RESUMO
Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes have distinct Plin gene members and additional protein forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to different types of lipid droplets that are composed of either triacylcerides or cholesterol esters. Furthermore, Plins with strong association preferences to triacylceride (or cholesterol ester) droplets can re-direct the relative intracellular triacylceride-cholesterol ester balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.
Assuntos
Proteínas de Transporte/metabolismo , Ésteres do Colesterol/metabolismo , Espaço Intracelular/metabolismo , Metabolismo dos Lipídeos , Fosfoproteínas/metabolismo , Triglicerídeos/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Ácidos Graxos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Perilipina-1 , Transporte Proteico , Ratos , Frações Subcelulares/metabolismoRESUMO
Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.
Assuntos
Adipócitos/metabolismo , Metabolismo Energético/fisiologia , Lipase/metabolismo , Lipólise/fisiologia , Fosfoproteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Células CHO , Proteínas de Transporte , Cricetinae , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Humanos , Lipase/genética , Masculino , Camundongos , Estresse Oxidativo/fisiologia , Perilipina-1 , Fosfoproteínas/genéticaRESUMO
The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.
Assuntos
Espaço Intracelular/metabolismo , Metabolismo dos Lipídeos , Família Multigênica , Fosfoproteínas/classificação , Terminologia como Assunto , Animais , Proteínas de Transporte , Evolução Molecular , Humanos , Perilipina-1 , Fosfoproteínas/genéticaRESUMO
Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein.
Assuntos
Lipólise , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Esterol Esterase/metabolismo , Animais , Western Blotting , Células CHO , Proteínas de Transporte , Cricetinae , Cricetulus , Imunofluorescência , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Perilipina-1 , Perilipina-2 , Perilipina-5 , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Proteínas/genética , Triglicerídeos/metabolismoRESUMO
Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A-phosphorylated HSL and perilipin A.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Metabolismo dos Lipídeos , Lipólise/genética , Fosfoproteínas/deficiência , Esterol Esterase/metabolismo , Animais , Células CHO , Proteínas de Transporte , Diferenciação Celular/fisiologia , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Fibroblastos/metabolismo , Camundongos , Perilipina-1 , Fosfoproteínas/genética , Fosforilação , Transporte Proteico/genética , Proteínas Recombinantes de FusãoRESUMO
The PAT family (originally named for Perilipin, ADFP and TIP47) now includes four members: Perilipins, ADFP, TIP47 and S3-12. Significant primary sequence homology and the ability to associate with lipid storage droplets (LSDs) are well conserved within this family and across species. In this study, we have characterized a novel PAT protein, lipid storage droplet protein 5 (LSDP5) of 463 residues. A detailed sequence analysis of all murine PAT proteins reveals that LSDP5, TIP47 and ADFP share the highest order of sequence similarity, whereas perilipin and S3-12 have more divergent carboxyl- and amino-termini, respectively. Ectopically-expressed YFP-LSDP5 or flag-LSDP5 fusion proteins associate with LSDs. In accord with recent published data for perilipin, forced expression of LSDP5 in CHO cells inhibits lipolysis of intracellular LSDs. The LSDP5 gene is primarily transcribed in cells that actively oxidize fatty acids, such as heart, red muscle and liver. Expression of LSDP5 is stimulated by ligand activation of peroxisomal proliferator-activated receptor alpha (PPARalpha), and significantly reduced in liver and heart in the absence of this transcription factor. PPARalpha is generally required for regulation of fatty acid metabolism during fasting, but fasting induces LSDP5 mRNA in liver even in the absence of PPARalpha.
Assuntos
Ácidos Graxos/metabolismo , Fosfoproteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte , Chlorocebus aethiops , Cromossomos Humanos Par 17 , Éxons , Jejum/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oxirredução , PPAR alfa/metabolismo , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Lipolysis involves the sequential breakdown of triglycerides into free fatty acids and glycerol. The extent of lipolysis is therefore a key determinant of the energy status of an individual and also dictates insulin resistance. Here, we describe a protocol for estimating lipolysis in murine adipocytes. Glycerol released during the lipolytic reaction is estimated radiometrically to determine the extent of lipolysis within the cell and the data are normalized to cell number.
Assuntos
Adipócitos/fisiologia , Lipólise , Adipócitos/citologia , Animais , Células Cultivadas , Glicerol/metabolismo , Camundongos , Triglicerídeos/metabolismoRESUMO
Perilipin protein coats the surface of intracellular lipid droplets and plays fundamental roles in lipid droplet formation and triacylglycerol hydrolysis. Perilipin is transcriptionally regulated through peroxisome proliferator-activated receptor and post-translationally stabilized by stored intracellular neutral lipids. In this study, we show that perilipin protein accumulates in transfected Chinese hamster ovary cells cultured in the presence of fatty acids but in turn is destabilized when lipid precursors for triacylglycerol synthesis are removed from culture serum. Adding fatty acids in the culture medium prevents the degradation of perilipin. Moreover, specific proteasome inhibitors, MG132, lactacystin, and ALLN, block the degradation, whereas inhibitors of other proteases are ineffective. Pulse-chase experiments confirm that perilipin is degraded through proteasome, a process that is inhibited by MG132 or ALLN and blunted by the addition of oleic acid. We have detected the co-immunoprecipitation of perilipin and ubiquitin, thus confirming that perilipin is conjugated to poly-ubiquitin and targeted for proteasomal degradation. Treatment with MG132 increases the expression of perilipin associated with lipid droplets as well as modestly throughout the cytosol. We conclude that the degradation of perilipin is mediated through an ubiquitination-proteasome pathway, which suggests another mode for the post-translational regulation of perilipin.
Assuntos
Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Células CHO , Proteínas de Transporte , Cricetinae , Meios de Cultura , Ácidos Graxos/metabolismo , Leupeptinas/farmacologia , Ácido Oleico/farmacologia , Perilipina-1 , Proteínas Recombinantes/metabolismo , Transfecção , Triglicerídeos/metabolismoRESUMO
The perilipin/ADRP/TIP47 (PAT) proteins localize to the surface of intracellular neutral lipid droplets. Perilipin is essential for lipid storage and hormone regulated lipolysis in adipocytes, and perilipin null mice exhibit a dramatic reduction in adipocyte lipid stores. A significant fraction of the approximately 200 amino acid N-terminal region of the PAT proteins consists of 11-mer helical repeats that are also found in apolipoproteins and other lipid-associated proteins. The C-terminal 60% of TIP47, a representative PAT protein, comprises a monomeric and independently folded unit. The crystal structure of the C-terminal portion of TIP47 was determined and refined at 2.8 A resolution. The structure consists of an alpha/beta domain of novel topology and a four-helix bundle resembling the LDL receptor binding domain of apolipoprotein E. The structure suggests an analogy between PAT proteins and apolipoproteins in which helical repeats interact with lipid while the ordered C-terminal region is involved in protein:protein interactions.
Assuntos
Proteínas de Ligação a DNA/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Metabolismo dos Lipídeos , Proteínas da Gravidez/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Perilipina-1 , Perilipina-2 , Perilipina-3 , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas da Gravidez/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Proteínas de Transporte VesicularRESUMO
Several of the aspartic acid protease inhibitors used to treat HIV infection increase basal lipolysis in adipocytes, but the cellular mechanisms leading to this augmentation are not well understood. We therefore studied the effects of chronic exposure to the HIV protease inhibitor, ritonavir, on the lipolytic cascade in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with ritonavir for 14 d (during and after differentiation) enhanced basal, isoproterenol (Iso)-stimulated, and cAMP analog-stimulated lipolysis. Enhancement of lipolysis was observed after Iso at concentrations between 0.1 and 10 mum. Despite a significant decrease in cyclic nucleotide phosphodiesterase (PDE)3B activity and protein levels, there were no changes in Iso-stimulated intracellular cAMP, protein kinase A (PKA) expression, or PKA activity. Ritonavir-augmented lipolysis was also observed under conditions that reversed the effect on PDE3B activity via preincubation with 1 mum (-)-N(6)-(2-phenylisopropyl)adenosine. In ritonavir-treated cells, protein expression of the lipid droplet-protective protein, perilipin, was significantly decreased, whereas there was no change in hormone-sensitive lipase. Activation of ERK1/2 by Iso did not play a role in the augmentation. We conclude that ritonavir decreases PDE3B and perilipin protein expression and affects both basal and catecholamine-stimulated lipolysis in 3T3-L1 adipocytes primarily through actions at sites downstream of PKA.
Assuntos
Adipócitos/fisiologia , Inibidores da Protease de HIV/farmacologia , Isoproterenol/farmacologia , Lipólise/efeitos dos fármacos , Ritonavir/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , CamundongosRESUMO
Activation of macrophages by TLR agonists enhances foam cell formation, but the underlying mechanisms are not understood. We examined the effects of TLR agonists on ADRP/ADFP, a protein associated with forming lipid droplets, and Mal1 a fatty acid-binding protein, in two mouse macrophage cell lines and human monocytes. Low doses of LPS, a TLR4 agonist increased both mRNA and protein levels of ADRP/ADFP and Mal1 in RAW 264.7 macrophages. Following pretreatment with Intralipid, fatty acids, or acetyl-LDL to increase triglyceride or cholesterol ester storage, LPS treatment still increased ADRP/ADFP and Mal1 mRNA levels. LPS also induced ADRP/ADFP and Mal1 in J774 macrophages and ADRP/ADFP in human monocytes. Zymosan, a fungal product that activates TLR2, poly-I:C, a viral mimetic that activates TLR3, and imiquimod, a TLR7 agonist, also increased ADRP/ADFP. Zymosan, but not poly-I:C or imiquimod, induced Mal1. In contrast, neither gene was induced by TNFalpha, IL-1beta, IL-6, or interferon-gamma. Thus TLR agonists induce ADRP/ADFP and Mal1, which likely contributes to macrophage triglyceride and cholesterol ester storage leading to foam cell formation.
Assuntos
Aterosclerose/imunologia , Proteínas de Ligação a Ácido Graxo/biossíntese , Macrófagos/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Receptores Toll-Like/agonistas , Aminoquinolinas/farmacologia , Animais , Ésteres do Colesterol/metabolismo , Humanos , Imiquimode , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Camundongos , Perilipina-2 , Poli I-C/farmacologia , Receptores Toll-Like/imunologia , Triglicerídeos/metabolismo , Zimosan/farmacologiaRESUMO
We investigated the role of LMNA in adipose tissue by developing a novel mouse model of lipodystrophy. Transgenic mice were generated that express the LMNA mutation that causes familial partial lipodystrophy of the Dunnigan type (FPLD2). The phenotype observed in FPLD-transgenic mice resembles many of the features of human FPLD2, including lack of fat accumulation, insulin resistance, and enlarged, fatty liver. Similar to the human disease, FPLD-transgenic mice appear to develop normally, but after several weeks they are unable to accumulate fat to the same extent as their wild-type littermates. One poorly understood aspect of lipodystrophies is the mechanism of fat loss. To this end, we have examined the effects of the FPLD2 mutation on fat cell function. Contrary to the current literature, which suggests FPLD2 results in a loss of fat, we found that the key mechanism contributing to the lack of fat accumulation involves not a loss, but an apparent inability of the adipose tissue to renew itself. Specifically, preadipocytes are unable to differentiate into mature and fully functional adipocytes. These findings provide insights not only for the treatment of lipodystrophies, but also for the study of adipogenesis, obesity, and insulin resistance.
Assuntos
Tecido Adiposo/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Mutação , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Sequência de Bases , Diferenciação Celular , Primers do DNA/genética , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Lipodistrofia Parcial Familiar/patologia , Lipólise/genética , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Termogênese/genética , Termogênese/fisiologiaRESUMO
Lipids have been shown to play a dual role in pancreatic beta-cells: a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse, and human islets of Langerhans as well as the rat clonal beta-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in beta-cells. To examine whether the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the beta-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 h. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared with control cells. Whereas glucose-stimulated insulin secretion was retained after palmitate exposure in cells overexpressing perilipin, it was completely abolished in control beta-cells. Thus, overexpression of perilipin appears to confer protection against the development of beta-cell dysfunction after prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.
Assuntos
Células Secretoras de Insulina/metabolismo , Fosfoproteínas/metabolismo , Adulto , Idoso , Animais , Proteínas de Transporte , Linhagem Celular , Feminino , Humanos , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Pessoa de Meia-Idade , Palmitatos/farmacologia , Perilipina-1 , Ratos , Ratos WistarRESUMO
OBJECTIVE: Sustained exposure to lipids is deleterious for pancreatic islet function. This could be mediated through increased pancreatic fat following increased dietary fat and in obesity, which has implications for the onset of type 2 diabetes. The aims of this study were to determine changes in extent and composition of pancreatic, hepatic, and visceral fat in mice fed a high-fat diet (HFD, 40% by weight) compared with a control diet (5% fat) of similar fatty acid composition, and to compare composition and extent of pancreatic fat in human type 2 diabetes. METHODS AND PROCEDURES: Mice were fed HFD for 3 or 15 weeks. Human postmortem pancreas was examined from subjects with type 2 diabetes (n = 9) and controls (n = 7). Tissue lipid content and composition were determined by gas chromatography and pancreatic adipocyte infiltration quantified by morphometry. RESULTS: Pancreatic triacylglycerol (TG) content was 20x greater (P < 0.05) in HFD mice and there were more pancreatic perilipin-positive adipocytes compared with controls after 15 weeks. The proportions of 18:1n -9 and 18:2n -6 in pancreatic TG and the 20:4n -6/18:2n -6 ratio in phospholipids, were higher (both P < 0.05) after HFD compared with controls. Human pancreatic TG content was correlated with the proportion of pancreatic perilipin-positive adipocytes (r = 0.64, P < 0.05) and associated with unsaturated fatty acid enrichment (P < 0.05). DISCUSSION: Adipocyte infiltration in pancreatic exocrine tissue is associated with high-fat feeding in mice and pancreatic TG content in humans. This alters the fatty acid milieu of the islet which could contribute to islet dysfunction.
Assuntos
Adipócitos/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Gordura Intra-Abdominal/metabolismo , Ácido Linoleico/metabolismo , Fígado/metabolismo , Pâncreas/metabolismo , Adipócitos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Transporte , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/patologia , Ácidos Graxos não Esterificados/metabolismo , Feminino , Humanos , Gordura Intra-Abdominal/enzimologia , Gordura Intra-Abdominal/patologia , Ácido Linoleico/administração & dosagem , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Pâncreas/enzimologia , Pâncreas/patologia , Perilipina-1 , Fosfoproteínas/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Fatores de Tempo , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: Accumulation of intracellular lipid droplets (LDs) in non-adipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. LDs are coated with perilipin, adipose differentiation-related protein, tail interacting protein of 47 kd (PAT) proteins that are thought to regulate LD turnover by modulating lipolysis. Our hypothesis is that PAT proteins modulate LD metabolism and therefore insulin resistance. RESEARCH DESIGN AND METHODS: We used a cell culture model (murine AML12 loaded with oleic acid) and small interfering RNA to directly assess the impact of PAT proteins on LD accumulation, lipid metabolism, and insulin action. PAT proteins associated with excess fat deposited in livers of diet-induced obese (DIO) mice were also measured. RESULTS: Cells lacking PAT proteins exhibited a dramatic increase in LD size and a decrease in LD number. Further, the lipolytic rate increased by approximately 2- to 2.5-fold in association with increased adipose triglyceride lipase (ATGL) at the LD surface. Downregulation of PAT proteins also produced insulin resistance, as indicated by decreased insulin stimulation of Akt phosphorylation (P < 0.001). Phosphoinositide-dependent kinase-1 and phosphoinositide 3-kinase decreased, and insulin receptor substrate-1 307 phosphorylation increased. Increased lipids in DIO mice livers were accompanied by changes in PAT composition but also increased ATGL, suggesting a relative PAT deficiency. CONCLUSIONS: These data establish an important role for PAT proteins as surfactant at the LD surface, packaging lipids in smaller units and restricting access of lipases and thus preventing insulin resistance. We suggest that a deficiency of PAT proteins relative to the quantity of ectopic fat could contribute to cellular dysfunction in obesity and type 2 diabetes.
Assuntos
Proteínas de Transporte/fisiologia , Hepatócitos/metabolismo , Proteínas de Membrana/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cromatografia em Camada Fina , Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Hepatócitos/citologia , Immunoblotting , Imuno-Histoquímica , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipólise/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Perilipina-2 , Perilipina-3 , RNA Interferente Pequeno/genéticaRESUMO
The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.
Assuntos
Adipócitos/metabolismo , Lipólise/fisiologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Contagem de Células , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Glicerol/metabolismo , Isoproterenol/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.
Assuntos
Pulmão/metabolismo , Proteínas de Membrana/fisiologia , Tensoativos/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Hidrólise , Lipídeos/química , Microscopia de Fluorescência , Perilipina-2 , Ratos , Triglicerídeos/metabolismoRESUMO
A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.
Assuntos
Epinefrina/farmacologia , Lipídeos/química , Esterol Esterase/metabolismo , Animais , Transporte Biológico , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Contração Muscular , Músculo Esquelético/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Fatores de Tempo , Vasoconstritores/farmacologiaRESUMO
Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.
Assuntos
Adipócitos/citologia , Tecido Adiposo/metabolismo , Diferenciação Celular , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/fisiologia , Proteínas da Gravidez/fisiologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Linhagem Celular , Cromatografia em Camada Fina , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Immunoblotting , Técnicas Imunoenzimáticas , Metabolismo dos Lipídeos , Lipólise , Espectrometria de Massas , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Perilipina-2 , Fosfolipídeos/metabolismo , Proteínas da Gravidez/genética , RNA Interferente Pequeno/farmacologia , Triglicerídeos/metabolismoRESUMO
Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.