RESUMO
Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.
Assuntos
Adipócitos/metabolismo , Gotículas Lipídicas/metabolismo , Perilipina-2/metabolismo , Perilipina-3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Tecido Adiposo/metabolismo , Animais , Animais Geneticamente Modificados , Homeostase , Metabolismo dos Lipídeos , Perilipina-2/genética , Perilipina-3/genética , Peixe-Zebra/metabolismoRESUMO
Lipid metabolism reprogramming is one of the adaptive events that drive tumor development and survival, and may account for resistance to chemotherapeutic drugs. Perilipins are structural proteins associated with lipophagy and lipid droplet integrity, and their overexpression is associated with tumor aggressiveness. Here, we sought to explore the role of lipid droplet-related protein perilipin-3 (PLIN3) in prostate cancer (PCa) chemotherapy. We investigated the role of PLIN3 suppression in docetaxel cytotoxic activity in PCa cell lines. Additional effects of PLIN3 depletion on autophagy-related proteins and gene expression patterns, apoptotic potential, proliferation rate, and ATP levels were examined. Depletion of PLIN3 resulted in docetaxel resistance, accompanied by enhanced autophagic flux. We further assessed the synergistic effect of autophagy suppression with chloroquine on docetaxel cytotoxicity. Inhibition of autophagy with chloroquine reversed chemoresistance of stably transfected shPLIN3 PCa cell lines, with no effect on the parental ones. The shPLIN3 cell lines also exhibited reduced Caspase-9 related apoptosis initiation. Moreover, we assessed PLIN3 expression in a series of PCa tissue specimens, were complete or partial loss of PLIN3 expression was frequently noted in 70% of the evaluated specimens. Following PLIN3 silencing, PCa cells were characterized by impaired lipophagy and acquired an enhanced autophagic response upon docetaxel-induced cytotoxic stress. Such an adaptation leads to resistance to docetaxel, which could be reversed by the autophagy blocker chloroquine. Given the frequent loss of PLIN3 expression in PCa specimens, we suggest that combination of docetaxel with chloroquine may improve the efficacy of docetaxel treatment in PLIN3-deficient cancer patients.
Assuntos
Autofagia/efeitos dos fármacos , Cloroquina/farmacologia , Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos , Perilipina-3/genética , Neoplasias da Próstata/tratamento farmacológico , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Masculino , Neoplasias da Próstata/genéticaRESUMO
Current endocrine therapy for prostate cancer (PCa) mainly inhibits androgen/androgen receptor (AR) signaling. However, due to increased intratumoural androgen synthesis and AR variation, PCa progresses to castration-resistant prostate cancer (CRPC), which ultimately becomes resistant to endocrine therapy. A search for new therapeutic perspectives is urgently needed. Methods: By screening lipid metabolism-related gene sets and bioinformatics analysis in prostate cancer database, we identified the key lipid metabolism-related genes in PCa. Bisulfite genomic Sequence Polymerase Chain Reaction (PCR) (BSP) and Methylation-Specific Polymerase Chain Reaction (PCR) (MSP) were preformed to detect the promoter methylation of ACSS3. Gene expression was analyzed by qRT-PCR, Western blotting, IHC and co-IP. The function of ACSS3 in PCa was measured by CCK-8, Transwell assays. LC/MS, Oil Red O assays and TG and cholesterol measurement assays were to detect the levels of TG and cholesterol in cells. Resistance to Enzalutamide in C4-2 ENZR cells was examined in a xenograft tumorigenesis model in vivo. Results: We found that acyl-CoA synthetase short chain family member 3 (ACSS3) was downregulated and predicted a poor prognosis in PCa. Loss of ACSS3 expression was due to gene promoter methylation. Restoration of ACSS3 expression in PCa cells significantly reduced LD deposits, thus promoting apoptosis by increasing endoplasmic reticulum (ER) stress, and decreasing de novo intratumoral androgen synthesis, inhibiting CRPC progression and reversing Enzalutamide resistance. Mechanistic investigations demonstrated that ACSS3 reduced LD deposits by regulating the stability of the LD coat protein perilipin 3 (PLIN3). Conclusions: Our study demonstrated that ACSS3 represses prostate cancer progression through downregulating lipid droplet-associated protein PLIN3.
Assuntos
Biomarcadores Tumorais/metabolismo , Coenzima A Ligases/metabolismo , Regulação Neoplásica da Expressão Gênica , Gotículas Lipídicas/metabolismo , Perilipina-3/antagonistas & inibidores , Perilipina-3/metabolismo , Neoplasias da Próstata/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Coenzima A Ligases/genética , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Perilipina-3/genética , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. Although lipid droplet (LD) binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 (Plin3) is involved in the regulation of beige adipocyte formation and thermogenic activities. In this study, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared with wild-type mice, Plin3 knockout mice were cold tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing peroxisome proliferator-activated receptor α (PPARα) activation. In adipocytes, Plin3 deficiency promoted PPARα target gene and uncoupling protein 1 expression and multilocular LD formation upon cold stimulus. Moreover, fibroblast growth factor 21 expression and secretion were upregulated, which was attributable to activated PPARα in Plin3-deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression.
Assuntos
Adipócitos Bege/metabolismo , Tecido Adiposo Branco/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise/genética , PPAR alfa/metabolismo , Perilipina-3/genética , Termogênese/genética , Animais , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/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
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
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.