Effects Of Betatrophin Expression On The Body’s Insulin Resistance And Related Signal Pathway

PART ONE CONSTRUCTION AND IDENTIFICATION OF MOUSE BETATROPHIN GENE EXPRESSION VECTOR IN MICEObjective To construct the mouse betatrophin gene expression vector and be used for the later cell transfection.Method Total RNA of liver in mice was extracted, Total RNA was reverse transcribed into c DNA, and the CDS sequence of Betatrophin was got by PCR. Through double enzyme digestion, connected to the eukaryotic expression vector of p IRES2-EGFP, Then builded into p IRES2-EGFP-Betatrophin. Finally, by double enzyme digestion and DNA sequencing to identify recombinant plasmid.Results The recombinant plasmid(p IRES2-EGFP-Betatrophin) and the recombinant plasmid of double enzyme digestion was by agarose gel electrophoresis, image showed that the former(p IRES2-EGFP-Betatrophin recombinant plasmid) appears in the corresponding to the location of the Marker 5897 bp; The latter appears two linear strip, respectively in the corresponding Marker 597 bp(Betatrophin CDS length sequence) location and 5.3 KB(p IRES2-EGFP linear stripe) position, preliminarily verified that recombinant plasmid constriction is successful. Sequencing results with Genbank betatrophin m RNA encoded by CDS sequence for alignment in the NCBI, comparing the result, it is completely consistent, it was proved that the gene of betatrophin c DNA is coming from mouse.Conclusions The construction of eukaryotic expression vector(p IRES2-EGFP-Betatrophin) is successful.PART TWO THE EXPRESSION OF BETATROPHIN GENE IN MICEObjective To explore Betatrophin expression differences in standard feeding and high-fat feeding C57 BL /6J mice tissues.Methods Take 5 high-fat feed(HFD) C57BL/6J mices of 28 weeks, 5standard feed(SD) C57BL/6J mices at the same age, and 5 Adiponectin knockout mice of HFD or SD mice, then killed. Liver, adipose tissue preserved in liquid nitrogen, later Total RNA were extracted and reversed transcription for c DNA, through RT-PCR to determine Betatrophin m RNA expression differences; Extracted protein by Western blot method to detect the protein expression differences.Results Betatrophin mRNA and protein expression of liver and fat in HFD mice was significantly higher(p < 0.05 or p < 0.01) than SD mice.Conclusion It indicates that, Betatrophin expression may be associated with the body’s insulin resistance.PART THREE EXPRESSION OF BETATROPHIN IN HEPATOCYTES AND THE EFFECT ON INSULIN SIGNAL PATHWAYObjective To explore Betatrophin gene expression in the state of insulin resistance in mouse primary hepatocytes, and the mechanisms of mouse primary hepatocytes overexpression Betatrophin to increase insulin resistance.Methods Hepatocytes were isolated from the C57BL/6J mouse and cultured. Grouping and using the increasing concentration of insulin for treating the hepatocytes; after a certain concentration of insulin treatment on hepatocytes for 24 hs, rosiglitazone and metformin were added in DMEM, then by PCR and Western blot detecting expression of betatrophin.Hepatocytes and macrophage RAW264.7 cocultured, the other group with rosiglitazone and metformin treating co-culture system, The same, PCR and Western blot detecting Betatrophin expression; In another set of experiment, mouse hepatocytes were treated with or without 1% of fasting serum from normal women(n=10) or metformin-treated PCOS women(pretreatment or post-treatment 3 months or 6 months)(n =10) for24 hr, respectively.Finally used overexpression plasmid(Ad-Betatrophin)and Si-RNA(Ad-sh Betatrophin) to transfect hepatocytes for 48 h, PCR and Western blot detect Betatrophin, P-Ins R and P-AKT expression.Results With the increasing of the insulin concentration, Betatrophin expression gradually increased(p < 0.05 or p < 0.01);primary hepatocytes treated by Ros or Met after certain concentration insulin effected 24 h, as a result,Betatrophin expression decreased significantly(p < 0.05 or p <0.01); Primary hepatocytes with macrophages264.7co-culture system, after rosiglitazone and metformin treament, Betatrophin expression significantly reduced(p < 0.05 or p < 0.01); P-Ins R, P- AKT expression significantly decreased(P < 0.01) when Betatrophin overexpression.and in contrast,P-Ins R, P- AKT expression significantly increased(P < 0.01).Conclusions Betatrophin gene expression may play a great role in the insulin resistance and increasing the insulin resistance through the PI3K/Akt signal pathway.