Fengqin Wang

Hypoglycemic and hypolipidemic properties of polysaccharides from Enterobacter cloacae Z0206 in KKAy mice.

The water-soluble polysaccharides (EPS) were isolated from culture broth of Enterobacter cloacae Z0206, and the hypoglycemic and hypolipidemic effects of EPS were investigated. In this study, KKAy mice were gavaged once daily with either distilled water or EPS (200 mg/kg body weight) for 6 weeks. Results showed that EPS possessed significant hypoglycemic effects. Improved oral glucose tolerance, reduced serum insulin levels as well as decreased serum triglycerides (TG), cholesterol (TC) and low density lipoprotein cholesterol (LDL-c) were observed after treatment with EPS. Furthermore, EPS upregulated the expression of glucokinase (GK), HSL (Hormone Sensitive Lipase), adipose triglyceride lipase (ATGL), carnitine palmitoyl transferase 1-α (CPT1-α), glucose transporter 2 (Glut2), adenosine monophosphate activated protein kinase (AMPK) and silent information regulator 1 (Sirt1), but downregulated the gene expression of glucose-6-phosphatase (G6P) and fatty acid synthase (FAS) in the liver. These results suggest that EPS exhibits hypoglycemic and hypolipidemic effects possibly through regulating AMPK- and SirT1-mediated effects on carbohydrate and lipid metabolism.

VIRMA mediates preferential m 6 A mRNA methylation in 3′UTR and near stop codon and associates with alternative polyadenylation

N6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

Structure characterization of a fucose-containing exopolysaccharide produced by Enterobacter cloacae Z0206.

A novel high molecular weight (1.1 × 10(6)Da) exopolysaccharide (EPS) produced by Enterobacter cloacae Z0206 strain was isolated by column chromatography. Complete hydrolysis of the EPS followed by gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) analyses showed that the EPS is composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid in the approximate molar ratio of 2:1:3:1:1. Partial acid hydrolysis of the purified EPS followed by gel permeation chromatography (GPC) yielded a hexasaccharide. A combination of chemical analysis coupled with mass spectrometry and 1D and 2D NMR spectroscopy applied to the oligosaccharide showed that the EPS comprises a heptasaccharide repeating unit.

Selenium-enriched exopolysaccharides improve skeletal muscle glucose uptake of diabetic KKAy mice via AMPK pathway

Selenium-enriched exopolysaccharides (EPS) produced by Enterobacter cloacae Z0206 have been proven to possess effect on reducing blood glucose level in diabetic mice. To investigate the specific mechanism, we studied the effects of oral supply with EPS on skeletal muscle glucose transportation and consumption in high-fat-diet-induced diabetic KKAy mice. We found that EPS supplementation increased expressions of glucose transporter 4 (Glut4), hexokinase 2 (hk2), phosphorylation of AMP-activated kinase subunit α2 (pAMPKα2), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and increased expression of characteristic protein of oxidative fibers such as troponin I and cytochrome c (Cytc). Furthermore, we found that EPS increased glucose uptake and expressions of pAMPKα2 and PGC-1α in palmitic acid (PA)-induced C2C12 cells. However, while EPS inhibited AMPKα2 with interference RNA (iRNA), effects of EPS on the improvement of glucose uptake diminished. These results indicated that EPS may improve skeletal muscle glucose uptake of diabetic KKAy mice through AMPKα2-PGC-1α pathway.

Selenium‑enriched exopolysaccharides produced by Enterobacter cloacae Z0206 alleviate adipose inflammation in diabetic KKAy mice through the AMPK/SirT1 pathway

Polysaccharides belong to a structurally diverse class of macromolecules, with the necessary flexibility for the precise regulatory mechanisms and high capacity for carrying biological information. On the basis of a previous study regarding the administration of selenium-enriched exopolysaccharides (Se-ECZ-EPS) produced by Enterobacter cloacae (E. cloacae) Z0206 which resulted in a reduction of blood glucose levels and showed significant anti-inflammatory and anti-diabetic effects, the present study was conducted to evaluate the effects and mechanism of EPS on the alleviation of fat inflammation in high-fat-diet (HFD) induced-diabetic KKAy mice. The HFD induced-diabetic KKAy mice were gavaged once daily with EPS (0.2 mg/g body weight) or distilled water, while the C57BL/6J mice were gavaged with distilled water. Six weeks later visceral adipose tissue (VAT) was collected for quantified polymerase chain reaction (qPCR) and western blot (WB) analysis. The results showed that following supplementation with EPS, interleukin (IL) 6, IL1β and tumor necrosis factor (TNF) α mRNA expression in VAT were significantly reduced, while Glut4, pAMPK and SirT1 protein expression were markedly increased when compared with KKAy mice gavaged with water. Furthermore, ATGL and HSL mRNA were also significantly decreased. Subsequently, 3T3-L1 adipocytes were treated with insulin to induce insulin resistance to determine the mechanism by which EPS affects inflammation. Following the treatment of adipocytes with 100 nM insulin for 8 h, IL6 and TNFα mRNA expression were significantly increased, while the content of glucose uptake and Glut4 protein expression were significantly decreased. When treated with 100 nM insulin and 0.1 mg/ml EPS, no significant change in IL6 and TNFα mRNA expression or glucose uptake were observed. However, when SirT1‑siRNA or AMPKα1-siRNA was transfected into the 3T3-L1 adipocytes prior to treatment with insulin and EPS, there was a significant increase in IL6 and TNFα mRNA abundance. In conclusion, VAT inflammation and lipolysis in HFD-induced KKAy mice were significantly decreased following EPS usage. Moreover, EPS may alleviate VAT inflammation primarily through the AMPK/SirT1 pathway.

Characterization, antioxidant property and cytoprotection of exopolysaccharide-capped elemental selenium particles synthesized by Bacillus paralicheniformis SR14

Instead of using existing methods to chemically synthesize elemental selenium particles (CheSePs), which first require separating and purifying polysaccharides or proteins and adding extra reducing agent, this study applied a novel method to directly assemble exopolysaccharide-capped biogenic elemental selenium particles (EPS-BioSePs) by Bacillus paralicheniformis SR14 during the metabolic process. Characterization by energy dispersive X-ray spectrometry (EDX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), size measurement and chemical composition analysis verified that EPS-BioSePs exhibited a monodispersed and homogeneous spherical structure 293.73±4.03nm in size. Compared to a widely used form of CheSePs stabilized and coated by bovine serum albumin, EPS-BioSePs exhibited better antioxidant properties on scavenging DPPH, superoxide and ABTS free radicals, but not hydroxyl radical. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells also indicated a significant cytoprotection of EPS-BioSePs against hydrogen peroxide-induced oxidative stress, as exhibited by cell viability reduction and suppression of ROS generation. These results suggested that this new form of selenium possessed great antioxidant property and cytoprotection and exopolysaccharide-producing bacteria could gradually become an appropriate choice to synthesize biogenic elemental selenium particles with potential applications as antioxidants.

Cathelicidin-WA polarizes E. coli K88-induced M1 macrophage to M2-like macrophage in RAW264.7 cells

ABSTRACT Immune cells ‐ macrophages induced by E. coli K88 will lead to a pro‐inflammatory response, which is important in host defense. Cathelicidin‐WA (CWA) is an efficient antimicrobial peptide (AMP) and can exert immunomodulatory properties. Many studies have demonstrated that AMP can modulate cellular subsets but whether CWA can regulate macrophage polarization by transferring E. coli K88‐induced M1 macrophage towards M2 one that of anti‐inflammation remains unclear. In this study, E. coli K88 increased the expression of pro‐inflammatory cytokines interleukin‐6, interleukin‐1&bgr;, tumor necrosis factor‐&agr; and chemokine CCL3 in RAW264.7 cells with a time‐dependent manner, as well as the expression of reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS). On this basis, CWA significantly decreased the pro‐inflammatory molecules but increased the anti‐inflammatory mediators interleukin‐4, interleukin‐10 and other M2‐related genes in E. coli K88‐induced macrophages. Western blot analysis indicated that CWA suppressed the expression of TLR‐4 and the phosphorylation of STAT1 and NF‐&kgr;B which modulated M1 macrophage while induced the phosphorylation of STAT6 which activated M2 macrophage. Double staining of M1‐specific CD86 and M2‐specific CD206 also proved the hypothesis. These results suggested that CWA might dampen the inflammation by modulating M1 phenotype to M2 phenotype in E. coli K88‐induced macrophages. HighlightsRAW264.7 cells showed a pro‐inflammatory response induced by E. coli K88.The pro‐inflammatory macrophage is analogous to M1 macrophage.CWA effectively suppresses the inflammation in E. coli K88‐induced macrophages.CWA might exert its immunomodulatory activities by switching M1 macrophages to M2 macrophages.

[Simultaneous determination of neutral sugars and uronic acid constituents in a novel bacterial polysaccharide using gas chromatography-mass spectrometry].

The purified novel bacterial polysaccharide was acid-hydrolyzed, followed by the subsequent derivatization using ethanethiol-trifluoroacetic acid and acetic anhydride-pyridine systems sequentially. Our findings differ from the previous reports in that the glucuronic acid was obtained through effective derivatization. The neutral sugars and glucuronic acid were analyzed using gas chromatography-mass spectrometry (GC-MS) with xyloses as an internal standard. The polysaccharide was found to be composed of fucose, glucose, glucuronic acid and galactose, with the relative molar ratio of 1.50: 1.0: 0.79: 2.06. The neutral sugars ratio was similar to the relative molar ratio for fucose, glucose and galactose of 1.76: 1.0: 1.98 through alditol acetates determined by GC. The percentages of glucuronic acid analyzed using either the carbazole and sulfuric acid method or the above method were 16.19% and 14.85%, respectively. These results indicate that it is practicable to use the derivatization method and GC-MS to quantitatively analyze neutral sugars and glucuronic acid simultaneously in polysaccharide. For GC-MS analysis, the procedure was developed for the simultaneous determination of the derivatives in 25 min, and was performed using an HP-5MS column. Molecular ion peaks were observed in the electron ionization (EI) mass spectra. The fragmentation mechanism for glucuronic acid derivative is discussed in detail.

MTCH2 promotes adipogenesis in intramuscular preadipocytes via an m6A-YTHDF1–dependent mechanism

Intramuscular fat is considered a potential factor that is associated with meat quality in animal production and insulin resistance in humans. N6‐methyladenosine (m6A) modification of mRNA plays an important role in regulating adipogenesis. However, the effects of m6A on the adipogenesis of intramuscular preadipocytes and associated mechanisms remain unknown. Here, we performed m6A sequencing to compare m6A methylome of the longissimus dorsi muscles (LDMs) between Landrace pigs (lean‐type breed) and Jinhua pigs (obese‐type breed with higher levels of intramuscular fat). Transcriptome‐wide m6A profiling of porcine LDMs was highly conserved with humans and mice. Furthermore, we identified a unique methylated gene in Jinhua pigs named mitochondrial carrier homology 2 (MTCH2). The m6A levels oiMTCHl mRNA were reduced by introducing a synonymous mutation, and adipogenesis test results showed that the MTCH2 mutant was inferior with regard to adipogenesis compared with the MTCH2 wild‐type. We then found that MTCH2 protein expression was positively associated with m6A levels, and an YTH domain family protein 1‐RNA immunoprecipitation‐quantitative PCR assay indicated that MTCH2 mRNA was a target of the YTH domain family protein 1. This study provides comprehensive m6A profiles of LDM transcriptomes in pigs and suggests an essential role for m6A modification of MTCH2 in intramuscular fat regulation.—Jiang, Q., Sun, B., Liu, Q., Cai, M., Wu, R., Wang, F., Yao, Y., Wang, Y., Wang, X. MTCH2 promotes adipogenesis in intramuscular preadipocytes via an m6A‐YTHDF1‐dependent mechanism. FASEB J. 33, 2971–2981 (2019). www.fasebj.org

Effect of soybean meal fermented with Bacillus subtilis BS12 on growth performance and small intestinal immune status of piglets

ABSTRACT This study was aimed to screen a high-efficient strain for degrading antigenic protein in soybean meal (SBM) and evaluate the effect of fermented soybean meal (FSBM) on growth performance of piglets. A Bacillus subtilis strain BS12 was selected through plate and fermentation experiment, which reduced 92.36% less glycinin and 88.44% less β-conglycinin in SBM. A total of 192 piglets were assigned to receive either a diet of SBM with antibiotics (the control group) or a diet containing 10% FSBM without antibiotics. The average daily gain and feed intake of pigs fed FSBM were superior (p < .10) to those fed the control diet. Reduced (p < .05) mRNA expression of pro-inflammatory cytokines was detected in the jejunum and ileum of pigs fed FSBM. These results demonstrated that a diet containing BS12 FSBM improved growth performance by reducing dietary inflammation in piglets.