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 Galpha(s) or Galpha(i) mediated (pertussis and cholera toxin insensitive), suggesting G(alphaq) as a candidate. Phospholipase C (PLC) is required, because the Ca(2+) 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/ERK(1/2)) pathway, with rapid, pronounced increases in ERK(1/2) 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 A(2) (cPLA(2))(-Ser505) phosphorylation (by MAPK/ERK(1/2)) was demonstrated by Western blot analysis. ASP signaling involves sequential activation of PI3K and PLC, with downstream activation of protein kinase C, Akt, MAPK/ERK(1/2), and cPLA(2), 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.

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 G(i)-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-Arg(77), and C3a des-Arg(77) (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 G(alpha 16). C3a des-Arg(77)/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-Arg(77)/ASP and C3a. Thus C5L2, a promiscuous complement fragment-binding protein with a high affinity site that binds C3a des-Arg(77)/ASP, may mediate the acylation-stimulating properties of this peptide.