Targeting the signaling pathway of acylation stimulating protein
2005; Elsevier BV; Volume: 47; Issue: 3 Linguagem: Inglês
10.1194/jlr.m500500-jlr200
ISSN1539-7262
AutoresMagdalena Maslowska, Helen Legakis, Farzad Assadi, Katherine Cianflone,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoAcylation 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. 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. Obesity is one of the most common health problems of our society (1Rossner S. Obesity: the disease of the twenty-first century.Int. J. Obes. Relat. Metab. Disord. 2002; 26: 2-4Google Scholar), and our biggest challenge is understanding how to limit its progress. Adipose tissue provides a long-term storage reservoir for energy surplus in the form of triglycerides (lipogenesis), which in turn can be mobilized (lipolysis) when necessary to provide energy for essential cellular processes (2Large V. Peroni O. Letexier D. Ray H. Beylot M. Metabolism of lipids in human white adipocyte.Diabetes Metab. 2004; 30: 294-309Google Scholar). Normally, the balance between lipogenesis and lipolysis is tightly controlled by numerous hormonal components. Predominance of the lipogenic state, whether driven by increases in food intake or lack of exercise, is dependent on the activation of specific intracellular enzymatic pathways. Regrettably, the continuous augmentation of adipose tissue stores leads to obesity, which, in turn, can lead to a number of metabolic perturbations, such as diabetes, coronary artery disease, and hypertension. In addition to being a storage organ, adipose tissue produces a variety of adipokines, some of which are closely involved in adipose tissue metabolism in an autocrine and paracrine manner (for review, see 3Guerre-Millo M. Adipose tissue hormones.J. Endocrinol. Invest. 2002; 25: 855-861Google Scholar). Acylation Stimulating Protein (ASP) is generated by adipose tissue through the interaction of Factor B and adipsin with complement C3 and is identical to C3adesArg (4Baldo A. Sniderman A.D. St-Luce S. Avramoglu R.K. Maslowska M. Hoang B. Monge J.C. Bell A. Mulay S. Cianflone K. The adipsin-acylation stimulating protein system and regulation of intracellular triglyceride synthesis.J. Clin. Invest. 1993; 92: 1543-1547Google Scholar). ASP production, along with its precursor proteins Factor B, adipsin, and C3, is increased during the differentiation of human and mouse adipocytes (5Cianflone K. Maslowska M. Differentiation induced production of ASP in human adipocytes.Eur. J. Clin. Invest. 1995; 25: 817-825Google Scholar, 6Peake P.W. O'Grady S. Pussell B.A. Charlesworth J.A. Detection and quantification of the control proteins of the alternative pathway of complement in 3T3-L1 adipocytes.Eur. J. Clin. Invest. 1997; 27: 922-927Google Scholar), a production that can be further augmented by insulin and dietary chylomicrons (7Maslowska M. Scantlebury T. Germinario R. Cianflone K. Acute in vitro production of ASP in differentiated adipocytes.J. Lipid Res. 1997; 38: 21-31Google Scholar). In vivo production of ASP in the adipose environment has been elegantly demonstrated by studying arterial-venous differences across an adipose tissue bed (8Saleh J. Summers L.K.M. Cianflone K. Fielding B.A. Sniderman A.D. Frayn K.N. 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: 884-891Google Scholar, 9Kalant D. Phelis S. Fielding B.A. Frayn K.N. Cianflone K. Sniderman A.D. Increased postprandial fatty acid trapping in subcutaneous adipose tissue in obese women.J. Lipid Res. 2000; 41: 1963-1968Google Scholar). Local adipose ASP production increased postprandially and correlated with plasma triglyceride (TG) clearance. This correlation of ASP with postprandial TG clearance has been demonstrated across a wide range of fasting ASP levels in men and woman (10Cianflone K. Zakarian R. Couillard C. Delplanque B. Despres J.P. Sniderman A.D. Fasting acylation stimulating protein is predictive of postprandial triglyceride clearance.J. Lipid Res. 2004; 45: 124-131Google Scholar). Moreover, the circulating levels of ASP are increased in obesity, with greater increases observed in women than in men (11Maslowska M. Vu H. Phelis S. Sniderman A.D. Rhode B.M. Blank D. Cianflone K. Plasma acylation stimulating protein, adipsin and lipids in non-obese and obese populations.Eur. J. Clin. Invest. 1999; 29: 679-686Google Scholar). Upon weight loss, ASP levels return to normal (12Cianflone K. Sniderman A.D. Kalant D. Marliss E.B. Gougeon R. Response of plasma ASP to a prolonged fast.Int. J. Obes. 1995; 19: 604-609Google Scholar, 13Sniderman A.D. Cianflone K. Eckel R.H. Levels of Acylation Stimulating Protein in obese women before and after moderate weight loss.Int. J. Obes. 1991; 15: 333-336Google Scholar, 14Faraj M. Havel P.J. Phelis S. Blank D. Sniderman A.D. Cianflone K. Plasma acylation-stimulating protein, adiponectin, leptin, and ghrelin before and after weight loss induced by gastric bypass surgery in morbidly obese subjects.J. Clin. Endocrinol. Metab. 2003; 88: 1594-1602Google Scholar). Studies have shown that ASP levels are also significantly higher in diabetes and cardiovascular disease (15Cianflone K. Xia Z. Chen L.Y. Critical review of Acylation Stimulating Protein physiology in humans and rodents.Biochim. Biophys. Acta. 2003; 1609: 127-143Google Scholar). ASP plays a key role in the regulation of lipid storage in that it stimulates the esterification of fatty acids onto a glycerol backbone, resulting in the augmentation of intracellular triglyceride depots in human preadipocytes, adipocytes, and skin fibroblasts (16Cianflone K. Maslowska M. Sniderman A.D. Acylation stimulating protein (ASP), an adipocyte autocrine: new directions.Semin. Cell Dev. Biol. 1999; 10: 31-41Google Scholar, 17Cianflone K. The Acylation Stimulating Protein pathway: clinical implications.Clin. Biochem. 1997; 30: 301-312Google Scholar). In stimulating triglyceride synthesis (TGS), ASP increases the activity of diacylglycerol acyltransferase (DGAT; the final enzyme in TGS) in membrane preparations from adipocytes (18Yasruel Z. Cianflone K. Sniderman A.D. Rosenbloom M. Walsh M. Rodriguez M.A. Effect of acylation stimulating protein on the triacylglycerol synthetic pathway of human adipose tissue.Lipids. 1991; 26: 495-499Google Scholar). ASP stimulates glucose transport in both adipocyte and muscle cells (19Maslowska M. Sniderman A.D. Germinario R. Cianflone K. ASP stimulates glucose transport in cultured human adipocytes.Int. J. Obes. Relat. Metab. Disord. 1997; 21: 261-266Google Scholar, 20Tao Y. Cianflone K. Sniderman A.D. Colby-Germinario S.P. Germinario R.J. Acylation-stimulating protein (ASP) regulates glucose transport in the rat L6 muscle cell line.Biochim. Biophys. Acta. 1997; 1344: 221-229Google Scholar) through the translocation of the glucose transporters GLUT1, GLUT4, and GLUT3 (21Germinario R. Sniderman A.D. Manuel S. Pratt S. Baldo A. Cianflone K. Coordinate regulation of triacylglycerol synthesis and glucose transport by Acylation Stimulating Protein.Metabolism. 1993; 42: 574-580Google Scholar). Finally, as with insulin, ASP also inhibits lipolysis (22Van Harmelen V. Reynisdottir S. Cianflone K. Degerman E. Hoffstedt J. Nilsell K. Sniderman A. Arner P. Mechanisms involved in the regulation of free fatty acid release from isolated human fat cells by acylation-stimulating protein and insulin.J. Biol. Chem. 1999; 274: 18243-18251Google Scholar), yet the effects of ASP and insulin are additive. Recently, C5L2, an orphan receptor, was identified as an ASP receptor (23Kalant D. Cain S.A. Maslowska M. Sniderman A.D. Cianflone K. Monk P.N. The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/Acylation-Stimulating Protein.J. Biol. Chem. 2003; 278: 11123-11129Google Scholar, 24Kalant D. Maclaren R. Cui W. Samanta R. Monk P.N. Laporte S.A. Cianflone K. C5L2 is a functional receptor for acylation-stimulating protein.J. Biol. Chem. 2005; 280: 23936-23944Google Scholar). C5L2 is a seven transmembrane Gprotein belonging to the C5a, C3a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP) family of receptors. In HEK-293 cells transfected with the receptor, ASP binds C5L2 with high affinity, and cells become responsive to ASP (but not insulin) for TGS and glucose transport (24Kalant D. Maclaren R. Cui W. Samanta R. Monk P.N. Laporte S.A. Cianflone K. C5L2 is a functional receptor for acylation-stimulating protein.J. Biol. Chem. 2005; 280: 23936-23944Google Scholar). However, how ASP interacts with the receptor to generate a signal is not well understood. Our initial study on ASP signaling demonstrated the involvement of protein kinase C (PKC) in ASP-stimulated TGS (25Baldo A. Sniderman A.D. St. Luce S. Zhang X.J. Cianflone K. Signal transduction pathway of acylation stimulating protein: involvement of protein kinase C.J. Lipid Res. 1995; 36: 1415-1426Google Scholar). Even though numerous studies have reported on the postreceptor signaling targets of the insulin pathway regulating glucose transport and lipolysis, the main components of the signaling cascade(s) resulting in the stimulation of TGS are unknown. The aim of this study was to identify the signaling pathway for ASP stimulation of TGS compared with insulin. Using murine 3T3-L1 preadipocytes as a cell model, we evaluated the involvement of the phospholipase C (PLC), PLD, PLA2, phosphatidylinositol 3-kinase (PI3K), Akt, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2) pathways. 3T3-L1 preadipocytes were obtained from the American Type Culture Collection (Manassas, VA). All tissue culture reagents, such as DMEM/F12, PBS, FBS, and trypsin, were from Gibco (Burlington, Ontario, Canada). Inhibitors used were pertussis toxin (PTX), isotetrandrine, PD098059, wortmannin, rapamycin, bisindolylmaleimide V, Akt inhibitor, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), arachidonyltrifluoromethyl ketone (AACOCF3; Calbiochem, La Jolla, CA), LY294002 (Promega, Madison, WI), and U73122 (Sigma, Oakville, Ontario, Canada). Stimulators of TGS, insulin, and phorbol 13-myristate 12-acetate (PMA), were from Sigma. Oleic acid[9,10-3H(N)] was from DuPont-New England Nuclear (Mississauga, Ontario, Canada). Thin-layer chromatography plates (silica gel 150A) came from Fisher (Nepean, Ontario, Canada). Organic solvents, scintillation vials, general chemicals, and tissue culture materials were from VWR (Montreal, Quebec, Canada). Scintillation fluid was from ICN (Costa Mesa, CA). Bio-Rad reagent for protein measurements was from Bio-Rad (Mississauga, Ontario, Canada). ASP was isolated and purified from human plasma as described previously (4Baldo A. Sniderman A.D. St-Luce S. Avramoglu R.K. Maslowska M. Hoang B. Monge J.C. Bell A. Mulay S. Cianflone K. The adipsin-acylation stimulating protein system and regulation of intracellular triglyceride synthesis.J. Clin. Invest. 1993; 92: 1543-1547Google Scholar). Each batch was verified for purity by ion-spray mass spectrometry at the McGill University Mass Spectrometry Unit. The activity of each ASP preparation was checked by its ability to stimulate TGS in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes, maintained at low passage number, were grown in DMEM/F12 supplemented with 10% FBS. TGS was evaluated as described in detail previously (16Cianflone K. Maslowska M. Sniderman A.D. Acylation stimulating protein (ASP), an adipocyte autocrine: new directions.Semin. Cell Dev. Biol. 1999; 10: 31-41Google Scholar, 17Cianflone K. The Acylation Stimulating Protein pathway: clinical implications.Clin. Biochem. 1997; 30: 301-312Google Scholar). At 80% confluence, the cells were plated at 7,000 cells/well on 24-well plates for experiments. On the 4th day after plating (at 100% cell confluence), preadipocytes were switched to serum-free medium (DMEM/F12) for 2 h followed by incubation with various inhibitors for 30 min (U73122, n-butanol, wortmannin, LY294002, rapamycin, bisindolylmaleimide V, PD98059, isotetrandrine, AACOCF3, or BAPTA-AM) or 4 h (PTX). Subsequently, the medium was changed to fresh serum-free DMEM/F12 supplemented with 100 μM [3H]oleate/BSA (5:1 molar ratio; average specific activity, 65 dpm/pmol), appropriate inhibitors, and stimulators (ASP, insulin, or PMA). Inhibitors were reconstituted and stored (working solution) according to the manufacturer's instructions and were added at appropriate concentrations from freshly prepared working solutions diluted in PBS. TGS was measured over 4 h at 37°C in the presence of inhibitors and hormones. Appropriate vehicle controls were used in each experiment. After the incubation period, radioactive medium was removed and the cells were washed two times in ice-cold PBS. The lipids were extracted for 1 h in 1 ml of heptane-isopropanol (3:2, v/v) and then rinsed with an additional 1 ml of the same solvent mix. Lipid extracts were evaporated to dryness in a centrifuge-evaporator (Canberra-Packard Canada) and redissolved in 100 μl of chloroform-methanol (2:1, v/v), and lipids were resolved by TLC in hexane-ethyl ether-acetic acid (75:25:1, v/v/v) with reference lipids run concurrently. Separated lipids were visualized with iodine vapor, and the spots corresponding to triglyceride were scraped into scintillation vials and counted by liquid scintillation counting (Beckman). Cell proteins were solubilized in 0.1 N NaOH for 3 h and measured by the method of Bradford (26Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Google Scholar). Note that, because TGS and lipolysis are ongoing, these experiments measure net TGS. Medium oleate depletion during the 4 h did not exceed 6%. Average basal TGS was 30–50 pmol/μg cell protein, which constitutes 50–70% of the radioactive lipids, with the remainder primarily in phospholipids. Monoacylglycerol, diacylglycerol (DAG), and free fatty acids constitute <5%. On average, TGS stimulation with ASP was 168.65 ± 24.17% (range, 132.7 ± 8.5% to 209.4 ± 17.0%), that with insulin was 233.71 ± 50.81% (range, 185.9 ± 15.1% to 308.4 ± 54.8%), and that with PMA was 167.23 ± 29.4% (range, 139.6 ± 10.7% to 198.2 ± 17.2%), where basal was always set as 100%. Cells were grown to confluence on 60 mm dishes, preincubated in serum-free medium for 2 h, and then stimulated with ASP or insulin in fresh serum-free medium for 0, 5, 15, 30, 45, and 60 min. The medium was quickly removed, and 500 μl of ice-cold lysing buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 1 mM PMSF, 200 μM orthovanadate, 20 mM β-glycerophosphate, 100 μM 4-(2-aminoethyl) benzenesulphonyl fluoride (irreversible serine protease inhibitor), 150 nM aprotinin, 1 μM E-64, and 1 μM leupeptin, pH 7.5) was added to the plates for 10 min at 4°C with gentle shaking. Total cell lysates were collected and centrifuged to remove particulate material (14,000 g for 10 min at 4°C). Aliquots of the supernatant were stored at −80°C for further analysis. Proteins were measured with the Bio-Rad assay. For Western blot analysis, Laemmli sample buffer was added to the aliquots of cell lysates and the samples were boiled for 3 min. Twenty-five microliters of cell lysate was loaded per lane, and the proteins were resolved by 10% SDS-PAGE. Gels were then transferred to a polyvinylidene difluoride membrane and were immunoblotted with the appropriate antibody to phosphorylated forms of intracellular proteins and reblotted with the antibodies directed to the nonphosphorylated proteins in question. The immobilized proteins were detected with the ECL Plus kit from Amersham Biosciences (Piscataway, NJ) using Kodak film. The results were normalized to basal TGS in each experiment (set as 100%) and are presented as means ± SEM. Differences were analyzed by two-way ANOVA for drug treatment and hormone treatment with the Bonferoni posthoc test, with P < 0.05 considered significant. Western immunoblots were quantified with the ChemiImager Ready System Alpha DigiDoc Imaging system (San Leonardo, CA). PTX was used to evaluate the involvement of the Gαi subunit in ASP action. Confluent 3T3-L1 preadipocytes were pretreated for 4 h with 100 ng/ml PTX before stimulation with ASP or insulin. As shown in Fig. 1A, PTX did not affect basal TGS (which is set as 100%). ASP stimulation of TGS is unaltered by PTX treatment (181.9 ± 14.6% for ASP alone vs. 216.3 ± 10.7% for ASP + PTX, where basal is set at 100%; P = NS). Insulin also stimulates TGS in 3T3-L1 preadipocytes (27Cammalleri C. Germinario R.J. The effects of protease inhibitors on basal and insulin-stimulated lipid metabolism, insulin binding, and signaling.J. Lipid Res. 2003; 44: 103-108Google Scholar) through the insulin receptor, which belongs to the family of tyrosine kinase receptors. As expected, PTX had no inhibitory effect on the TGS stimulatory action of insulin (302.1 ± 2.1% for insulin alone vs. 346.1 ± 10.6% for insulin + PTX; P = NS). Our data demonstrating the rapid biphasic increases in intracellular DAG and PKC translocation by ASP (25Baldo A. Sniderman A.D. St. Luce S. Zhang X.J. Cianflone K. Signal transduction pathway of acylation stimulating protein: involvement of protein kinase C.J. Lipid Res. 1995; 36: 1415-1426Google Scholar) are suggestive of PLC and/or PLD involvement in ASP signaling (28Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.Science. 1992; 258: 607-614Google Scholar). This was tested using specific inhibitors and Ca2+ chelators. The effect of U73122, a potent and widely used inhibitor of PLC (29Yule D.I. Williams J.A. U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.J. Biol. Chem. 1992; 267: 13830-13835Google Scholar), was tested. As shown in Fig. 1B, U73122 resulted in a slight but significant decrease in basal TGS at the highest concentration only (35.2% inhibition; P < 0.001). The ASP stimulatory effect was gradually blocked in a concentration-dependent manner, leading to a complete loss of ASP stimulatory activity at the highest concentration tested (P < 0.001 at 20 μM U73122). On the other hand, insulin-stimulated TGS (308.4 ± 51.5%; P < 0.0001) was inhibited by only 7.1% at 20 μM U73122 (data not shown). These data suggest that although both ASP and insulin can stimulate TGS, ASP, in contrast to insulin, mediates its effects via PLC. The Ca2+-chelating agent BAPTA-AM is a cell-permeable molecule that becomes hydrolyzed and trapped inside the cell as an active chelator, thus chelating only intracellular Ca2+ concentration ([Ca2+]). As shown in Fig. 1C, basal TGS decreased slightly but significantly with increasing BAPTA concentrations (32.8% inhibition at 25 μM BAPTA-AM; P < 0.001). ASP-stimulated TGS (184.1 ± 23%; P < 0.001) was inhibited significantly by 84.1% and 93.1% (5 and 25 μM, respectively; P < 0.001). Similarly, insulin stimulated TGS by 203.8 ± 9.6%, an effect that was attenuated by 56.6% and 62.8% with 5 and 25 μM BAPTA-AM, respectively (P < 0.001 compared with insulin alone). The involvement of PLD in ASP signaling was evaluated using n-butanol, a primary alcohol, which serves as an artificial substrate for PLD, generating phosphatidylalcohol instead of phosphatidic acid (30Liscovitch M. Czarny M. Fiucci G. Tang X. Phospholipase D: molecular and cell biology of a novel gene family.Biochem. J. 2000; 345: 401-415Google Scholar). As the generated phosphatidylalcohol can no longer be converted to DAG by the enzyme phosphatidic acid phosphatase, n-butanol effectively prevents PLD-generated second messengers. In the absence of n-butanol, ASP-stimulated TGS was 265.1 ± 11.1% (where basal was set as 100%; P < 0.0001); however, the ASP effect was not inhibited at any n-butanol concentrations tested (Fig. 1D). The involvement of PI3K was analyzed using the two inhibitors wortmannin (cell-permeable, irreversible) and LY294002 (reversible). Both compounds inhibit the catalytic site of PI3K and have been used extensively to demonstrate the role of PI3K in insulin action on glucose transport (31Carlsen J. Christiansen K. Grunnet N. Vinten J. Involvement of PI 3-kinase and activated ERK in facilitating insulin-stimulated triacylglycerol synthesis in hepatocytes.Cell. Signal. 1999; 11: 713-717Google Scholar, 32Clarke J.F. Young P.W. Yonezawa K. Kasuga M. Holman G.D. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.Biochem. J. 1994; 300: 631-635Google Scholar). As shown in Fig. 2A, wortmannin alone had no effect on basal TGS. The ASP stimulatory effect (258.3 ± 24.7% for ASP alone; P < 0.0001) was inhibited by 70% (down to 148.1 ± 6.7% for ASP + 100 nM wortmannin; P < 0.0001). Inhibition was evident at all concentrations tested. Wortmannin also inhibited up to 80% of the insulin-stimulated TGS (360.8 ± 20.1% for insulin alone vs. 156.1 ± 6.4% for insulin + 100 nM wortmannin; P < 0.0001). Similarly, with LY294002 (Fig. 2B), ASP-stimulated TGS (187.4 ± 24.1%; P < 0.001) was reduced in a concentration-dependent manner down to basal TGS levels at 25 μM LY294002 (P < 0.01 vs. ASP alone). A similar effect was observed on insulin-stimulated TGS, although complete inhibition was already observed with 10 μM LY294002 (P < 0.001). A slight inhibition of basal TGS with increasing concentrations of LY294002 was negligible compared with those for ASP- and insulin-stimulated TGS. Once activated, PI3K can stimulate the activation of 3-phosphoinositide-dependent protein kinase-1 (PDK-1) (which activates Akt), PKC, Ras, and others. Western blot analysis showed that PDK-1 phosphorylation on serine 241 (Ser241) from 0 to 60 min remains constant after ASP or insulin stimulation (data not shown). On the other hand, Akt, the immediate target of PDK-1, is rapidly phosphorylated on Ser473, an event necessary for activation. As shown in Fig. 2D, phosphorylation of Akt on Ser473 (Akt-P) by ASP is rapid, reaching its maximum between 5 and 10 min and diminishing to basal levels by 45 min. Insulin activation of Akt phosphorylation was as rapid (5 min), but Akt remained phosphorylated over the entire time course, with a slight decrease at 60 min (Fig. 2E). Furthermore, treatment of cells with the Akt inhibitor 1l-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O]octadecylcarbonate (Fig. 2C) resulted in the complete elimination of ASP stimulation in a concentration-dependent manner (P < 0.0001). Basal TGS was only slightly affected at the highest concentration of inhibitor (23.8% inhibition at 10 μM; P < 0.05). We have shown previously that PKC is implicated in ASP signaling and that phorbol 12-myristate 13-acetate (PMA), a known PKC activator, also stimulates TGS (25Baldo A. Sniderman A.D. St. Luce S. Zhang X.J. Cianflone K. Signal transduction pathway of acylation stimulating protein: involvement of protein kinase C.J. Lipid Res. 1995; 36: 1415-1426Google Scholar). PKC is regulated through phosphorylation of the newly synthesized PKC by PDK-1 (33Dutil E.M. Toker A. Newton A.C. Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1).Curr. Biol. 1998; 8: 1366-1375Google Scholar) and activation by Ca2+ and lipids. 3T3-L1 preadipocytes were incubated with PMA (to stimulate TGS via PKC activation), and various inhibitors were tested. Although wortmannin and LY294002 inhibited both the ASP and insulin effects (Fig. 2A, B), the TGS stimulatory action of PMA was unaffected by LY294002 (Table 1; P = NS). Furthermore, PMA treatment did not induce Akt phosphorylation (data not shown), suggesting that PKC acts downstream of PI3K as a stimulator of TGS. On the other hand, intracellular [Ca2+] chelation with 25 μM BAPTA-AM decreased PMA stimulation of TGS from 145.5% to 105.3%, an inhibition of 88.5% (Table 1; P < 0.005).TABLE 1.Effect of inhibitors on PMA stimulation of TGSInhibitorConcentrationNo InhibitorInhibitor−PMA+PMAPaFor PMA versus without PMA (no inhibitor present).−PMA+PMAPbFor PMA = inhibitor versus PMA and no inhibitor.LY29400250 μM100 ± 3.6163.9 ± 16.30.00151.9 ± 7.9134.4 ± 20.1NSBAPTA-AM25 μM100 ± 4.5145.5 ± 19.50.00665.4 ± 3.9105.3 ± 13.00.05Rapamycin50 nM100 ± 4.0198.5 ± 17.20.000469.9 ± 4.4203.1 ± 12.3NSBisindolylmaleimide V25 μM100 ± 5.5139.6 ± 10.70.0228.9 ± 4.4111.4 ± 11.4NSPD9805925 μM100 ± 3.5289.3 ± 52.50.00187.3 ± 9.4160.3 ± 32.90.002BAPTA-AM, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; PMA, phorbol 13-myristate 12-acetate; TGS, triglyceride synthesis. Confluent cells were pretreated with serum-free medium containing the indicated inhibitors at the indicated concentrations for 30 min and then stimulated with 20 nM PMA for an additional 4 h. All inhibitors were present throughout the experiment. TGS was measured as picomoles of [3H]oleate incorporated into TG. The results were normalized to basal TGS in each of the experiments performed and are presented as percentage TGS stimulation, where basal (no addition and no treatment) was set as 100%. The appropriate amount of vehicle was included in the medium for each of the inhibitors tested. The data were analyzed using two-way ANOVA followed by the Bonferoni posthoc test.a For PMA versus without PMA (no inhibitor present).b For PMA = inhibitor versus PMA and no inhibitor. Open table in a new tab BAPTA-AM, 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; PMA, phorbol 13-myristate 12-acetate; TGS, triglyceride synthesis. Confluent cells were pretreated with serum-free medium containing the indicated inhibitors at the indicated concentrations for 30 min and then stimulated with 20 nM PMA for an additional 4 h. All inhibitors were present throughout the experiment. TGS was measured as picomoles of [3H]oleate incorporated into TG. The results were normalized to basal TGS in each of the experiments performed and are presented as percentage TGS stimulation, where basal (no addition and no treatment) was set as 100%. The appropriate amount of vehicle was included in the medium for each of the inhibitors tested. The data were analyzed using two-way ANOVA followed by the Bonferoni posthoc test. The mamalian target of rapaycin (mTOR) pathway, which involves the activation of p70 S6 kinase and 4E binding protein (gene transcription/RNA translation pathway), is a well-characterized downstream target of PI3K through Akt. We evaluated the mTOR pathway using two specific inhibitors, rapamycin (an inhibitor of mTOR) and bisindolylmaleimide V (a p70 S6 kinase inhibitor). Ra
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