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• W2021492010 abstract "An abundant supply of extracellular nutrients is believed to be sufficient to suppress catabolism of cellular macromolecules. Here we show that, despite abundant extracellular nutrients, interleukin-3-deprived hematopoietic cells begin to catabolize intracellular lipids. Constitutive Akt activation blunts the increased β-oxidation that accompanies growth factor withdrawal, and in growth factor-replete cells, phosphatidylinositol 3-kinase (PI3K) signaling is required to suppress lipid catabolism. Surprisingly, PI3K and Akt exert these effects by suppressing expression of the β-oxidation enzyme carnitine palmitoyltransferase 1A (CPT1A). Cells expressing a short hairpin RNA against CPT1A fail to induce β-oxidation in response to growth factor withdrawal and are unable to survive glucose deprivation. When CPT1A is constitutively expressed, growth factor stimulation fails to repress β-oxidation. As a result, both net lipid synthesis and cell proliferation are diminished. Together, these results demonstrate that modulation of CPT1A expression by PI3K-dependent signaling is the major mechanism by which cells suppress β-oxidation during anabolic growth. An abundant supply of extracellular nutrients is believed to be sufficient to suppress catabolism of cellular macromolecules. Here we show that, despite abundant extracellular nutrients, interleukin-3-deprived hematopoietic cells begin to catabolize intracellular lipids. Constitutive Akt activation blunts the increased β-oxidation that accompanies growth factor withdrawal, and in growth factor-replete cells, phosphatidylinositol 3-kinase (PI3K) signaling is required to suppress lipid catabolism. Surprisingly, PI3K and Akt exert these effects by suppressing expression of the β-oxidation enzyme carnitine palmitoyltransferase 1A (CPT1A). Cells expressing a short hairpin RNA against CPT1A fail to induce β-oxidation in response to growth factor withdrawal and are unable to survive glucose deprivation. When CPT1A is constitutively expressed, growth factor stimulation fails to repress β-oxidation. As a result, both net lipid synthesis and cell proliferation are diminished. Together, these results demonstrate that modulation of CPT1A expression by PI3K-dependent signaling is the major mechanism by which cells suppress β-oxidation during anabolic growth. Single-cell eukaryotes like yeast autonomously regulate their uptake of nutrients from the extracellular environment. In such organisms, cellular metabolism is regulated primarily in response to nutrient availability (1Lohr D. Venkov P. Zlatanova J. FASEB J. 1995; 9: 777-787Crossref PubMed Scopus (331) Google Scholar). In contrast, it has been proposed that mammalian cells do not take up and metabolize nutrients without instruction from extracellular signals. These signals, which include cytokines and other lineage-specific growth factors, stimulate signal transduction pathways that orchestrate cellular metabolism through effects on gene expression and enzyme kinetics. Together, these signal-induced changes function to direct uptake and utilization of nutrients, channeling metabolites into biosynthetic pathways (2Hedeskov C.J. Biochem. J. 1968; 110: 373-380Crossref PubMed Scopus (84) Google Scholar, 4Vander Heiden M.G. Plas D.R. Rathmell J.C. Fox C.J. Harris M.H. Thompson C.B. Mol. Cell. Biol. 2001; 21: 5899-5912Crossref PubMed Scopus (425) Google Scholar). One important example of this phenomenon occurs during lymphocyte stimulation, where receptor-induced signaling directly increases glucose transport, glycolysis, lactate production, and synthesis of lipids (5Cooper E.H. Barkhan P. Hale A.J. Br. J. Haematol. 1963; 9: 101-111Crossref PubMed Scopus (82) Google Scholar, 9Kenny J.J. Martinez-Maza O. Fehniger T. Ashman R.F. J. Immunol. 1979; 122: 1278-1284PubMed Google Scholar). Many of the metabolic changes elicited by growth factors result from activation of the phosphatidylinositol 3-kinase (PI3K)/Akt 4The abbreviations used are: PI3K, phosphatidylinositol 3′ kinase; IL-3, interleukin-3; CPT I, carnitine palmitoyltransferase I; CPT1A, carnitine palmitoyltransferase I, isoform A; myrAkt, myristoylated Akt; IRES, internal ribosomal entry site; GFP, green fluorescent protein; shRNA, short hairpin RNA; qPCR, quantitative reverse-transcriptase-PCR; ACC, acetyl-CoA carboxylase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BSA, bovine serum albumin. 4The abbreviations used are: PI3K, phosphatidylinositol 3′ kinase; IL-3, interleukin-3; CPT I, carnitine palmitoyltransferase I; CPT1A, carnitine palmitoyltransferase I, isoform A; myrAkt, myristoylated Akt; IRES, internal ribosomal entry site; GFP, green fluorescent protein; shRNA, short hairpin RNA; qPCR, quantitative reverse-transcriptase-PCR; ACC, acetyl-CoA carboxylase; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BSA, bovine serum albumin. signaling system. In this pathway, binding of a growth factor to its surface receptor induces activation of the lipid kinase PI3K, which phosphorylates phosphatidylinositol species (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4542) Google Scholar). End products of PI3K activity recruit the serine/threonine kinase Akt to the plasma membrane where it becomes a substrate for phosphorylation and activation by phosphoinositide-dependent protein kinase 1 and other regulatory kinases (10Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4542) Google Scholar). The metabolic effects of growth factor-induced PI3K/Akt stimulation include increases in glucose import and glycolysis, which fuel the bioenergetic and biosynthetic activities of growing cells (11Plas D.R. Talapatra S. Edinger A.L. Rathmell J.C. Thompson C.B. J. Biol. Chem. 2001; 276: 12041-12048Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Cantrell D.A. Curr. Opin. Immunol. 2003; 15: 294-298Crossref PubMed Scopus (22) Google Scholar). Constitutive activation of PI3K and/or Akt through a variety of genetic mechanisms is a common transforming event in human cancer, including some 25% of breast carcinoma and 30% of colon carcinoma (14Luo J. Manning B.D. Cantley L.C. Cancer Cell. 2003; 4: 257-262Abstract Full Text Full Text PDF PubMed Scopus (1128) Google Scholar, 17Samuels Y. Wang Z. Bardelli A. Silliman N. Ptak J. Szabo S. Yan H. Gazdar A. Powell S.M. Riggins G.J. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Velculescu V.E. Science. 2004; 304: 554Crossref PubMed Scopus (2852) Google Scholar). Cancer cells with constitutive Akt activity have a growth factor-independent ability to take up glucose and channel it into biosynthetic pathways, adopting a glucose-driven metabolism long known to characterize rapidly proliferative tumors (18Warburg O. Klin. Wochenschr. Berl. 1925; 4: 534-536Crossref Scopus (104) Google Scholar, 19Plas D.R. Thompson C.B. Oncogene. 2005; 24: 7435-7442Crossref PubMed Scopus (330) Google Scholar). Therefore, Akt activation supports anabolic metabolism in non-transformed cells and contributes to autonomous growth in tumor cells. The net synthesis of lipids, particularly phospholipids for daughter cell membranes, is required for cell proliferation. Inhibition of lipid synthesis is an effective strategy to suppress proliferation of various cell types (20Kuhajda F.P. Jenner K. Wood F.D. Hennigar R.A. Jacobs L.B. Dick J.D. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6379-6383Crossref PubMed Scopus (570) Google Scholar, 21Pizer E.S. Wood F.D. Heine H.S. Romantsev F.E. Pasternack G.R. Kuhajda F.P. Cancer Res. 1996; 56: 1189-1193PubMed Google Scholar). Recent evidence has shown that stimulation of PI3K/Akt increases cellular synthesis of fatty acids from glucose and other precursors (22Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar, 24Van de S e T. De Schrijver E. Heyns W. Verhoeven G. Swinnen J.V. Cancer Res. 2002; 62: 642-646PubMed Google Scholar). Fatty acids are important metabolic intermediates, because they can either be used for lipid synthesis and protein modification (e.g. palmitoylation, myristoylation, and synthesis of glycerophosphatidylinositol anchors), or they can be degraded through mitochondrial β-oxidation, which produces substrates that maintain ATP generation through oxidative phosphorylation. In the liver, the rate of β-oxidation is determined by the malonyl-CoA-dependent allosteric regulation of carnitine palmitoyltransferase I (CPT I), an enzyme located on the outer mitochondrial membrane that esterifies long chain fatty acids to carnitine, thereby initiating mitochondrial import (25McGarry J.D. Mannaerts G.P. Foster D.W. J. Clin. Invest. 1977; 60: 265-270Crossref PubMed Scopus (493) Google Scholar, 28Foster D.W. Ann. N. Y. Acad. Sci. 2004; 1033: 1-16Crossref PubMed Scopus (162) Google Scholar). Such dynamic regulation of lipid synthesis/degradation allows hepatocytes to oscillate between postprandial lipid synthesis and fasting-induced lipid degradation. Whether lipid metabolism is similarly regulated in cells undergoing a growth factor-induced proliferative response has not been investigated. Therefore, we have examined the regulation of β-oxidation during cell growth and survival of hematopoietic cells. In the quiescent state, hematopoietic cells oxidize fatty acids obtained from the extracellular milieu and from intracellular lipids, and these activities allow them to survive glucose withdrawal. When stimulated to proliferate, hematopoietic cells commit to net lipid synthesis by suppressing β-oxidation and concomitantly inducing lipid synthesis. The ability to suppress β-oxidation is required for these cells to achieve maximal rates of proliferation. Surprisingly, this suppression results primarily from modulation of CPT1A expression rather than its enzyme activity. These results identify a novel mechanism used to modulate lipid metabolism in proliferating cells. Cell Culture and Reagents—IL-3 dependent, bax–/– bak–/– cells have been described (29Lum J.J. Bauer D.E. Kong M. Harris M.H. Li C. Lindsten T. Thompson C.B. Cell. 2005; 120: 237-248Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar) and were used here to avoid the confounding variable of apoptotic cell death. In other experiments, IL-3-dependent FL5.12 cells were studied. The IL-3-dependent, bax–/– bak–/– cells were cultured in RPMI 1640 medium containing l-glutamine (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gemini); 3 ng/ml recombinant murine IL-3 (BD Pharmingen); HEPES buffer (9 mm, Invitrogen); β-mercaptoethanol (50 μm, Sigma); penicillin G and streptomycin sulfate (91 units/ml and 91 μg/ml, respectively, Invitrogen). All cultures was performed at 37 °C in 5% CO2. To withdraw cells from IL-3, they were washed three times and resuspended in complete medium lacking IL-3. To restimulate cells, they were pelleted and resuspended in complete medium containing IL-3. Cell size and concentration were determined using a Coulter Z2 particle analyzer or a hemocytometer. The PI3K inhibitor LY294002 (Cell Signaling) was diluted in Me2SO to make a stock, and then diluted further in culture medium. Lipid Metabolism Assays—To measure lipid synthesis, cells were pelleted and resuspended in medium supplemented with [14C]glucose or palmitic acid (Sigma-Aldrich). For Fig. 1A, cells were cultured with 5.8 μCi of d-[6-14C]glucose/106 cells or 0.013 μCi of palmitic acid-[carboxy-14C]/106 cells. Prior to addition to culture medium, palmitate was complexed to a 10% solution of essentially fatty acid free bovine serum albumin (BSA, Sigma) by vortexing for 1 min. For other lipid synthesis assays, cells were cultured with d-[U-14C6]glucose. Cells were plated at an initial density of 0.3–0.5 × 106/ml and incubated for 24 h, then counted using a hemocytometer and harvested in triplicate aliquots. Labeled cells were washed three times in phosphate-buffered saline and lysed in 0.4 ml of Triton X-100 (0.5% in H2O). Lipids were extracted using a modification of the Bligh-Dyer method (30Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar) in which 1 ml of methanol, 2 × 1 ml of chloroform, and 1 ml of H2O were sequentially added, with vortexing. Samples were centrifuged, and the organic phase was transferred to a new tube, evaporated to dryness under a nitrogen stream, and resuspended in 50 μl of chloroform. To quantitate lipid synthesis, resuspended samples were transferred to scintillation vials with 2.5 ml of EcoLume (ICN Radiochemicals) and counted on a 1450 Microbeta Jet or Microbeta Trilux scintillation counter (Wallac). For fractionation of lipid species, samples were spotted onto glass microfiber chromatography paper impregnated with silicic acid (Varian) and separated by chromatography in hexanes:diethyl ether:acetic acid (170:30:1) with size standards. Lipid species were visualized with iodine vapor, cut from the paper, and transferred to scintillation vials for quantitation of radioactivity. In Fig. 1A, “total” lipid was determined by summing the activity from individual species, and bars represent lipid synthesis for the entire culture. β-Oxidation of fatty acids was measured by modifying previously described assays (31Brivet M. Slama A. Saudubray J.M. Legrand A. Lemonnier A. Ann. Clin. Biochem. 1995; 32: 154-159Crossref PubMed Scopus (47) Google Scholar, 32Buzzai M. Bauer D.E. Jones R.G. Deberardinis R.J. Hatzivassiliou G. Elstrom R.L. Thompson C.B. Oncogene. 2005; 24: 4165-4173Crossref PubMed Scopus (298) Google Scholar). Cells were pelleted and resuspended in medium supplemented with [9,10-3H]palmitate complexed to BSA by vortexing a mixture of the palmitate and a 10% BSA solution at a 1:2 volume ratio. A total of 3.3 μl of [9,10-3H]palmitate and 6.7 μl of BSA were used per 1 ml of cell culture medium. Each sample used 0.5 × 106 cells in 1 ml of medium supplemented with the [9,10-3H]palmitate-BSA mixture and cultured for 24 h in 24-well plates. After 24 h, supernatant was applied to ion-exchange columns (Dowex 1X8–200, Sigma), and tritiated water was recovered by eluting with 2.5 ml of H2O. A 0.75-ml aliquot was then used for scintillation counting. For each data point, eight samples were prepared, four cultured without and four with 0.2 mm (+)-etomoxir sodium (Sigma), an inhibitor of mitochondrial long-chain fatty acid oxidation. To determine β-oxidation rate, a ranked pair analysis was used to determine the difference between oxidation counts in the absence and presence of etomoxir. The outlier among the four resulting values was discarded, and average and standard deviations were determined for the three remaining values. To measure β-oxidation of endogenous lipid, 75 × 106 cells were labeled in 150 ml of IL-3-containing medium supplemented with a [9,10-3H]palmitate-BSA mixture. This mixture was prepared by adding 40 μCi of [9,10-3H]palmitate to 1 ml of a20 μm unlabeled palmitate stock in a solution of 10% essentially fatty acid-free BSA. This mixture was added to 150 ml of pre-warmed medium for culture. Cells were cultured for 2 days, and then harvested and washed three times in phosphate-buffered saline. The supernatant from the third wash contained negligible radioactivity. The labeled, washed cells were then cultured at a density of 0.25 × 106 cells/ml in medium either containing or lacking IL-3 and containing or lacking etomoxir (0.2 mm). On subsequent days, aliquots of 0.5 ml were withdrawn from each flask, pelleted to remove cells, and applied to ion-exchange columns as above for determination of radioactivity. When cell density in IL-3-containing cultures exceeded 0.6 × 106/ml, more medium was added to culture, and total dilution was used to correct oxidation counts. DNA Constructs—The murine Akt-1 containing a Src myristoylation sequence fused to the N terminus (myrAkt) was described elsewhere (33Edinger A.L. Thompson C.B. Mol. Biol. Cell. 2002; 13: 2276-2288Crossref PubMed Scopus (473) Google Scholar). The Akt1 open reading frame from this vector was subcloned into the expression vector, pBabe-IRES-GFP to create the vector pBabe-myrAkt-IRES-GFP. The CPT1A cDNA was amplified from murine liver cDNA and cloned into PCR2.1-TOPO (Invitrogen). The insert was then cloned as an EcoRI fragment into the retroviral construct MIGR1-GFP, creating MIGR1-GFP-CPT1A. Short hairpin RNA (shRNA) experiments used a published approach to express an shRNA under control of the human U6 RNA polymerase III promoter (34Fox C.J. Hammerman P.S. Cinalli R.M. Master S.R. Chodosh L.A. Thompson C.B. Genes Dev. 2003; 17: 1841-1854Crossref PubMed Scopus (273) Google Scholar). Briefly, a PCR product was generated from the plasmid pEF6-hU6 using primers that also contained a hairpin sequence against the CPT1A 19-mer 5′-GGCATAAACGCAGAGCATT-3′. This PCR product was cloned into pEF6-TOPO, and then the promoter and hairpin were shuttled to a pBabe-puro-derived construct (34Fox C.J. Hammerman P.S. Cinalli R.M. Master S.R. Chodosh L.A. Thompson C.B. Genes Dev. 2003; 17: 1841-1854Crossref PubMed Scopus (273) Google Scholar). Cell Lines—The myrAkt-overexpressing clones and empty vector control cells for Figs. 2 and 3 were generated by electroporating IL-3-dependent bax–/– bak–/– cells with 8 μgof pBabe-myrAkt-IRES-GFP by Nucleofector Solution V (Amaxa) using program T20. Five days later, green fluorescent protein (GFP)-positive cells were isolated by fluorescence-activated cell sorting to generate a bulk population of myrAkt-expressing cells. Stable clones were isolated by fluorescence-activated cell sorting with sorting into 96-well round bottom plates, and clones expressing high levels of myrAkt were identified by Western blotting. The shRNA clones and vector controls from Fig. 6 were generated in a similar fashion. Clones were screened by withdrawing cells from IL-3 and performing Western blots against CPT1A. To generate CPT1A-overexpressing and vector control IL-3-dependent cells (Fig. 7), we prepared retrovirus by co-transfecting 293T cells (Lipofectamine, Invitrogen) with MIGR1-GFP or MIGR1-GFP-CPT1A and a helper virus plasmid. Two days later, supernatants containing retroviral particles were collected. To infect IL-3-dependent cells, 3 × 106 cells were collected and resuspended in 1 ml of retrovirus-containing medium. Polybrene was added to a final concentration of 8 μg/ml, and the sample was centrifuged at 2200 rpm for 90 min. Cells were cultured at 37 °C for 5 h, then given fresh complete medium. On the third day after infection, GFP-positive cells were harvested by fluorescence-activated cell sorting.FIGURE 3IL-3 stimulation of PI3K/Akt suppresses expression of CPT1A. A, cells were withdrawn from IL-3 for 3 days and then re-stimulated. Total RNA was harvested at the time points indicated and analyzed by quantitative reverse transcription-PCR (qPCR) for CPT1A abundance. Bars are average of three amplification reactions, with the CPT1A/β-actin ratio arbitrarily set to 1.0 for RNA collected from unstimulated cells (time 0). B, protein lysates were also collected from cells after a 3-day IL-3 withdrawal (time 0), or at various time points after re-stimulation (m, minutes; h, hours). Lysates were blotted and probed for CPT1A and for β-actin. As a control, protein was also collected from cells that had not been deprived of IL-3 (C). C, IL-3 withdrawn cells were re-stimulated with IL-3 in the absence or presence of the PI3K inhibitor LY294002 (LY). Phosphorylation of Akt at Ser-473 is normally observed within 15 min of IL-3 stimulation; LY abolished this phosphorylation, but not phosphorylation of STAT5, which is independent of PI3K activity (top four blots). After 48 h, protein was again collected and analyzed for CPT1A expression (bottom two blots). D, a vector clone and two clones overexpressing myrAkt were withdrawn from IL-3 for 1 day, and CPT1A abundance was compared with IL-3-stimulated cells by Western blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Increased CPT1A expression and β-oxidation are required for survival during withdrawal of glucose and growth factors. A, IL-3-dependent clones expressing an shRNA against CPT1A (shRNA-1 and -2) had decreased abundance of CPT1A (top) and β-oxidation (bottom) when withdrawn from IL-3. Parental cells and a vector control clone (vec-1) are shown for comparison. Average β-oxidation activity and standard deviations are shown for three independent assays. B, parental IL-3-dependent cells, a vector control clone, and two shRNA clones were cultured in medium containing low glucose (0.3 mm) and no IL-3. Viability was determined by exclusion of propidium iodide at the indicated times. Each data point reflects the average and standard deviation for three cultures processed in parallel.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Constitutive CPT1A expression increases β-oxidation rate and decreases lipid synthesis and cell proliferation. A, IL-3-dependent cells were infected with a retroviral vector containing the mouse CPT1A cDNA (MIGR1-CPT1A). Abundance of CPT1A protein (top) and rate of β-oxidation (bottom) were determined in the presence of IL-3 and compared with cells infected with a control retrovirus (MIGR1-vec). B, vector control and CPT1A-overexpressing cells were assayed for their ability to convert [14C]glucose into lipids during IL-3 stimulation. Cells were cultured in the presence of [14C]glucose for 24 h, then lipids were harvested and analyzed by scintillation counting. Bars represent the average and standard deviations of three measurements. C, cells infected with MIGR1-vec (open circles) or MIGR1-CPT1A (filled circles) were analyzed for proliferation rate soon after retroviral infection. Cumulative population doublings were calculated over 6 days of culture.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of Microarray Data—Microarray data using cDNA samples from the IL-3-dependent cell line FL5.12 were reported previously (34Fox C.J. Hammerman P.S. Cinalli R.M. Master S.R. Chodosh L.A. Thompson C.B. Genes Dev. 2003; 17: 1841-1854Crossref PubMed Scopus (273) Google Scholar). These experiments used cDNA samples prepared from triplicate cultures grown in the presence of IL-3 or 12 h after IL-3 withdrawal. cDNAs were hybridized to Affymetrix murine 11K oligonucleotide microarrays. Western Blotting—Protein lysates were made by lysing cells in radioimmunoprecipitation buffer supplemented with Complete protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitors (Cocktails 1 and 2, Sigma). Protein was quantitated using BCA Protein Assay reagent (Pierce). Protein electrophoresis used NuPAGE 4–12% Bis-Tris gels (Invitrogen). The following primary antibodies were used: polyclonal total Akt and phospho-Akt (Sre-473, Cell Signaling Technology); monoclonal β-actin (Sigma); phospho-STAT5 A/B (Tyr-694/699, Upstate Biotech); and polyclonal CPT1A (a gift from Victor Zammit, Highland Research Institute, Scotland, UK). Horseradish peroxidase-conjugated secondary antibodies were anti-sheep (Upstate Biotech) anti-rabbit and anti-mouse (Amersham Biosciences). Proteins were detected with ECL Plus (Amersham Biosciences). Quantitative Reverse Transcription-PCR—For quantitative reverse transcriptase-PCR (qPCR) analysis, total cellular RNA was prepared using Tri reagent (Sigma). Complementary DNA was generated from 1 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen). Amplifications and data generation were performed using TaqMan probe sets, a 7900HT Sequence Detection System, and the SDS2.1 software (Applied Biosystems). Amplifications for β-actin and CPT1A were performed in triplicate, and β-actin mRNA abundance was used to normalize CPT1A mRNA abundance. Standard deviations were generated using the formula, [(RQ)(ΔCt S.D.)/average ΔCt)]. Lymphocyte Experiments—Adult female C57/Bl6 mice were sacrificed according to established mouse care protocols and in accordance with the Institutional Animal Care and Use Committee at The University of Pennsylvania. The spleen and lymph nodes (inguinal, axillary, and mesenteric) were harvested, crushed in Hanks' basic salt solution, and washed once in phosphate-buffered saline lacking Ca2+ and Mg2+, supplemented with 1% fetal calf serum. T lymphocytes were purified by negative selection using StemSep Mouse T-cell enrichment mixture (Stem Cell Technologies) and an AutoMACS separator (Miltenyi Biotec). After enrichment, T lymphocytes were either utilized directly for RNA, protein, and β-oxidation analysis, or were plated into wells pretreated with plate-bound anti-CD3 (Ebioscience) and anti-CD28 antibodies (BD Pharmingen) at 1 μg/ml each. Plated cells were cultured for 2 days, and then recombinant IL-2 was added to the medium at a concentration of 50 units/ml (PeproTech). After 2 more days, cells were harvested for RNA, protein, and β-oxidation analysis. To measure β-oxidation in T lymphocytes, 0.5 × 106 cells were plated into RPMI supplemented with [9,10-3H]palmitate-BSA as outlined above under “Lipid Metabolism Assays.” After a 4-h culture period, the medium was applied to ion-exchange resin as above. Growth Factor Stimulation Simultaneously Increases Lipid Synthesis and Suppresses Fatty Acid Oxidation—The cytokine growth factor IL-3 stimulates phospholipid synthesis by activating the PI3K/Akt system (35Bauer D.E. Hatzivassiliou G. Zhao F. Andreadis C. Thompson C.B. Oncogene. 2005; 24: 6314-6322Crossref PubMed Scopus (387) Google Scholar). Free fatty acids used for phospholipid synthesis might come from the extracellular pool, from an intracellular pool generated by de novo fatty acid synthesis using glucose and other substrates, or from both. To compare the effects of IL-3 signaling on lipogenesis using these two fatty acid pools, we cultured IL-3-dependent cells with 14C-labeled glucose or palmitate in the presence or absence of IL-3 and measured lipid synthesis. In the absence of IL-3, cells made a small amount of lipid using extracellular free fatty acids, but there was essentially no synthesis from glucose (Fig. 1A). By contrast, cells stimulated with IL-3 synthesized a larger amount of lipid using either substrate. IL-3 increased lipid synthesis from glucose by >100-fold (131.7 ± 28), whereas the increase in synthesis from palmitate was much less dramatic (3.2 ± 1). Under all conditions tested, phospholipid was the predominant lipid species synthesized, accounting for >75% of recovered 14C-containing lipid. A maximal rate of cell proliferation involves net synthesis of macromolecules and the avoidance of simultaneous synthesis and degradation of metabolic intermediates (“futile cycling”). Most cell types can degrade fatty acids through mitochondrial β-oxidation. We therefore tested whether growth factor stimulation affected the rate of β-oxidation. In absence of growth factor stimulation, IL-3-dependent cells exit the cell cycle and undergo progressive atrophy; re-introduction of IL-3 stimulates these quiescent cells to grow and re-enter the cell cycle (29Lum J.J. Bauer D.E. Kong M. Harris M.H. Li C. Lindsten T. Thompson C.B. Cell. 2005; 120: 237-248Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar, 36Rathmell J.C. Vander Heiden M.G. Harris M.H. Frauwirth K.A. Thompson C.B. Mol. Cell. 2000; 6: 683-692Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar). Cells were cultured in the absence of IL-3 for 3 days and then re-stimulated by IL-3 addition. Cell growth, proliferation, de novo lipid synthesis, and fatty acid oxidation were examined periodically over the course of the experiment. Quiescent cells regained maximal size within 2 days of IL-3 stimulation and had begun to proliferate exponentially by the third day (data not shown). Lipid synthesis increased within the first day of IL-3 stimulation and was at maximal levels by the third day, coinciding with the onset of exponential proliferation (Fig. 1B). By contrast, the quiescent cells had a relatively high rate of β-oxidation of the fatty acid palmitate, and this was rapidly and persistently suppressed by introduction of the growth factor (Fig. 1B). By 72 h after IL-3 re-addition, the oxidation rate was suppressed to baseline levels. Growth Factor Suppression of β-Oxidation Requires Stimulation of PI3K—The effects of IL-3 on lipid synthesis have been reported to require activation of PI3K (35Bauer D.E. Hatzivassiliou G. Zhao F. Andreadis C. Thompson C.B. Oncogene. 2005; 24: 6314-6322Crossref PubMed Scopus (387) Google Scholar); therefore, we examined the role of this signaling pathway in suppressing β-oxidation. The PI3K inhibitor LY294002 increased β-oxidation rate in a dose-dependent fashion despite IL-3 stimulation (Fig. 2A). Because many metabolic effects of PI3K stimulation, including induction of lipid synthesis, are dependent on activation of Akt, we next examined the role of Akt in suppressing β-oxidation. To that end, a constitutively active allele of Akt was transfected stably into IL-3-dependent cells. Two independent clones expressing this construct maintained Akt phosphorylation (Fig. 2B) and continued to suppress β-oxidation (Fig. 2C) after IL-3 withdrawal. Together, these results demonstrate that activation of the" @default.
• W2021492010 created "2016-06-24" @default.
• W2021492010 creator A5013924501 @default.
• W2021492010 creator A5047684237 @default.
• W2021492010 creator A5088551754 @default.
• W2021492010 date "2006-12-01" @default.
• W2021492010 modified "2023-10-10" @default.
• W2021492010 title "Phosphatidylinositol 3-Kinase-dependent Modulation of Carnitine Palmitoyltransferase 1A Expression Regulates Lipid Metabolism during Hematopoietic Cell Growth" @default.
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