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. (J. Clin. Invest. 1993.
The orphan receptor C5L2 has recently been described as a high affinity binding protein for complement fragments C5a and C3a that, unlike the previously described C5a receptor (CD88), couples only weakly to
Through their capacity to store fatty acids as triacylglycerol molecules, adipocytes serve a vital physiologic role. This study presents further evidence that this process can be modulated in human adipocytes by the adipsin/acylation stimulating protein (ASP) pathway and suggests a novel function for the product of this system--ASP. The data demonstrate the following: (1) ASP stimulates triacylglycerol synthesis within adipocytes, and this occurs to a greater extent in differentiating than undifferentiated cells (242% +/- 32% vs 168% +/- 11%, p < 0.01, respectively, at an ASP concentration of 88 ng/mL; (2) ASP does not affect the Km for triacylglycerol synthesis but does substantially increase Vmax; (3) when ASP is generated in vitro through incubation of its precursor proteins under appropriate conditions, triacylglycerol synthesis increases to the same extent as when plasma-purified ASP is added to the medium; (4) human adipocytes contain mRNA for the specific serine protease adipsin and the two precursor proteins C3 and factor B required to interact for the production of ASP; and (5) the extent to which cultured differentiating adipocytes produce ASP is proportional to the degree to which they have accumulated triacylglycerol mass during differentiation (r2 = 0.7523, p < 0.0005). These findings provide the first evidence for the existence of the adipsin/ASP pathway in human adipocytes, and this may markedly enhance our understanding of the processes which regulate triacylglycerol clearance from plasma.
The present data suggest involvement of the ASP/adipsin pathway in the pathogenesis of obesity.
Acylation stimulating protein (ASP) is a potent stimulator of adipocyte triacylglycerol storage. In vivo studies have shown that ASP production by adipocytes increases locally after a fat meal. Initial in vitro studies demonstrated increased production of ASP in the presence of chylomicrons (CHYLO). The present aim was to define the CHYLO component responsible. None of the apoproteins tested (AI, AII, AIV, CI, CII, CIII, and E) were capable of stimulating C3 (the precursor protein) or ASP production. Rather, the active component is a nonlipid, loosely associated, trypsin-sensitive molecule. High pressure liquid chromatography fractionation of the CHYLO infranate proteins identified the critical protein as transthyretin (TTR), which binds retinolbinding protein and complexes thyroxine and retinol. Addition of TTR alone, with lipid emulsion, or with respun CHYLO to human differentiated adipocytes had little effect on C3 and ASP production. By contrast, when transthyretin was added to CHYLO, C3 and ASP production were substantially enhanced up to 75-and 7.5-fold respectively, compared with the effect of native CHYLO alone. Finally, a polyclonal antibody against TTR could inhibit stimulation of C3 and ASP production by CHYLO (by 98 and 100%, respectively) and by CHYLO infranate proteins (by 99 and 94%, respectively). We hypothesize that TTR mediates the transfer of the active components from CHYLO to adipocytes, which then stimulates increased C3 and ASP production. Thus the CHYLO provides the physiologic trigger of the ASP pathway.Adipose tissue is recognized as an active organ releasing hormones, enzymes, and proteins such as lipoprotein lipase (1), cholesteryl ester transfer protein (2), apoprotein E (3), angiotensinogen (4), estrogen (5), tumor necrosis factor (6), and leptin (7). Both human and murine adipose tissue have been shown to synthesize and secrete complement C3, factor B, and adipsin, proteins involved in the alternate complement pathway (8 -12). Furthermore, complement C3, factor B, and adipsin have been shown to be synthesized and secreted in a differentiation-dependent manner by adipocytes (10 -12). The interaction of these three proteins results in the cleavage of complement C3 generating the bioactive protein acylation stimulating protein (ASP/C3adesArg) 1 (9 -11), which was initially recognized as a small basic protein present in human plasma (13). ASP can actively stimulate triacylglycerol synthesis in human adipocytes through a coordinated effect on translocation of the glucose transporters (GLUT1, GLUT3, and GLUT4) (14 -16) and increases in the activity of the enzyme diacylglycerol acyltransferase. These effects of ASP are mediated through the diacylglycerol protein kinase C pathway (17) via specific interaction with the cell surface. The most responsive target to the action of ASP is adipose tissue, which is also known as the primary tissue for storage and release of energy. Within the cell, free fatty acids are enzymatically esterified to a glycerol-3-phosphate backbone to form triacylglyce...
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