GLP-1 Induces The Expression Of FNDC5 Derivatives That Execute Lipolytic Actions

Glucagon-like peptide(GLP-1)is a peptide hormone secreted by intestinal epithelial L cells,which plays a very important role in regulating glucose-dependent insulin synthesis and secretion.Moreover,it can treat obesity by maintaining satiety and by regulating adipogenesis and lipid metabolism.Therefore,a variety of GLP-1 receptor agonists(GLP-1RAs)have been produced for the treatment of diabetes and other metabolic disorders.However,studies on GLP1 are mainly focused on glucose metabolism,and the underlying mechanism by which these drugs regulate the body weight in obese patients remains incompletely understood.Irisin,derived from transmembrane protein fibronectin type III domain containing 5(FNDC5),is a recently identified peptide hormone that is released from several tissues in the body including skeletal muscle,adipocytes,and pancreatic beta cells.Induced by exercise,irisin is involved in converting white adipose tissue(WAT)into brown adipose tissue(BAT)and in regulation of thermogenesis.It has also been reported as a marker for inflammation and various metabolic disorders.Irisin has gained much interest for its potential use as a therapeutic to treat metabolic and endocrine disorders;however,there has been great conflict regarding its expression,cleavage,circulating levels,detection,excretion,and designation.We realized that the anti-obesity effects of GLP-1 and GLP-1RAs notably overlap with those of FNDC5/irisin.Both GLP-1 and irisin inhibit adipocyte differentiation,while stimulating the browning and lipolysis of WAT.At present,the relationship and the functions between GLP-1 and FNDC5/irisin has not been reported.Objective:To prove that GLP-1 induces the expression of FNDC5 in pancreatic β cells;To characterize the molecular mechanism of GLP-1 induced FNDC5 activation and expression in pancreatic beta cells;To determine the functional relationship between GLP-1 and FNDC5 and effects on lipolysis;To confirm the existence of secretable FNDC5(sFNDC5)isoform,and to clarify the biological function of this isoform by in vitro cell experiments and in vivo animal experiments.Materials and methods:1.RNA extraction and qRT-PCR detected the expression of related genes.2.Western blot was used to detect the expression of various proteins in cells and primary mouse tissues.3.Cloning the human FNDC5 promoter sequence into the pGL4.1 luciferase vector.4.Generating FNDC5 knock-out cell lines by using the CRISPR-Cas9 system.5.The secretion levels of irisin in cell culture supernatant were detected by ELISA.6.Expression and purification of sFNDC5 from Pichia.7.Animal experiment to observe the lipid metabolism effects of r-sFNDC5 in high fat dietinduced obese mouse model.8.All data are presented as the means±SEM.The statistical significance was analyzed by using GraphPad Prism 7.0 software,and a comparison between two groups was performed using one-way ANOVA analysis followed by an unpaired Student’s t-test.P<0.05 was considered to be statistically significant.Results:1.GLP-1 upregulates FNDC5 expression in beta cells.(1)qRT-PCR and Western blot showed that GLP-1 upregulates FNDC5 expression in both human βLox5 cells and rat INS1 cells.(2)The secreted levels of irisin in the culture medium GLP-1-treated βLox5 cell were upregulated.(3)By treating βLox5 cells with GLP-1 in combination with or without various signaling pathway inhibitors,we found that PKA,AKT and ERK signaling pathways mediate GLP-1 induction of FNDC5 expression.(4)GLP-1 upregulates FNDC5 gene and protein expression in both mouse pancreas and brain tissue.2.The molecular mechanism of GLP-1 induced FNDC5 activation and expression.(1)Bioinformatics analysis identified a putative binding site element of cAMP response element-binding protein(CREB)at promoter region of the human fndc5 gene,which is highly conserved in rats,mice,pigs,and chimpanzees.(2)We clone the human FNDC5 proximal promoter into the pGL4 luciferase vector upstream of the luciferase gene.HEK293 cells transfected with the FNDC5 promoter-luciferase reporter plasmid and serial concentrations of the CREB expression plasmid.The luciferase signal increased with the dose of the CREB gene in co-transfected HEK293 cells.(3)We create several 5’ truncated constructs by using the sub-cloning technique,HEK293 cells were transfected with a CREB expression plasmid and a plasmid with luciferase under the control of three different lengths of the FNDC5 promoter.The result showed that the signal increase depended on the presence of the predicted CREB binding site in the FNDC5 promoter.3.The functional relationship between GLP-1 and FNDC5 and effects on lipolysis and autophagy.(1)Generating FNDC5 knock-out cell lines.(2)FNDC5 protein was not expressed in its KO βLox5 cells.GLP-1 failed to increase the secretion of irisin in FNDC5 KO βLox5 cells.(3)GLP-1 failed to activate the expression of lipolysis and autophagy-related genes and protein in FNDC5 KO βLox5 cells.4.Discovery of secretory FNDC5.(1)We identified a new FNDC5 isoform transcript from rat INS-1 cell lines by qRT-PCR analysis.(2)The two bands were sequenced,and the results showed that the long band was the FNDC5 sequence currently reported(membrane-bound FNDC5,mFNDC5),while the short band was a novel secreted FNDC5(sFNDC5)isoform without the transmembrane region.(3)We also detected both mFNDC5 and sFNDC5 transcripts in all tested organs of Sprague Dawley rats.5.Expression and purified recombinant sFNDC5.(1)High quantity of r-sFNDC5 can be using a Pichia pastoris expression system and the protein was purified from the clear medium using a Ni-NTA chromatography column.(2)SDS-PAGE analysis showed r-sFNDC5 have five bands range from 18 to 27 kDa.(3)Treatment of the purified r-sFNDC5 with PNGase F led to a single band at around 18 kDa.6.R-sFNDC5 could induced the expression of lipolysis-related genes but not autophagy-related genes in fndc5 KO βLox5 cells.7.The anti-obesity activities of GLP-1 and sFNDC5 in mature 3T3-L1 adipocytes.(1)R-sFNDC5 strongly induced the expression of browning and lipolysis related genes and protein in a dose-dependent manner,while GLP-1 showed only a weak activity.(2)Activation of ERK1/2 and AKT signaling pathways in mature 3T3-L1 adipocytes by rsFNDC5 and GLP-1 at indicated concentrations.8.R-sFNDC5 attenuated diet-induced obesity,glucose metabolism/insulin sensitivity,and dyslipidemia.(1)Compared with the saline control,both r-sFNDC5 and r-irisin reduced the weight of mouse epididymal fat and the body weight of high fat diet(HFD)mice.(2)Both r-sFNDC5 and r-irisin significantly improved glucose tolerance in obese mice as demonstrated in the glucose tolerance test(GTT)and significantly reduced levels of fasting insulin in the insulin tolerance test(ITT).(3)These two secreted FNDC5 derivatives further decreased the serum levels of cholesterol,triglyceride,and free fatty acid(FFA).(4)R-sFNDC5 and r-irisin upregulated the expression of UCP-1 in subcutaneous and epididymal fat from different mice groups.(5)Both r-sFNDC5 and r-irisin promote fat oxidation in the liver,as indicated by decreased hepatic lipid accumulation and increased expression of fatty acid β-oxidation genes.Conclusion:1.GLP-1 upregulated the expression and secretion of FNDC5 in β-cells.2.GLP-1 exerts its lipolysis effects on pancreatic β-cells by inducing the expression of FNDC5.3.There is a secretable FNDC5 isoform in rat INS-1 cells.4.R-sFNDC5 possesses potent lipolysis actions on pancreatic β cells,adipocytes,and the dietinduced obese mouse model.