Detection and quantification of the control proteins of the alternative pathway of complement in 3T3-L1 adipocytes.
The complement peptide C3a desarg is identical to acylation-stimulating protein (ASP), a human plasma protein that potently stimulates adipocyte triacylglycerol synthesis and glucose transport. Both human and murine adipocytes express mRNA and/or protein for the complement components C3 and factors B and D (adipsin) required to generate ASP. However, the regulatory mechanisms controlling this process are unknown. We have established a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique to demonstrate the presence in mouse 3T3-L1 adipocytes of mRNA for all components of the alternative pathway, including the control proteins factors I and H, CR1 and properdin. On differentiation, mRNA for C3 (fivefold) and factor D (> 50-fold) increased, whereas stimulation with tumour necrosis factor (TNF)-alpha and interleukin (IL) 1 beta led to eightfold increases in factor B mRNA. Metabolic labelling followed by immunoprecipitation showed that factor B protein is normally present in small quantities, and is greatly increased by cytokine stimulation. The larger quantities of C3 and H proteins present were little affected, whereas levels of C3a increased on cytokine stimulation. These results suggest that the rate-limiting step in the cytokine-induced production of ASP in adipocytes is factor B synthesis.
Peake
P W
PW
Department of Medicine, Prince Henry Hospital, Sydney, Australia.
O'Grady
S
S
Pussell
B A
BA
Charlesworth
J A
JA
D016475:3T3 Cells; D017667:Adipocytes / Q000737:chemistry*; D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D015926:Complement C3a / Q000032:analysis* / Q000235:genetics; D011415:Complement Factor B / Q000032:analysis* / Q000235:genetics; D011416:Complement Factor D; D017242:Complement Factor H / Q000032:analysis / Q000235:genetics; D003170:Complement Pathway, Alternative; D007375:Interleukin-1 / Q000494:pharmacology; D051379:Mice; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012697:Serine Endopeptidases / Q000032:analysis* / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001798:Blood Proteins; C493495:CFH protein, human; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C044541:complement C3a, des-Arg-(77)-; D015926:Complement C3a; D017242:Complement Factor H; D012697:Serine Endopeptidases; C493488:CFD protein, human; D011416:Complement Factor D; C493491:complement factor D, mouse; D011415:Complement Factor B