OBJECTIVE: To investigate the regulatory role of miRNA-26a in vascular smooth muscle cell (VSMC) calcification by regulating connective tissue growth factor (CTGF). METHODS: Rat thoracic aorta VSMCs (A7r5 cells) with induced calcification were treated with AR234960 agonist or transfected with miR-26a mimic, or with both treatments. Alizarin red staining was used to determine calcium deposition, and phosphatase (ALP) activity in the cells was measured. The mRNA and protein expressions of miR-26a, OPG, OPN, BMP-2 and collagen Ⅱ were detected using qPCR and Western blotting. The binding of miR-26a to CTGF was verified using dual luciferase reporter gene assay. RESULTS: After induced calcification, A7r5 cells showed gradually decreased miR-26a expression (P < 0.05) and progressively increased CTGF expression (P < 0.05) with the extension of induction time. Treatment of the cells with AR234960 obviously increased calcification in the cells, while transfection with miR-26a mimic significantly reduced cell calcification. The calcifying cells showed significantly increased ALP activity and expressions of OPN, BMP-2 and collagen Ⅱ (P < 0.05) and lowered OPG expression (P < 0.05), and treatment with AR234960 did not produce obvious effects on these changes (P > 0.05). Transfection with miR-26a mimic resulted in significantly decreased ALP activity and expressions OPN, BMP-2 and collagen Ⅱ expression (P < 0.05) and increased OPG expression (P < 0.05) in the calcifying cells. These effects of miR-26a mimic was significantly attenuated by treatment of the cells with AR234960 (P < 0.05). The result of luciferase reporter gene assay confirmed the binding of miR-26a to CTGF. CONCLUSION miRNA-26a can effectively alleviate vascular calcification by lowering the level of CTGF, reducing ALP activity and the expressions of OPN, BMP-2 and collagen Ⅱ, and increasing the expression of OPG.
Animals ; Calcium/metabolism* ; Cells, Cultured ; Connective Tissue Growth Factor/pharmacology* ; MicroRNAs/metabolism* ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Phosphoric Monoester Hydrolases/pharmacology* ; RNA, Messenger/metabolism* ; Rats ; Sulfones ; Vascular Calcification

OBJECTIVE: To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. METHODS: Wild-type (WT) mice and systemic CD36 knockout (CD36^-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. RESULTS: CD36^-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36^-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36^-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36^-/- mice (P>0.05). In the muscle tissue of CD36^-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36^-/- mice. CONCLUSION CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.
Animals ; Gene Deletion ; Histones/genetics* ; Insulin ; Insulin Receptor Substrate Proteins/metabolism* ; Insulin Resistance/genetics* ; Membrane Cofactor Protein/genetics* ; Mice ; Mice, Knockout ; Muscles/metabolism* ; Phosphoric Monoester Hydrolases/metabolism* ; Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger/metabolism* ; Receptor, Insulin/metabolism* ; Tyrosine/genetics* ; Up-Regulation

Mitochondrial dysfunction is a hallmark of metabolic diseases such as obesity, type 2 diabetes mellitus, neurodegenerative diseases, and cancers. Dysfunction occurs in part because of altered regulation of the mitochondrial pyruvate dehydrogenase complex (PDC), which acts as a central metabolic node that mediates pyruvate oxidation after glycolysis and fuels the Krebs cycle to meet energy demands. Fine-tuning of PDC activity has been mainly attributed to post-translational modifications of its subunits, including the extensively studied phosphorylation and de-phosphorylation of the E1α subunit of pyruvate dehydrogenase (PDH), modulated by kinases (pyruvate dehydrogenase kinase [PDK] 1-4) and phosphatases (pyruvate dehydrogenase phosphatase [PDP] 1-2), respectively. In addition to phosphorylation, other covalent modifications, including acetylation and succinylation, and changes in metabolite levels via metabolic pathways linked to utilization of glucose, fatty acids, and amino acids, have been identified. In this review, we will summarize the roles of PDC in diverse tissues and how regulation of its activity is affected in various metabolic disorders.
Acetylation ; Amino Acids ; Citric Acid Cycle ; Diabetes Mellitus, Type 2 ; Fatty Acids ; Glucose ; Glycolysis ; Metabolic Diseases ; Metabolic Networks and Pathways ; Metabolism* ; Mitochondria ; Neurodegenerative Diseases ; Obesity ; Oxidative Phosphorylation ; Oxidoreductases ; Phosphoric Monoester Hydrolases ; Phosphorylation ; Phosphotransferases ; Protein Processing, Post-Translational ; Pyruvate Dehydrogenase Complex* ; Pyruvic Acid*

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignancies in the world today, with a high mortality rate. CDX2 is a Drosophila caudal-related homeobox transcription factor that plays an important role in GC. Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is an important tumor suppressor which is widely expressed in normal human tissues. The aim of the study was to determine the relationship and mechanism between CDX2 and PTEN in invasion and migration of GC cells.

METHODSpcDNA3-CDX2 plasmids were transfected into MGC-803 cells to up-regulate CDX2 protein, and small interfering RNA-CDX2 was transfected to down-regulate CDX2. The influence of CDX2 or PTEN on cell migration and invasion was measured by invasion, migration and wound healing assays. Western blotting assay and immunofluorescence were used to detect the expression of CDX2, PTEN, phosphorylation of Akt, E-cadherin and N-cadherin. Statistical significance was determined by one-way analysis of variance.

RESULTSThe results showed that CDX2 reduced the migration and invasion of GC cells (P < 0.05), and inhibited the activity of Akt through down-regulating PTEN expression (P < 0.05). CDX2 also restrained epithelial-mesenchymal transition of GC cells.

CONCLUSIONSCDX2 inhibited invasion and migration of GC cells by PTEN/Akt signaling pathway, and that may be used for potential therapeutic target.

CDX2 Transcription Factor ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Chromosomes, Human, Pair 10 ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; physiology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Microfilament Proteins ; genetics ; metabolism ; PTEN Phosphohydrolase ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Tensins ; Wound Healing ; genetics ; physiology

Compartments of soil microorganism and enzymes between stereoscopic cultivation (three storeys) and field cultivation (CK) of Panax notoginseng were carried out, and the effects on P. notoginseng agronomic characters were also studied. Results show that concentration of soil microorganism of stereoscopic cultivation was lower than field cultivation; the activity of soil urea enzyme, saccharase and neutral phosphatase increased from lower storey to upper storey; the activity of soil urea enzyme and saccharase of lower and upper storeys were significantly lower than CK; agronomic characters of stereoscopic cultivated P. notoginsengin were inferior to field cultivation, the middle storey with the best agronomic characters among the three storeys. The correlation analysis showed that fungi, actinomycetes and neutral phosphatase were significantly correlated with P. notoginseng agronomic characters; concentration of soil fungi and bacteria were significantly correlated with the soil relative water content; actinomycete and neutral phosphatase were significantly correlated with soil pH and relative water content, respectively; the activities of soil urea enzyme and saccharase were significantly correlated with the soil daily maximum temperature difference. Inconclusion, The current research shows that the imbalance of soil microorganism and the acutely changing of soil enzyme activity were the main reasons that caused the agronomic characters of stereoscopic cultivated P. notoginseng were worse than field cultivation. Thus improves the concentration of soil microorganism and enzyme activity near to field soil by improving the structure of stereoscopic cultivation is very important. And it was the direction which we are endeavoring that built better soil ecological environment for P. notoginseng of stereoscopic cultivation.
Hydrogen-Ion Concentration ; Panax notoginseng ; growth & development ; Phosphoric Monoester Hydrolases ; metabolism ; Soil ; chemistry ; Soil Microbiology ; beta-Fructofuranosidase ; metabolism

Impaired glucose homeostasis is one of the risk factors for causing metabolic diseases including obesity, type 2 diabetes, and cancers. In glucose metabolism, pyruvate dehydrogenase complex (PDC) mediates a major regulatory step, an irreversible reaction of oxidative decarboxylation of pyruvate to acetyl-CoA. Tight control of PDC is critical because it plays a key role in glucose disposal. PDC activity is tightly regulated using phosphorylation by pyruvate dehydrogenase kinases (PDK1 to 4) and pyruvate dehydrogenase phosphatases (PDP1 and 2). PDKs and PDPs exhibit unique tissue expression patterns, kinetic properties, and sensitivities to regulatory molecules. During the last decades, the up-regulation of PDKs has been observed in the tissues of patients and mammals with metabolic diseases, which suggests that the inhibition of these kinases may have beneficial effects for treating metabolic diseases. This review summarizes the recent advances in the role of specific PDK isoenzymes on the induction of metabolic diseases and describes the effects of PDK inhibition on the prevention of metabolic diseases using pharmacological inhibitors. Based on these reports, PDK isoenzymes are strong therapeutic targets for preventing and treating metabolic diseases.
Acetyl Coenzyme A ; Decarboxylation ; Diabetes Mellitus, Type 2 ; Glucose ; Homeostasis ; Humans ; Isoenzymes ; Mammals ; Metabolic Diseases ; Metabolism ; Obesity ; Oxidoreductases* ; Phosphoric Monoester Hydrolases ; Phosphorylation ; Phosphotransferases* ; Pyruvate Dehydrogenase Complex ; Pyruvic Acid* ; Risk Factors ; Up-Regulation

OBJECTIVETo investigate the potential substitution effect of hOGG1 and hMTH1 on oxidative DNA damage, based on gene-deficient cell strains models.

METHODShOGG1 and hMTH1 gene deficient cell strains models were established by Human embryonic lung fibroblasts (HFL) cells. After HFL cells being exposed to 100 µmol/L H₂O₂ for 12 h, HPLC-EC detecting technique and RT-PCR method were adopted to analyze the genetic expression level of 8-oxo-dG (7, 8-dihydro-8-oxoguanine).

RESULTSThe gene-deficient cell strains models of hOGG1 and hMTH1 were obtained by infecting target cells with high titer of lentivirus. The mRNA expression level of hOGG1 was 0.09 ± 0.02, 91% lower than it in normal HFL cells, which was 1.00 ± 0.04. As the same, the mRNA expression level of hMTH1 (0.41 ± 0.04) also decreased by 60% compared with it in normal HFL cells (1.02 ± 0.06). After induced by 100 µmol/L H₂O₂ for 12 h, the genetic expression level of hMTH1 in hOGG1 gene-deficient cells (1.26 ± 0.18) increased 25% compared with it in control group (1.01 ± 0.07). Meanwhile, the genetic expression level of hOGG1 in hMTH1 gene-deficient cells (1.54 ± 0.25) also increased by 52%. The DNA 8-oxo-dG levels in hOGG1 gene-deficient cells (2.48 ± 0.54) was 3.1 times compared with it in the control group (0.80 ± 0.16), the difference showed statistical significance (P < 0.01). Whereas the 8-oxo-dG levels in hMTH1 gene-deficient cells (1.84 ± 0.46) was 2.3 times of it in the control group, the difference also showed statistical significance (P < 0.01).

CONCLUSIONBased on gene-deficient HFL cells models, a synergetic substitution effect on DNA damage and repair activity by both hOGG1 and hMTH1 were firstly discovered when induced by oxidation. The substitution effect of hOGG1 were stronger than that of hMTH1.

Cell Line ; DNA Damage ; DNA Glycosylases ; genetics ; DNA Repair ; DNA Repair Enzymes ; genetics ; Fibroblasts ; metabolism ; Humans ; Oxidative Stress ; genetics ; Phosphoric Monoester Hydrolases ; genetics

Glucose homeostasis is tightly controlled by the regulation of glucose production in the liver and glucose uptake into peripheral tissues, such as skeletal muscle and adipose tissue. Under prolonged fasting, hepatic gluconeogenesis is mainly responsible for glucose production in the liver, which is essential for tissues, organs, and cells, such as skeletal muscle, the brain, and red blood cells. Hepatic gluconeogenesis is controlled in part by the concerted actions of transcriptional regulators. Fasting signals are relayed by various intracellular enzymes, such as kinases, phosphatases, acetyltransferases, and deacetylases, which affect the transcriptional activity of transcription factors and transcriptional coactivators for gluconeogenic genes. Protein arginine methyltransferases (PRMTs) were recently added to the list of enzymes that are critical for regulating transcription in hepatic gluconeogenesis. In this review, we briefly discuss general aspects of PRMTs in the control of transcription. More specifically, we summarize the roles of four PRMTs: PRMT1, PRMT 4, PRMT 5, and PRMT 6, in the control of hepatic gluconeogenesis through specific regulation of FoxO1- and CREB-dependent transcriptional events.
Acetyltransferases ; Adipose Tissue ; Arginine* ; Brain ; Erythrocytes ; Fasting ; Gluconeogenesis ; Glucose* ; Homeostasis ; Liver ; Metabolism* ; Methyltransferases* ; Muscle, Skeletal ; Phosphoric Monoester Hydrolases ; Phosphotransferases ; Protein-Arginine N-Methyltransferases ; Transcription Factors

Field experiments were conducted in Shangluo pharmaceutical base in Shaanxi province to study the effect of nitrogen, phosphorus and potassium in different fertilization levels on Platycodon grandiflorum soil microorganism and activities of soil enzyme, using three-factor D-saturation optimal design with random block design. The results showed that N0P2K2, N2P2K0, N3P1K3 and N3P3K1 increased the amount of bacteria in 0-20 cm of soil compared with N0P0K0 by 144.34%, 39.25%, 37.17%, 53.58%, respectively. The amount of bacteria in 2040 cm of soil of N3P1K3 increased by 163.77%, N0P0K3 increased the amount of soil actinomycetes significantly by 192.11%, while other treatments had no significant effect. N2P0K2 and N3P1K3 increased the amounts of fungus significantly in 0-20 cm of soil compared with N0P0K0, increased by 35.27% and 92.21%, respectively. N3P0K0 increased the amounts of fungus significantly in 20-40 cm of soil by 165.35%, while other treatments had no significant effect. All treatments decrease soil catalase activity significantly in 0-20 cm of soil except for N2P0K2, and while N2P2K0 and NPK increased catalase activity significantly in 2040 cm of soil. Fertilization regime increased invertase activity significantly in 2040 cm of soil, and decreased phosphatase activity inordinately in 0-20 cm of soil, while increased phosphatase activity in 2040 cm of soil other than N1P3K3. N3P0K0, N0P0K3, N2P0K2, N2P2K0 and NPK increased soil urease activity significantly in 0-20 cm of soil compared with N0P0K0 by 18.22%, 14.87%,17.84%, 27.88%, 24.54%, respectively. Fertilization regime increased soil urease activity significantly in 2040 cm of soil other than N0P2K2.
Bacteria ; enzymology ; growth & development ; isolation & purification ; metabolism ; Bacterial Proteins ; analysis ; metabolism ; Catalase ; analysis ; metabolism ; Fertilizers ; analysis ; Fungal Proteins ; analysis ; metabolism ; Fungi ; enzymology ; growth & development ; isolation & purification ; metabolism ; Nitrogen ; metabolism ; Phosphoric Monoester Hydrolases ; analysis ; metabolism ; Phosphorus ; metabolism ; Potassium ; metabolism ; Soil ; chemistry ; Soil Microbiology ; Urease ; analysis ; metabolism

OBJECTIVETo screen obesity-related protein biomarkers of young men using differential proteomic method and OffGel electrophoresis.

METHODSTen male obese volunteers with the age of 18 - 44 years were selected. The control group was matched with the ratio of 1:1 considering the factors of age and gender etc. Two milliliter venous blood was collected after 8 hours fasting. Albumin and IgG were removed from the plasma samples with highly specific immune-affinity method. Then the peptide-mixed samples were separated by pI with OffGel electrophoresis after solution digestion. Further separation and identification were performed by Nano HPLC-Chip-MS/MS system. The different proteins between the two groups were compared.

RESULTSOverall, 332 and 301 proteins were identified in the obesity and control groups, respectively. There were 43 proteins with significant differences between the two groups, 17 of which were in the control group and 26 in the obesity group. Protein function annotation results showed that the level of adiponectin was lower while the level of C-reaction protein and three other phosphatases were higher in the obesity group compared with the control group.

CONCLUSIONAdiponectin, C-reaction protein and three phosphatases were closely related to obesity of young men.

Adiponectin ; blood ; Adolescent ; Adult ; Biomarkers ; blood ; C-Reactive Protein ; metabolism ; China ; epidemiology ; Humans ; Male ; Obesity ; blood ; epidemiology ; Phosphoric Monoester Hydrolases ; blood ; Proteomics ; Young Adult