as anti‐miR agents. The results clearly indicate that backbone‐modified PNAs are good candidates for the development of very efficient drugs based on anti‐miR activity, due to their enhanced bioavailabilities, and that overall anti‐miR performance is a combination of cellular uptake and RNA binding.     

Interaction between miR4749 and Human Serum Albumin as Revealed by Fluorescence,FRET, Atomic Force Spectroscopy and Computational Modelling

The interaction of Human Serum Albumin (HSA) with the microRNA, miR4749, was investigated by Atomic Force Spectrscopy (AFS), static and time-resolved fluorescence spectroscopy and by computational methods. The formation of a HSA/miR4749 complex with an affinity of about 10⁴ M⁻¹ has been assessed through a Stern–Volmer analysis of steady-state fluorescence quenching of the lone Trp residue (Trp214) emission of HSA. Förster Resonance Energy Transfer (FRET) measurements of fluorescence lifetime of the HSA/miR4749 complex were carried out in the absence and in the presence of an acceptor chromophore linked to miR4749. This allowed us to determine a distance of 4.3 ± 0.5 nm between the lone Trp of HSA and the dye bound to miR4749 5p-end. Such a distance was exploited for a screening of the possible binding sites between HSA and miR4749, as predicted by computational docking. Such an approach, further refined by binding free energy calculations, led us to the identification of a consistent model for the structure of the HSA/miR4749 complex in which a positively charged HSA pocket accommodates the negatively charged miRNA molecule. These results designate native HSA as a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.     

Live‐Cell MicroRNA Imaging through MnO2 Nanosheet‐Mediated DD‐A Hybridization Chain Reaction

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live‐cell miRNA imaging using a biodegradable MnO₂ nanosheet‐mediated DD‐A FRET hybridization chain reaction (HCR). The MnO₂ nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO₂ nanosheets are degraded by cellular GSH. Then, the target miR‐21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (d onor) and one TAMRA (a cceptor) into close proximity to generate significantly enhanced DD‐A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies.     

Maternal Flaxseed Oil During Lactation Enhances Bone Development in Male Rat Pups

Flaxseed oil is an alpha linolenic acid source important in the growth and body development stage; furthermore, this acid acts on adipose tissue and bone health. The aim of this study was to evaluate body composition, fatty acid composition, hormone profile, retroperitoneal adipocyte area and femur structure of pups at weaning, whose mothers were fed a diet containing flaxseed oil during lactation. After birth, pups were randomly assigned: control (C, n = 12) and flaxseed oil (FO, n = 12), rats whose mothers were treated with diet containing soybean or flaxseed oil. At 21 days, the pups were weaned and body mass, length, body composition, biochemical parameter, leptin, osteoprotegerin, osteocalcin, fatty acids composition, intra‐abdominal fat mass and femur structure were analyzed. FO showed (p < 0.05): higher body mass (+12 %) and length (+9 %); body fat mass (g, +45 %); bone mineral density (+8 %), bone mineral content (+55 %) and bone area (+35 %), osteocalcin (+173 %) and osteoprotegerin (+183 %). Arachidonic acid was lower (p < 0.0001), alpha‐linolenic and eicosapentaenoic were higher (p < 0.0001). Intra‐abdominal fat mass was higher (+25 %), however, the retroperitoneal adipocytes area was lower (?44 %). Femur mass (+10 %), distance between epiphyses (+4 %) and bone mineral density (+13 %) were higher. The study demonstrates that adequate flaxseed oil content during a lactation diet plays an important role in the development of pups.     

Revealing the Anti-Tumor Effect of Artificial miRNA p-27-5p on Human Breast Carcinoma Cell Line T-47D

microRNAs (miRNAs) cause mRNA degradation or translation suppression of their target genes. Previous studies have found direct involvement of miRNAs in cancer initiation and progression. Artificial miRNAs, designed to target single or multiple genes of interest, provide a new therapeutic strategy for cancer. This study investigates the anti-tumor effect of a novel artificial miRNA, miR P-27-5p, on breast cancer. In this study, we reveal that miR P-27-5p downregulates the differential gene expressions associated with the protein modification process and regulation of cell cycle in T-47D cells. Introduction of this novel artificial miRNA, miR P-27-5p, into breast cell lines inhibits cell proliferation and induces the first "gap" phase (G1) cell cycle arrest in cancer cell lines but does not affect normal breast cells. We further show that miR P-27-5p targets the 3'-untranslated mRNA region (3'-UTR) of cyclin-dependent kinase 4 (CDK4) and reduces both the mRNA and protein level of CDK4, which in turn, interferes with phosphorylation of the retinoblastoma protein (RB1). Overall, our data suggest that the effects of miR p-27-5p on cell proliferation and G1 cell cycle arrest are through the downregulation of CDK4 and the suppression of RB1 phosphorylation. This study opens avenues for future therapies targeting breast cancer.