In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Bioreactors ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

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

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.
Adenoviridae ; genetics ; metabolism ; Adiponectin ; biosynthesis ; genetics ; Animals ; Cell Line ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the renoprotective effect of adiponectin in streptozotocin (STz)-induced diabetic rats and explore its association with oxidation stress.

METHODSType 2 diabetes mellitus was induced in rats by high-lipids and high-sucrose feeding and intraperitoneal STZ injection. The recombinant plasmid pIRES2-EGFP-gAd expressing globular adiponectin was intraperitoneally injected in the rats mediated by liposome. Thirty-two Wistar rats were randomized into 4 groups, namely the normal control group (NC), diabetic group without any therapy (DM), diabetic group treated with pIRES2-EGFP-gAd (DA) and diabetic group treated with pIRES2-EGFP (DP). After the corresponding treatments for 8 weeks, the blood glucose, HbA1c and urine albumin excretion rate (UAER) were measured, and the kidneys were collected to determine the production of reactive oxygen species (ROS) and assess renal pathologies. Immunohistochemistry and Western blot were employed to determine the protein levels of endothelial nitric oxide synthesis (eNOS) and phosphorylated AMP-activated protein kinase (pAMPK).

RESULTSUAER and ROS production increased significantly in DM group as compared with that in the control group (P<0.05), while no significant differences were found in UARE among the DM, DA, and DP groups (P>0.05). Blood glucose level, HbA1c and ROS were significantly decreased in DA group in comparison with those in DM group (P<0.05). Glomerular hypetrophy, mesangial expansion, basal membrane thickening, tubular epithelial cells cavitation and exfoliation, and mononuclear lymphocyte infiltration occurred in DM group, while these changes were ameliorated in gAd transfection group. The renal expression levels of eNOS and p-AMPK proteins in DM group were significantly lower than those in the control group (P<0.05) and gAd transfection group (P<0.05).

CONCLUSIONSThe renoprotective effect of adiponectin may be at least partially mediated by the activation of the AMPK signaling passway, ROS production inhibition, relief of the oxidative stress, and up-regulation of eNOS expression in the renal tissue of diabetic rats.

Adiponectin ; biosynthesis ; genetics ; pharmacology ; Animals ; Antioxidants ; pharmacology ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Diabetic Nephropathies ; metabolism ; prevention & control ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Oxidative Stress ; drug effects ; Protective Agents ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Transfection

In order to get soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFalpha, we constructed the fusion gene sTNFRII-gAD, which encoded human sTNFR II and the globular domain of adiponectin (gAD), and then expressed it in mammalian cells and analyzed its anti-TNFalpha activity. First, sTNFRII cDNA was obtained by RT-PCR from the total RNA of human peripheral blood lymphocytes, and fused in frame with gAD gene. Then, the fusion gene sTNFRII-gAD was cloned into the expression vector pAAV2neo to result in the plasmid pAAV2neo-sTNFRII-gAD. By immunofluorescent staining with monoclonal antibody either against TNFRII or against adiponectin, we demonstrated that the pAAV2neo-s7NFRII-gAD-transiently-transfected BHK-21S cells were positive. To obtain G418-resistant BHK-21S/pAAV2neo-sTNFRII-gAD cells, we cultured the transfected BHK-21S cells above in 10% FBS containing DMEM media with 800 microg/mL G418 for 15 days, and changed the serum-containing culture media to a serum-free chemically defined media so as to change the cells culturing style from adhesion to suspension. 24 hours later, we harvested the supernatant of the culture for sTNFRII-gAD fusion protein characterization and anti-TNFalpha activity analysis. With monoclonal antibody either against TNFRII or against adiponectin, the Western blotting analysis showed that the sTNFRII-gAD fusion protein was expressed and existed as monomer, trimer and multimer forms in the supernatant. The bioactivity assay demonstrated that the sTNFRII-gAD fusion protein had the ability to neutralize TNFalpha so as to inhibit the cytotoxicity of TNFalpha on L929 cells. Put together, this study has laid the groundwork for large-scale preparation of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Cells, Cultured ; Cricetinae ; Humans ; Protein Structure, Tertiary ; genetics ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Solubility ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

Adiponectin is an adipokine predominantly synthesized and secreted by adipocytes in the white adipose tissue, and it can lower the blood glucose level and increase free fatty acid oxidation. In the current study, we developed the globular domain of adiponectin (gapM1) to treat type II diabetes. In both flask and fermentor, we cultivated Pichia pastoris expressing recombinant gapM1 and established the purification procedure by using gel filtration and anion exchange chromatography. To evaluate the biological activity of recombinant gapM1, we used rat type II diabetes model fed high-fat diet in combination with low-dose STZ (Streptozocin) induction. We purified 200 mg gapM1 with purity of 96% from 10 liters of supernatant. The recombinant gapM1 significantly lowered blood glucose (34.2%), serum triglyceride (79.6%) and total cholesterol (62.1%) in type II diabetes induced rat. Therefore, the recombinant human gapM1 is successfully expressed in Pichia pastoris and effectively treated type II diabetes in rat models.
Adiponectin ; biosynthesis ; genetics ; pharmacology ; therapeutic use ; Animals ; Diabetes Mellitus, Experimental ; drug therapy ; Genetic Vectors ; Hypoglycemic Agents ; pharmacology ; therapeutic use ; Male ; Pichia ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; therapeutic use

Adiponectin ; blood ; metabolism ; Antiviral Agents ; therapeutic use ; Aspartate Aminotransferases ; blood ; Fatty Liver ; blood ; pathology ; Female ; Genotype ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Hepatitis C, Chronic ; blood ; drug therapy ; virology ; Humans ; Insulin Resistance ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; blood ; pathology ; Male ; Metabolic Syndrome ; blood ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Viral ; blood ; Receptors, Adiponectin ; genetics ; metabolism

OBJECTIVETo construct and express the recombinant human adiponectin (gAd) global domain.

METHODSgAd complementary DNA (cDNA) was obtained from human fat tissue by RT-PCR. The PCR product was cloned into the vector pMD18-T and the prokaryotic expression vector pET32a(+). The recombinant vector was identified by digestion with double restriction endonucleases SalI and EcoRI, PCR and sequence analysis. The recombinant plasmid containing gAd gene was transformed into E. coli BL21 (DE3), and the expression of the fusion protein His-gAd was induced by IPTG.

RESULTSThe gAd cDNA of 412 bp was obtained from the total RNA of the fat tissue and verified by sequence analysis.

CONCLUSIONThe recombinant plasmid could stably express the 34-kD fusion protein His-gAd in the engineered bacteria in the form of inclusion bodies.

Adiponectin ; biosynthesis ; genetics ; Adult ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Female ; Genetic Vectors ; genetics ; Humans ; Prokaryotic Cells ; cytology ; metabolism ; Recombinant Proteins ; biosynthesis

Gene of human adiponectin (ADPN) was cloned by PCR-driven overlap extension. The ADPN gene was linked into pGEM-T vector. After the sequence was determined, the ADPN gene was subcloned into expression vector pPIC3.5K to yield the recombinant expression vector pPIC3.5K-ADPN. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation, then the recombinant strain was identified by PCR and Southern blotting. After induction by methanol, ADPN was expressed in GS115, then the protein was identified by Western blotting. The results showed that the ADPN was expressed successfully. The optimum conditions of expression were 30 degrees C and 1% methanol inducing 48 h.
Adiponectin ; biosynthesis ; genetics ; Cloning, Molecular ; Electroporation ; Humans ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; biosynthesis ; genetics ; Temperature

OBJECTIVE: To investigate the effect of adiponectin on the osteoblast differentiation and its signal transduction. METHODS: Adipopnectin receptor (AdipoR) was detected by immunoblot analysis. Alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. Osteocalcin was measured by a specific radioimmunoassay kit, and the extent of mineralized matrix was determined. RNA interference was used to down-regulate the expression of AdipoR1 in human osteoblasts, and the effect of adiponectin on osteoblast differentiation was investigated. RESULTS: Only AdipoR1 protein was detected in human osteoblasts. Adiponectin could promote osteoblast differentiation, and result in a dose-dependent increase in ALP activity, osteocalcin secretion, and an increase in mineralized nodules. Suppression of AdipoR1 with siRNA could abolish the adiponectin induced ALP expression. Adiponectin could induce the activation of p38 and JNK, but not ERK1/2 in osteoblasts, and the pretreatment of osteoblasts with the p38 inhibitor (SB203580) could block the adiponectin-induced ALP activity. CONCLUSION Adiponectin can induce human osteoblast differentiation via AdipoR1/p38 pathway.
Adiponectin ; pharmacology ; Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; analysis ; RNA, Small Interfering ; genetics ; Receptors, Adiponectin ; biosynthesis ; Signal Transduction