Using Adipocyte-and Intestine Epithelium-Specific Knockout Mice To Study Expression And Function Of Metrnl

BackgroudAdipokines plays an important role in regulating functions of adipose tissue and metabolism homeostasis.Metrnl which was recently found in our lab was expressed in various tissues,especially in white adipose tissue and mucosal tissues.Metrnl was reported to be a novel neurotrophic factor with promoting neurite outgrowth and neuroblast migration.And Metrnl expression was upregulated during adipogenesis and obesity.Based on study of Metrnl expression profiles in different tissues,we decided to focus on adipose tissue and intestine to explore potential functions of Metrnl with tissue-specific Metrnl knockout mice.Methods1.Sensitivity,precision and specifity of mouse ELISA development kit for Metrnl was studied to establish a stable and accurate method for Metrnl detection.2.Adipocyte-specific Metrnl knockout mice was generated by mating Metrnlloxp/loxp mice and Fabp4-Cre mie.3.Under normal diet,Metrnl mRNA expression in tissues of Adipocyte-specific Metrnl knockout mice and wide type mice was detected by real time-PCR and serum Metrnl was performed by ELISA.And monitoring energy expenditure and performing glucose tolerance test was executed.Under high fat diet,monitoring energy expenditure and performing glucose tolerance test and insulin tolerance test was executed.Hematoxylin and eosin(H&E)staining of white adipose tissue and relative expression of genes related to lipid metabolism and thermogenesis of brown adipose tissue was performed.4.Metrnl expression profile was researched through immunohistochemical staining of human tissues microarray and relative expreesion of Metrnl in tissues of C57BL/6 mice.5.Intestine epithelium-specific Metrnl knockout mice was generated by mating Metrnlloxp/loxp mice and Villin-Cre mie.6.Body weight and colon length of Intestine epithelium-specific Metrnl knockout mice and wide type mice was measured,H&E staining of colon and Metrnl level detection in serum and gut fluid was performed.Periodic acid-Schiff(PAS)staining for intestine mucus was performed and we further detected the expression of antimicrobial peptides,such as regenerating islet-derived 3 gamma(Reg3g),lactotransfferin,serum amyloid A-3(SAA3)and regenerating islet-derived 3 beta(Reg3b).Results1.A stable and accurate method for detecting Metrnl concentration was established.The method has high sensitivity,strong accuracy with 5-15% of variable coefficient,and is just used to detect mouse Metrnl.Additionally,the method of sampling mouse blood must be the same in order to comparability of Metrnl.2.Adipocyte-specific Metrnl knockout mice was generated and verified by significant reduce of Metrnl mRNA expreesion in adipose tissue.Metrnl expression was reduced by 57% in adipose tissue of Adipocyte-specific Metrnl knockout mice,whereas no difference was found in liver,muscle,heart and brain.And serum Metrnl had a reduce tendency in mice with deletion of Metrnl in adipocyte.3.Compared with wide type mice,Adipocyte-specific Metrnl knockout mice displayed no change in energy expenditure and glucose tolerance under normal diet.Under high fat diet,contrary to wide type mice,white adipocyte size and insulin sensitivity significantly decreased in mice with deficency of Metrnl in adipocyte,and no difference was found in relative expression of genes related to lipid metabolism and thermogenesis of brown adipose tissues between two groups.4.Immunohistochemistry for human tissues microarray and real-time PCR for tissues of C57BL/6 mice indicated that Metrnl expression profile was nearly consistent in human and mice,and highest expression of Metrnl in intestine epithelium was confirmed.5.Intestine epithelium-specific Metrnl knockout mice was generated and verified by significant reduce of Metrnl mRNA expreesion in intestine epithelium.Metrnl expression was reduced by 96% in intestine epithelium of Intestine epithelium-specific Metrnl knockout mice,whereas no difference was existed in liver,muscle,heart and adipose tissue.And in mice with deficiency of Metrnl in intestine epithelium,serum Metrnl had a reduce tendency and notable reduce existed in Metrnl level of gut fluid.6.Compared with wide type mice,no remarkable differences were displayed in body weight and intestine histology in mice with deletion of Metrnl in intestine epithelium.Mucus secretion and mucin2 relative expression of Intestine epithelium-specific Metrnl knockout mice displayed no difference compared with wide type mice.However,deletion of Metrnl in intestine epithelium significantly decreased relative expreesion of antimicrobial peptides such as Reg3 g and lactotransferrin,and reduced relative expreesion of some antimicrobial peptides such as SAA3 and Reg3 b.ConclusionsDeletion of Metrnl in adipocyte did not result in significant decrease in serum Metrnl level;deficency of Metrnl in intestine epitheliun did not significantly reduce serum Metrnl level and resulted in robust decrease in Metrnl level in gut fluid.Adipocyte Metrnl can elevate insulin sensitivity;intestine epithelium Metrnl can play a local role in regulating expression of antimicrobial peptides,and Metrnl may be involved in intestine immune responses.