Role Of ?1-adrenergic Receptor In Increased Lipolysis In Cancer Cachexia

Part I Implication of Lipolysis-pathway Receptors in Lipolysis in Patients with Cancer CachexiaObjectives:To detect the expression of lipolysis-pathway receptors of adipocytes and to elucidate their implications in cancer cachectic patients.Methods:A total of 34 patients were recruited, including 12 cancer cachexia patients, 13 cancer controls and 9 nonmalignant controls. Gene expression levels of (31-adrenergic receptor (ADRB1),?2-adrenergic receptor (ADRB2), (33-adrenergic receptor (ADRB3), a2C-adrenergic receptor (ADRA2C), natriuretic peptide receptor A (NPRA), insulin receptor (INSR), and HSL were determined in adipose samples of 34 patients by Real-time PCR. Protein levels of AD RBI and HSL were detected by Western blot. ADRB1 Localization was performed by immunofluorescence staining.Results:mRNA levels of both ADRB1 and HSL were-50% elevated selectively in the cachexia group (P=0.022; P=0.013), whereas mRNA levels of the other receptors remained unchanged. ADRB1 protein level showed a 1.5 fold and 3 fold increase in the cachexia group as compared with cancer controls and nonmalignant controls (P< 0.001; P<0.001). ADRB1 was confirmed as a membrane protein in adipocytes by immuno fluorescence staining. HSL protein expression was 2-2.5 fold increased selectively in cancer cachexia group (P<0.001; P=0.005). There was a positive correlation between protein level of ADRB 1 and HSL (r=0.474, P=0.005), as well as between ADRB1 level and lipolytic rate (r=0.406, P=0.002; r=0.435, P=0.01).Conclusion:We found increased ADRB1 expression in adipocytes of cancer cachectic patients, which is positively correlated with elevated HSL expression and increased lipolysis rate in this pathological setting. Part II Establishment of Cell Model with Human ADRB1 Gene Overexpression Based on 3T3-L1 Cell LineObjectives:To establish an adipocyte model with stable overexpression of human ADRB1 gene based on 3T3-L1 cell line with lentiviral vector.Methods:Human ADRB1 cDNA and lentiviral vector (pLVX-IRES-ZsGreenl) were ligated using recombinant DNA technology. The recombinant vector and packaging plasmids were co-transfected into 293T cells for virus packaging. Packed virus particles were secreted into the medium, which was collected 48 hours after transfection and applied for infection, through which ADRB1 gene were stably transfected into 3T3-L1 preadipocyte cell line. Finally, stably transfected cell line was selected with fluorescence-activated cell sorting (FACS).Results:All procedures were correct including cloning of ADRB1 construct, lentiviral packaging and infection, screening of stably transfected 3T3-L1 cell line. ADRB1 gene was integrated into host cell genome with viral DNA, so that ADRB1 protein was stably overexpressed on 3T3-L1 preadipocyte cell membrane. Western blot displayed a significantly elevated protein level of ADRB1 compared with the mock group, which demonstrated that model establishment is correct.Conclusion:We have established an adipocyte model with stable overexpression of ADRB1 based on 3T3-L1 cell line, which laid the foundation for further studies by mimicing the pathological changes of adipocytes in cancer cachectic patients. Part III Implications of ADRB1 Gene Overexpression in Fat Consumption in Cancer CachexiaObjectives:to further investigate the implications of ADRB1 gene overexpression in fat consumption in cancer cachexia.Methods:An overall study of overexpression of ADRB1 gene in fat consumption in cancer cachexia and the potential mechanism was carried out through inducing 3T3-L1 prcadipocyte with ADRB1 overexpression differentiate into adipocyte by MDI-based criteria. In the study, its impact on lipolysis was evaluated by detecting protein level of HSL; that in adipocyte differentiation evaluated by oil red staining and detection of protein level of 422(aP2), molecular marker in the process; that in triglyceride (TG) synthesis evaluated by the detection of protein level of fatty acid synthase, key enzyme in fat synthesis pathway.Results:3T3-L1 differentiated completely in the control and mock group, but didn't in ADRB1 overexpression group, which could be attenuated by ADRB1 inhibitor with dose-effect relationship. Increased protein level of HSL and decreased level of FAS were detected in ADRB1 overexpression group, compared with the control and mock group, which could be attenuated by ADRB1 inhibitor.Conclusion:ADRB1 gene overexpression leads to fat consumption in cancer cachexia through inhibition of TG synthesis, stimulation of TG breakdown, and inhibition of adipocyte differentiation.