Generation And Insulin Sensitivity Study Of Transgenic Mice With Adipose-Specific Overexpression Of Subfatin

Adipose tissue had been considered the largest energy reservoir of the body for a very long time before people gradually realized that it also serves as a highly active endocrine organ in the 1990s. The biologically active peptides and proteins produced by adipose tissue are termed adipokines, which are involved in the regulation of many important pathophysiological processes.Obesity, which is characterized by excessive adipose tissue, has become a global health problem, and is defined as the major risk factor for many serious diseases of mankind. With this understanding, to find out the physiological functions of adipokines and the causal connection between adipokines and obesity is of great value to find effective treatment for pathological consequences caused by obesity. In the present, significant progress has been made in the study of adipokines. For example, leptin alone or combined with low-dose insulin is proposed to treat type 1 diabetes patients. Another example is the demonstration of how visfatin could serve as the therapeutic target for stroke.In the past twenty years, more and more adipokines have been identified, with a total number of about 100. Our lab identified a novel adipokine using caloric restriction animal model in 2008, and we named it Subfatin.According to our preliminary experimental results, Subfatin abundantly expresses in white adipose tissue, especially in subcutaneous adipose tissue. Its expression in the white adipose tissue is down regulated in caloric-restricted rats, but is up regulated in diet-induced obesity mice. Its expression was persistently increased during the process of preadipocyte differentiation. Hence, we suppose Subfatin is closely related to the adipose biology and obesity.Transgenic animal model with adipose-specific overexpression of an adipokine is a reliable and effective tool to explore the function of the adipokine in adipose biology and metabolic homeostasis in vivo. Hence, we try to generate transgenic mice with adipose-specific overexpression of Subfatin and then study the insulin sensitivity function in these transgenic mice.Methods1. Construction and sequencing of pBluescript â…¡ SK (+)-aP2-Subfatin vector(1) Subfatin cDNA-his6 was acquired from plasmid pIRES-EGFP-Subfatin. Then it was inserted into a plasmid called pBS aP2 promoter (5.4 kb) poly A so as to prepare pBluescript â…¡SK (+)-aP2-Subfatin vector which would be used to direct adipose-specific Subfatin expression.(2) pBluescript â…¡ SK (+)-aP2-Subfatin was verified by sequencing. 2. Generation of transgenic mice(1) Microinjected the vector fragment containing the aP2 promoter, Subfatin cDNA, and poly A into C57BL/6 mouse zygotes.(2) Transferred the zygotes into the oviducts of a C57BL/6 pseudopregnant female mouse to generate the FO offsprings.(3) Selected the transgenic founders according to a PCR-based DNA identification result, and mate them with C57BL/6 mice to obtain F1 offsprings. And identified the genotype of the F1 offsprings. 3. Validation of the Subfatin transgenic mice(1) RT-PCR and Real-time PCR were used to identify the transcription of Subfatin.(2) Immunohistochemical method was used to detect Subfatin protein in white adipose tissue.(3) ELISA assay was used to determine the serum Subfatin concentration.4. Insulin sensitivity study of Subfatin transgenic mice(1) Body weight and food intake were measured respectively under the conditions of normal chow diet and high fat diet.(2) GTT was performed under the conditions of normal chow diet and high fat diet.(3) ITT and HIEG clamp test were performed under the condition of high fat diet.Results1. The sequencing result of pBluescript â…¡ SK (+) -aP2-Subfatin vector showed that the sequences of aP2 Promoter and Subfatin open reading frame in the vector were correct, indicating the vector was successfully constructed.2. The genotyping results of the FO offsprings showed that four transgenic founders were obtained.3. According to RT-PCR result, exogenous Subfatin (Subfatin-his6) was specifically expressed in white adipose tissue and brown adipose tissue rather than other tissues in transgenic mice.4. The Real-time PCR result showed higher Subfatin transcription level in white adipose tissue of transgenic mice than that of wild type mice, which indicated Subfatin overexpressed in white adipose tissue of transgenic mice.5. The immunohistochemical result indicated Subfatin protein overexpression in white adipose tissue of transgenic mice.6. The ELISA result showed a higher serum concentration of Subfatin protein in transgenic mice than that in wild type mice. This result was consistent with the secretion characteristics of Subfatin and also indicated that Subfatin was overexpressed in transgenic mice.7. When fed normal chow diet or high fat diet, transgenic mice showed no significant differences to wild type mice in body weight and food intake.8. According to the result of GTT, transgenic mice showed no significant difference to wild type mice in insulin sensitivity when fed normal chow diet, but had better insulin sensitivity when fed high fat diet.9. Both the results of ITT and HIEG clamp test showed that transgenic mice had obviously better insulin sensitivity than wild type mice when fed high fat diet.In conclusion, we successfully generated a strain of transgenic mice with adipose-specific overexpression of Subfatin. Using GTT, ITT and HIEG clamp test, we demonstrated an insulin sensitization of adipose Subfatin in a high fat diet model.