ARL15 Regulates The Secretion Of Adiponectin And Mediates The Adiponectin Receptor Signaling Pathway

BackgroundAdiponectin,also known as Acrp30,Apm1,AdipoQ or GBP28,is specifictly secreted by adipocyte cells.Clinical studies and basic experiments has conf IRmed that the close relationship between the low expression levels of the Adiponectin and the obesity related diseases(such as insulin resistance,type 2 diabetes and atherosclerosis heart disease).Adiponectin acts like a good factor,plays a crucial role in the regulation of energy metabolisman,anti-atherosclerosis and so on.As a insulin sensitizer,Adiponectin is a promising therapeutic approach.However,it has not been thoroughly explored in the context of pharmacological intervention.Recently,a small-molecule activator of the Adiponectin receptor,AdipoRon,has been proved to ameliorate insulin resistance,so understanding of Adiponectin and its downstream signaling pathways will provide a guide for the development of novel drugs in the treatment of obesity-related metabolic diseases.With the large-scale sequencing results of susceptibility disease genes came out,and the arrival of the post genome era,many genes have been labeled new associations with diseases.In terms of Adiponectin,a recent research of GWAS on plasma Adiponectin level shows that: in addition to the Adiponectin gene encoding the Adipoq site,lead SNP rs266717 is significantly related to the level of Adiponectin,a new gene named as ARL15(rs4311394)is also relevance to the plasma level of Adiponectin.And patients who carried the G alleles in rs4311394 loci not only have the high risk of having cardiovascular disease and diabetes,and accompanied with multiple metabolic disorders.Another study about screening susceptibility genes of the European obese children also showed that ARL15 gene exists genomic copy number variation,which increases the incidence rate of children's obesity.Lately in the north Indian population a GWAS studie about rheumatoid arthritis has also found that ARL15 is a new loci of non-human histocompatibility antigens(HLA)in rheumatoid arthritis(RA)susceptibilitly genes.In addition to aboved messages,we have researched susceptibility genes in type 2 diabetes complicated with atherosclerosis,we also found that ARL15 gene alleles significantly have different incidence rates of atherosclerosis.The above evidence suggested us ARL15 may be an important genetic loci,and may be associated with inflammation,obesity and diabetes.Thought little known about the ARL15 function,it is been proved widely expressed in white adipose tissue,skeletal muscle,liver,pancreas,heart,kidney,stomach,pituitary,placental tissue.And its structure analyssuggests it may belongs to the Small GTP binding protein family.The biological functions of this family is responsible for intracellular vesicles transportation and involved in intracellular signal transduction.So we assume that ARL15 may be involved in regulating Adiponectin and its receptors signaling pathways.Methods:1.In the state of normal or insulin resistance,test the m RNA and the protein levels of ARL15 and Adiponectin during the course of adipocyte differentiation.Using Immunofluorescence locate both the Adiponectin and the ARL15 proteins in the adipocytes derived from 3T3-L1 cells;2.In the state of normal or insulin resistance,test the expressions of ARL15 protein and the expressions of Adipo R1 and AMPK \ PGC-1 ?,which is as the key downstream molecules of Adiponectin receptors signaling pathways in skeletal muscle cells;3.In the state of physiological/insulin resistance,test the expressions of ARL15 protein and the expressions of IRS-1which is as the key downstream molecules of insulin signaling pathway in skeletal muscle cells;4.After the ARL15 overexpression or knock down,study the change of Adiponectin secretion,Adiponectin receptor signaling pathways and insulin signaling pathway during the normal physiological state and insulin stimulating state.Results:1.ARL15 expresses both in the preadipocyte and mature adipocyte.Followed the adipocyte differention,mRNA and protein levels of ARL15 increases accompanied with increasing mRNA and protein levels of Adiponectin;Immunofluorescence experimenct shows that ARL15 co-localizates with Adiponectin in the cell plasma;2.In the mature fat cells,insulin upregulates the ARL15 expression,while TNF?negatively regulates the ARL15 expression;3.ARL15 expresses in mature skeletal muscle cells;Normally ARL15 raises the expression of AdipoR1 and activates the downstream molecular of AdipoR1,such as AMPK and PGC-1 ?;4.We have successfully built both a stable cell lines of overexpression ARL15 gene and stable cell lines in which ARL15 has been knocked down with s IRNA in C2C12 myoblast,both provide the technical support for subsequent experiments;5.By overexpression and interference on ARL15,it has been proved that ARL15 promote the Adiponectin-Adiponectin receptors signaling pathways through raising AdipoR1 expression in C2C12 myotube;6.Using overexpression and the interference of gene modification on ARL15,ARL15 can phosphorylate the IRS-1,which improves insulin sensitivity in skeletal muscle cells.Conclusions:ARL15 is likely to be a new molecule to enhance the expression of the Adiponectin during adipogenesis of 3T3-L1 cells and potentiates insulin sensitivity by upregulate the Adiponectin receptor1 and improve the phosphorylation of IRS-1 in C2C12.