RESUMO
Perilipin-2 (PLIN2) can anchor to lipid droplets (LDs) and play a crucial role in regulating nascent LDs formation. Bimolecular fluorescence complementation (BiFC) and flow cytometry were examined to verify the PLIN2-CGI-58 interaction efficiency in bovine adipocytes. GST-Pulldown assay was used to detect the key site arginine315 function in PLIN2-CGI-58 interaction. Experiments were also examined to research these mutations function of PLIN2 in LDs formation during adipocytes differentiation, LDs were measured after staining by BODIPY, lipogenesis-related genes were also detected. Results showed that Leucine (L371A, L311A) and glycine (G369A, G376A) mutations reduced interaction efficiencies. Serine (S367A) mutations enhanced the interaction efficiency. Arginine (R315A) mutations resulted in loss of fluorescence in the cytoplasm and disrupted the interaction with CGI-58, as verified by pulldown assay. R315W mutations resulted in a significant increase in the number of LDs compared with wild-type (WT) PLIN2 or the R315A mutations. Lipogenesis-related genes were either up- or downregulated when mutated PLIN2 interacted with CGI-58. Arginine315 in PLIN2 is required for the PLIN2-CGI-58 interface and could regulate nascent LD formation and lipogenesis. This study is the first to study amino acids on the PLIN2 interface during interaction with CGI-58 in bovine and highlight the role played by PLIN2 in the regulation of bovine adipocyte lipogenesis.
Assuntos
Arginina , Gotículas Lipídicas , Animais , Bovinos , Perilipina-2/genética , Perilipina-2/química , Perilipina-2/metabolismo , Arginina/genética , Arginina/metabolismo , Gotículas Lipídicas/metabolismo , Mutação , Adipócitos/metabolismo , Metabolismo dos LipídeosRESUMO
Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be coactivated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein coactivation on a per-residue level. We identified the region from residues N209-N215 in ATGL as essential for coactivation by CGI-58. ATGL variants with amino acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 coactivation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the coactivation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for coactivation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58-mediated coactivation of ATGL.
Assuntos
Inteligência Artificial , Lipase , Animais , Camundongos , Lipase/metabolismo , Lipólise/fisiologia , Triglicerídeos/metabolismo , Aminoácidos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismoRESUMO
Cell death-inducing DNA fragmentation factor-like effector C (CIDEC) expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, E186X, a single-nucleotide CIDEC variant is associated with lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. We found that Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet-induced glucose intolerance. Furthermore, we revealed the role of CIDEC in lipid metabolism using transcriptomics and lipidomics. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in high-fat diet-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, we demonstrated that CIDEC regulates the enzymatic activity of adipose triglyceride lipase via interacting with its activator, CGI-58, to reduce free fatty acid release and lipotoxicity. In addition, we confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans, and treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid metabolism and whole-body glucose homeostasis. In summary, our findings identify human CIDEC as a potential 'drug' or a 'druggable' target to reverse obesity-induced lipotoxicity and glucose intolerance.
Assuntos
Intolerância à Glucose , Resistência à Insulina , Animais , Colesterol , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos não Esterificados , Glucose , Intolerância à Glucose/genética , Intolerância à Glucose/prevenção & controle , Humanos , Resistência à Insulina/genética , Lipase/genética , Metabolismo dos Lipídeos , Lipoproteínas LDL/metabolismo , Camundongos , Nucleotídeos/metabolismo , Obesidade/genética , Proteínas/metabolismo , Transgenes , TriglicerídeosRESUMO
Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.
Assuntos
Expressão Gênica , Lipase , Acilação , Animais , Células HEK293 , Humanos , Hidrólise , Lipase/biossíntese , Lipase/química , Lipase/genética , Lipase/isolamento & purificação , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , SpodopteraRESUMO
The Chanarin-Dorfman syndrome (CDS) is a rare, autosomal recessively inherited genetic disease. This syndrome is associated with a decrease in the lipolysis activity in multiple tissue cells because of recessive mutations in the abhydrolase domain containing 5 (ABHD5) gene, which leads to the accumulation of lipid droplets in multiple types of cells. Major clinical symptoms in patients with CDS include ichthyosis and intracytoplasmic lipid droplets. The variability of clinical symptoms in patients with CDS depends on a large number of mutations involved. In this syndrome, liver involvement is an important cause of mortality and morbidity. This review aims to summarize the demographic characteristic, clinical symptoms, liver involvement and mutations in CDS patients in the literature to date.
Assuntos
Eritrodermia Ictiosiforme Congênita , Erros Inatos do Metabolismo Lipídico , Doenças Musculares , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Humanos , Eritrodermia Ictiosiforme Congênita/diagnóstico , Eritrodermia Ictiosiforme Congênita/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Doenças Musculares/genéticaRESUMO
Comparative gene identification-58 (CGI-58), also known as α/ß-hydrolase domain-containing 5 (ABHD5), is a member of a large family of proteins containing an α/ß-hydrolase-fold. CGI-58 is well-known as the co-activator of adipose triglyceride lipase (ATGL), which is a key enzyme initiating cytosolic lipid droplet lipolysis. Mutations in either the human CGI-58 or ATGL gene cause an autosomal recessive neutral lipid storage disease, characterized by the excessive accumulation of triglyceride (TAG)-rich lipid droplets in the cytoplasm of almost all cell types. CGI-58, however, has ATGL-independent functions. Distinct phenotypes associated with CGI-58 deficiency commonly include ichthyosis (scaly dry skin), nonalcoholic steatohepatitis, and hepatic fibrosis. Through regulated interactions with multiple protein families, CGI-58 controls many metabolic and signaling pathways, such as lipid and glucose metabolism, energy balance, insulin signaling, inflammatory responses, and thermogenesis. Recent studies have shown that CGI-58 regulates the pathogenesis of common metabolic diseases in a tissue-specific manner. Future studies are needed to molecularly define ATGL-independent functions of CGI-58, including the newly identified serine protease activity of CGI-58. Elucidation of these versatile functions of CGI-58 may uncover fundamental cellular processes governing lipid and energy homeostasis, which may help develop novel approaches that counter against obesity and its associated metabolic sequelae.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Homeostase , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Humanos , Lipase/metabolismo , LipóliseRESUMO
BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition and the most common liver disease worldwide, affecting more than one-third of the population. So far there have been no reports on mendelian inheritance in families with NAFLD. METHODS: We performed whole-exome or targeted next-generation sequencing on patients with autosomal dominant NAFLD. RESULTS: We report a heritable form of NAFLD and/or dyslipidemia due to monoallelic ABHD5 mutations, with complete clinical expression after the fourth decade of life, in 7 unrelated multiplex families encompassing 39 affected individuals. The prevalence of ABHD5-associated NAFLD was estimated to be 1 in 1,137 individuals in a normal population. CONCLUSION: We associate a Mendelian form of NAFLD and/or dyslipidemia with monoallelic ABHD5 mutations. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a common multifactorial disorder with a strong genetic component. Inherited forms of NAFLD have been suspected but, their molecular pathogenesis has not been disclosed. Here we report a heritable form of NAFLD with clinical expression after 40 years of age, associated with monoallelic ABHD5 mutations.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Dislipidemias/genética , Predisposição Genética para Doença , Mutação , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Idoso , Alelos , Dislipidemias/complicações , Feminino , Frequência do Gene , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Linhagem , Prevalência , Sequenciamento do Exoma , Adulto JovemRESUMO
The aim of the present study was to investigate the time and intensity dependent effects of exercise on the heart components of the lipolytic complex. Wistar rats ran on a treadmill with the speed of 18 m/min for 30 min (M30) or 120 min (M120) or with the speed of 28 m/min for 30 min (F30). The mRNA and protein expressions of the compounds adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58), G0/G1 switch gene 2 (G0S2), hormone sensitive lipase (HSL) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were examined by real-time PCR and Western blot, respectively. Lipid content of free fatty acids (FFA), diacylglycerols (DG) and triacylglycerols (TG) were estimated by gas liquid chromatography. We observed virtually no changes in the left ventricle lipid contents and only minor fluctuations in its ATGL mRNA levels. This was in contrast with its right counterpart i.e., the content of TG and DG decreased in response to both increased duration and intensity of a run. This occurred in tandem with increased mRNA expression for ATGL, CGI-58 and decreased expression of G0S2. It is concluded that exercise affects behavior of the components of the lipolytic system and the lipid content in the heart ventricles. However, changes observed in the left ventricle did not mirror those in the right one.
Assuntos
Ventrículos do Coração/metabolismo , Lipólise , Esforço Físico , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Ácidos Graxos não Esterificados/metabolismo , Lipase/genética , Lipase/metabolismo , Masculino , Especificidade de Órgãos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Wistar , Esterol Esterase/genética , Esterol Esterase/metabolismo , Triglicerídeos/metabolismoRESUMO
Mutations in the gene encoding comparative gene identification 58 (CGI-58), also known as α ß hydrolase domain-containing 5 (ABHD5), cause neutral lipid storage disorder with ichthyosis (NLSDI). This inborn error in metabolism is characterized by ectopic accumulation of triacylglycerols (TAG) within cytoplasmic lipid droplets in multiple cell types. Studies over the past decade have clearly demonstrated that CGI-58 is a potent regulator of TAG hydrolysis in the disease-relevant cell types. However, despite the reproducible genetic link between CGI-58 mutations and TAG storage, the molecular mechanisms by which CGI-58 regulates TAG hydrolysis are still incompletely understood. It is clear that CGI-58 can regulate TAG hydrolysis by activating the major TAG hydrolase adipose triglyceride lipase (ATGL), yet CGI-58 can also regulate lipid metabolism via mechanisms that do not involve ATGL. This review highlights recent progress made in defining the physiologic and biochemical function of CGI-58, and its broader role in energy homeostasis. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Metabolismo Energético , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Animais , Humanos , Hidrólise , Eritrodermia Ictiosiforme Congênita/genética , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Lipase/genética , Lipase/metabolismo , Gotículas Lipídicas/patologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Triglicerídeos/genéticaRESUMO
Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for ß-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp(21) and Trp(25)) and right (harboring Trp(29)) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp(21) and Trp(25) and two adjacent leucines. Trp(29) serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Humanos , Lipase/genética , Deleção de Sequência , Triglicerídeos/genética , Triglicerídeos/metabolismoRESUMO
Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria; however, this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10; age 51-53 days old) were progressively treadmill trained at a 10% incline for 8 wk ending with 25 m/min for 1 h compared with control. Sciatic nerve stimulation for hind-limb muscle contraction (and lipolysis) was administered for 30 min to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red gastrocnemius (RG), and white gastrocnemius were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG-isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction; therefore, any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content but through more complicated intracellular mechanisms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Lipase/metabolismo , Contração Muscular , Músculo Esquelético/enzimologia , Condicionamento Físico Animal , Resistência Física , Animais , Estimulação Elétrica , Lipólise , Masculino , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/inervação , Oxirredução , Ratos Sprague-Dawley , Nervo Isquiático/fisiologia , Fatores de Tempo , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
Survival in a terrestrial, dry environment necessitates a permeability barrier for regulated permeation of water and electrolytes in the cornified layer of the skin (the stratum corneum) to minimize desiccation of the body. This barrier is formed during cornification and involves a cross-linking of corneocyte proteins as well as an extensive remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by various hydrolytic enzymes generates ceramides, cholesterol, and non-esterified fatty acids for the extracellular lipid lamellae in the stratum corneum. However, the important role of epidermal triacylglycerol (TAG) metabolism during formation of a functional permeability barrier in the skin was only recently discovered. Humans with mutations in the ABHD5/CGI-58 (α/ß hydrolase domain containing protein 5, also known as comparative gene identification-58, CGI-58) gene suffer from a defect in TAG catabolism that causes neutral lipid storage disease with ichthyosis. In addition, mice with deficiencies in genes involved in TAG catabolism (Abhd5/Cgi-58 knock-out mice) or TAG synthesis (acyl-CoA:diacylglycerol acyltransferase-2, Dgat2 knock-out mice) also develop severe skin permeability barrier dysfunctions and die soon after birth due to increased dehydration. As a result of these defects in epidermal TAG metabolism, humans and mice lack ω-(O)-acylceramides, which leads to malformation of the cornified lipid envelope of the skin. In healthy skin, this epidermal structure provides an interface for the linkage of lamellar membranes with corneocyte proteins to maintain permeability barrier homeostasis. This review focuses on recent advances in the understanding of biochemical mechanisms involved in epidermal neutral lipid metabolism and the generation of a functional skin permeability barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Animais , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Epiderme , Humanos , Eritrodermia Ictiosiforme Congênita/genética , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Camundongos , Camundongos Knockout , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Permeabilidade , Dermatopatias Metabólicas/genética , Dermatopatias Metabólicas/metabolismo , Dermatopatias Metabólicas/patologia , Triglicerídeos/genéticaRESUMO
BACKGROUND & AIMS: Adipose tissue (AT)-derived fatty acids (FAs) are utilized for hepatic triacylglycerol (TG) generation upon fasting. However, their potential impact as signaling molecules is not established. Herein we examined the role of exogenous AT-derived FAs in the regulation of hepatic gene expression by investigating mice with a defect in AT-derived FA supply to the liver. METHODS: Plasma FA levels, tissue TG hydrolytic activities and lipid content were determined in mice lacking the lipase co-activator comparative gene identification-58 (CGI-58) selectively in AT (CGI-58-ATko) applying standard protocols. Hepatic expression of lipases, FA oxidative genes, transcription factors, ER stress markers, hormones and cytokines were determined by qRT-PCR, Western blotting and ELISA. RESULTS: Impaired AT-derived FA supply upon fasting of CGI-58-ATko mice causes a marked defect in liver PPARα-signaling and nuclear CREBH translocation. This severely reduced the expression of respective target genes such as the ATGL inhibitor G0/G1 switch gene-2 (G0S2) and the endocrine metabolic regulator FGF21. These changes could be reversed by lipid administration and raising plasma FA levels. Impaired AT-lipolysis failed to induce hepatic G0S2 expression in fasted CGI-58-ATko mice leading to enhanced ATGL-mediated TG-breakdown strongly reducing hepatic TG deposition. On high fat diet, impaired AT-lipolysis counteracts hepatic TG accumulation and liver stress linked to improved systemic insulin sensitivity. CONCLUSIONS: AT-derived FAs are a critical regulator of hepatic fasting gene expression required for the induction of G0S2-expression in the liver to control hepatic TG-breakdown. Interfering with AT-lipolysis or hepatic G0S2 expression represents an effective strategy for the treatment of hepatic steatosis.
Assuntos
Tecido Adiposo/metabolismo , Jejum/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/genética , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Fígado/metabolismo , Animais , Western Blotting , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fatores de Crescimento de Fibroblastos/biossíntese , Genes de Troca , Fígado/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , RNA/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1ß. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Fígado Gorduroso/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/deficiência , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Animais , Fígado Gorduroso/patologia , Feminino , Fígado/patologia , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND & AIMS: Autophagy-related 14 (ATG14) is a key regulator of autophagy. ATG14 is also localized to lipid droplet; however, the function of ATG14 on lipid droplet remains unclear. In this study, we aimed to elucidate the role of ATG14 in lipid droplet homeostasis. METHODS: ATG14 loss-of-function and gain-of-function in lipid droplet metabolism were analyzed by fluorescence imaging in ATG14 knockdown or overexpression hepatocytes. Specific domains involved in the ATG14 targeting to lipid droplets were analyzed by deletion or site-specific mutagenesis. ATG14-interacting proteins were analyzed by co-immunoprecipitation. The effect of ATG14 on lipolysis was analyzed in human hepatocytes and mouse livers that were deficient in ATG14, comparative gene identification-58 (CGI-58), or both. RESULTS: Our data show that ATG14 is enriched on lipid droplets in hepatocytes. Mutagenesis analysis reveals that the Barkor/ATG14 autophagosome targeting sequence (BATS) domain of ATG14 is responsible for the ATG14 localization to lipid droplets. Co-immunoprecipitation analysis illustrates that ATG14 interacts with adipose triglyceride lipase (ATGL) and CGI-58. Moreover, ATG14 also enhances the interaction between ATGL and CGI-58. In vitro lipolysis analysis demonstrates that ATG14 deficiency remarkably decreases triglyceride hydrolysis. CONCLUSIONS: Our data suggest that ATG14 can directly enhance lipid droplet breakdown through interactions with ATGL and CGI-58.
Assuntos
Lipase , Gotículas Lipídicas , Camundongos , Animais , Humanos , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Lipólise , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Homeostase , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismoRESUMO
Comparative gene identification-58 (CGI-58) is an activating protein of triacylglycerol (TAG) lipase. It has a variety of catalytic activities whereby it may play different roles in diverse organisms. In this study, a homolog of CGI-58 in Phaeodactylum tricornutum (PtCGI-58) was identified. PtCGI-58 was localized in mitochondria by GFP fusion protein analysis, which is different from the reported subcellular localization of CGI-58 in animals and plants. Respectively, PtCGI-58 overexpression resulted in increased neutral lipid content and TAG accumulation by 42-46% and 21-32%. Likewise, it also increased the relative content of eicosapentaenoic acid (EPA), and in particular, the EPA content in TAGs almost doubled. Transcript levels of genes involved in de novo fatty acid synthesis and mitochondrial ß-oxidation were significantly upregulated in PtCGI-58 overexpression strains compared with wild-type cells. Our findings suggest that PtCGI-58 may mediate the breakdown of lipids in mitochondria and the recycling of acyl chains derived from mitochondrial ß-oxidation into TAG biosynthesis. Moreover, this study potentially illuminates new functions for CGI-58 in lipid homeostasis and provides a strategy to enrich EPA in algal TAGs.
Assuntos
Lipase , Triglicerídeos/metabolismoRESUMO
Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/ß hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.
Assuntos
Acil Coenzima A , Transferência Ressonante de Energia de Fluorescência , Acil Coenzima A/metabolismo , Lipólise/fisiologia , Lipase/genética , Ácidos GraxosRESUMO
The α/ß-Hydrolase domain-containing protein 5 (ABHD5; also known as comparative gene identification-58, or CGI-58) is the causative gene of the Chanarin-Dorfman syndrome (CDS), a disorder mainly characterized by systemic triacylglycerol accumulation and a severe defect in skin barrier function. The clinical phenotype of CDS patients and the characterization of global and tissue-specific ABHD5-deficient mouse strains have demonstrated that ABHD5 is a crucial regulator of lipid and energy homeostasis in various tissues. Although ABHD5 lacks intrinsic hydrolase activity, it functions as a co-activating enzyme of the patatin-like phospholipase domain-containing (PNPLA) protein family that is involved in triacylglycerol and glycerophospholipid, as well as sphingolipid and retinyl ester metabolism. Moreover, ABHD5 interacts with perilipins (PLINs) and fatty acid-binding proteins (FABPs), which are important regulators of lipid homeostasis in adipose and non-adipose tissues. This review focuses on the multifaceted role of ABHD5 in modulating the function of key enzymes in lipid metabolism.
RESUMO
The Chanarin-Dorfman syndrome (CDS) is a rare, autosomal recessively inherited genetic disease, whch is associated with a decrease in the lipolysis activity in multiple tissue cells. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears, skeletal muscle and central nervous system. Mutations in ABHD5/CGI58 gene have been confirmed to be associated with CDS. We performed whole exome sequencing on a Chinese CDS patient with skin ichthyosis features mimicking lamellar ichthyosis, ectropion, sensorineural hearing loss, and lipid storage in peripheral blood neutrophils. A novel homozygous missense mutation (p.L154R) in ABHD5 gene was detected in this patient. Genotype-phenotype analysis in reported CDS patients revealed no particular correlation. Our findings further enrich the reservoir of ABHD5 mutations in CDS.
RESUMO
Tiaoganquzhi Decoction (TGQZD) is a traditional Chinese herbal formulation demonstrated to be a clinically effective treatment for nonalcoholic fatty liver disease (NAFLD), although details concerning its clinical mechanism are poor. This study aimed to explore the mechanism of TGQZD on improvement of inflammatory damage and dyslipidemia caused by NAFLD through the CGI-58/ROS/NLRP3 inflammasome pathway. In our research, the in vivo protective effects of TGQZD on HFD-induced liver injury in rats and in vitro using lipopolysaccharide (LPS)+palmitate (PA)-stimulated HepG-2 cells model. Histological changes were evaluated by hematoxylin-eosin and Oil Red O staining. Inflammatory cytokines and protein expression were analyzed by ELISA, Real time PCR and western blotting. Liver function, blood lipids, free fatty acids (FFA), and reactive oxygen species (ROS) were determined by biochemical detection. Our results indicated that TGQZD exhibited anti-inflammatory activity, reduced the severity of NAFLD and ameliorated the pathological changes. Further, TGQZD improved liver function and lipid metabolism in NAFLD rats. TGQZD lowered serum aspartate aminotransferase, alanine aminotransferase, triglyceride, and total cholesterol levels. TGQZD suppressed the formulation of FFA and ROS. It also reduced the expression and release of the inflammatory cytokine interleukin-1ß by promoting CGI-58 expression and inhibiting the expression of FFA, TNF-α, and the NLRP3 inflammasome induced by ROS. TGQZD exhibited anti-inflammatory effects via the CGI-58, ROS and NLRP3 inflammasome pathway in vivo and in vitro, respectively. Our findings demonstrated that TGQZD is a useful and effective therapeutic agent for treating NAFLD via promotion of CGI-58 to inhibit the expression of ROS-induced NLRP3 inflammasome.