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1.
Biomed Pharmacother ; 136: 111260, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33465676

ABSTRACT

OBJECTIVE: Cryptotanshinone (CPT) and dihydrotanshinone (DHT) are diterpenoid anthraquinone compounds extracted from traditional Chinese herbal medicine (TCM). Recent studies have shown that CPT regulates the signal transduction pathways via microRNA (miRNA) alterations. However, few studies have investigated the role of DHT in miRNA alterations affecting cell-signaling pathways. This study aimed to investigate the miRNA alterations and post-transcriptional regulation activities of DHT in comparison to CPT. METHODS: HepG2 and HT-29 cells were treated with DHT or CPT for 72 h. MiRNA, transcription factor encoding mRNA, and downstream gene expression were determined using real-time quantitative PCR. Protein expression was analyzed using western blotting. RESULTS: The results revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via miR-15a-5p, miR-27a-5p, miR-100-5p, and miR-200a-5p alterations.In silico target predictions showed that downregulation of epidermal growth factor receptor (EGFR) mRNA expression by DHT might also suppress the expression of STAT family proteins and lead to anti-proliferation effects. We also found that, compared to CPT, DHT might possess higher potency in cell growth regulation via multi-miRNA and transcription factor alterations. CONCLUSION: This study revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via alterations in miRNAs and transcription factors. In addition, the findings of this study suggest that DHT is more potent than CPT in cancer chemopreventive activities. Therefore, DHT at a low dose is a TCM compound with less toxic side effects and may contribute to the development of natural medicine as a potential cancer chemopreventive agent.


Subject(s)
Anticarcinogenic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Colonic Neoplasms/drug therapy , Furans/pharmacology , Liver Neoplasms/drug therapy , MicroRNAs/metabolism , Phenanthrenes/pharmacology , Quinones/pharmacology , Transcriptome/drug effects , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , HT29 Cells , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , MicroRNAs/genetics , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Molecules ; 23(2)2018 Jan 25.
Article in English | MEDLINE | ID: mdl-29370116

ABSTRACT

N-Butylidenephthalide (BP), which is extracted from a traditional Chinese medicine, Radix Angelica Sinensis (danggui), displays antitumor activity against various cancer cell lines. The purpose of this study was to investigate the cytotoxic and radiosensitizing effect of BP and the underlying mechanism of action in human breast cancer cells. BP induces apoptosis in breast cancer cells, which was revealed by the TUNEL assay; the activation of caspase-9 and PARP was detected by western blot. In addition, BP-induced G2/M arrest was examined by flow cytometry and the expression levels of the G2/M regulatory protein were detected by western blot. BP also suppresses the migration and invasion of breast cancer cells, which was tested by wound healing and the matrigel invasion assay; the involvement of EMT-related gene expressions was detected by real-time PCR. Furthermore, BP enhanced the radiosensitivity of breast cancer cells, which was measured by the colony formation assay and comet assay, where the foci of γ-H2AX after radiation significantly increased in BP pretreated cells and was evidenced by immunocytochemistry staining and western blot. The homologous recombination (HR) repair protein Rad51 was down-regulated after BP pretreatment. These results indicate that BP might be a potential chemotherapeutic and radiosensitizing agent for breast cancer therapy.


Subject(s)
Antineoplastic Agents/pharmacology , Phthalic Anhydrides/pharmacology , Radiation-Sensitizing Agents/pharmacology , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Breast Neoplasms , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , Female , Humans , Phthalic Anhydrides/chemistry , Radiation Tolerance/drug effects , Radiation-Sensitizing Agents/chemistry
3.
Mol Biol Rep ; 42(5): 927-35, 2015 May.
Article in English | MEDLINE | ID: mdl-25421647

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression. MiR-1908 is a recently identified miRNA that is highly expressed in human adipocytes. However, it is not known what role of miR-1908 is involved in the regulation of human adipocytes. In this study, we demonstrate that the level of miR-1908 increases during the adipogenesis of human multipotent adipose-derived stem (hMADS) cells and human preadipocytes-visceral. Overexpression of miR-1908 in hMADS cells inhibited adipogenic differentiation and increased cell proliferation, suggesting that miR-1908 is involved in the regulation of adipocyte cell differentiation and metabolism, and, thus, may have an effect on human obesity.


Subject(s)
Adipocytes/physiology , Adipogenesis/physiology , MicroRNAs/physiology , Adipogenesis/genetics , Cell Differentiation/genetics , Cell Proliferation/genetics , Gene Expression Regulation , Humans , MicroRNAs/genetics
4.
Macromol Biosci ; 13(8): 1100-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23682013

ABSTRACT

Microwell chips (25 mm × 25 mm) are fabricated to select proper substrates for growing three-dimensional (3D) spheroids from mesenchymal stem cells (MSCs). Different bio-macromolecules and their combinations are immobilized on the chip by air plasma treatment and by polyelectrolyte interaction. Only a small number of MSCs (≈10(5) ) are needed for each chip. The expression level of N-cadherin, a cell-adhesion molecule, is used as an indicator for cell-cell interactions. MSC spheroids expressing the highest N-cadherin level also show the greatest osteogenic potential. The microwell chip may be used as an efficient platform to screen bio-macromolecules that enhance the differentiation potential of MSCs.


Subject(s)
Cell Culture Techniques/methods , Cell Differentiation/physiology , Mesenchymal Stem Cells/metabolism , Adipose Tissue/cytology , Adipose Tissue/metabolism , Animals , Cadherins/biosynthesis , Cell Adhesion , Cell Communication , Cell Proliferation , Humans , Mesenchymal Stem Cells/cytology , Osteogenesis , Polymers/metabolism , Rats , Rats, Sprague-Dawley , Spheroids, Cellular/metabolism
5.
J Bioenerg Biomembr ; 44(5): 579-86, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22820890

ABSTRACT

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.


Subject(s)
Antioxidants/pharmacology , Apoptosis Regulatory Proteins/biosynthesis , Glucose/metabolism , Insulin Receptor Substrate Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Thioctic Acid/pharmacology , 3T3-L1 Cells , Animals , Apoptosis Regulatory Proteins/genetics , Gene Expression , Glucose/genetics , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin Resistance/genetics , Mice , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Transport/drug effects , Protein Transport/genetics , Proto-Oncogene Proteins c-akt/genetics , Reactive Oxygen Species , Signal Transduction/genetics
6.
J Bioenerg Biomembr ; 44(1): 225-32, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22249831

ABSTRACT

To explore the effects of Lyrm1 knockdown on the mitochondrial function of 3 T3-L1 adipocytes using small interfering RNA (siRNA). 3 T3-L1 preadipocytes were infected with either a negative control (NC) expression lentivirus or a Lyrm1-shRNA expression lentivirus and induced to differentiate. The knockdown efficiency of Lrym1-specific shRNA in 3 T3-L1 cells was evaluated by real-time PCR. The ultrastructure of the mitochondria in adipocytes was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers and Ucp2 mRNA were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The mitochondrial membrane potential and ROS levels of cells were analyzed with a FACScan flow cytometer using Cell Quest software. Cells transfected with lentiviral-Lyrm1-shRNA showed a significantly reduced transcription of Lyrm1 mRNA compared with NC cells. The size and ultrastructure of mitochondria in Lyrm1 knockdown adipocytes was similar to those of the NC cells. There was no significant difference in mtDNA copy number between the two groups. The total level of ATP production, mitochondrial membrane potential and Ucp2 mRNA expression levels were dramatically increased in adipocytes transfected with Lyrm1 RNAi. Furthermore, the level of ROS was dramatically decreased in Lyrm1 knockdown adipocytes. Knockdown of the Lyrm1 gene in adipocytes resulted in dramatically increased cellular ATP production, mitochondrial membrane potentials and levels Ucp2 mRNA, while ROS levels were significantly decreased. These results imply that mitochondrial function is improved in adipocytes after the knockdown of Lyrm1.


Subject(s)
Adenosine Triphosphate/biosynthesis , Adipocytes/metabolism , Apoptosis Regulatory Proteins/metabolism , Mitochondria/metabolism , Obesity/metabolism , 3T3-L1 Cells , Animals , Apoptosis Regulatory Proteins/genetics , Flow Cytometry , Gene Dosage , Gene Knockdown Techniques , Lentivirus , Membrane Potential, Mitochondrial , Mice , Microscopy, Confocal , Microscopy, Electron, Transmission , Mitochondria/ultrastructure , Plasmids/genetics , RNA Interference , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction
7.
J Bioenerg Biomembr ; 43(3): 247-55, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21647634

ABSTRACT

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002 mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.


Subject(s)
Antibodies, Monoclonal/pharmacology , Insulin Resistance/physiology , Insulin/pharmacology , Membrane Proteins/immunology , Mitochondria/metabolism , Oxidoreductases/immunology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Adenosine Triphosphate/biosynthesis , Adipocytes/drug effects , Adipocytes/immunology , Adipocytes/metabolism , Antibodies, Monoclonal/immunology , Glucose/metabolism , Glucose Transporter Type 4/metabolism , Humans , Insulin Receptor Substrate Proteins/metabolism , Insulin Resistance/immunology , Membrane Potential, Mitochondrial , Mitochondria/drug effects , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism
8.
J Bioenerg Biomembr ; 43(2): 109-18, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21607879

ABSTRACT

Obesity, which is caused by energy uptake being greater than energy expenditure, is widely prevalent today. Currently, only a limited number of efficient interventional strategies are available for the prevention of obesity. Previous studies have shown that UCP4 transcription occurs at a considerable level in mouse skeletal muscle; however, the exact functions of UCP4 remain unclear. In this study, we investigated the effect of UCP4 on mitochondrial function and insulin sensitivity in mature L6 myocytes. UCP4 overexpression in L6 myocytes induced increased mitochondrial carnitine palmitoyltransferase 1A (CPT1A) and decreased citrate synthase (CS) mRNA in the basal condition (i.e., in the absence of insulin). UCP4 overexpression significantly improved insulin sensitivity, increased tyrosine phosphorylation of IRS-1 in the presence of insulin, and significantly reduced intracellular triglyceride (TG). Additionally, intracellular ATP content and mitochondrial membrane potential were downregulated. We also observed that intracellular ROS, mitochondrial morphology, and mitochondrial mtDNA copy number were maintained upon UCP4 expression, with no change in mitochondrial fusion and fission. In summary, our findings provide evidence to show that UCP4 overexpression reduced the insulin sensitivity and mitochondrial fatty acid oxidation of L6 myocytes. These findings support the notion that UCPs are ideal targets for treatment of insulin resistance.


Subject(s)
Fatty Acids/metabolism , Insulin Resistance , Ion Channels/biosynthesis , Membrane Potential, Mitochondrial , Mitochondria, Muscle/metabolism , Mitochondrial Proteins/biosynthesis , Muscle Fibers, Skeletal/metabolism , Animals , Cell Line , Fatty Acids/genetics , Insulin/metabolism , Ion Channels/genetics , Mice , Mitochondrial Proteins/genetics , Mitochondrial Uncoupling Proteins , Obesity/genetics , Obesity/metabolism , Oxidation-Reduction , Rats , Reactive Oxygen Species/metabolism , Triglycerides/genetics , Triglycerides/metabolism
9.
Mol Med Rep ; 4(3): 519-23, 2011.
Article in English | MEDLINE | ID: mdl-21468601

ABSTRACT

The aim of this study was to investigate whether the early endosome antigen 1 (EEA1) and/or PI3K pathway is involved in the molecular mechanisms underlying the effects of the six-transmembrane protein of prostate 4 (STEAP4; also called STAMP2 and TIARP) on the insulin sensitivity of human adipocytes. Our data demonstrated that siRNA-mediated STEAP4 deficiency significantly decreased insulin-stimulated glucose transport in mature human adipocytes by decreasing GLUT4 translocation to the plasma membrane through attenuated Akt phosphorylation. We further found that EEA1 may not be involved in the mechanisms underlying the effects of STEAP4 on insulin-stimulated glucose uptake and GLUT4 translocation, as indicated by the results that i) STEAP4 does not alter the effects of EEA1 on insulin-stimulated glucose uptake and GLUT4 translocation; ii) STEAP4 does not modify the expression of EEA1 protein; and iii) STEAP4 does not interact with EEA1 according to FRET analysis. In conclusion, this study revealed that the knockdown of STEAP4 inhibits insulin-stimulated glucose transport and GLUT4 translocation via the attenuated phosphorylation of Akt, independent of the effects of EEA1.


Subject(s)
Gene Knockdown Techniques , Glucose Transporter Type 4/metabolism , Glucose/metabolism , Insulin/pharmacology , Membrane Proteins/metabolism , Oxidoreductases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Vesicular Transport Proteins/metabolism , Adipocytes/drug effects , Adipocytes/enzymology , Biological Transport/drug effects , Humans , Phosphorylation/drug effects
10.
Int J Mol Med ; 26(6): 803-11, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21042773

ABSTRACT

We previously identified the six-transmembrane epithelial antigen of prostate (STEAP) 4 as a novel plasma membrane protein that is up-regulated in obese patients and may play a significant role in the development of human obesity. In this study, a STEAP4-specific antibody was used to characterize the biological functions of the STEAP4 protein in human adipocytes. Cell viability assays (Trypan Blue exclusion), CCK-8 assays and cell cycle analysis showed that the STEAP4 antibody inhibited pre-adipocyte proliferation. Morphological observations by electron microscopy and confocal laser microscopy, annexin V-FITC labeling, caspase-3 and caspase-8 activity assays as well as data from quantitative real-time RT-PCR (qPCR) further determined that the STEAP4 antibody could promote apoptosis in pre-adipocytes. Based on quantitative Oil Red O staining and the expression profiles of specific markers, we demonstrated that the STEAP4 antibody did not affect adipogenesis, but the 2-deoxy-d-[3H]-glucose uptake tests showed that it induced the insulin-stimulated glucose uptake in mature human adipocytes. In conclusion, our results demonstrated that the STEAP4 antibody does not influence human adipocyte differentiation, but it is likely that the STEAP4 protein regulates proliferation and apoptosis and plays an important role in modulating the insulin sensitivity of human adipocytes.


Subject(s)
Adipocytes/drug effects , Antibodies, Monoclonal/pharmacology , Glucose/metabolism , Membrane Proteins/immunology , Oxidoreductases/immunology , Adipocytes/cytology , Adipocytes/metabolism , Analysis of Variance , Antibodies, Monoclonal/immunology , Apoptosis/drug effects , Cell Growth Processes/drug effects , Cells, Cultured , Humans , Immunohistochemistry , Sincalide/metabolism
11.
J Radiat Res ; 51(4): 473-9, 2010.
Article in English | MEDLINE | ID: mdl-20679743

ABSTRACT

Resveratrol, a member of a class of polyphenolic compounds known as flavonols, has been extensively studied for its anticancer, antiviral, anti-inflammatory, and neuroprotective roles. Caenorhabidits elegans is a well-established animal for investigating responses to radiation. We found that resveratrol may provide protection against hazardous radiation. Pre-treatment with resveratrol extended both the maximum and mean life span of irradiated C. elegans. Resveratrol acted as a strong radical scavenger and regulated superoxide dismutase (SOD) expression. In addition, resveratrol was shown to be capable of alleviating gamma-ray radiation exposure-induced reduction in mitochondrial SOD expression. Ultimately, a correlation may exist between dietary intake of trace amounts of resveratrol and anti-aging effects. A specific response mechanism may be activated after the administration of resveratrol in irradiated animals. Our results suggest the protective effect of resveratrol is due to its strong ability to protect from oxidative stress and protective effects in mitochondria. Therefore, resveratrol is potentially an effective protecting agent against irradiative damage.


Subject(s)
Caenorhabditis elegans/drug effects , Caenorhabditis elegans/radiation effects , Radiation-Protective Agents/pharmacology , Stilbenes/pharmacology , Animals , Animals, Genetically Modified , Antioxidants/pharmacology , Base Sequence , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , DNA Primers/genetics , DNA, Helminth/genetics , DNA, Mitochondrial/genetics , Free Radical Scavengers/pharmacology , Gamma Rays/adverse effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/radiation effects , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Resveratrol , Sirtuins/genetics , Sirtuins/metabolism , Superoxide Dismutase/metabolism
12.
Eur J Endocrinol ; 160(2): 177-84, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19022914

ABSTRACT

OBJECTIVE: To characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects. METHODS AND RESULTS: RT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes. CONCLUSIONS: By increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.


Subject(s)
Adipocytes/cytology , Apoptosis/physiology , Obesity/genetics , Proteins/genetics , Proteins/metabolism , Stem Cells/cytology , Stem Cells/physiology , 3T3-L1 Cells , Adipose Tissue/cytology , Adipose Tissue/physiology , Adult , Amino Acid Sequence , Animals , Antibodies , Apoptosis Regulatory Proteins , Base Sequence , COS Cells , Cell Differentiation/physiology , Cell Division/physiology , Chlorocebus aethiops , Gene Expression Regulation , HeLa Cells , Homeostasis/physiology , Humans , Mice , Middle Aged , Molecular Sequence Data , NIH 3T3 Cells , Obesity/metabolism , Proteins/immunology , Rabbits
13.
Acta Pharmacol Sin ; 30(1): 120-4, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19079291

ABSTRACT

AIM: NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese patients. The purpose of this study was to investigate the effects of NYGGF4 on basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes and to understand the underlying mechanisms. METHODS: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D-[3H]glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt, ERK1/2, p38, and JNK. RESULTS: NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK. CONCLUSION: NYGGF4 regulates the functions of IRS-1 and Akt, decreases GLUT4 translocation and reduces glucose uptake in response to insulin. These observations highlight the potential role of NYGGF4 in glucose homeostasis and possibly in the pathogenesis of obesity.


Subject(s)
3T3-L1 Cells/metabolism , Carrier Proteins/metabolism , Glucose/metabolism , Animals , Carrier Proteins/genetics , Glucose Transporter Type 4/genetics , Glucose Transporter Type 4/metabolism , Humans , Insulin/metabolism , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Mice , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology
14.
Peptides ; 29(11): 2052-60, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18652865

ABSTRACT

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.


Subject(s)
Adipose Tissue/metabolism , Receptors, Neuropeptide Y/genetics , Adipocytes, White/drug effects , Animals , Eating/drug effects , Gene Expression Profiling , Insulin/blood , Male , Oligonucleotide Array Sequence Analysis , Oligonucleotides, Antisense/pharmacology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction
15.
Acta Pharmacol Sin ; 29(5): 587-92, 2008 May.
Article in English | MEDLINE | ID: mdl-18430367

ABSTRACT

AIM: To determine the relationship between six-transmembrane epithelial antigen of the prostate 4 (STEAP4) expression and obesity. METHODS: RT-PCR and immunoblot analyses were performed to determine the differential expressions of STEAP4 mRNA and protein, respectively, in human omental adipose tissue from obese patients and normal weight controls. The expression pattern of STEAP4 mRNA in various human tissues was determined by RT-PCR. The subcellular localization of the STEAP4 protein in human adipose tissue was confirmed by immunohistochemistry. Finally, we confirmed that cultured human omental adipose tissue undergoes TNF-alpha-mediated regulation of the STEAP4 expression. RESULTS: STEAP4 mRNA and protein levels were downregulated in omental adipose tissue from obese patients relative to normal controls. The STEAP4 expression was most abundant in human adipose tissue. An immunohistochemical analysis confirmed that STEAP4 was associated with the plasma membrane of adipocytes. The STEAP4 expression was induced by TNF-alpha in a dose-dependent manner in human adipose tissue. CONCLUSION: STEAP4 was abundantly expressed in human adipose tissue, and the STEAP4 expression was significantly downregulated in obese patients. STEAP4 localized to the plasma membrane of adipocytes, and the STEAP4 expression was induced by TNF-alpha in adipose tissue. These data suggest that STEAP4 may play a significant role in the development of human obesity.


Subject(s)
Down-Regulation/drug effects , Membrane Proteins/metabolism , Obesity/metabolism , Oxidoreductases/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adipose Tissue/chemistry , Adipose Tissue/metabolism , Down-Regulation/physiology , Gene Expression , Humans , Immunohistochemistry , Male , Membrane Proteins/genetics , Obesity/genetics , Omentum/metabolism , Oxidoreductases/genetics , Proteins/metabolism , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/genetics
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 24(3): 251-5, 2007 Jun.
Article in Chinese | MEDLINE | ID: mdl-17557231

ABSTRACT

OBJECTIVE: To investigate the effect of resistin overexpression on 3T3-L1 adipocyte lipid and glucose metabolism. METHODS: Expression vector for rat resistin gene was constructed and transfected into 3T3-L1 adipocytes. Cell differentiation and lipid accumulation was determined by Oil Red O staining. Differentiation marker genes (pref-1, C/EBPalpha, FAS) and glucose transporter 4 (GLUT4) gene mRNA expressions were evaluated by reverse transcription-PCR (RT-PCR). Triglyceride (TG) and free fatty acids (FFAs) in adipocytes were measured by colorimetric kit. RESULTS: (1) In resistin-overexpressed adipocytes, the lipid droplets presented at the second day which was earlier than the control cells. (2) The expression of C/EBPalpha and FAS genes in resistin-overexpressed adipocytes were up-regulated and the pref-1 was down-regulated compared with that of the control cells. (3) In resistin-overexpressed adipocytes, cellular TG and FFAs levels were significantly increased (P<0.05). (4) There was no difference in the expression of GLUT4 gene between 3T3-L1 adipocytes and resistin-overexpressed adipocytes (P> 0.05). CONCLUSION: Overexpression of resistin can affect 3T3-L1 adipocyte lipid metabolism and thereby result in obesity and insulin resistance, but have no effect on GLUT4 gene expression.


Subject(s)
Adipocytes/metabolism , Gene Expression , Lipid Metabolism/genetics , Resistin/genetics , Resistin/metabolism , 3T3-L1 Cells , Animals , Cell Differentiation/genetics , Fatty Acids, Nonesterified/metabolism , Glucose Transporter Type 4/genetics , Mice , Rats , Triglycerides/metabolism
17.
Biochem Biophys Res Commun ; 352(2): 469-78, 2007 Jan 12.
Article in English | MEDLINE | ID: mdl-17129576

ABSTRACT

To identify differentially expressed genes between obese individuals and normal control, we have undertaken suppression subtractive hybridization (SSH). Omental adipose tissues were obtained via abdominal surgery for appendicitis in both 13 obese subjects [BMI (body mass index) >30 kg/m2] and 13 normal subjects (BMI >18 and <25 kg/m2). Following SSH, about one thousand clones were sequenced and found to derive from 426 different genes. These predominately expressed genes included genes involved in lipid metabolism, cytokines, signal transduction, GLUT4 translocation, cell cycle and growth, cytoskeleton, and others. Although more detailed analyses are necessary, it is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of obesity.


Subject(s)
Adipose Tissue/metabolism , Gene Expression Profiling/methods , In Situ Hybridization/methods , Obesity/metabolism , Omentum/metabolism , Proteome/metabolism , Gene Expression Regulation , Humans , Male , Middle Aged
18.
Gene ; 379: 132-40, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16815647

ABSTRACT

A novel gene named NYGGF4, which was expressed at a higher level in obese subjects, was isolated and characterized. It is a 1527-bp cDNA, containing 753 nucleotides of an ORF (open reading frame) predicting 250 amino acids with a molecular mass of 28.27 kDa. Amino acid sequence analysis revealed NYGGF4 has a phosphotyrosine-binding (PTB) domain. Northern blot analysis revealed NYGGF4 is expressed primarily in adipose tissue, heart, and skeletal muscle but not in any other tissue examined. Confocal imagery analyses with green fluorescent protein-tagged protein transiently expressed in 3T3-L1 preadipocytes and 293-T cells show that NYGGF4 localizes in the cytoplasm. Furthermore, ectopic expression of NYGGF4 dramatically increases the proliferation of 3T3-L1 peadipocytes without affecting adipocytic differentiation. And the NYGGF4 expression 3T3-L1 cells had a greater number of cells in S-phase. Our data suggest that NYGGF4 might be a signaling molecule and could play a role in cell growth and adipogenesis process.


Subject(s)
Adipocytes/cytology , Carrier Proteins/genetics , Phosphotyrosine/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Proliferation , DNA, Complementary/analysis , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Profiling , Humans , Mice , Molecular Sequence Data , Obesity/genetics , Obesity/metabolism , Phenotype , Phosphotyrosine/genetics , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , S Phase , Sequence Analysis , Species Specificity , Transfection
19.
Life Sci ; 79(15): 1428-35, 2006 Sep 05.
Article in English | MEDLINE | ID: mdl-16716360

ABSTRACT

Uncoupling proteins are a family of mitochondrial proteins involved in energy metabolism. We previously showed that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. However, the effect of UCP4 on adipocytes is unclear. In this work, we established a stable preadipocyte cell line overexpressing UCP4 to observe the direct effect of UCP4 on adipocytes. Cells overexpressing UCP4 showed significantly attenuated differentiation of preadipocytes into adipocytes. During differentiation, expression of adipogenesis-associated markers such as fatty acid synthetase, peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, adipocyte lipid binding protein and lipoprotein lipase were downregulated. Preadipoctes expressing UCP4 grew faster and more of them stayed in S phase compared to control cells. In addition, UCP4 overexpression protected preadipocytes from apoptosis induced by serum deprivation. Our results demonstrate that overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes.


Subject(s)
Adipocytes/cytology , Adipogenesis/genetics , Cell Differentiation/genetics , Gene Expression Regulation, Developmental , Ion Channels/genetics , Ion Channels/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , 3T3-L1 Cells , Animals , Apoptosis/genetics , CCAAT-Enhancer-Binding Protein-alpha , Cell Line , Cell Proliferation , Fatty Acid Synthases/metabolism , Mice , Mitochondrial Uncoupling Proteins , PPAR gamma/metabolism , Rats , Transcriptional Activation
20.
Chin Med J (Engl) ; 119(6): 496-503, 2006 Mar 20.
Article in English | MEDLINE | ID: mdl-16584648

ABSTRACT

BACKGROUND: Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor. METHODS: Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide. RESULTS: Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation. CONCLUSION: RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.


Subject(s)
Adipocytes/drug effects , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Peptide Library , Resistin/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/isolation & purification , Mice , Molecular Sequence Data , Rats , Resistin/antagonists & inhibitors
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