The adipsin-acylation stimulating protein system and regulation of intracellular triglyceride synthesis.

We have previously characterized an activity from human plasma that markedly stimulates triglyceride synthesis in cultured human skin fibroblasts and human adipocytes. Based on its in vitro activity we named the active component acylation stimulating protein (ASP). The molecular identity of the active serum component has now been determined. NH2-terminal sequence analysis, ion spray ionization mass spectroscopy, and amino acid composition analysis all indicate that the active purified protein is a fragment of the third component of plasma complement, C3a-desArg. As well, reconstitution experiments with complement factors B, D, and complement C3, the components necessary to generate C3a, have confirmed the identity of ASP as C3a. ASP appears to be the final effector molecule generated by a novel regulatory system that modulates the rate of triglyceride synthesis in adipocytes.

Infection of human monocyte-derived macrophages by human immunodeficiency virus mediated by cell-to-cell transmission.

We have infected ten-day-old primary cultures of human monocyte-derived macrophages (MDM) with HIV-1 by cocultivation with chronically infected monocytic cell lines. This work has involved the U-937 monocytoid cell line, chronically infected with the HIV-IIIB strain of HIV-1 (U-937HIV IIIB) as well as a number of cell clones, termed UHC, which were derived from U-937HIV IIIB by limiting dilution. Cell-free virus, derived from each of U-937HIV IIIB cells and the UHC1 clone were noninfectious for MDM, as determined by failure to express viral p24 antigen (Ag). In contrast, viral p24 Ag production was detected in MDM that had been cocultivated with U-937HIV IIB, and with each of three UHC clones that produced infectious virus. Infection, in each case, was confirmed by polymerase chain reaction detection via the amplification of proviral DNA. In contrast, cocultivation with the UHC15.7 clone, which fails to cleave viral gp160 to its gp120 and gp41 products or the UHC8 clone, which lacks functional reverse transcriptase, did not lead to infection of MDM. Pretreatment of MDM for 2 hr with 1 microM AZT completely prevented infection by culture fluids containing HIVada, a macrophage-tropic virus, but did not affect infection mediated by cocultivation. These results suggest that cell-to-cell transmission of HIV-1, among monocyte-derived macrophages, can be mediated by proviral DNA. Moreover, gp120 at the surface of infected cells may play an important role in this process, since cell-to-cell HIV transmission could not be demonstrated with the UHC clone that is defective in cleavage of the viral envelope glycoprotein gp160.

Signal transduction pathway of acylation stimulating protein: involvement of protein kinase C.

Acylation Stimulating Protein (ASP) was recently purified to homogeneity from human plasma and shown to be identical to C3adesArg. ASP stimulates triglycerides synthesis in human skin fibroblasts and primary human adipocytes. In vitro differentiation of human preadipocytes to mature fat cells results in increased expression and accumulation of ASP in the medium. These differentiated human adipocytes are also much more responsive to ASP than preadipocytes. The object of this study was to investigate the signal transduction pathway by which ASP causes triglyceride synthesis (TGS) to increase in human cultured fibroblasts and adipocytes. No evidence was found for a protein kinase A-mediated response. ASP action was consistent with a protein kinase C (PKC)-mediated pathway in that: 1) the effect of ASP on TGS was mimicked by 1-10 nM phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC; (202% ASP vs. 178% PMA stimulation); 2) the effect of PMA and ASP were non-additive with respect to TGS; 3) staurosporine (50 nM) and GF109203X (bisindolymaleimide) at 1 microM, both competitive inhibitors of the ATP-binding site on PKC, inhibited both ASP and PMA stimulation of TGS (-59% and -65% for ASP and -84% and -99% for PMA, respectively); 4) Calphostin C (0.8 microM) which interacts with the regulatory domain of PKC also inhibited the ASP- and PMA-mediated stimulation of PKC (-76% +/- 11% inhibition for ASP and -99% +/- 20% inhibition for PMA), although in all cases the inhibition of PMA-stimulated triglyceride synthesis was greater; 5) ASP caused a time-dependent increase in intracellular diacylglycerol accumulation; and finally 6) stimulation by ASP caused an increase in PKC activity and a time-dependent translocation of PKC (maximal effect at 30 min) from the soluble intracellular compartment to a membrane-bound fraction (basal activity 22% in the membrane-bound fraction, ASP 54%, P < 0.05 and PMA 69% P < 0.0025). Taken together, the data are consistent with the conclusion that ASP acts to stimulate triglyceride synthesis via activation of the protein kinase C pathway.