Magdalena Maslowska

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.

The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating protein

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 Gi-like G proteins (Cain, S. A., and Monk, P. N. (2002) J. Biol. Chem. 277, 7165–7169). Here we demonstrate that C5L2 binds the metabolites of C4a and C3a, C4a des-Arg77, and C3a des-Arg77 (also known as the acylation-stimulating protein or ASP) at a site distinct from the C5a binding site. The binding of these metabolites to C5L2 does not stimulate the degranulation of transfected rat basophilic leukemia cells either through endogenous rat G proteins or when co-transfected with human Gα16. C3a des-Arg77/ASP and C3a can potently stimulate triglyceride synthesis in human skin fibroblasts and 3T3-L1 preadipocytes. Here we show that both cell types and human adipose tissue express C5L2 mRNA and that the human fibroblasts express C5L2 protein at the cell surface. This is the first demonstration of the expression of C5L2 in cells that bind and respond to C3a des-Arg77/ASP and C3a. Thus C5L2, a promiscuous complement fragment-binding protein with a high affinity site that binds C3a des-Arg77/ASP, may mediate the acylation-stimulating properties of this peptide.

Impaired response of fibroblasts from patients with hyperapobetalipoproteinemia to acylation-stimulating protein.

Acylation-stimulating protein (ASP) is a small, basic, human plasma protein that markedly stimulates triglyceride synthesis in human adipocytes and cultured human skin fibroblasts. The present studies examine the response to ASP of cultured skin fibroblasts from normal subjects patients with hyperapobetalipoproteinemia, patients with familial hypercholesterolemia, and patients with hypertriglyceridemia without hyperapobetalipoproteinemia. Triglyceride synthesis induced by ASP did not differ significantly among the normals, the patients with familial hypercholesterolemia, and the patients with hypertriglyceridemia with normal low density lipoprotein (LDL) apolipoprotein B levels; however, on average, it was markedly reduced in the patients with hyperapobetalipoproteinemia. In all groups studied, evidence of specific saturable binding of radioiodinated ASP was present. Binding, however, was significantly reduced in the groups with hyperapobetalipoproteinemia whereas the other three groups were indistinguishable. By contrast, LDL-specific binding was reduced only in the patients with familial hypercholesterolemia. There was a significant direct relation between the degree of ASP binding and the triglyceride synthesis inducible by ASP. In addition, with the exception of the patients with familial hypercholesterolemia, there was an inverse relation between both ASP-specific binding and ASP-induced triglyceride synthesis in fibroblasts to LDL levels in plasma whereas no relation was evident to plasma high density lipoprotein and very low density lipoprotein.

Of mice and men (and women) and the acylation-stimulating protein pathway.

The storage and release of energy by adipocytes is of fundamental biologic importance. Not surprisingly, therefore, the rate at which these processes occur can be modulated by a variety of physiologic molecules. A newly recognized participant is produced by adipocytes themselves: acylation-stimulating protein (ASP). This article focuses on the most recent in-vivo evidence regarding how the ASP pathway may influence energy storage and release. In brief, the rate at which triglycerides are cleared from plasma (i.e. the rate at which they are hydrolysed) is determined by lipoprotein lipase and insulin, which is the principal hormone that regulates lipoprotein lipase. By contrast, the ASP pathway modulates the rate at which fatty acids are taken up and converted to triglycerides by adipocytes. Under certain circumstances, however, reduction of activity of the ASP pathway may negatively impact on the first step of the process. ASP also influences the rate at which fatty acids are released by adipocytes, and it is clear that insulin and ASP interact in a variety of ways that involve energy storage and release. Accordingly, to understand the impact of any intervention on energy storage and release by adipocytes, the effects of both insulin and ASP must be taken into account.

Targeting the signaling pathway of acylation stimulating protein

Acylation stimulating protein (ASP; C3adesArg) stimulates triglyceride synthesis (TGS) and glucose transport in preadipocytes/adipocytes through C5L2, a G-protein-coupled receptor. Here, ASP signaling is compared with insulin in 3T3-L1 cells. ASP stimulation is not Gαs or Gαi mediated (pertussis and cholera toxin insensitive), suggesting Gαq as a candidate. Phospholipase C (PLC) is required, because the Ca2+ chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester and the PLC inhibitor U73122 decreased ASP stimulation of TGS by 93.1% (P < 0.0.001) and 86.1% (P < 0.004), respectively. Wortmannin and LY294002 blocked ASP effect by 69% (P < 0.001) and 116.1% (P < 0.003), respectively, supporting phosphatidylinositol 3-kinase (PI3K) involvement. ASP induced rapid, transient Akt phosphorylation (maximal, 5 min; basal, 45 min), which was blocked by Akt inhibition, resembling treatment by insulin. Downstream of PI3K, mamalian target of rapaycin (mTOR) is required for insulin but not ASP action. By contrast, both ASP and insulin activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2) pathway, with rapid, pronounced increases in ERK1/2 phosphorylation, effects partially blocked by PD98059 (64.7% and 65.9% inhibition, respectively; P < 0.001). Time-dependent (maximal, 30 min) transient calcium-dependent phospholipase A2 (cPLA2)-Ser505 phosphorylation (by MAPK/ERK1/2) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK1/2, and cPLA2, all of which leads to an effective and prolonged stimulation of TGS.

Novel roles for acylation stimulating protein/C3adesarg : A review of recent in vitro and in vivo evidence

Recent experimental evidence is shedding more light on the physiological actions of acylation-stimulating protein (ASP)/C3adesArg. The role of ASP in regulating lipid metabolism has primarily focused on its participation in the stimulation of triglyceride synthesis (TGS) and glucose transport. Although there is no doubt that ASP, an adipocyte-produced hormone, plays a key physiological role, accumulating evidence suggests that the effects of ASP go beyond its acute effects on lipid metabolism. In this review, we present novel findings of ASP/C3adesArg effects on preadipocyte differentiation. In 3T3-L1 and 3T3-F442A cells, ASP can substitute for insulin and enhance differentiation as measured by intracellular lipid droplet accumulation, clonal expansion, and increased expression of differentiation markers. Specifically, ASP increased basal TGS by 250% after 9 days differentiation, with similar effects induced by insulin. With ASP treatment, expression of C/EBPdelta was up-regulated early in differentiation (day 2) and decreased thereafter. Expression of PPARgamma and late markers of differentiation, such as adipsin and diacylglycerol acyltransferase-1, were also increased. Effects on clonal expansion were indicated by a twofold increase in [(3)H] thymidine incorporation in 3T3-L1 cells compared to treatment with IBMX + DX alone. Further, the effects of ASP extended beyond adipose tissue to endocrine effects on hormone secretion of insulin (pancreatic cells); cytokines TNFalpha, IL-1beta, and IL-6 (myeloid cells); prolactin, growth hormone, and adrenocorticotropin (pituitary cells). Finally, the potential implication of C5L2, the newly discovered ASP receptor, and its expression profile in various tissues are discussed relative to ASP function.