Acylation Stimulating Protein Production Research Articles

Complement‐mediated chronic inflammation is associated with diabetic microvascular complication

Chronic inflammation is characteristic of type 2 diabetes mellitus (T2DM). Obesity-activated adipocytes release adipocytokines, which induce the secretion of proinflammatory cytokines, resulting in vascular endothelial dysfunction and organ injury. C3a is a candidate to induce tissue inflammation. We investigated the association between diabetic microangiopathy and complement-mediated inflammation in 32 obese T2DM patients and 32 normal donors. Plasma levels of complement components and their activation intermediates were examined and related to the level of complication. An incubation study of post-prandial serum was carried out to measure the in vitro production of acylation stimulating protein (ASP/C3a desArg) by chylomicron. Plasma levels of C3, C4, factor B, iC3b, Bb, and ASP were significantly increased in T2DM patients. Levels of C4d and membrane attack complex (C5b-9) were not significantly elevated. The activation rate of these factors indicated that only the early phase of alternative complement pathway was excessively activated. A statistical study revealed close correlation between ASP, body mass index, and highly sensitive C-reactive protein. Plasma ASP was significantly increased in the macroalbuminuric and proliferative retinopathy patient groups. An incubation study revealed that ASP was produced after the in vitro incubation of post-prandial serum from a T2DM patient with hyperchylomicronaemia. Activation of the alternative complement pathway occurs in obese T2DM patients and is enhanced in the post-prandial hyperchylomicronic condition, which induces overproduction of ASP and C3a-mediated tissue inflammation. Therefore, complement-mediated inflammation may contribute to the acceleration of diabetic microangiopathy in addition to the development of macroangiopathy.

Inflammatory Markers and Adipokines Alter Adipocyte-derived ASP Production Through Direct and Indirect Immune Interaction

Obesity and related metabolic diseases are associated with chronic low-grade inflammation, characterized by increased pro-inflammatory proteins. Several studies have demonstrated increases in acylation stimulating protein (ASP) and its precursor protein C3 in obesity, diabetes and dyslipidemia. To evaluate the effects of acute inflammatory factors and adipokines on ASP production and potential mechanisms of action, 3T3-L1 adipocytes were treated for 24 h with adipokines, cytokines, macrophage-conditioned media and direct co-culture with J774 macrophages. ASP and C3 in the media were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride stores). Leptin, adiponectin, IL-10, LPS and TNF-α increased ASP production (151%, 153%, 190%, 318%, 134%, P<0.05, respectively,). C5a and RANTES (Regulated and normal T cell expressed and secreted) decreased ASP production ( - 34%, - 47%, P<0.05), which was also associated with a decrease in the precursor protein C3 ( - 39% to - 51%, P<0.01), while keratinocyte chemoattractant (KC; murine IL-8 ortholog) had no effect on ASP and C3 secretion. By contrast, apelin, omentin and visfatin also decreased ASP ( - 27%, - 49%, - 22%, P<0.05), but without changes in precursor protein C3 secretion. Macrophage-conditioned media alone had little effect on C3 or ASP, while co-culture of adipocytes with macrophages markedly increased ASP and C3 production (272%, 167%, P<0.05). These in vitro results suggest various metabolic hormones and inflammatory factors can affect ASP production through increased precursor C3 production and/or by changing the rate of C3 conversion to ASP. As an adipokine, ASP could constitute a new link between adipocytes and macrophages.

Plasma Levels of Acylation-Stimulating Protein Are Strongly Predicted by Waist/Hip Ratio and Correlate with Decreased LDL Size in Men

The association of abdominal obesity with cardiovascular risk is often linked to altered secretion of adipose-derived factors and an abnormal lipid profile including formation of atherogenic small dense low density lipoprotein particles (sdLDL). Acylation-stimulating protein (ASP) is an adipose-derived hormone that exhibits potent lipogenic effects. Plasma ASP levels increase in obesity; however, the association of ASP levels with body fat distribution is not yet established, and no study to date has investigated the association of ASP with LDL size. In this study, we examined the association of ASP levels with abdominal obesity measures and the lipid profile including LDL size in 83 men with a wide range of abdominal girths. Regression analysis showed that waist/hip ratio was the main predictor of ASP levels (β = 0.52, P < 0.0001), significantly followed by decreased LDL size. BMI and TG levels, although positively correlated with ASP levels, were excluded as significant predictors in regression analysis. No correlation was found with LDL-C or apoB levels. ASP levels were 62.5% higher in abdominally obese compared to nonobese men. Waist/hip ratio presenting as the main predictor of ASP levels, suggests increased ASP production by abdominal fat which, as proposed previously, may result from resistance to ASP function causing delayed TG clearance and subsequent formation of atherogenic sdLDL.

Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes

In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

Hormone and pharmaceutical regulation of ASP production in 3T3‐L1 adipocytes

Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

Cord Blood ASP Is Predicted by Maternal Lipids and Correlates With Fetal Birth Weight

The acylation stimulating protein (ASP) is a potent lipogenic adipokine that correlates with postprandial triglyceride (TG) clearance and is linked to the pathophysiology of obesity and related disorders. To investigate ASP levels in cord blood and its relation to maternal and cord blood lipid parameters and fetal birth weight. Thirty nondiabetic pregnant women, their newborns, and thirty-three nonpregnant controls were included in this study. Fasting maternal and cord blood ASP, TGs, nonesterified fatty acids (NEFAs), cholesterol, glucose levels, in addition to maternal BMI and fetal birth weight were measured. No significant difference was found between cord blood ASP (16.3 +/- 0.96 nmol/l) and ASP levels in the adult controls (15.7 +/- 1.0 nmol/l). Cord blood ASP, however, was lower than maternal plasma ASP levels (25.4 +/- 1.6 nmol/l, P < 0.001). Yet, lipid levels in cord blood, particularly TGs were markedly decreased compared to control and maternal TG levels (threefold and 7.4-fold, P < 0.001 respectively). Maternal TGs significantly correlated with fetal birth weight (r = 0.54, P = 0.002). Multiple regression analysis showed that maternal TGs (beta = 0.57, P = 0.01) and NEFAs (beta = 0.43, P = 0.024) predicted 45% variation in cord blood ASP levels, independent of all measured maternal and cord blood parameters. Cord blood ASP showed a positive correlation with fetal birth weight (r = 0.524, P = 0.037) in neonates above average fetal birth weight of the studied population. This is the first study investigating ASP in cord blood. We suggest that maternal hypertriglyceridemia is associated with increased fetal ASP production, thus enhancing fetal fat storage independent of maternal glucose variations in nondiabetic women.

Rosiglitazone decreases postprandial production of acylation stimulating protein in type 2 diabetics

BackgroundWe evaluated plasma ASP and its precursor C3 in type 2 diabetic men with/without rosiglitazone (ROSI) treatment compared to healthy non-obese men. We tested (1) whether plasma ASP or C3 are altered postprandially in subcutaneous adipose tissue or forearm muscle effluent assessed by arteriovenous (A-V) differences in healthy lean men and older obese diabetic men and (2) whether treatment with ROSI changes the arteriovenous gradient of ASP and/or C3.MethodsIn this ongoing placebo-controlled, crossover, double-blinded study, AV differences following a mixed meal were measured in diabetic men (n = 6) as compared to healthy men (n = 9).ResultsPostprandial arterial and adipose venous TG and venous NEFA were increased in diabetics vs. controls (p < 0.05–0.0001). ROSI treatment decreased postprandial arterial TG (p < 0.001), adipose venous NEFA (p < 0.005), reduced postprandial glucose (p < 0.0001) and insulin concentrations (p < 0.006). In healthy men, there was no change in postprandial C3, but an increase in adipose venous ASP vs. arterial ASP (p < 0.02), suggesting ASP production, with no change in forearm muscle. In older, obese diabetic subjects, arterial C3 was greater than in controls (p < 0.001). Arterial C3 was greater than venous C3 (p < 0.05), an effect that was lost with ROSI treatment. In diabetics, postprandial venous ASP was greater than arterial (p < 0.05), indicating ASP production, an effect that was lost with ROSI treatment (p < 0.01).ConclusionIncreased postprandial venous production of ASP is specific for adipose tissue (absent in forearm muscle). Increased postprandial C3 and ASP in diabetic subjects is consistent with an ASP resistant state, this state is partially normalized by treatment with ROSI.

Chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to overproduction of acylation stimulating protein.

Acylation stimulating protein (ASP) is a fragment of the third component of complement (C3) that is generated in the presence of chylomicron, and plays a role in the synthesis of triacylglycerol by transporting free fatty acids into adipocytes. However, the precise mechanism of ASP generation, especially the role of chylomicron in ASP generation, is unknown. We examined the mechanism through which chylomicron induces ASP generation. Ultracentrifugationally separated chylomicron was incubated with normal human serum (NHS) under various conditions, and the amounts of complement activation products and ASP in the incubation mixture were determined by enzyme-linked immunosorbent assay (ELISA). Upon incubation of NHS with various amounts of chylomicron for 120 min, ASP was generated in a dose-dependent manner. The time course of the production of ASP was similar to the time course of the C3 tick-over phenomenon that occurred by depletion of factor H from the serum. The complement activation induced by chylomicron was different from the usual complement activation that occurs under the regulation of factor H and factor I with respect to the time course and the amount of ASP produced. Our results indicate that chylomicron accelerates C3 tick-over by regulating the role of factor H, leading to the overproduction of ASP.

Abstracts for Poster Presentation

Abstracts for Poster Presentation

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.

Regulation of Complement C3 Expression by the Bile Acid Receptor FXR

The farnesoid X receptor (FXR; NR1H4) is an intracellular bile acid-sensing transcription factor that plays a critical role in the regulation of synthesis and transport of bile acids as well as lipid metabolism. Although the reciprocal relationship between bile acid and triglyceride levels is well known, the mechanism underlying this link is not clearly defined. In this study, we demonstrate that FXR regulates the expression of at least two secreted factors, complement component C3 and FGF15, the rat ortholog of FGF19, known to influence lipid metabolism. The analysis of the human complement C3 gene reveals the presence of functional FXR response elements in the proximal promoter of C3. Furthermore, rats given a single dose of an FXR agonist exhibit an increase in the plasma concentration of complement C3 protein. These studies demonstrate a mechanism by which FXR, a nuclear receptor with a limited tissue expression pattern, regulates secretion of factors that ultimately can affect lipid metabolism in an endocrine or paracrine manner.

Acylation-stimulating protein precursor proteins in adipose tissue in human obesity

Recent reports have suggested a link between acylation-stimulating protein (ASP) and complement C3 with obesity, insulin resistance, coronary artery disease, and hyperlipidemia. Our aim was to examine the mRNA expression of C3 and other factors related to ASP production (such as factor B and adipsin) in adipose tissue. The influence of gender and obesity was examined in subcutaneous (SC) and omental (OM) tissues from 16 males and 16 females with body mass index (BMI) from 20 to 54 kg/m 2. The results demonstrate that factor B mRNA expression is higher in males than females in both SC and OM tissues. In female SC tissue, C3 and adipsin mRNA decrease with increasing BMI ( r = 0.557, P = .025 and r = 0.717 P = .002, respectively), with no change in factor B. By contrast, in males there was a pronounced increase in C3, adipsin, and factor B in OM tissue with increasing BMI ( r = 0.759 P = .001, r = 0.650 P = .006, and r = 0.568 P = .022, respectively). Of note, however, in both men and women there was a marked increase in the OM/SC ratio of C3 and adipsin with increasing BMI. These results suggest that in female SC adipose tissue, there is downregulation of factors related to ASP production in obesity, perhaps to limit further expansion of adipose tissue. In males, there is increased expression in OM tissue. In addition, relative OM/SC expression increases with obesity and these changes may contribute to the development of visceral adipose tissue.

Regulation by retinoic acid of acylation-stimulating protein and complement C3 in human adipocytes

Acylation-stimulating protein (ASP), a product of complement C3, stimulates triacylglycerol synthesis in adipocytes. Previous studies have identified transthyretin, associated with chylomicrons, as a stimulator of C3 and ASP production. Since both transthyretin and chylomicrons transport retinyl ester/retinol, our goal was to investigate whether retinoic acid (RA) could be a potential hormonal mediator of the effect. Inhibitors of protein synthesis and protein secretion eliminated the stimulatory effects of chylomicrons on both C3 and ASP production in human differentiated adipocytes, suggesting that de novo protein synthesis and secretion are both required. Incubation with chylomicrons increased C3 mRNA levels (37±1.5%). RA alone or with chylomicrons had a stimulatory effect on C3 production (29-fold at 16.6nM RA) and ASP production. An RA receptor antagonist blocked stimulation of C3 mRNA and C3 secretion by both RA and chylomicrons. Finally, RA and chylomicrons activated a 1.8kb C3-promoter–luciferase construct transfected into 3T3-F442 and 3T3-L1 cells (by 41±0.2% and 69±0.3% respectively), possibly via RA receptor half-sites identified by sequence analysis. This is the first evidence documenting stimulation by RA of the C3 gene. Thus we propose RA as a novel cellular trigger in chylomicrons that subsequently results in increased ASP production by adipocytes after a meal.

Regulation by retinoic acid of acylation-stimulating protein and complement C3 in human adipocytes.

Acylation-stimulating protein (ASP), a product of complement C3, stimulates triacylglycerol synthesis in adipocytes. Previous studies have identified transthyretin, associated with chylomicrons, as a stimulator of C3 and ASP production. Since both transthyretin and chylomicrons transport retinyl ester/retinol, our goal was to investigate whether retinoic acid (RA) could be a potential hormonal mediator of the effect. Inhibitors of protein synthesis and protein secretion eliminated the stimulatory effects of chylomicrons on both C3 and ASP production in human differentiated adipocytes, suggesting that de novo protein synthesis and secretion are both required. Incubation with chylomicrons increased C3 mRNA levels (37+/-1.5%). RA alone or with chylomicrons had a stimulatory effect on C3 production (29-fold at 16.6 nM RA) and ASP production. An RA receptor antagonist blocked stimulation of C3 mRNA and C3 secretion by both RA and chylomicrons. Finally, RA and chylomicrons activated a 1.8 kb C3-promoter-luciferase construct transfected into 3T3-F442 and 3T3-L1 cells (by 41+/-0.2% and 69+/-0.3% respectively), possibly via RA receptor half-sites identified by sequence analysis. This is the first evidence documenting stimulation by RA of the C3 gene. Thus we propose RA as a novel cellular trigger in chylomicrons that subsequently results in increased ASP production by adipocytes after a meal.

Enhanced triglyceride clearance with intraperitoneal human acylation stimulating protein in C57BL/6 mice.

Acylation stimulating protein (ASP), a novel adipocyte-derived autocrine protein, stimulates triglyceride synthesis and glucose transport in vitro in human and murine adipocytes. In vitro, chylomicrons increase ASP and precursor complement C3 production in adipocytes. Furthermore, in vivo, ASP production from human adipose tissue correlates positively with triglyceride clearance postprandially. The aim of the present study was to determine if intraperitoneally injected ASP accelerated triglyceride clearance in vivo after a fat load in C57Bl/6 mice. ASP increased the triglyceride clearance with a reduction of the triglyceride area under the curve over 6 h (AUC(0-6)) from 102.6 +/- 30.0 to 61.0 +/- 14.5 mg. dl(-1). h(-1) (P < 0.05), especially in the latter postprandial period (AUC(3-6); 56.2 +/- 18.0 vs. 24.9 +/- 8.9 mg. dl(-1). h(-1), P < 0.025). ASP also reduced plasma glucose both in the mice with accelerated plasma triglyceride clearance and in those with relatively delayed triglyceride clearance (P < 0.025). Therefore, ASP alters postprandial triglyceride and glucose metabolism.

Chylomicron-specific Enhancement of Acylation Stimulating Protein and Precursor Protein C3 Production in Differentiated Human Adipocytes

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 retinol-binding 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.

Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period

The objective of this study was to determine whether Acylation Stimulating Protein (ASP) is generated in vivo by human adipose tissue during the postprandial period. After a fat meal, samples from 12 subjects were obtained (up to 6 h) from an arterialized hand vein and an anterior abdominal wall vein that drains adipose tissue. Veno-arterial (V-A) gradients across the subcutaneous adipose tissue bed were calculated. The data demonstrate that ASP is produced in vivo (positive V-A gradient) with maximal production at 3–5 h postprandially. The plasma triacylglycerol (TAG) clearance was evidenced by a negative V-A gradient. It increased substantially after 3 h and remained prominant until the final time point. There was, therefore, a close temporal coordination between ASP generation and TAG clearance. In contrast, plasma insulin and non-esterified fatty acid (NEFA) had an early (1–2 h) postprandial change. Fatty acid incorporation into adipose tissue (FIAT) was calculated from V-A glycerol and non-esterified fatty acid (NEFA) differences postprandially. FIAT was negative during the first hour, implying net fat mobilization. FIAT then became increasingly positive, implying net fat deposition, and overall followed the same time course as ASP and TAG clearance. There was a direct positive correlation between total ASP production and total FIAT (r = 0.566, P < 0.05). These data demonstrate that ASP is generated in vivo by human adipocytes and that this process is accentuated postprandially, supporting the concept that ASP plays an important role in clearance of TAG from plasma and fatty acid storage in adipose tissue.—Saleh, J., L. K. M. Summers, K. Cianflone, B. A. Fielding, A. D. Sniderman, and K. N. Frayn. Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period. J. Lipid Res. 1998. 39: 884–891.

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.

Acute in vitro production of acylation stimulating protein in differentiated human adipocytes.

We have previously shown that in normolipidemic healthy adults, plasma acylation stimulating protein (ASP) increases postprandially and is produced in vitro by cultured differentiated human adipocytes. The present studies were undertaken to examine the influence of specific plasma components on endogenous ASP production in cultured human adipocytes. The results demonstrate that neither glucose nor fatty acids (over a wide range of concentrations) had any substantial effect on ASP production. Insulin increased ASP production up to 2-fold (208% +/- 18%, P < 0.01). However, the most profound increase in ASP was generated by the addition of chylomicrons to the cell culture medium. Chylomicrons (CHYLO) obtained from postprandial plasma increased ASP production in a time- and concentration-dependent manner, producing up to a 150-fold increase in ASP at the highest concentration of CHYLO tested (500 microg triacylglycerol/mL medium (P < 0.001)). By contrast, very low (VLDL), high (HDL), and low density lipoproteins (LDI) had only marginal effects. The effects on ASP parallelled the changes in adipocyte C3 secretion (the precursor protein of ASP). As with ASP, glucose, oleate, insulin, and hepatic lipoproteins (VLDL, LDL, and HDL) had little or no effect on C3 secretion. In contrast, CHYLO had an even greater effect on C3 secretion than on ASP generation. Finally, the effects of CHYLO on generation of ASP and C3 were not dependent on lipolysis of CHYLO by lipoprotein lipase (LPL). These results are consistent with the changes in plasma ASP seen postprandially, and suggests a role of ASP as a positive feedback regulator of triacylglycerol synthesis in adipose tissue.

Differentiation‐induced production of ASP in human adipocytes

Acylation Stimulating Protein (ASP) is a human plasma protein that stimulates both triacylglycerol synthesis and glucose transport. ASP is identical to C3adesArg and is generated by the interaction of factor B, complement C3 and adipsin. We have demonstrated that mature fat cells express messages for factors B, complement C3 and adipsin; that human pre-adipocytes, when cultured under differentiating conditions to produce adipocytes, generate ASP in the culture medium; and that human adipocytes also become more responsive to ASP as they differentiate. The aim of this study, therefore, was to examine the temporal production of ASP during adipocyte differentiation in relation to other adipose specific factors involved in lipogenesis. The results demonstrate that (i) there was little ASP production by differentiating adipocytes over the first 7 days, with a marked increase in ASP thereafter (up to sixfold); (ii) this increase was paralleled by large increases in the message level of factor B and complement C3 and moderate increases in adipsin message; (iii) increases in lipoprotein lipase (LPL) message and glycerol-3-phosphate dehydrogenase (GPDH) activity (both key enzymes for substrate supply for triacylglycerol synthesis) occurred earlier than the increase in ASP; and (iv) in spite of the increase in LPL and GPDH, triacylglycerol synthetic capacity only markedly increases following the increase in ASP production in adipocytes. Although the present study cannot be interpreted as showing causality with respect to triacylglycerol synthesis, it does point to an important role for ASP in human adipose tissue physiology.