RESUMEN
It has been suggested that some cancer cells rely upon fatty acid oxidation (FAO) for energy. Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1) with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 µM) have an off-target effect of inhibiting complex I of the electron transport chain. Surprisingly, however, when FAO was reduced further by genetic knockdown of CPT1, the proliferation rate of these same cells decreased nearly 2-fold and could not be restored by acetate or octanoic acid supplementation. Moreover, CPT1 knockdowns had altered mitochondrial morphology and impaired mitochondrial coupling, whereas cells in which CPT1 had been approximately 90% inhibited by etomoxir did not. Lipidomic profiling of mitochondria isolated from CPT1 knockdowns showed depleted concentrations of complex structural and signaling lipids. Additionally, expression of a catalytically dead CPT1 in CPT1 knockdowns did not restore mitochondrial coupling. Taken together, these results suggest that transport of at least some long-chain fatty acids into the mitochondria by CPT1 may be required for anabolic processes that support healthy mitochondrial function and cancer cell proliferation independent of FAO.
Asunto(s)
Carnitina O-Palmitoiltransferasa/fisiología , Proliferación Celular/fisiología , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Línea Celular Tumoral , Transporte de Electrón/efectos de los fármacos , Ácidos Grasos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno , Interferencia de ARNRESUMEN
Cellular proliferation requires the formation of new membranes. It is often assumed that the lipids needed for these membranes are synthesized mostly de novo. Here, we show that proliferating fibroblasts prefer to take up palmitate from the extracellular environment over synthesizing it de novo. Relative to quiescent fibroblasts, proliferating fibroblasts increase their uptake of palmitate, decrease fatty acid degradation, and instead direct more palmitate to membrane lipids. When exogenous palmitate is provided in the culture media at physiological concentrations, de novo synthesis accounts for only a minor fraction of intracellular palmitate in proliferating fibroblasts as well as proliferating HeLa and H460 cells. Blocking fatty acid uptake decreased the proliferation rate of fibroblasts, HeLa, and H460 cells, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Our results suggest that cells scavenging exogenous lipids may be less susceptible to drugs targeting glycolysis and de novo lipid synthesis.
Asunto(s)
Ácidos Grasos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Lípidos de la Membrana/metabolismo , Células 3T3-L1 , Animales , Línea Celular Tumoral , Proliferación Celular , Células HeLa , Humanos , RatonesRESUMEN
BACKGROUND: The chemical instability of antiangiogenic fumagillin, combined with its poor retention during intravascular transit, requires an innovative solution for clinical translation. We hypothesized that an Sn-2 lipase-labile fumagillin prodrug, in combination with a contact-facilitated drug delivery mechanism, could be used to address these problems. METHODS: α(v)ß(3)-targeted and nontargeted nanoparticles with and without fumagillin in the prodrug or native forms were evaluated in vitro and in vivo in the Matrigel™ (BD Biosciences, CA, USA) plug model of angiogenesis in mice. RESULTS: In vitro experiments demonstrated that the new fumagillin prodrug decreased viability at least as efficacious as the parent compound, on an equimolar basis. In the Matrigel mouse angiogenesis model, α(v)ß(3)-fumagillin prodrug decreased angiogenesis as measured by MRI (3T), while the neovasculature was unaffected with the control nanoparticles. CONCLUSION: The present approach resolved the previously intractable problems of drug instability and premature release in transit to target sites.
Asunto(s)
Ciclohexanos/uso terapéutico , Ácidos Grasos Insaturados/uso terapéutico , Lipasa/uso terapéutico , Nanomedicina , Neovascularización Patológica/terapia , Profármacos/uso terapéutico , Animales , Disponibilidad Biológica , Células Cultivadas , Ciclohexanos/farmacocinética , Ácidos Grasos Insaturados/farmacocinética , Humanos , Ratones , Ratas , Sesquiterpenos/farmacocinética , Sesquiterpenos/uso terapéuticoRESUMEN
Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress, thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic myocardium.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Metabolismo Energético , Proteínas de la Membrana/metabolismo , Miocardio/enzimología , Miocitos Cardíacos/enzimología , Fosfatidilgliceroles/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Fosfatidilgliceroles/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Identification and quantification of unsaturated fatty acid (FA) isomers in a biological system are significant in the study of lipid metabolism and catabolism, membrane biophysics, and pathogenesis of diseases but are challenging in lipidomics. We developed a novel approach for identification and quantitation of unsaturated FA isomers by exploiting two facts: (1) unsaturated FA anions yield fragment ion(s) from loss of CO(2) or H(2)O from the anions upon collision-induced dissociation; and (2) the fragment ions yielded from discrete FA isomers have distinct profiles of the fragment ion intensity vs. collision conditions. These distinct profiles likely result from the differential interactions of the negative charge of the fragment ion with the electron clouds of the double bonds due to their different distances in discrete FA isomers. The novel approach was also extended to analyze the double bond isomers of FA chains present in phospholipids by multistage tandem mass spectrometry. Collectively, we developed a new approach for identification and quantification of the double bond isomers of endogenous FA species or FA chains present in intact phospholipid species. We believe that this approach should further advance the lipidomic power for identification of the biochemical mechanisms underlying metabolic diseases.
Asunto(s)
Ácidos Grasos Insaturados/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Dióxido de Carbono/metabolismo , Grasas de la Dieta , Isomerismo , Ratones , Agua/metabolismoRESUMEN
Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A2 (iPLA2) dual signature nucleotide ((G/A)XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA2/lipase family members designated iPLA2epsilon, iPLA2zeta, and iPLA2eta (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA2epsilon, iPLA2zeta, and iPLA2eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, a mechanism-based suicide substrate inhibitor of all known iPLA2s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50=0.1-0.5 microm). Quantitative PCR revealed dramatically increased expression of iPLA2epsilon and iPLA2zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA2 isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.