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Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.
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Resistência à Insulina , Obesidade , Masculino , Camundongos , Animais , Obesidade/complicações , Obesidade/genética , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Dieta Hiperlipídica/efeitos adversos , Lisossomos/metabolismo , Lipídeos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Alpha/beta hydrolase domain-containing 5 (ABHD5), also termed CGI-58, is the key upstream activator of adipose triglyceride lipase (ATGL), which plays an essential role in lipid metabolism and energy storage. Mutations in ABHD5 disrupt lipolysis and are known to cause the Chanarin-Dorfman syndrome. Despite its importance, the structure of ABHD5 remains unknown. In this work, we combine computational and experimental methods to build a 3D structure of ABHD5. Multiple comparative and machine learning-based homology modeling methods are used to obtain possible models of ABHD5. The results from Gaussian accelerated molecular dynamics and experimental data of the apo models and their mutants are used to select the most likely model. Moreover, ensemble docking is performed on representative conformations of ABHD5 to reveal the binding mechanism of ABHD5 and a series of synthetic ligands. Our study suggests that the ABHD5 models created by deep learning-based methods are the best candidate structures for the ABHD5 protein. The mutations of E41, R116, and G328 disturb the hydrogen bonding network with nearby residues and suppress membrane targeting or ATGL activation. The simulations also reveal that the hydrophobic interactions are responsible for binding sulfonyl piperazine ligands to ABHD5. Our work provides fundamental insight into the structure of ABHD5 and its ligand-binding mode, which can be further applied to develop ABHD5 as a therapeutic target for metabolic disease and cancer.
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Dysregulation of adipose tissue plasmalogen metabolism is associated with obesity-related metabolic diseases. We report that feeding mice a high-fat diet reduces adipose tissue lysoplasmalogen levels and increases transmembrane protein 86 A (TMEM86A), a putative lysoplasmalogenase. Untargeted lipidomic analysis demonstrates that adipocyte-specific TMEM86A-knockout (AKO) increases lysoplasmalogen content in adipose tissue, including plasmenyl lysophosphatidylethanolamine 18:0 (LPE P-18:0). Surprisingly, TMEM86A AKO increases protein kinase A signalling pathways owing to inhibition of phosphodiesterase 3B and elevation of cyclic adenosine monophosphate. TMEM86A AKO upregulates mitochondrial oxidative metabolism, elevates energy expenditure, and protects mice from metabolic dysfunction induced by high-fat feeding. Importantly, the effects of TMEM86A AKO are largely reproduced in vitro and in vivo by LPE P-18:0 supplementation. LPE P-18:0 levels are significantly lower in adipose tissue of human patients with obesity, suggesting that TMEM86A inhibition or lysoplasmalogen supplementation might be therapeutic approaches for preventing or treating obesity-related metabolic diseases.
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Plasmalogênios , Termogênese , Adipócitos/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/fisiologia , Homeostase , Humanos , Hidrolases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Plasmalogênios/metabolismo , Termogênese/fisiologiaRESUMO
Alpha/beta hydrolase domain-containing protein 5 (ABHD5) is a highly conserved protein that regulates various lipid metabolic pathways via interactions with members of the perilipin (PLIN) and Patatin-like phospholipase domain-containing protein (PNPLA) protein families. Loss of function mutations in ABHD5 result in Chanarin-Dorfman Syndrome (CDS), characterized by ectopic lipid accumulation in numerous cell types and severe ichthyosis. Recent data demonstrates that ABHD5 is the target of synthetic and endogenous ligands that might be therapeutic beneficial for treating metabolic diseases and cancers. However, the structural basis of ABHD5 functional activities, such as protein-protein interactions and ligand binding is presently unknown. To address this gap, we constructed theoretical structural models of ABHD5 by comparative modeling and topological shape analysis to assess the spatial patterns of ABHD5 conformations computed in protein dynamics. We identified functionally important residues on ABHD5 surface for lipolysis activation by PNPLA2, lipid droplet targeting and PLIN-binding. We validated the computational model by examining the effects of mutating key residues in ABHD5 on an array of functional assays. Our integrated computational and experimental findings provide new insights into the structural basis of the diverse functions of ABHD5 as well as pathological mutations that result in CDS.
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1-Acilglicerol-3-Fosfato O-Aciltransferase/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Biologia Computacional/métodos , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Mutação , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Humanos , Ligantes , Gotículas Lipídicas/química , Conformação ProteicaRESUMO
ABHD5 is an essential coactivator of ATGL, the rate-limiting triglyceride (TG) lipase in many cell types. Importantly, ABHD5 also functions as a tumor suppressor, and ABHD5 mRNA expression levels correlate with patient survival for several cancers. Nevertheless, the mechanisms involved in ABHD5-dependent tumor suppression are not known. We found that overexpression of ABHD5 induces cell cycle arrest at the G1 phase and causes growth retardation in a panel of prostate cancer cells. Transcriptomic profiling and biochemical analysis revealed that genetic or pharmacological activation of lipolysis by ABHD5 potently inhibits mTORC1 signaling, leading to a significant downregulation of protein synthesis. Mechanistically, we found that ABHD5 elevates intracellular AMP content, which activates AMPK, leading to inhibition of mTORC1. Interestingly, ABHD5-dependent suppression of mTORC1 was abrogated by pharmacological inhibition of DGAT1 or DGAT2, isoenzymes that re-esterify fatty acids in a process that consumes ATP. Collectively, this study maps out a novel molecular pathway crucial for limiting cancer cell proliferation, in which ABHD5-mediated lipolysis creates an energy-consuming futile cycle between TG hydrolysis and resynthesis, leading to inhibition of mTORC1 and cancer cell growth arrest.
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1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , Lipólise/fisiologia , Metabolismo/fisiologia , RNA-SeqRESUMO
Significant advances have been made towards understanding the role of immune cell-tumor interplay in either suppressing or promoting tumor growth, progression, and recurrence, however, the roles of additional stromal elements, cell types and/or cell states remain ill-defined. The overarching goal of this NCI-sponsored workshop was to highlight and integrate the critical functions of non-immune stromal components in regulating tumor heterogeneity and its impact on tumor initiation, progression, and resistance to therapy. The workshop explored the opposing roles of tumor supportive versus suppressive stroma and how cellular composition and function may be altered during disease progression. It also highlighted microenvironment-centered mechanisms dictating indolence or aggressiveness of early lesions and how spatial geography impacts stromal attributes and function. The prognostic and therapeutic implications as well as potential vulnerabilities within the heterogeneous tumor microenvironment were also discussed. These broad topics were included in this workshop as an effort to identify current challenges and knowledge gaps in the field.
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OBJECTIVE: Beclin1 is a core molecule of the macroautophagy machinery. Although dysregulation of macroautophagy is known to be involved in metabolic disorders, the function of Beclin1 in adipocyte metabolism has not been investigated. In the present study, we aimed to study the role of Beclin1 in lipolysis and mitochondrial homeostasis of adipocytes. METHODS: Autophagic flux during lipolysis was examined in adipocytes cultured in vitro and in the adipose tissue of mice. Adipocyte-specific Beclin1 knockout (KO) mice were used to investigate the activities of Beclin1 in adipose tissues. RESULTS: cAMP/PKA signaling increased the autophagic flux in adipocytes differentiated from C3H10T1/2 cells. In vivo autophagic flux was higher in the brown adipose tissue (BAT) than that in the white adipose tissue and was further increased by the ß3 adrenergic receptor agonist CL316243. In addition, surgical denervation of BAT greatly reduced autophagic flux, indicating that sympathetic nerve activity is a major regulator of tissue autophagy. Adipocyte-specific KO of Beclin1 led to a hypertrophic enlargement of lipid droplets in BAT and impaired CL316243-induced lipolysis/lipid mobilization and energy expenditure. While short-term effects of Beclin1 deletion were characterized by an increase in mitochondrial proteins, long-term Beclin1 deletion led to severe disruption of autophagy, resulting in mitochondrial loss, and dramatically reduced the expression of genes involved in lipid metabolism. Consequently, adipose tissue underwent increased activation of cell death signaling pathways, macrophage recruitment, and inflammation, particularly in BAT. CONCLUSIONS: The present study demonstrates the critical roles of Beclin1 in the maintenance of lipid metabolism and mitochondrial homeostasis in adipose tissues.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Proteína Beclina-1/genética , Deleção de Genes , Lipólise/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Adipócitos/ultraestrutura , Tecido Adiposo Marrom/metabolismo , Animais , Autofagia/genética , Proteína Beclina-1/metabolismo , Linhagem Celular , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Variações do Número de Cópias de DNA , Imunidade , Metabolismo dos Lipídeos , Camundongos , Camundongos Knockout , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Transdução de Sinais , Termogênese/genéticaRESUMO
A central function of adipose tissue is in the management of systemic energy homeostasis that is achieved through the co-ordinated regulation of energy storage and mobilization, adipokine release, and immune functions. With the dramatic increase in the prevalence of obesity and obesity-related metabolic disease over the past 30 years, there has been extensive interest in targeting adipose tissue for therapeutic benefit. However, in order for this goal to be achieved it is essential to establish a comprehensive atlas of adipose tissue cellular composition and define mechanisms of intercellular communication that mediate pathologic and therapeutic responses. While traditional methods, such as fluorescence-activated cell sorting (FACS) and genetic lineage tracing, have greatly advanced the field, these approaches are inherently limited by the choice of markers and the ability to comprehensively identify and characterize dynamic interactions among stromal cells within the tissue microenvironment. Single cell RNA sequencing (scRNAseq) has emerged as a powerful tool for deconvolving cellular heterogeneity and holds promise for understanding the development and plasticity of adipose tissue under normal and pathological conditions. scRNAseq has recently been used to characterize adipose stem cell (ASC) populations and has provided new insights into subpopulations of macrophages that arise during anabolic and catabolic remodeling in white adipose tissue. The current review summarizes recent findings that use this technology to explore adipose tissue heterogeneity and plasticity.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Branco , Macrófagos/metabolismo , Células Estromais/citologia , Adipócitos/imunologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/imunologia , Tecido Adiposo Branco/metabolismo , Animais , Comunicação Celular , Citometria de Fluxo , Humanos , Macrófagos/citologia , Obesidade/metabolismo , Obesidade/terapia , Análise de Sequência de RNA , Análise de Célula Única , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
OBJECTIVE: This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS: Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the ß3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFRα+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS: Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS: These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.
Assuntos
Tecido Adiposo Branco/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Adipócitos Marrons/metabolismo , Adipogenia , Animais , Antagomirs/metabolismo , Diferenciação Celular , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Dioxóis/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células RAW 264.7 , Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The accumulation of neutral lipids in intracellular lipid droplets has been associated with the formation and progression of many cancers, including prostate cancer (PCa). Alpha-beta Hydrolase Domain Containing 5 (ABHD5) is a key regulator of intracellular neutral lipids that has been recently identified as a tumor suppressor in colorectal cancer, yet its potential role in PCa has not been investigated. Through mining publicly accessible PCa gene expression datasets, we found that ABHD5 gene expression is markedly decreased in metastatic castration-resistant PCa (mCRPC) samples. We further demonstrated that RNAi-mediated ABHD5 silencing promotes, whereas ectopic ABHD5 overexpression inhibits, the invasion and proliferation of PCa cells. Mechanistically, we found that ABHD5 knockdown induces epithelial to mesenchymal transition, increasing aerobic glycolysis by upregulating the glycolytic enzymes hexokinase 2 and phosphofrucokinase, while decreasing mitochondrial respiration by downregulating respiratory chain complexes I and III. Interestingly, knockdown of ATGL, the best-known molecular target of ABHD5, impeded the proliferation and invasion, suggesting an ATGL-independent role of ABHD5 in modulating PCa aggressiveness. Collectively, these results provide evidence that ABHD5 acts as a metabolic tumor suppressor in PCa that prevents EMT and the Warburg effect, and indicates that ABHD5 is a potential therapeutic target against mCRPC, the deadly aggressive PCa.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Aerobiose , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Respiração Celular , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Glicólise , Células HEK293 , Humanos , Lipase/metabolismo , Masculino , Mitocôndrias/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Consumo de Oxigênio , Neoplasias da Próstata/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Triglicerídeos/metabolismoRESUMO
Current knowledge regarding acute regulation of adipocyte lipolysis is largely based on receptor-mediated activation or inhibition of pathways that influence intracellular levels of cAMP, thereby affecting protein kinase A (PKA) activity. We recently identified synthetic ligands of α-ß-hydrolase domain containing 5 (ABHD5) that directly activate adipose triglyceride lipase (ATGL) by dissociating ABHD5 from its inhibitory regulator, perilipin-1 (PLIN1). In the current study, we used these novel ligands to determine the direct contribution of ABHD5 to various aspects of lipolysis control in white (3T3-L1) and brown adipocytes. ABHD5 ligands stimulated adipocyte lipolysis without affecting PKA-dependent phosphorylation on consensus sites of PLIN1 or hormone-sensitive lipase (HSL). Cotreatment of adipocytes with synthetic ABHD5 ligands did not alter the potency or maximal lipolysis efficacy of the ß-adrenergic receptor (ADRB) agonist isoproterenol (ISO), indicating that both target a common pool of ABHD5. Reducing ADRB/PKA signaling with insulin or desensitizing ADRB suppressed lipolysis responses to a subsequent challenge with ISO, but not to ABHD5 ligands. Lastly, despite strong treatment differences in PKA-dependent phosphorylation of HSL, we found that ligand-mediated activation of ABHD5 led to complete triglyceride hydrolysis, which predominantly involved ATGL, but also HSL. These results indicate that the overall pattern of lipolysis controlled by ABHD5 ligands is similar to that of isoproterenol, and that ABHD5 plays a central role in the regulation of adipocyte lipolysis. As lipolysis is critical for adaptive thermogenesis and in catabolic tissue remodeling, ABHD5 ligands may provide a means of activating these processes under conditions where receptor signaling is compromised.
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1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/efeitos dos fármacos , Piperazinas/farmacologia , Tiazepinas/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Feminino , Hidrólise , Insulina/farmacologia , Ligantes , Lipólise , Camundongos , Perilipina-1/metabolismo , Fosforilação , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Esterol Esterase/metabolismo , Triglicerídeos/metabolismoRESUMO
OBJECTIVE: Fibroblast growth factor 21 (FGF21) shows great potential for the treatment of obesity and type 2 diabetes, as its long-acting analogue reduces body weight and improves lipid profiles of participants in clinical studies; however, the intracellular mechanisms mediating these effects are poorly understood. AMP-activated protein kinase (AMPK) is an important energy sensor of the cell and a molecular target for anti-diabetic medications. This work examined the role of AMPK in mediating the glucose and lipid-lowering effects of FGF21. METHODS: Inducible adipocyte AMPK ß1ß2 knockout mice (iß1ß2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received long-acting FGF21 for two weeks. RESULTS: Consistent with previous studies, FGF21 treatment significantly reduced body weight, adiposity, and liver lipids in HFD fed mice. To add, FGF21 improved circulating lipids, glycemic control, and insulin sensitivity. These effects were independent of adipocyte AMPK and were not associated with changes in browning of white (WAT) and brown adipose tissue (BAT). Lastly, we assessed whether FGF21 exerted its effects through the AMPK/ACC axis, which is critical in the therapeutic benefits of the anti-diabetic medication metformin. ACC DKI mice had improved glucose and insulin tolerance and a reduction in body weight, body fat and hepatic steatosis similar to WT mice in response to FGF21 administration. CONCLUSIONS: These data illustrate that the metabolic improvements upon FGF21 administration are independent of adipocyte AMPK, and do not require the inhibitory action of AMPK on ACC. This is in contrast to the anti-diabetic medication metformin and suggests that the treatment of obesity and diabetes with the combination of FGF21 and AMPK activators merits consideration.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Glucose/metabolismo , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adipócitos/metabolismo , Animais , Homeostase , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Proteínas Quinases/genéticaRESUMO
Perilipin (PLIN) proteins constitute an ancient family important in lipid droplet (LD) formation and triglyceride metabolism. We identified an additional PLIN clade (plin6) that is unique to teleosts and can be traced to the two whole genome duplications that occurred early in vertebrate evolution. Plin6 is highly expressed in skin xanthophores, which mediate red/yellow pigmentation and trafficking, but not in tissues associated with lipid metabolism. Biochemical and immunochemical analyses demonstrate that zebrafish Plin6 protein targets the surface of pigment-containing carotenoid droplets (CD). Protein kinase A (PKA) activation, which mediates CD dispersion in xanthophores, phosphorylates Plin6 on conserved residues. Knockout of plin6 in zebrafish severely impairs the ability of CD to concentrate carotenoids and prevents tight clustering of CD within carotenoid bodies. Ultrastructural and functional analyses indicate that LD and CD are homologous structures, and that Plin6 was functionalized early in vertebrate evolution for concentrating and trafficking pigment.
Assuntos
Gotículas Lipídicas/metabolismo , Perilipina-1/metabolismo , Peixe-Zebra , Animais , Carotenoides/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Perilipina-1/genética , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Alpha-beta hydrolase domain-containing 5 (ABHD5), the defective gene in human Chanarin-Dorfman syndrome, is a highly conserved regulator of adipose triglyceride lipase (ATGL)-mediated lipolysis that plays important roles in metabolism, tumor progression, viral replication, and skin barrier formation. The structural determinants of ABHD5 lipolysis activation, however, are unknown. We performed comparative evolutionary analysis and structural modeling of ABHD5 and ABHD4, a functionally distinct paralog that diverged from ABHD5 ~500 million years ago, to identify determinants of ABHD5 lipolysis activation. Two highly conserved ABHD5 amino acids (R299 and G328) enabled ABHD4 (ABHD4 N303R/S332G) to activate ATGL in Cos7 cells, brown adipocytes, and artificial lipid droplets. The corresponding ABHD5 mutations (ABHD5 R299N and ABHD5 G328S) selectively disrupted lipolysis without affecting ATGL lipid droplet translocation or ABHD5 interactions with perilipin proteins and ABHD5 ligands, demonstrating that ABHD5 lipase activation could be dissociated from its other functions. Structural modeling placed ABHD5 R299/G328 and R303/G332 from gain-of-function ABHD4 in close proximity on the ABHD protein surface, indicating they form part of a novel functional surface required for lipase activation. These data demonstrate distinct ABHD5 functional properties and provide new insights into the functional evolution of ABHD family members and the structural basis of lipase regulation.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Lipólise/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Adipócitos Marrons/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Expressão Gênica , Técnicas de Silenciamento de Genes , Lipase/metabolismo , Gotículas Lipídicas , Lisofosfolipase/química , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The higher prevalence of obesity-related metabolic disease in males suggests that female sex hormones provide protective mechanisms against the pathogenesis of metabolic syndrome. Because browning of white adipose tissue (WAT) is protective against obesity-related metabolic disease, we examined sex differences in ß3-adrenergic remodeling of WAT in mice. METHODS: Effects of the ß3-adrenergic receptor agonist CL316,243 (CL) on browning of white adipose tissue were investigated in male and female C57BL mice. The role of ovarian hormones in female-specific browning was studied in control female C57BL mice and mice with ovarian failure induced by 4-vinylcyclohexene diepoxide treatment for 15 days. RESULTS: We found that treatment with CL-induced upregulation of brown adipocyte markers and mitochondrial respiratory chain proteins in gonadal WAT (gWAT) of female mice, but was without effect in males. In contrast, CL treatment was equally effective in males and females in inducing brown adipocyte phenotypes in inguinal WAT. The tissue- and sex-specific differences in brown adipocyte recruitment were correlated with differences in sympathetic innervation, as determined by tyrosine hydroxylase immunostaining and western blotting. Levels of the neurotrophins NGF and BDNF were significantly higher in gWAT of female mice. CL treatment significantly increased NGF levels in gWAT of female mice but did not affect BDNF expression. In contrast, estradiol treatment doubled BDNF expression in female adipocytes differentiated in vitro. Ovarian failure induced by 4-vinylcyclohexene diepoxide treatment dramatically reduced BDNF and TH expression in gWAT, eliminated induction of UCP1 by CL, and reduced tissue metabolic rate. CONCLUSIONS: Collectively, these data demonstrate that female mice are more responsive than males to the recruitment of brown adipocytes in gonadal WAT and this difference corresponds to greater levels of estrogen-dependent sympathetic innervation.
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De novo brown adipogenesis involves the proliferation and differentiation of progenitors, yet the mechanisms that guide these events in vivo are poorly understood. We previously demonstrated that treatment with a ß3-adrenergic receptor (ADRB3) agonist triggers brown/beige adipogenesis in gonadal white adipose tissue following adipocyte death and clearance by tissue macrophages. The close physical relationship between adipocyte progenitors and tissue macrophages suggested that the macrophages that clear dying adipocytes might generate proadipogenic factors. Flow cytometric analysis of macrophages from mice treated with CL 316,243 identified a subpopulation that contained elevated lipid and expressed CD44. Lipidomic analysis of fluorescence-activated cell sorting-isolated macrophages demonstrated that CD44+ macrophages contained four- to five-fold higher levels of the endogenous peroxisome-proliferator activated receptor gamma (PPARγ) ligands 9-hydroxyoctadecadienoic acid (HODE), and 13-HODE compared with CD44- macrophages. Gene expression profiling and immunohistochemistry demonstrated that ADRB3 agonist treatment upregulated expression of ALOX15, the lipoxygenase responsible for generating 9-HODE and 13-HODE. Using an in vitro model of adipocyte efferocytosis, we found that IL-4-primed tissue macrophages accumulated lipid from dying fat cells and upregulated expression of Alox15. Furthermore, treatment of differentiating adipocytes with 9-HODE and 13-HODE potentiated brown/beige adipogenesis. Collectively, these data indicate that noninflammatory removal of adipocyte remnants and coordinated generation of PPARγ ligands by M2 macrophages provides localized adipogenic signals to support de novo brown/beige adipogenesis.
Assuntos
Adipócitos Brancos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Comunicação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipócitos Brancos/patologia , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Morte Celular , Linhagem Celular , Separação Celular/métodos , Técnicas de Cocultura , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Ligantes , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Fagocitose/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), α-ß hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas de Transporte/metabolismo , Lipólise/genética , Fosfoproteínas/metabolismo , Proteínas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Células 3T3-L1 , Acil Coenzima A/metabolismo , Adipócitos/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Ligantes , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Perilipina-1 , Perilipina-5 , Fosfoproteínas/genética , Proteínas/genéticaRESUMO
This work investigated how cold stress induces the appearance of brown adipocytes (BAs) in brown and white adipose tissues (WATs) of adult mice. In interscapular brown adipose tissue (iBAT), cold exposure increased proliferation of endothelial cells and interstitial cells expressing platelet-derived growth factor receptor, α polypeptide (PDGFRα) by 3- to 4-fold. Surprisingly, brown adipogenesis and angiogenesis were largely restricted to the dorsal edge of iBAT. Although cold stress did not increase proliferation in inguinal white adipose tissue (ingWAT), the percentage of BAs, defined as multilocular adipocytes that express uncoupling protein 1, rose from undetectable to 30% of total adipocytes. To trace the origins of cold-induced BAs, we genetically tagged PDGFRα(+) cells and adipocytes prior to cold exposure, using Pdgfra-Cre recombinase estrogen receptor T2 fusion protein (CreER(T2)) and adiponectin-CreER(T2), respectively. In iBAT, cold stress triggered the proliferation and differentiation of PDGFRα(+) cells into BAs. In contrast, all newly observed BAs in ingWAT (5207 out of 5207) were derived from unilocular adipocytes tagged by adiponectin-CreER(T2)-mediated recombination. Surgical denervation of iBAT reduced cold-induced brown adipogenesis by >85%, whereas infusion of norepinephrine (NE) mimicked the effects of cold in warm-adapted mice. NE-induced de novo brown adipogenesis in iBAT was eliminated in mice lacking ß1-adrenergic receptors. These observations identify a novel tissue niche for brown adipogenesis in iBAT and further define depot-specific mechanisms of BA recruitment.
Assuntos
Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Temperatura Baixa/efeitos adversos , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Tecido Adiposo Marrom/inervação , Tecido Adiposo Marrom/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Norepinefrina/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Adrenérgicos beta 1/deficiência , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , SimpatectomiaRESUMO
Adipocyte lipolysis can increase the production of inflammatory cytokines such as interleukin-6 (IL-6) that promote insulin resistance. However, the mechanisms that link lipolysis with inflammation remain elusive. Acute activation of ß3-adrenergic receptors (ADRB3) triggers lipolysis and up-regulates production of IL-6 in adipocytes, and both of these effects are blocked by pharmacological inhibition of hormone-sensitive lipase. We report that stimulation of ADRB3 induces expression of sphingosine kinase 1 (SphK1) and increases sphingosine 1-phosphate production in adipocytes in a manner that also depends on hormone-sensitive lipase activity. Mechanistically, we found that adipose lipolysis-induced SphK1 up-regulation is mediated by the c-Jun N-terminal kinase (JNK)/activating protein-1 signaling pathway. Inhibition of SphK1 by sphingosine kinase inhibitor 2 diminished the ADRB3-induced IL-6 production both in vitro and in vivo. Induction of IL-6 by ADRB3 activation was suppressed by siRNA knockdown of Sphk1 in cultured adipocytes and was severely attenuated in Sphk1 null mice. Conversely, ectopic expression of SphK1 increased IL-6 expression in adipocytes. Collectively, these data demonstrate that SphK1 is a critical mediator in lipolysis-triggered inflammation in adipocytes.
Assuntos
Adipócitos/citologia , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipólise , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esfingolipídeos/química , Espectrometria de Massas em TandemRESUMO
Adult adipose tissue contains a large supply of progenitors that can renew fat cells for homeostatic tissue maintenance and adaptive growth or regeneration in response to external challenges. However, the in vivo mechanisms that control adipocyte progenitor behavior are poorly characterized. We recently demonstrated that recruitment of adipocyte progenitors by macrophages is a central feature of adipose tissue remodeling under various adipogenic conditions. Catabolic remodeling of white adipose tissue by ß3-adrenergic receptor stimulation requires anti-inflammatory M2-polarized macrophages to clear dying adipocytes and to recruit new brown adipocytes from progenitors. In this Extra Views article, we discuss in greater detail the cellular elements of adipogenic niches and report a strategy to isolate and characterize the subpopulations of macrophages and adipocyte progenitors that actively participate in adrenergic tissue remodeling. Further characterization of these subpopulations may facilitate identification of new cellular targets to improve metabolic and immune function of adipose tissue.