The Study Of Localization Mechanism And Function Of Irisin

Irisin, a post-translation processing product of fibronectin type HI domain-containing protein 5 (FNDC5),can promote browning of white adipocytes by combining with unknown receptor of white adipocytes. At present, the subcellular localization mechanism of irisin is still in its infancy, in addition its role in promoting browning of white adipocytes should be further explored. In present study, to understand the impact of signal peptide and transmembrane domain on subcellular localization of irisin, the localization function of signal peptide and transmembrane domain were researched; to reveal the subcellular localization mechanism of irisin, N-linked glycosylation modification and cleavage of FNDC5 was analyzed; to preliminary exploration of the mechanism of irisin promoting browning of white adipocytes,white adipocytes were treated with prokaryotic expressed GST-irisin.(1) Detection of localization function of FNDC5 signal peptide and transmembrane domainTo detect localization function of signal peptide and transmembrane domain, and understand the effect on localization of irisin, signal peptide of FNDC5 was analyzed by program of Phobius.Transmembrane domain of FNDC5 was analyzed by program of SignalP 4.1 Server. According to analysis results, signal peptide expression vector pEGFP-SP, transmembrane domain expression vector pEGFP-TM, and FNDC5 expression vector pFNDC5-EYFP were constructed. The subcellular localization of the fusion proteins were observed by transfection of HeLa cells, respectively. Subcellular localization results showed that EGFP-SP located in endoplasmic reticulum, EGFP-TM located in cell membrane, FNDC5-EYFP emitted yellow fluorescence in the cell membrane. The above results showed that signal peptide had function of endoplasmic reticulum localization; irisin could be located in the cell membrane by transmembrane domain.(2) Research of cleavage of FNDC5 and secretion of irisinTo explore the possible of cleavage of FNDC5 protein, and verify that how irisin secreted to the extracellular matix, expression vectors of pEGFP-FNDC5 and pDsred2-FNDC5-EGFP were constructed. By transfection of HeLa cells, the subcellular localization of cleavage products of the fusion protein was observed. By Western blotting detection using GFP antibody, the relative molecular weight of cleavage products of EGFP-FNDC5, FNDC5-EYFP, and Dsred2-FNDC5-EGFP protein were analyzed, respectively. The results showed that signal peptide and transmembrane domain can be cleaved from FNDC5. After undergoing cleavage of signal peptide, irisin was localized in the cell membrane under the action of transmembrane domain. Irisin was finally secreted into the extracellular matrix by cleavage of transmembrane domain. Therefore, cleavage of FNDC5 is a premise of secretion of irisin.(3) Detection of N-linked glycosylation modification of FNDC5For research the N-linked glycosylation modification of FNDC5, total proteins from muscle was digested by glycoside bydrolase PNGase F or Endo H, respectively. Monoclonal antibody of irisin was used for Western blotting detection of the digested protein. Results showed that FNDC5 from muscle tissue was PNGase F and Endo H sensitive glycoprotein. Therefore, FNDC5 is N-linked glycosylation modified glycoprotein.(4) Effect of N-linked glycosylation modification of FNDC5 on irisin secretionTo explore the relationship between N-linked glycosylation and irisin secretion, N-linked glycosylation sites of FNDC5 was analyzed by NetNGlyc1.0 Server program. According to the analysis results, mutation of N-linked glycosylation sites of FNDC5 vectors were constructed, and the subcellular localization of sites mutation fusion protein was observated. GFP antibodies were used for Western blotting detection. Subcellular localization of FNDC5 fusion protein with mutation in 36 amino acid residues was retained in the endoplasmic reticulum; subcellular localization of FNDC5 fusion protein with mutation in 81 amino acid residues was similar as wild type FNDC5 fusion protein. The results of Western blotting showed that compared with wild type FNDC5 fusion protein, relative molecular weight of FNDC5 fusion protein with mutation in 36 or 81 amino acid residues were reduced, by PNGase F digestion, the relative molecular weights of wild type FNDC5 fusion protein and FNDC5 fusion protein with mutation in 36 amino acid residues or 81 amino acid residues were reduced to 51 kDa. Therefore,36 and 81 amino acid residues are N-linked glycosylation sites of FNDC5. N-linked glycosylation of 36 amino acid residues of FNDC5 is an important guarantee for signal peptide cleavage of FNDC5 and irisin secretion.(5) Isolation and characterization of white adipocytesIn order to a preliminary cell preparation for exploration the mechanism of irisin in browning of white adipocytes, white and brown adipocytes were isolated from inguinal white adipose tissues and interscapular region adipose tissues of C57BL/6 mice by collagenase digestion method, respectively. White adipocytes were characterized by immunofluorescence staining, quantitative real-time PCR (qPCR), and Western blotting analysis as control of brown adipocytes. The results of cell immunofluorescence staining showed that white adipocytes and brown adipocytes were UCP-1 positive, white adipocytes was ASC-1 positive, brown adipocytes was ASC-1 negative. The results of qPCR showed that expression of UCP-1 in white adipocytes was 0.47-fold of that in the brown adipocytes (P<0.05), expression of a-tubulin in white adipocytes was 1.41-fold of that in the brown adipocytes (P>0.05). The results of Western blotting showed that UCP-1 expression in white adipocytes was 0.49-fold of that in brown adipocytes (P<0.01), expression of a-tubulin was 1.53-fold of that in brown adipocytes (P>0.05), and ASC-1 expression was not detected in brown adipocytes.Therefore, the cells were confirmed as white dipocytes, which can be used for further research of irisin effect on adipose.(6) Detection of FNDC5 expression profile of miceIn order to understand tissue expression of FNDC5, and lay a preliminary foundation for cloning of FNDC5, the expression of FNDC5 in cardiac, muscle, liver, spleen, lung, kidney, white adipose, and brown adipose tissue of the mice were detected through qPCR and Western blotting technology. The results of qPCR showed that expression of FNDC5 in cardiac tissue was significantly higher than that in muscle, lung, liver, spleen, kidney, white adipose tissue, and brown adipose tissue (all P<0.01). Western blotting showed that FNDC5 bands appeared in the cardiac and muscle tissue samples. FNDC5 was highly expressed in heart and muscle tissue.(7) Prokaryotic expression of FNDC5 and irisinFor purification FNDC5 and irisin protein, PGEX-4T-1-FNDC5 and PGEX-4T-1-irisin were constructed by recombinant FNDC5 and irisin gene with glutathione transferase (GST) tag by the way of C-terminal fusion, respectively. PGEX-4T-1-FNDC5 and PGEX-4T-1-irisin were transformed into BL21(DE3)pLysS. After induced by 1.0 mmol/L IPTG at 37?, GST-FNDC5 and GST-irisin proteins were purified by glutathione beads, and subjected to Coomassie Blue staining and Western blotting analyses. The results demonstrated that IPTG could induce the expression of GST-FNDC5 and GST-irisin proteins. In addition, antibodies, which used in this research, could specifically bind with FNDC5 and irisin.(8) Preliminary exploration of the mechanism of irisin promoting browning of white adipocytesIn order to study mechanism of irisin promoting browning of white adipocytes, white adipocytes were treated with different concentrations of GST-irisin (10 ng/mL, 100 ng/mL, and 1 ng/mL), respectively. After 6 h treatment, the expression of UCP-1 was detected by qPCR. Compared with a control group (0 ng/mL), the expression of UCP-1 was increased in three treatment group.The expression of UCP-1 was significant difference between the 10 ng/mL treatment group and the 100 ng/mL treatment group or 1?g/mL treatment group (all P<0.05).The expression of UCP-1 was no significant difference between 100 ng/mL treatment group and 1?g/mL treatment group (P>0.05). Therefore, the best used concentration of GST-irisin was 100 ng/mL. In order to determine the molecular mechanism of irisin promoting browning of white adipocytes, white adipocytes were treated with 100 ng/mL of GST-irisin, phosphorylation of protein kinase p38, ERK, and AKT were detected by Western blotting. The results showed that phosphorylation of p38 and ERK protein but not AKT protein were activated by 100 ng/mL of GST-irisin. Therefore, the effects of irisin on browning of white adipocytes were related to phosphorylation of p38 and ERK protein, unrelated to the phosphorylation of AKT protein.