The Study Of Adiponectin In The Signal Pathway Of Liver Glucose Metabolic In L-02 Cells

Objectives:To obtain the expression of recombinant adiponection protein which has biologic activity in human hepatocyte line named L-02. And to study the signaling pathway of adiponectin modulates gluconeogenesis in human hepatocyte L-02.Methods:①To extract the total RNA from human adipose tissue. We got the adiponectin cDNA by RT-PCR. Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR. Construct recombinant clone vector pMD18-T/ADPN, and identify the sequence. Extract the encoding fragment of human ADPN gene, ligated into eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO, construct adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN.②After transformation, recombinant expression vector pCDNA3.1/CT-GFP-TOPO-ADPN is introduced into TOP10 Competent cells. Choose the white colony to verify. Extract adiponectin recombination vector named pCDNA3.1-ADPN.③The cultured L-02 cells are transfected with pCDNA3.1-ADPN by liposome, after sieving by G418. Immunofluorescence observes transfection efficiency. The expression product in cell lysate and culture supernatant was identified by SDS-PAGE,western blot and immunohistochemical method.④Using 0mmol/L,5 mmol/L,15mmol/L and 30mmol/L glucose to simulate no glucose,low glucose, middle glucose, high glucose environment. Treat transfect ion cells and control cells with the above-mentioned conditions. Determine cultivate glucose by enzymology; detect the recombination adiponectin protein's activity.⑤Determine phospho-AMPK, CREB, phospho-CREB in normal L-02 group, transfected group, high glucose treatment group, and hungry group by western blot, observed the TORC2 localization in the four group, glucose dehydrogenase couple way detectes Glucose-6-phosphorate (G6P) activity.Results:①Successfully construct the adiponectin clone vector pMD18-T/ADPN and adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN. ADPN gene was inserted in correct location.②We can see the green fluorescence in the transfected pCDNA3.1/CT-GFP- TOPO-AND cells by the immunofluorescence microscope; the transfection efficiency is about 60%.③Recombinant protein secretes into culture medium. The MW of recombinant protein is about 57kD. Western blot proves that expressed protein could combine with ADPN polyclonal antibody.④There is obvious glucose difference between transfection group and control group (p<0.05), which treated with high glucose for 24 h. There is no obvious among no glucose group, low glucose group and middle glucose group (p>0.05).⑤Compare with other groups, there is stronger p-AMPK expression in the transfect ion group (p<0.05), TORC2 was mainly localization in cytoplasm, the G6P activity was cut down significant (p<0.05), but there is no significant in the expression of CREB,p-CREB (p>0.05).Conclusions:Adiponectin eukaryotic expression vector was successfully constructed. It can express solubility and secrete adiponectin protein in human hepatocytes L-02. The recombinant protein has biology activity.The mechanism of inhibitin hepar glyconeogenesis may involve activing AMPK, depressing the TORC2 entry the nuclear, and cutting down the G6P activity. The signaling pathway may be adiponectin-AMPK-CREB-TORC2- glyconeogenesis key enzyme.