Objective: To investigate the influence of high fat diet-induced obesity (HFDIO) on the differentially methylated region (DMR) of the imprinted gene and global genome methylation of sperm DNA. METHODS: We performed bisulfite sequencing on the DMR of the imprinted gene and global genome methylation of sperm DNA in the mouse model of HFDIO. RESULTS: No statistically significant differences were found between the HFDIO model and normal control mice in MEG3-IG (93.73 vs 97.26%, P = 0.252), H19 (98.00 vs 97.83%, P = 0.920), IGF2 (97.34 vs 96.25%, P =0.166), IGF2R (1.43 vs 1.11%, P = 0.695), PEG3 (0.19 vs 0.38%, P = 0.537), MEST (0.23 vs 0.68%, P = 0.315), NNAT (0.31 vs 0.00%, P = 0.134), or SNRPN (1.88 vs 3.13%, P = 0.628). A total of 8 942 DMRs were detected across the sperm genome (P <0.05). Gene functional enrichment analysis indicated that the enriched terms with the largest numbers of genes were the metabolic process (n = 1 482), RNA synthesis (n = 779), and transcription (n = 767). CONCLUSIONS The methylation level underwent no significant change in the DMRs of the imprinted genes from the mice with HFDIO, but the CG methylation of the genes involved in the metabolic process, RNA synthesis and transcription were significantly altered.
Animals ; DNA Methylation ; Diet, High-Fat ; Genome ; Genomic Imprinting ; Insulin-Like Growth Factor II ; Male ; Mice ; Obesity ; genetics ; metabolism ; RNA ; biosynthesis ; Spermatozoa ; metabolism

OBJECTIVETo explore the mechanism of electroacupuncture on improving insulin resistance of rat from aspects of morphology and function of mitochondrial in quadriceps femoris.

METHODSForty-eight 8-week Wistar rats (female and male in half) were randomly divided into a normal group (16 rats, group A), a model control group (16 rats, group B), a model plus electroacupuncture (EA) group (8 rats, group C) and a model plus sham acupoint EA group (8 rats, group D). Group A was given with basic diet while high-fat diet was applied in the group B, group C and group D for 8 weeks to establish model of insulin resistance. After the model establishment, "Guanyuan" (CV 4), "Zhongwan" (CV 12), "Zusanli" (ST 36) and "Fenglong" (ST 30) were selected according to acupoint combination of manifestation-root in the group C, while four points in non-meridian area where 1 to 2 mm next to the acupoints used in group C were selected in the group D. The treatment was given 15 min per time with 1 mA of intensity and 2 Hz in frequency, 5 times per week for totally 8 weeks. The transmission electron microscope was adopted to observe mitochondria structure, and chemical colorimetry was used to test the activity of adenosine triphosphate (ATP) synthase and phosphomolybdic acid colorimetry was applied to measure the content of ATP.

RESULTSAfter the treatment, the body mass was (401.63 +/- 109.81) g in the group B, which was significantly higher than (305.88 +/- 62.72) g in the group A (P < 0.05); morphological structure of mitochondrion was damaged, showing swelling and deformation; the activity of ATP synthase was decreased (P < 0.05) and the content of ATP in tissue of quadriceps femoris was also obviously lowered (P < 0.05). The body mass was (294.13 +/- 53.78) g in the group C, which was significantly lower than that in the group B (P < 0.05); the damaged mitochondrion was restored and merged among each other; the activity of ATP synthase was increased (P < 0.05); the content of ATP in tissue of quadriceps femoris was obviously lifted (P < 0.05). The results in group D were not different from those in group B.

CONCLUSIONThe electroacupuncture with manifestation-root acupoint combination could improve the recovery of damaged structure of mitochondrion and promote the merge among each other, which could enhance oxidizing capacity, lower body mass and improve synthetic rate of ATP.

Acupuncture Points ; Adenosine Triphosphate ; biosynthesis ; Animals ; Diabetes Mellitus, Type 2 ; metabolism ; therapy ; Electroacupuncture ; Female ; Humans ; Insulin ; metabolism ; Insulin Resistance ; Male ; Mitochondria ; enzymology ; metabolism ; ultrastructure ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Quadriceps Muscle ; metabolism ; ultrastructure ; Rats ; Rats, Wistar

The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria (MFMC) on PPARγ (Peroxisome Proliferator Activated Receptor gamma) and GLUT-4 (Glucose transporter-4) with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL(-1) were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC (500 μg·mL(-1)) was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARγ gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide (CHX), which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARγ and GLUT-4 in vitro.
Biological Transport ; Dose-Response Relationship, Drug ; Fruit ; Gene Expression ; drug effects ; Glucose ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Hypoglycemic Agents ; pharmacology ; In Vitro Techniques ; Insulin ; metabolism ; Momordica ; Muscle Fibers, Skeletal ; drug effects ; PPAR gamma ; metabolism ; Plant Extracts ; pharmacology ; Protein Biosynthesis ; Protein Synthesis Inhibitors ; pharmacology ; Rosiglitazone ; Thiazolidinediones ; pharmacology ; Up-Regulation

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology

To construct, express and purify Exendin-4 analogue and detect its biological activity in vivo. Insert gene sequence into fusion partner ofpED plasmid which is helped to purification, entitled the new recombinant plasmid 5 Exendin-4 analogue polypeptide gene and fusion partner gene was linked by acid hydrolysisgene, transformed to E. coli BL21 and the fusion protein was induced by lactose. After acid hydrolysis, the Exendin-4 analogue polypeptide separated from fusion chaperon. Anion charge chromatography were used to further purification. 6 to 8 week-old ICR mice were injected (s.c) with Exendin-4 analogue, blood glucose and plasma insulin level was detected in different period after oral glucose tolerance test. The results show that high expression of inclusion body was induced by lactose, which accounted for 40% of germ proteins, the Exendin-4 analogue was obtained with the purity of 91.8% after being purified by anion charge chromatography. Bioactivity assay showed that the level of blood glucose of mouse which treated with exendin-4 analogue was obviously decreased to normal (P < 0.01), and the level of plasma insulin was increased obviously (P < 0.01).
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Transfer Techniques ; Hypoglycemic Agents ; metabolism ; pharmacology ; Insulin ; blood ; Male ; Mice ; Mice, Inbred ICR ; Peptides ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Venoms ; biosynthesis ; genetics ; pharmacology

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.
3T3-L1 Cells ; *Adipocytes/drug effects/metabolism ; Adiponectin/biosynthesis ; Adipose Tissue/metabolism ; Animals ; Endoplasmic Reticulum Stress/drug effects ; Insulin Resistance/genetics ; Mice ; Mitochondria/drug effects/*metabolism/pathology ; Mitochondrial Turnover/drug effects/genetics ; *Nitric Oxide Synthase Type II/genetics/metabolism ; Nuclear Respiratory Factor 1 ; Obesity/genetics/metabolism ; Palmitic Acid/pharmacology ; Thiazolidinediones/pharmacology

OBJECTIVETo investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine ÷ cytokines and insulin-dependent glucose uptake in human differentiated adipocytes.

METHODSAdipose tissues were obtained from patients undergoing liposuction. Stromal vascular cells were extracted and differentiated into adipocytes.A specific ligand for NOD1, was administered to human adipocytes in culture. Nuclear factor-κB transcriptional activity and proinflammatory chemokine ÷ cytokines production were determined by reporter plasmid assay and enzyme-linked immunosorbent assay, respectively.Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[³H] glucose uptake assay. Furthermore, chemokine ÷ cytokine secretion and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon stimulation of NOD1 ligand were analyzed.

RESULTSNuclear factor-κB transcriptional activity and monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and IL-8 secretion in human adipocytes were markedly increased stimulated with NOD1 ligand (all P<0.01).Insulin-induced glucose uptake was decreased upon the activation of NOD1 (P<0.05).NOD1 gene silencing by siRNA reduced NOD1 ligand-induced MCP-1,IL-6, and IL-8 release and increased insulin-induced glucose uptake (all P<0.05).

CONCLUSIONNOD1 activation in adipocytes might be implicated in the onset of insulin resistance.

Adipocytes ; metabolism ; Adult ; Cells, Cultured ; Cytokines ; biosynthesis ; Female ; Glucose ; metabolism ; Humans ; Inflammation ; etiology ; Insulin Resistance ; Ligands ; Middle Aged ; NF-kappa B ; physiology ; Nod1 Signaling Adaptor Protein ; physiology

OBJECTIVETo optimize the culture media by adding the growth factors required to maintain tenocytes survival and promote their differentiation without fetal bovine serum (FBS) supplementation, in order for the approach to be used for any future tendon tissue engineering.

METHODSThe human tenocytes were cultured in α-MEM media by adding FBS at various concentrations and supplementing both insulin-like growth factor 1 (IGF-1) and transforming growth factor β-3 (TGFβ-3). A number of growth factors were selected that could support tenocytes expansion at reduced differentiated state with the minimum FBS. By employing fractional factorial design, different treatment groups went through AlamarBlue(TM) tests to evaluate the cell number growth whilst collagen quantification by real time RT-PCR technique and tenocyte differentiation were also studied.

RESULTSThe tenocytes cultured for 14 days with 0% FBS, 50 ng/ml IGF-1 and 10 ng/ml TGFβ-3 maintained survival over 14 days, the Cell count were 6228.68 ± 43.87. They were higher than the other experimental groups, but less than 10% FBS control group (13 576.74 ± 286.75, t = 41.29, P < 0.05). The tenocytes cultured in the treated group also showed enhanced collagen synthesis ((0.322 ± 0.003) ng, t = 4.13 - 5.93, P < 0.05).

CONCLUSIONThese findings have shown for the first time that human tenocytes could be maintained survival for a long period of time in the culture media without FBS, having this approach a suitable one for tendon tissue engineering.

Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Culture Media ; pharmacology ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Tendons ; cytology ; drug effects ; metabolism ; Transforming Growth Factor beta3 ; pharmacology

Diabetes mellitus has become one of the most common chronic diseases, thereby posing a major challenge to global health. Characterized by high levels of blood glucose (hyperglycemia), diabetes usually results from a loss of insulin-producing β-cells in the pancreas, leading to a deficiency of insulin (type 1 diabetes), or loss of insulin sensitivity (type 2 diabetes). Both types of diabetes have serious secondary complications, such as microvascular abnormalities, cardiovascular dysfunction, and kidney failure. Various complex factors, such as genetic and environmental factors, are associated with the pathophysiology of diabetes. Over the past two decades, the role of small, single-stranded noncoding microRNAs in various metabolic disorders, especially diabetes mellitus and its complications, has gained widespread attention in the scientific community. Discovered first as an endogenous regulator of development in the nematode Caenorhabditis elegans, these small RNAs post-transcriptionally suppress mRNA target expression. In this review, we discuss the potential roles of different microRNAs in diabetes and diabetes-related complications.
Animals ; Diabetes Complications ; genetics ; metabolism ; Diabetes Mellitus ; genetics ; metabolism ; Glucose ; metabolism ; Homeostasis ; genetics ; Humans ; Insulin ; metabolism ; MicroRNAs ; biosynthesis ; genetics ; metabolism

OBJECTIVETo explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.

METHODSThe mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.

RESULTSThe expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.

CONCLUSIONSThe recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; therapy ; Diphtheria Toxin ; biosynthesis ; genetics ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Genomic Imprinting ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection