RESUMO
Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.
Assuntos
Células Lúteas , Perilipina-2 , Progesterona , Animais , Feminino , Bovinos , Progesterona/metabolismo , Perilipina-2/metabolismo , Perilipina-2/genética , Células Lúteas/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Perilipina-3/metabolismo , Corpo Lúteo/metabolismo , Células CultivadasRESUMO
Perilipins (PLINs) constitute an evolutionarily conserved family of proteins that specifically associate with the surface of lipid droplets (LDs). These proteins function in LD biogenesis and lipolysis and help to stabilize the surface of LDs. PLINs are typically composed of three different protein domains. They share an N-terminal PAT domain of unknown structure and function, a central region containing 11-mer repeats that form amphipathic helices, and a C-terminal domain that adopts a 4-helix bundle structure. How exactly these three distinct domains contribute to PLIN function remains to be determined. Here, we show that the N-terminal PAT domain of PLIN3 binds diacylglycerol (DAG), the precursor to triacylglycerol, a major storage lipid of LDs. PLIN3 and its PAT domain alone bind liposomes with micromolar affinity and PLIN3 binds artificial LDs containing low concentrations of DAG with nanomolar affinity. The PAT domain of PLIN3 is predicted to adopt an amphipathic triangular shaped structure. In silico ligand docking indicates that DAG binds to one of the highly curved regions within this domain. A conserved aspartic acid residue in the PAT domain, E86, is predicted to interact with DAG, and we found that its substitution abrogates high affinity binding of DAG as well as DAG-stimulated association with liposome and artificial LDs. These results indicate that the PAT domain of PLINs harbor specific lipid-binding properties that are important for targeting these proteins to the surface of LDs and to ER membrane domains enriched in DAG to promote LD formation.
Assuntos
Diglicerídeos , Perilipina-3 , Diglicerídeos/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise , Perilipina-1 , Perilipina-2/metabolismo , Perilipina-3/química , Perilipina-3/metabolismo , Domínios Proteicos , Proteínas/metabolismo , HumanosRESUMO
The indigenous microbial milieu within tumorous tissues exerts a pivotal influence on the genesis and advancement of gastric cancer (GC). This investigation scrutinizes the functions and molecular mechanisms attributed to Prevotella intermedia in the malignant evolution of GC. Isolation of P. intermedia from paired GC tissues was undertaken. Quantification of P. intermedia abundance in 102 tissues was accomplished using quantitative real-time PCR (qRT-PCR). Assessment of the biological effects of P. intermedia on GC cells was observed using culture medium supernatant. Furthermore, the protein profile of GC cells treated with tumor-derived P. intermedia was examined through label-free protein analysis. The functionality of perilipin 3 (PLIN3) was subsequently confirmed using shRNA. Our investigation revealed that the relative abundance of P. intermedia in tumor tissues significantly surpassed that of corresponding healthy tissues. The abundance of P. intermedia exhibited correlations with tumor differentiation (p = 0.006), perineural invasion (p = 0.004), omentum majus invasion (p = 0.040), and the survival duration of GC patients (p = 0.042). The supernatant derived from tumor-associated P. intermedia bolstered the proliferation, clone formation, migration, and invasion of GC cells. After indirect co-cultivation with tumor-derived P. intermedia, dysregulation of 34 proteins, including PLIN3, was discerned in GC cells. Knockdown of PLIN3 mitigated the malignancy instigated by P. intermedia in GC cells. Our findings posit that P. intermedia from the tumor microenvironment plays a substantial role in the malignant progression of GC via the modulation of PLIN3 expression. Moreover, the relative abundance of P. intermedia might serve as a potential biomarker for the diagnosis and prognosis of GC.
Assuntos
Neoplasias Gástricas , Humanos , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Perilipina-3 , Prevotella intermedia , Prognóstico , Neoplasias Gástricas/patologia , Microambiente TumoralRESUMO
BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Gotículas Lipídicas/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Oncogenes , Perilipina-3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Microambiente TumoralRESUMO
Rho GTPases are key regulators of the actin-based cytoskeleton; Rab GTPases are key regulators of membrane traffic. We report here that the atypical Rho GTPase family member, RhoBTB3, binds directly to Rab9 GTPase and functions with Rab9 in protein transport from endosomes to the trans Golgi network. Gene replacement experiments show that RhoBTB3 function in cultured cells requires both RhoBTB3's N-terminal, Rho-related domain and C-terminal sequences that are important for Rab9 interaction. Biochemical analysis reveals that RhoBTB3 binds and hydrolyzes ATP rather than GTP. Rab9 binding opens the autoinhibited RhoBTB3 protein to permit maximal ATP hydrolysis. Because RhoBTB3 interacts with TIP47 on membranes, we propose that it may function to release this cargo selection protein from vesicles to permit their efficient docking and fusion at the Golgi.
Assuntos
Endossomos/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Proteínas rho de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Perilipina-3 , Proteínas da Gravidez/metabolismo , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Perilipin 3 (PLIN3), a lipid droplet-associated protein, is found to be highly expressed in human cancers. This study aimed to investigate the biological functions and underlying mechanism of PLIN3 in lung adenocarcinoma (LUAD). METHODS: To analyse PLIN3 expression in normal and cancerous tissues, relevance between PLIN3 expression and survival prognosis, and to predict the pathways related to PLIN3, bioinformatic analysis was performed. In A549 and H1299 cells, qRT-PCR or western blotting was used to determine mRNA/protein expression of PLIN3, PD-L1, and c-Myc. In A549 and H1299 cells, CCK-8 assay, EdU, and flow cytometry were used to assess cell viability, proliferation, and apoptosis. Chip and luciferase reporter assays were performed to verify the binding of PD-L1 with c-Myc. The functions of PLIN3 were examined in vivo in a xenograft tumor model. RESULTS: In LUAD tissues and cells, PLIN3 expression was downregulated. A shorter survival time was observed in patients with high PLIN3 expression than in patients with low PLIN3 expression. Silencing of PLIN3 inhibited cell proliferation, PD-L1 expression, and Myc pathway, as well as induced apoptosis in LUAD cells. c-Myc acts as a transcription factor of PD-L1. Moreover, the inhibitory actions of PLIN3 silencing on c-Myc and PD-L1 expression as well as cell proliferation and stimulatory action of PLIN3 silencing on cell apoptosis were reversed by c-Myc overexpression. In vivo, PLIN3 silencing inhibited the growth of xenograft tumour and reduced PLIN3, PD-L1, and c-Myc protein expression. CONCLUSION: Silencing of PLIN3 inhibited tumour growth by regulating the Myc/PD-L1 pathway.
1. Silencing of PLIN3 inhibited tumour growth in vivo and in vitro.2. Silencing of PLIN3 inhibited PD-L1 expression and Myc pathway in LUAD cells.3. c-Myc acted as a transcription factor of PD-L1.4. Silencing of PLIN3 exerted anti-tumour effects by regulating c-Myc/PD-L1.
Assuntos
Adenocarcinoma de Pulmão , Adenocarcinoma , Neoplasias Pulmonares , Humanos , Adenocarcinoma de Pulmão/genética , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Pulmonares/metabolismo , Perilipina-3RESUMO
PURPOSE: Smaller lipid droplet morphology and GLUT 4 protein expression have been associated with greater muscle oxidative capacity and glucose uptake, respectively. The main purpose of this study was to determine the effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT4, perilipin 3, and perilipin 5 expressions. METHODS: Twenty healthy men (age 24.0 ± 1.0 years, BMI 23.6 ± 0.4 kg/m2) were recruited for the study. The participants were subjected to an acute bout of exercise on a cycle ergometer at 50% VO2max until they reached a total energy expenditure of 650 kcal. The study was conducted after an overnight fast. Vastus lateralis muscle biopsies were obtained before and immediately after exercise for immunohistochemical analysis to determine lipid, perilipin 3, perilipin 5, and GLUT4 protein contents while GLUT 4 mRNA was quantified using RT-qPCR. RESULTS: Lipid droplet size decreased whereas total intramyocellular lipid content tended to reduce (p = 0.07) after an acute bout of endurance exercise. The density of smaller lipid droplets in the peripheral sarcoplasmic region significantly increased (0.584 ± 0.04 to 0.638 ± 0.08 AU; p = 0.01) while larger lipid droplets significantly decreased (p < 0.05). GLUT4 mRNA tended to increase (p = 0.05). There were no significant changes in GLUT 4, perilipin 3, and perilipin 5 protein levels. CONCLUSION: The study demonstrates that exercise may impact metabolism by enhancing the quantity of smaller lipid droplets over larger lipid droplets.
Assuntos
Gotículas Lipídicas , Perilipina-5 , Masculino , Humanos , Adulto Jovem , Adulto , Perilipina-1/metabolismo , Gotículas Lipídicas/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Perilipina-5/metabolismo , Perilipina-3/metabolismo , Músculo Esquelético/fisiologia , Lipídeos , RNA Mensageiro/metabolismo , Metabolismo dos Lipídeos/fisiologiaRESUMO
BACKGROUND AND AIMS: NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD. APPROACH AND RESULTS: We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation. CONCLUSIONS: These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD.
Assuntos
Autofagia , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Perilipina-3/metabolismo , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
For rapid and unlimited cell growth and proliferation, cancer cells require large quantities of nutrients. Many metabolic pathways and nutrient uptake systems are frequently reprogrammed and upregulated to meet the demand from cancer cells, including the demand for lipids. The lipids for most adult normal cells are mainly acquired from the circulatory system. Whether different cancer cells adopt identical mechanisms to ensure sufficient lipid supply, and whether the lipid demand and supply meet each other, remains unclear, and was investigated in lung cancer cells. Results showed that, despite frequent upregulation in de novo lipogenesis and the lipid transporter system, different lung cancer cells adopt different proteins to acquire sufficient lipids, and the lipid supply frequently exceeds the demand, as significant amounts of lipids stored in the lipid droplets could be found within lung cancer cells. Lipid droplet surface protein, PLIN3, was found frequently overexpressed since the early stage in lung cancer tissues. Although the expression is not significantly associated with a specific gender, age, histology type, disease stage, and smoking habit, the frequently elevated expression of PLIN3 protein indicates the importance of lipid droplets for lung cancer. These lipid droplets are not only for nutrient storage, but are also crucial for tumor growth and proliferation, as well as survival in starvation. These results suggest that manipulation of lipid droplet formation or TG storage in lung cancer cells could potentially decrease the progression of lung cancer. Further exploration of lipid biology in lung cancer could help design novel treatment strategies.
Assuntos
Neoplasias Pulmonares , Inanição , Adulto , Humanos , Gotículas Lipídicas/metabolismo , Perilipina-3/metabolismo , Metabolismo dos Lipídeos , Proliferação de Células , Proteínas de Membrana/metabolismo , Inanição/metabolismo , Neoplasias Pulmonares/metabolismo , Lipídeos/fisiologiaRESUMO
Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.
Assuntos
Autofagia/fisiologia , Desenvolvimento Embrionário/fisiologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Animais , Técnicas de Cultura de Células , Citometria de Fluxo , Immunoblotting , Lipólise/fisiologia , Camundongos , Microscopia de Fluorescência , Perilipina-3/metabolismoRESUMO
KEY POINTS: We have recently shown that a high-fat, high-calorie (HFHC) diet decreases whole body glucose clearance without impairing skeletal muscle insulin signalling, in healthy lean individuals. These diets are also known to increase skeletal muscle IMTG stores, but the effect on lipid metabolites leading to skeletal muscle insulin resistance has not been investigated. This study measured the effect of 7 days' HFHC diet on (1) skeletal muscle concentration of lipid metabolites, and (2) potential changes in the perilipin (PLIN) content of the lipid droplets storing intramuscular triglyceride (IMTG). The HFHC diet increased PLIN3 protein expression and redistributed PLIN2 to lipid droplet stores in type I fibres. The HFHC diet increased IMTG content in type I fibres, while lipid metabolite concentrations remained the same. The data suggest that the increases in IMTG stores assists in reducing the accumulation of lipid metabolites known to contribute to skeletal muscle insulin resistance. ABSTRACT: A high-fat, high-calorie (HFHC) diet reduces whole body glucose clearance without impairing skeletal muscle insulin signalling in healthy lean individuals. HFHC diets also increase skeletal muscle lipid stores. However, unlike certain lipid metabolites, intramuscular triglyceride (IMTG) stored within lipid droplets (LDs) does not directly contribute to skeletal muscle insulin resistance. Increased expression of perilipin (PLIN) proteins and colocalisation to LDs has been shown to assist in IMTG storage. We aimed to test the hypothesis that 7 days on a HFHC diet increases IMTG content while minimising accumulation of lipid metabolites known to disrupt skeletal muscle insulin signalling in sedentary and obese individuals. We also aimed to identify changes in expression and subcellular distribution of proteins involved in IMTG storage. Muscle biopsies were obtained from the m. vastus lateralis of 13 (11 males, 2 females) healthy lean individuals (age: 23 ± 2.5 years; body mass index: 24.5 ± 2.4 kg m-2 ), following an overnight fast, before and after consuming a high-fat (64% energy), high-calorie (+47% kcal) diet for 7 days. After the HFHC diet, IMTG content increased in type I fibres only (+101%; P < 0.001), whereas there was no change in the concentration of either total diacylglycerol (P = 0.123) or total ceramides (P = 0.150). Of the PLINs investigated, only PLIN3 content increased (+50%; P < 0.01) solely in type I fibres. LDs labelled with PLIN2 increased (+80%; P < 0.01), also in type I fibres only. We propose that these adaptations of LDs support IMTG storage and minimise accumulation of lipid metabolites to protect skeletal muscle insulin signalling following 7 days' HFHC diet.
Assuntos
Dieta Hiperlipídica , Resistência à Insulina , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Perilipinas/metabolismo , Triglicerídeos/análise , Adulto , Feminino , Humanos , Masculino , Perilipina-2 , Perilipina-3 , Adulto JovemRESUMO
Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg-1·min-1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (-53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers (P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.
Assuntos
Atletas , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/biossíntese , Músculo Esquelético/fisiologia , Perilipinas/metabolismo , Adulto , Ciclismo/fisiologia , Biópsia , Exercício Físico/fisiologia , Humanos , Masculino , Fibras Musculares de Contração Lenta/fisiologia , Perilipina-2/genética , Perilipina-2/metabolismo , Perilipina-3/genética , Perilipina-3/metabolismo , Perilipina-5/genética , Perilipina-5/metabolismo , Resistência Física , Triglicerídeos/metabolismo , Adulto JovemRESUMO
Hepatic lipid accumulation is the most common pathological characteristic of alcoholic liver disease (ALD). In mammalian cells, excess neutral lipids are stored in lipid droplets (LDs). As a member of perilipin family proteins, Plin3 was recently found to regulate the LD biogenesis. However, the roles and mechanism of Plin3 in ALD progression remain unclear. Herein, we found that alcohol stimulated Plin3 expression in both mouse livers and cultured AML12 mouse hepatic cells, which was accompanied by excess LD accumulation in hepatocytes. The elevations of Plin3 in alcohol-treated hepatocytes paralleled with the levels of both PPARα and γ, and the protein degradation of Plin3 was also reduced after alcohol exposure. Moreover, Plin3 knockdown increased cellular sensitivity to alcohol-induced apoptosis, endoplasmic reticulum (ER) stress, and inflammatory cytokines release, including TNF-α, IL-1, and IL-6ß. Notably, alcohol exacerbated triglycerides (TG) accumulation in the ER and caused ER dilation in Plin3-knockdown AML12 cells. Finally, we observed that Plin3 interacted with dynein subunit Dync1i1 and mediated the colocalization of LDs and microtubules, while high concentration of alcohol disrupted microtubules and caused dispersion of excess small LDs in cytoplasm. Summarily, Plin3 promotes lipid export from the ER and reduces ER lipotoxic stress, thereby, protecting against alcoholic liver injury. Moreover, Plin3 could be an adapter protein mediating LD transport by microtubules. This study explored the roles of Plin3 in alcohol-induced hepatic injury, suggesting Plin3 as a potential target for the prevention of ALD progression.
Assuntos
Retículo Endoplasmático/metabolismo , Etanol/efeitos adversos , Hepatócitos/citologia , Perilipina-3/metabolismo , Animais , Linhagem Celular , Dineínas do Citoplasma/metabolismo , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Camundongos , Modelos Biológicos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Perilipina-3/genética , Triglicerídeos/metabolismoRESUMO
The presence of lipid droplets (LD) and the utilization of fatty acids (FA) as a source of energy are Sertoli cell (SC) putative characteristics. It is well known that SCs can phagocyte and degrade apoptotic germ cells (AGC) resulting in increasing lipid content and ATP levels. A relationship between the regulation of lipid storage and of lipid oxidation in SC might be envisaged. The aim of this study was to analyze whether AGC and FA are able to simultaneously regulate molecular mechanisms involved in lipid storage and in FA oxidation in SC. The experimental model utilized in this study consisted in SC cultures obtained from 20-day-old rats that were co-cultured with AGC or treated with palmitic acid (PA, 500 µM) for 24 and 48 h. AGC and PA increase LD, triacylglycerol (TAG) content and mRNA levels of Plin1, Plin2, Plin3 (proteins involved in TAG storage). Simultaneously, AGC and PA rise the extent of FA oxidation and mRNA levels of Cpt1 and Lcad (proteins involved in FA degradation). Results also show that peroxisome proliferator-activated receptor (PPAR) transcriptional activity, transcription factor which participate in lipid metabolism regulation, increases by AGC and PA treatment in SC. Additionally, the presence of a PPARg antagonist decreases the upregulation of LD content and Plin1 expression. Similarly, the presence of a PPARb/d antagonist reduces the increase in FA oxidation and Cpt1 mRNA levels. Altogether these results suggest that AGC and FA, which probably generate PPAR ligands, regulate lipid storage and fatty acid utilization, contributing to the energy homeostasis in the seminiferous tubules.
Assuntos
Apoptose , Comunicação Celular , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/farmacologia , Células de Sertoli/efeitos dos fármacos , Espermatozoides/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Técnicas de Cocultura , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Oxirredução , Ácido Palmítico/metabolismo , Perilipina-1/genética , Perilipina-1/metabolismo , Perilipina-2/genética , Perilipina-2/metabolismo , Perilipina-3/genética , Perilipina-3/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos Sprague-Dawley , Células de Sertoli/metabolismo , Transdução de Sinais , Espermatozoides/patologia , Triglicerídeos/metabolismoRESUMO
Lipid droplets (LDs) are the main energy resource for porcine preimplantation embryonic development. PLIN3 has been implicated in LD formation and regulation. Therefore, this study aimed to detect the dynamic pattern of PLIN3 in pig oocytes and cumulus cells (CC) during in vitro maturation (IVM), and to determine the relationship between PLIN3 and LD content. IVM with cumulus-enclosed oocytes (CEO), cumulus-denuded oocytes (DO) and the CCs denuded from the corresponding oocytes (DCC) was performed in porcine follicular fluid (PFF) or PFF-free optimized medium. DO and the DCC were cultured together under the same conditions as described above, while the DO was named DTO and the DCC was named DTCC in this group. Firstly, our results revealed LDs distributed widely in oocytes and CC, while the PLIN3 protein coated these LDs and spread out ubiquitously in the cytoplasm. Secondly, not only the mRNA level but also at protein level of PLIN3 in immature naked oocytes (IO) was higher than that in matured CEO, DO and DTO. Although PLIN3 was expressed at lower levels in CC from immature oocytes (ICC), the protein level of PLIN3 was comparably higher in the ECC and DCC groups. The triglyceride (TG) content in CEO and DO was significantly less abundant compared with that in IO. Therefore, our results indicated that co-culturing of oocytes and CC might affect PLIN3 expression levels in CC but not in oocytes. Lipid accumulation in pig oocytes during maturation might be affected by PLIN3 cross-talk between oocytes and CC.
Assuntos
Células do Cúmulo/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Perilipina-3/metabolismo , Animais , Técnicas de Cocultura , Feminino , Líquido Folicular , Oócitos/metabolismo , Perilipina-3/genética , Suínos , Triglicerídeos/metabolismoRESUMO
Hepatitis C virus (HCV) is an enveloped RNA virus responsible for 170 million cases of viral hepatitis worldwide. Over 50% of chronically infected HCV patients develop hepatic steatosis, and steatosis can be induced by expression of HCV core protein (core) alone. Additionally, core must associate with cytoplasmic lipid droplets (LDs) for steatosis development and viral particle assembly. Due to the importance of the LD as a key component of hepatic lipid storage and as a platform for HCV particle assembly, it seems this dynamic subcellular organelle is a gatekeeper in the pathogenesis of viral hepatitis. Here, we hypothesized that core requires the host LD scaffold protein, perilipin (PLIN)3, to induce hepatic steatosis. To test our hypothesis in vivo, we have studied core-induced hepatic steatosis in the absence or presence of antisense oligonucleotide-mediated knockdown of PLIN3. PLIN3 knockdown blunted HCV core-induced steatosis in transgenic mice fed either chow or a moderate fat diet. Collectively, our studies demonstrate that the LD scaffold protein, PLIN3, is essential for HCV core-induced hepatic steatosis and provide new insights into the pathogenesis of HCV.
Assuntos
Fígado Gorduroso/genética , Hepatite C/metabolismo , Fígado/metabolismo , Perilipina-3/genética , Animais , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Genótipo , Hepacivirus , Hepatite C/genética , Hepatite C/patologia , Hepatite C/virologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Gotículas Lipídicas/virologia , Metabolismo dos Lipídeos/genética , Fígado/patologia , Fígado/virologia , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Perilipina-3/antagonistas & inibidoresRESUMO
Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs inSaccharomyces cerevisiae,demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targetingin vivoandin vitro Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.
Assuntos
Proteínas de Transporte/química , Gotículas Lipídicas/química , Proteínas de Membrana/química , Fosfoproteínas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Perilipina-1 , Perilipina-2 , Perilipina-3 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Alinhamento de Sequência , Transgenes , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Hepatitis C Virus (HCV) promotes lipid droplet (LD) formation and perturbs the expression of the LD associated PAT proteins ADRP and TIP47, to promote its own lifecycle. HCV enhances TIP47 and suppresses ADRP by displacing it from LD surface in infected cell models. We have previously shown that suppression of TIP47 by miR-148a and miR-30a decreased intracellular LDs and HCV RNA. Thus, this study aimed at examining whether this microRNA-mediated suppression of HCV would limit HCV-dependent displacement of ADRP from LDs. ADRP expression was examined in 21 HCV-infected liver biopsies and 9 healthy donor liver tissues as well as in HCV-infected Huh7 cells using qRT-PCR. miR-148a and miR-30a expression was manipulated using specific oligos in JFH-1 infected, oleic acid treated cells, to study their impact on ADRP expression using qRT-PCR, and immunofluorescence microscopy. Intracellular HCV RNA was assessed using qRT-PCR. ADRP is down regulated in patients as well as HCVcc-JFH-I infected cell models. Forcing the expression of both miRNAs induced ADRP on the mRNA and protein levels. This study shows that HCV suppresses hepatic ADRP expression in infected patients and cell lines. Forcing the expression of miR-148a and miR-30a limits the suppressive effect of HCV on ADRP. J. Med. Virol. 89:653-659, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Hepacivirus/patogenicidade , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Perilipina-2/antagonistas & inibidores , Adulto , Biópsia , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Hepatite C/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Perilipina-3/metabolismo , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: A fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas. METHODS: Examined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data. RESULTS: We found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3' end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3' genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified. CONCLUSIONS: We have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3' end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.
Assuntos
Cistadenocarcinoma Seroso/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Perilipina-3/genética , Fosfoproteínas Fosfatases/genética , Idoso , Carcinogênese/genética , Carcinoma Epitelial do Ovário , Aberrações Cromossômicas , Cistadenocarcinoma Seroso/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Fusão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Mutação , Neoplasias Epiteliais e Glandulares/patologia , Proteínas de Fusão Oncogênica/genética , Neoplasias Ovarianas/patologia , Transcriptoma/genéticaRESUMO
Lipid droplets (LDs) are a conserved feature of most organisms. Vertebrate adipocytes have evolved to efficiently store and release lipids for the whole organism from a single droplet. Perilipin 1, the most abundant lipid-coat protein in adipocytes, plays a key role in regulating lipolysis. In other tissues such as liver and muscle, LDs serve very different biological functions, buffering surplus lipids for subsequent oxidation or export. These tissues express perilipins 2 or 3, rather than perilipin 1. We sought to understand the role of perilipins 2 and 3 in regulating basal lipolysis. Bimolecular fluorescence complementation studies suggested that whereas perilipin 1 prevents the activation of adipose tissue triacylglycerol lipase by its coactivator, AB-hydrolase domain containing-5 (ABHD5), perilipins 2 and 3 do so less effectively. These differences are mediated by a conserved region within the carboxy terminus of perilipin 1 that binds and stabilizes ABHD5 by retarding its degradation by the proteosome. Chimeric proteins generated by fusing the carboxy terminus of perilipin 1 to the amino terminus of perilipins 2 or 3 stabilize ABHD5 and suppress basal lipolysis more effectively than WT perilipins 2 or 3. Furthermore, knockdown of perilipin 1 in adipocytes leads to replacement of perilipin 2 on LDs. In these cells we observed reduced ABHD5 expression and LD localization and a corresponding increase in basal lipolysis. Collectively these data suggest that whereas perilipin 1 potently suppresses basal lipolysis in adipocytes, perilipins 2 and 3 facilitate higher rates of basal lipolysis in other tissues where constitutive traffic of fatty acids via LDs is a necessary step in their metabolism.