Effects of lipoic acid on lipolysis in 3T3-L1 adipocytes

Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser⁵⁶³ and at Ser⁶⁶⁰, which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E₂ released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E₂.     

Endothelin-1 induces lipolysis in 3T3-L1 adipocytes

Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release in the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase that was time and dose dependent. With a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h of exposure. This effect was inhibited by the ETA receptor antagonist BQ-610 (10 microM) but not by the ETB receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI 3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ-22536 (100 microM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor dexamethasone (100 nM), the PKC inhibitor H-7 (6 microM), or the PI 3-kinase inhibitor wortmannin (100 nM) also had no effect. ET-1-induced lipolysis was blocked by inhibition of extracellular signal-regulated kinase (ERK) activation using PD-98059 (75 microM), whereas a p38 MAPK inhibitor (SB-203580; 20 microM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and that involves ERK activation.     

Activin B inhibits lipolysis in 3T3-L1 adipocytes

Activin B, consisting of two inhibin βB (INHBB) subunits, is a hormone known to affect gonadal function, reproduction and fetal development. We have reported that INHBB and activin B receptors are highly expressed in adipocytes suggesting that activin B may have local effects in adipose tissue. In this study, we investigate the effect of activin B on lipolysis, measured as release of non-esterified fatty acids and free glycerol. Recombinant activin B decreased lipolysis in a concentration-dependent manner and increased intracellular triglyceride content in 3T3-L1 adipocytes. siRNA-mediated knock-down of INHBB expression increased lipolysis, and this effect was abolished by addition of recombinant activin B. In line with its inhibitory effect on lipolysis, activin B caused a down regulation of the expression of adipose triglyceride lipase and hormone sensitive lipase, key genes involved in lipolysis. In summary, we suggest that activin B is a novel adipokine that inhibits lipolysis in a paracrine or autocrine manner.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

Irisin regulates thermogenesis and lipolysis in 3T3-L1 adipocytes

BackgroundAdipose tissue plays a pivotal role in the development and progression of the metabolic syndrome which along with its complications is an epidemic of the 21st century. Irisin is an adipo-myokine secreted mainly by skeletal muscle and targeting, among others, adipose tissue. In brown adipose tissue it upregulates uncoupling protein-1 (UCP1) which is responsible for mitochondrial non-shivering thermogenesis.MethodsHere we analyzed the effects of irisin on the metabolic activity of 3T3-L1 derived adipocytes through a mitochondrial flux assay. We also assessed the effects of irisin on the intracellular signaling through Western Blot. Finally, the gene expression of ucp1 and lipolytic genes was examined through RT-qPCR.ResultsIrisin affects mitochondrial respiration and lipolysis in a time-dependent manner through the regulation of PI3K-AKT pathway. Irisin also induces the expression of UCP1 and the regulation of NF-κB, and CREB and ERK pathways.ConclusionOur data supports the role of irisin in the induction of non-shivering thermogenesis, the regulation of energy expenditure and lipolysis in adipocytes.General significanceIrisin may be an attractive therapeutic target in the treatment of obesity and related metabolic disorders.     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

Intracellular fatty acid downregulates ob gene expression in 3T3-L1 adipocytes

The effect of intracellular free fatty acid (FFA) accumulation on ob gene expression in adipocytes was examined. In fully differentiated 3T3-L1 adipocytes, triacsin C, a specific acyl CoA synthetase inhibitor with a K(i) of 8.97 microM, inhibited ob gene expression by 20% at 5 x 10(-5)M. At this concentration, triacsin C induced accumulation of intracellular FFA. Treatment with both chylomicron and triacsin C reduced ob gene expression more than treatment with triacsin C alone. Treatment with 2-bromopalmitate, a poorly metabolizable palmitate analog, reduced ob gene expression by 50% at 10(-4)M, but palmitate at the same concentration had no effect. This is the first demonstration that the ob gene is downregulated by intracellular FFA accumulation, thereby raising the possibility that ob product is regulated in response to lipolysis.     

Differential inhibition of lipolysis by 2-bromopalmitic acid and 4-bromocrotonic acid in 3T3-L1 adipocytes

Two inhibitors of fatty acid oxidation, 2-bromopalmitic acid (Br-C16) and 4-bromocrotonic acid (Br-C4) were examined for their effect on lipolysis in 3T3-L1 adipocytes. Both agents inhibited in a dose-dependent manner the rate of oxidation of exogenously added [1-¹⁴C]palmitate with similar time-courses, reaching a plateau at 3–9 h. While Br-C16 at 50 μM and 100 μM inhibited palmitate oxidation by approximately 40% and 60%, respectively, pretreatment with both concentrations inhibited lipolysis in washed cells in an almost identical manner. The magnitude of inhibition increased with time of pretreatment. On the other hand, like inhibition of fatty acid oxidation, inhibition of lipolysis by Br-C4 pretreatment was dose-dependent with maximal inhibition reached after 3 h pretreatment. The finding that isoproterenol- and dibutyryl cAMP-stimulated lipolysis were similarly suppressed by either Br-C4 or Br-C16 pretreatment, suggesting that a step distal to cAMP formation was involved. In addition, while the inhibitory effect of Br-C16 was not significantly influenced, the inhibition of lipolysis caused by Br-C4 was attenuated by pretreating cells with crotonic acid, octanoate, or palmitate. The longer chain-length of the fatty acids the cells were exposed, the stronger attenuation of the inhibition caused by Br-C4 was observed. Moreover, whereas pretreatment with Br-C16 was without effect, pretreatment with Br-C4 significantly decreased hormone-sensitive lipase (HSL) activity in cell extracts, albeit to an extent much smaller than its inhibitory effect on lipolysis. In conclusion, these results indicate that irreversible inhibition of lipolysis by Br-C16 or Br-C4 cannot be attributed to their effect on fatty acid oxidation. Some factor capable of modulating HSL activity seems to be involved.     

Neuropeptide Y potentiates beta-adrenergic stimulation of lipolysis in 3T3-L1 adipocytes

Recently, we have shown that neuropeptide Y (NPY) is produced and upregulated in visceral adipose tissue of an early-life programmed rat model of central obesity. Moreover, we have demonstrated that NPY promotes proliferation of adipocyte precursor cells and contributes to the pathogenesis of obesity. However, the role of NPY in regulating adipocyte metabolism is poorly understood. The present study was designed to examine the effects of NPY on adipocyte metabolic function using 3T3-L1 adipocytes as an in vitro cell model system. We found that although it did not affect basal lipolysis, NPY potentiated isoproterenol (a β-adrenergic receptor agonist) stimulated lipolysis. Furthermore, this potentiation occurred upstream of adenylyl cyclase, since NPY did not enhance forskolin (an activator of adenylyl cyclase) stimulated lipolysis. In addition, NPY also augmented isoproterenol-stimulated phosphorylation of hormone sensitive lipase. In contrast, NPY did not alter the expression of several key lipolytic and lipogenic enzymes/proteins. Taken together, our results revealed a novel cross talk between the NPY and β-adrenergic signaling pathways in regulating lipolysis. Thus, the present findings add a new dimension to the dynamic role NPY plays in regulating energy balance.     

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.     

CRISPR/Cas9敲除PLIN1基因增强3T3-L1脂肪细胞的脂解作用

应用CRISPR/Cas9技术敲除3T3-L1前脂肪细胞plin1,观察PLIN1缺失对脂肪细胞中脂肪水解的影响并探究可能机制。常规培养3T3-L1前脂肪细胞,电穿孔法转染plin1敲除载体,嘌呤霉素培养基挑选plin1敲除细胞,观察转染及筛选后的细胞存活率。"鸡尾酒"法诱导3T3-L1前脂肪细胞分化,酶法测定甘油和TG含量,油红O染色观察脂滴形态及数目的变化。Western blotting检测PLIN1、PPARγ、Fsp27和脂肪酶的蛋白表达;RT-PCR检测PLIN1和脂肪酶的mRNA表达。对照组细胞诱导分化后,微小脂滴数目较少,单房脂滴数目较多并围绕细胞核呈环型排列。相较于对照组,敲除组细胞诱导分化后微小脂滴数目增加,单房脂滴体积缩小,数目减少;细胞中PLIN1mRNA及蛋白表达被显著抑制(P0.05);甘油水平显著上升(0.0984±0.0076),TG含量显著下降(0.031 0±0.005 3);HSL和ATGL两种脂肪酶的mRNA及蛋白表达均升高(P0.05);PPARγ和Fsp27的表达未有明显变化。上述结果表明plin1敲除后通过暴露脂滴中脂质以及上调脂肪酶等效应增强了3T3-L1脂肪细胞的脂解作用。     

Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis In 3T3-L1 adipocytes

AMP-activated protein kinase (AMPK) is a phylogenetically conserved intracellular energy sensor that has been implicated as a major regulator of glucose and lipid metabolism in mammals. However, its possible role in mediating or influencing the adrenergic control of lipolysis in adipocytes remains uncertain. In this study, we utilized the murine cultured preadipocyte line 3T3-L1 to examine this question. Treatment of adipocytes with isoproterenol or forskolin promoted the phosphorylation of AMPK at a critical activating Thr-172 residue in a dose- and time-dependent manner. This correlated well with a stimulation of the activity of AMPK, as measured in the immune complex. Analogs of cAMP mimicked the effect of isoproterenol and forskolin on AMPK phosphorylation. Treatment of adipocytes with insulin reduced both basal and forskolin-induced AMPK phosphorylation via a pathway dependent on phosphatidylinositol 3'-kinase. Overexpression of a dominant-inhibitory mutant of AMPK blocked isoproterenol-induced lipolysis by approximately 50%. These data indicate that there exists a novel pathway by which cAMP can lead to the activation of AMPK, and in adipocytes, this is required for maximal activation of lipolysis.     

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.     

Lipoic acid inhibits adiponectin production in 3T3-L1 adipocytes

Lipoic acid (LA) is a naturally occurring compound with antioxidant properties. Recent attention has been focused on the potential beneficial effects of LA on obesity and related metabolic disorders. Dietary supplementation with LA prevents insulin resistance and upregulates adiponectin, an insulin-sensitizing adipokine, in obese rodents. The aim of this study was to investigate the direct effects of LA on adiponectin production in cultured adipocytes, as well as the potential signaling pathways involved. For this purpose, fully differentiated 3T3-L1 adipocytes were treated with LA (1–500 μM) during 24 h. The amount of adiponectin secreted to media was detected by ELISA, while adiponectin mRNA expression was determined by RT-PCR. Treatment with LA induced a dose-dependent inhibition on adiponectin gene expression and protein secretion. Pretreatment with the PI3K inhibitor LY294002 inhibited adiponectin secretion and mRNA levels, and significantly potentiated the inhibitory effect of LA on adiponectin secretion. The AMPK activator AICAR also reduced adiponectin production, but surprisingly, it was able to reverse the LA-induced inhibition of adiponectin. The JNK inhibitor SP600125 and the MAPK inhibitor PD98059 did not modify the inhibitory effect of LA on adiponectin. In conclusion, our results revealed that LA reduces adiponectin secretion in 3T3-L1 adipocytes, which contrasts with the stimulation of adiponectin described after in vivo supplementation with LA, suggesting that an indirect mechanism or some in vivo metabolic processing is involved.     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H₂O₂-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.     

Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes

Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of ³[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.     

Fas activates lipolysis in a Ca2+-CaMKII-dependent manner in 3T3-L1 adipocytes

Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca²⁺/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca²⁺ chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes.     

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.