Establishment Of Screening Model For Beta3 Adrenoceptor Agonists And Screening Of Potential Beta3 Adrenoceptor Agonists

BACKGROUNDS:Diabetes mellitus(DM) is a metabolic disorder of multiple etiologies, and about 90% - 95% of all diabetes patients belong to Type 2 diabetes mellitus(Type 2 DM).With rising of the prevalence in the world,DM has been another chronic non-communicable disease which harms people's health severely following cardio-cerebral vascular diseases and tumors.As a systemic endocrine and metabolic disease,obesity not only damages shape,but also is associated with multiple diseases,such as Hyperlipidema,Atherosclerosis,Coronary Heart Disease,DM,etc.It has been explored that obesity is an independent risk factor,and above 90% of Type 2 DM patients companied with obesity.Therefore,research and discovery of new drugs for both obesity and Type 2 DM would be an attractive approach.As a subclass ofβ-adrenergic receptors(β-AR),β3-adrenoceptor(β3-AR) is found on the cell surface of both white and brown adipocytes and is non-sensitive to classicalβ-AR antagonists.It has been explored thatβ3-AR agonists are effective for obesity and Type 2 DM by stimulating lipolysis in white adipose tissue(WAT) and thermogenesis in brown adipose tissure(BAT),improving insulin sensitivity and glucose uptake etc.Therefore,β3-AR is maybe the double treatment targets for obesity and Type 2 DM.Because of the significant difference ofβ3-AR gene between human and rodent in structure and function,only the compounds based on humanβ3-AR screening model can be reliably foreseened their efficacy and safety in clinical trials.AIMS:The aims of present study are to establish a molecular and cellular screening model system for humanβ3-AR agonists to lay foundations for the following screening forβ3-AR agonists with higher potency and selection.In order to discover aβ3-AR agonist for obesity and/or Type 2 DM, a series of new compounds will be screened for theβ3-AR agonistic activities by the screening models.METHODS:(1) Aβ3-AR gene cDNA amplified from pENTR_ADRB3-Stop,a plasmid carried with humanβ3-AR gene by Polymerase Chain Reaction(PCR) was digested by the restriction enzymes of KpnⅠand XbaⅠ.An eukaryotic expression plasmid, pcDNA3.1(+)-β3-AR plasmid, was constructed by inserted theβ3-AR cDNA fragment into pcDNA3.1(+) vector digested by the same restriction enzymes.Then the plasmid was transfected into HEK293 cells,and the expression ofβ3-AR mRNA and protein were detected by RT-PCR and immunocytochemistry respectively.(2) The expression ofβ1-AR,β2-AR orβ3-AR receptor proteins in CHO-K1 cells stably transfected with humanβ1-AR,β2-AR orβ3-AR target gene were detected by immunocytochemistry,immunofluence,Western Blotting and radioligand binding assay respectively. (3) pCRE-luc and pRL-TK plasmids were transiently cotransfected into CHO-K1 cells stably expressed with humanβ3-AR at the ratio of 10:1 to establish a screening model based on dual-luciferase reporter gene assay.After the valiablity of the model was identified by isoprenaline stimulation,theβ3-AR agonistic activities of a series of compounds were tested with the model.(4) The expression ofβ1-AR,β2-AR orβ3-AR in SW872 preadipocytes were detected by immunocytochemistrical stain,and insulin resistance in differentiated SW872 adipocytes was induced by 1μM dexamethasone.(5)The pharmacological activities of Betaphrine,a derivative of Phenylethanolamine were tested by proliferation and differentiation model of SW872 preadipocytes,insulin resistance model of SW872 adipocytes and screening model forβ3-AR agonists based on dual-lucifease reporter gene assay.RESULTS:(1)The pcDNA3.1(+)-β3-AR plasmid was identified to be constructed successfully by restriction digestion and DNA sequencing.β3-AR mRNA and protein were successfully expressed in HEK293 cells transfected with the pcDNA3.1(+)-β3-AR plasmid detected by RT-PCR and immunocytochemistry.(2)Three types of CHO-K1 cells stably transfected withβ1-AR,β2-AR orβ3-AR gene respectively were showed to be positive immune response by immunocytochemistrical stain,and obvious fluorescence were mainly located on membrane and in cytoplasm.Expected protein lanes were obtained from the three type of CHO-K1 cells when detected by Western Blotting assay, and 125I-Cyanopindolol, a non-selective radioligand forβ-AR,bound specifically with the three types of cells.(3)Compared with control,the activities of luciferase reporter genes significantly increased when stimulated with isoprenaline 48 h after CHO-K1 cells stably expressed with humanβ3-AR were cotransfected with pCRE-luc and pRL-TK(P<0.01),which indicated that the cell model kept good reponse toβ3-AR agonists and could be used as a screening model forβ3-AR agonists.With the screening model,8 from 20 new compounds showed higher potency forβ3-AR agonistic activities were selected out for further study.(4)SW872 preadipocytes expressedβ1-AR,β2-AR andβ3-AR detected by immunocytochemistrical stain,and they could differentiate into adipocytes when induced by 0.6μM Oleic acid for 3 days.The glucose concentration in culture medium significantly increased by coincubated with 1μM Dexamethasone for 4 days compared with control(P<0.01),and it could be prevented by Pioglitazone,a PPARγagonist,in a manner of concentration-dependance.(5)Betaphrine significantly promoted the proliferation of SW872 preadipocytes,decreased lipid accumulation during the differentiation prosess of SW872 preadipocytes,improved the insulin resistance in SW872 adipocytes and increased the activities of luciferase reporter genes in a manner of concentration-dependance,all these effects could be prevented by propranolol.CONCLUSIONS:(1)pcDNA3.1(+)-β3-AR,an eukaryotic expression vector,has been successfully constructed and expressed in eukaryotic cells. (2)CHO-K1 cells stably transfected with humanβ1-AR,β2-AR orβ3-AR gene express their receptor proteins respectively,and can be used as screening models for highly potent and selectiveβ3-AR agonists.(3)The screening model forβ3-AR agonists based on dual-luciferase reporter genes keeps good reponse toβ3-AR agonists,which shows that the model is valiable and can be used as a screening model forβ3-AR agonists.(4)An insulin resistance model in SW872 adipocytes has been successfully established by the induction of Dexamethasone.(5)Betaphrine is a high potentβ3-AR agonist.It's anti-obesity and anti-diabetic effects maybe associated with promoting lipolysis,improving insulin resistance etc,and it may be a new drug for obesity and Type 2 DM.