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.