RESUMO
BACKGROUND: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice. METHODS: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group. RESULTS: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice. CONCLUSIONS: This study unveils the new role of catalase and its role in lysosomal function during aging. Video abstract.
Assuntos
Fibroblastos , Lisossomos , Masculino , Camundongos , AnimaisRESUMO
Recent evidence has linked the lysosomal cholesterol accumulation in Niemann-Pick type C1 with anomalies associated with primary ciliogenesis. Here, we report that perturbed intracellular cholesterol distribution imposed by lysosomal cholesterol accumulation during TMEM135 depletion is closely associated with impaired ciliogenesis. TMEM135 depletion does not affect the formation of the basal body and the ciliary transition zone. TMEM135 depletion severely blunts Rab8 trafficking to the centrioles without affecting the centriolar localization of Rab11 and Rabin8, the upstream regulators of Rab8 activation. Although TMEM135 depletion prevents enhanced IFT20 localization at the centrioles, ciliary vesicle formation is not affected. Furthermore, enhanced IFT20 localization at the centrioles is dependent on Rab8 activation. Supplementation of cholesterol in complex with cyclodextrin rescues Rab8 trafficking to the centrioles and Rab8 activation, thereby recovering primary ciliogenesis in TMEM135-depleted cells. Taken together, our data suggest that TMEM135 depletion prevents ciliary vesicle elongation, a characteristic of impaired Rab8 function. Our study thus reveals a previously uncharacterized effect of erroneous intracellular cholesterol distribution on impairing Rab8 function and primary ciliogenesis.
Assuntos
Colesterol , Cílios , Proteínas rab de Ligação ao GTP , Centríolos/metabolismo , Colesterol/metabolismo , Cílios/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Slc25a17 is known as a peroxisomal solute carrier, but the in vivo role of the protein has not been demonstrated. We found that the zebrafish genome contains two slc25a17 genes that function redundantly, but additively. Notably, peroxisome function in slc25a17 knockdown embryos is severely compromised, resulting in an altered lipid composition. Along the defects found in peroxisome-associated phenotypic presentations, we highlighted that development of the swim bladder is also highly dependent on Slc25a17 function. As Slc25a17 showed substrate specificity towards coenzyme A (CoA), injecting CoA, but not NAD+ , rescued the defective swim bladder induced by slc25a17 knockdown. These results indicated that Slc25a17 acts as a CoA transporter, involved in the maintenance of functional peroxisomes that are essential for the development of multiple organs during zebrafish embryogenesis. Given high homology in protein sequences, the role of zebrafish Slc25a17 may also be applicable to the mammalian system.
Assuntos
Coenzima A/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Sacos Aéreos/crescimento & desenvolvimento , Sacos Aéreos/metabolismo , Sequência de Aminoácidos , Animais , Coenzima A/genética , Sequência Conservada , Evolução Molecular , Proteínas de Membrana/genética , Peixe-ZebraRESUMO
Primary cilium is a microtubule structure that emanates from the surface of most human cells. Primary cilia assemble during the resting stage (G0 phase) and disassemble with cell cycle progression. Defects associated with the control of the assembly or disassembly of the primary cilium have been implicated in various human diseases, including ciliopathy and cancer. Although studies have suggested the interplay between activation of autophagy and ciliogenesis, any direct mechanism between autophagy abatement and disassembly of primary cilium remains elusive. In this study, we found that the gradual abatement in autophagy during serum-restimulation was a dynamic process and significantly correlated with the disassembly of primary cilium in human retinal pigmented epithelial (RPE1) cells. Although autophagy activity was gradually decreased during serum-restimulation, the alteration in autophagy under the same condition prevented the disassembly of the primary cilium. Autophagy inhibitors such as chloroquine, U18666A and 3-methyladenine (3-MA) retained both the number of ciliated cells and cilium length. In contrast, rapamycin treatment during serum-restimulation maintained the number of ciliated cells with shortened cilia. Taken together, alteration in autophagy during serum-restimulation prevent the disassembly of the primary cilium, and autophagy modulators may serve as useful compounds for studying mechanistic details related to the disassembly of the primary cilium and ciliopathy.
Assuntos
Autofagia , Cílios/metabolismo , Epitélio Pigmentado da Retina/citologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Cílios/efeitos dos fármacos , Humanos , Soro/metabolismo , Sirolimo/farmacologiaRESUMO
Peroxisomes are dynamic and multifunctional organelles involved in various cellular metabolic processes, and their numbers are tightly regulated by pexophagy, a selective degradation of peroxisomes through autophagy to maintain peroxisome homeostasis in cells. Catalase, a major peroxisome protein, plays a critical role in removing peroxisome-generated reactive oxygen species (ROS) produced by peroxisome enzymes, but the contribution of catalase to pexophagy has not been reported. Here, we investigated the role of catalase in peroxisome degradation during nutrient deprivation. Both short interfering RNA-mediated silencing of catalase and pharmacological inhibition by 3-aminotriazole (3AT) decreased the number of peroxisomes and resulted in the downregulation of peroxisomal proteins, such as PMP70 and PEX14 under serum starvation. In addition, treatment with 3AT induced NBR1-dependent autophagy and PEX5 ubiquitination in the absence of serum, which was accompanied by accumulation of ROS. Co-treatment with antioxidant agent N-acetyl-l-cysteine (NAC) prevented ROS accumulation and pexophagy by modulating peroxisome protein levels and the association of NBR1, a pexophagy receptor with peroxisomes. Taken together, these findings demonstrate that catalase plays an important role in pexophagy during nutrient deprivation.
Assuntos
Catalase/metabolismo , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Soro/metabolismo , Autofagia , Catalase/antagonistas & inibidores , Linhagem Celular , Células Hep G2 , Humanos , UbiquitinaçãoRESUMO
Cisplatin is an alkylating agent that interferes with DNA replication and kills proliferating carcinogenic cells. Several studies have been conducted to attenuate the side effects of cisplatin; one such side effect in cancer patients undergoing cisplatin chemotherapy is ototoxicity. However, owing to a lack of understanding of the precise mechanism underlying cisplatin-induced side effects, management of cisplatin-induced ototoxicity remains unsolved. We investigated the protective effects of fenofibrate, a PPAR-α activator, on cisplatin-induced ototoxicity. Fenofibrate prevented cisplatin-induced loss of hair cells and improved cell viability; moreover, fenofibrate significantly attenuated the threshold of auditory brainstem responses (ABR) in cisplatin-injected mice. Fenofibrate significantly increased PPAR-α, PPAR-γ, and PGC-1α expression, which consequently resulted in increased number and functional enzyme levels of peroxisomes and mitochondria, and markedly decreased phospho-p53 (S15), activated caspase-3, cleaved-PARP, and NF-κB p65 nuclear translocation, which reduced NADPH oxidase isoform (NOX3 and NOX4) expression, thereby decreasing reactive oxygen species (ROS) production in cisplatin-treated tissues ex vivo. Taken together, these results indicate that fenofibrate rescues cisplatin-induced ototoxicity by maintaining peroxisome and mitochondria number and function, reducing inflammation, and decreasing ROS levels. Our findings suggest that fenofibrate administration might serve as an effective therapeutic agent against cisplatin-induced ototoxicity.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/antagonistas & inibidores , Cisplatino/toxicidade , Otopatias/induzido quimicamente , Otopatias/prevenção & controle , Fenofibrato/uso terapêutico , Hipolipemiantes/uso terapêutico , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cóclea/patologia , Otopatias/patologia , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Peroxissomos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
ß-lapachone (ß-L) is a substrate of reduced nicotinamide adenine dinucleotide (NADH): quinone oxidoreductase 1 (NQO1). NQO1 reduces quinones to hydroquinones using NADH as an electron donor and consequently increases the intracellular NAD+/NADH ratio. The activation of NQO1 by ß-L has beneficial effects on several metabolic syndromes, such as obesity, hypertension, and renal injury. However, the effect of ß-L on bone metabolism remains unclear. Here, we show that ß-L might be a potent inhibitor of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. ß-L inhibited osteoclast formation in a dose-dependent manner and also reduced the expression of osteoclast differentiation marker genes, such as tartrate-resistant acid phosphatase (Acp5 or TRAP), cathepsin K (CtsK), the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2), osteoclast-associated receptor (Oscar), and dendritic cell-specific transmembrane protein (Dc-stamp). ß-L treatment of RANKL-induced osteoclastogenesis significantly increased the cellular NAD+/NADH ratio and resulted in the activation of 5' AMP-activated protein kinase (AMPK), a negative regulator of osteoclast differentiation. In addition, ß-L treatment led to significant suppression of the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß), which can stimulate osteoclastogenesis. ß-L treatment downregulated c-Fos and nuclear factor of activated T-cells 1 (NFATc1), which are master transcription factors for osteoclastogenesis. Taken together, the results demonstrated that ß-L inhibits RANKL-induced osteoclastogenesis and could be considered a potent inhibitor of RANKL-mediated bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.
Assuntos
Naftoquinonas/química , Osteoclastos/citologia , Ligante RANK/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Doenças Ósseas/metabolismo , Diferenciação Celular , Sobrevivência Celular , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , NAD/química , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteogênese , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Pexophagy is the selective degradation of peroxisomes for maintaining peroxisome homeostasis within cells. Peroxisome dynamics and pexophagy are important events required to maintain the quality control of peroxisomes, thereby preventing peroxisome-associated diseases. To identify novel pexophagy modulators, we developed a cell-based screening system and selected 2,2'-dipyridyl (2,2-DP) as a candidate molecule. 2,2-DP treatment induced peroxisome degradation as evidenced by an increased number of low-pH autolysosomes originating from peroxisomes and a decrease in the expression of peroxisomal proteins such as catalase, Pex14, and PMP70. The phenotype was defined as pexophagy, because 2,2-DP induced autophagy and inhibition of autophagy significantly reduced the degree of peroxisome degradation. Mechanistically, 2,2-DP-dependent pexophagy seemed to be mediated by iron chelation, since another iron chelator displayed a similar effect on pexophagy, but a copper chelator did not. Notably, iron replenishment prevented 2,2-DP-mediated pexophagy. Taken together, our results suggest that 2,2-DP treatment disrupts peroxisome dynamics and promotes pexophagy through iron depletion.
Assuntos
2,2'-Dipiridil/administração & dosagem , Autofagia/fisiologia , Quelantes de Ferro/administração & dosagem , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
Cobalt is an essential heavy metal that is necessary for the formation of vitamin B12 (hydroxocobalamin). However, exposure to excess cobalt for a prolonged period can harm the human body, causing pulmonary fibrosis, blindness, deafness, and peripheral neuropathy. 3-Aminotriazole (3-AT) is a catalase inhibitor that is often used to investigate the physiological effects of catalase. The present study found that injection of 3-AT in mice significantly reduced CoCl2-induced hearing impairment. In cultured organ of Corti explants from rats, 3-AT treatment protected hair cells from CoCl2-induced cytotoxicity. To determine the mechanism by which 3-AT protected from CoCl2-induced ototoxicity, we used the HEI-OC1 auditory cell line. Pretreatment with 10 mM 3-AT attenuated CoCl2-induced accumulation of ROS and induction of proinflammatory cytokine expression. Interestingly, these protective effects of 3-AT did not require catalase activity, as demonstrated by a series of experiments using RNA interference-mediated catalase knockdown in HEI-OC1 cells and using catalase-deficient mouse embryonic fibroblasts. Our results demonstrated the mechanisms of CoCl2-induced ototoxicity that may provide better ways to prevent the ototoxic effect of cobalt exposure.
Assuntos
Amitrol (Herbicida)/farmacologia , Cobalto/toxicidade , Células Ciliadas Auditivas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Catalase/antagonistas & inibidores , Catalase/metabolismo , Linhagem Celular , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/induzido quimicamente , Perda Auditiva/prevenção & controle , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Órgão Espiral/citologia , Órgão Espiral/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Testes de Toxicidade/métodosRESUMO
Even though bortezomib, a proteasome inhibitor, is a powerful chemotherapeutic agent used to treat multiple myeloma (MM) and other lymphoma cells, recent clinical reports suggest that the proteasome inhibitor therapy may be associated with severe bilateral hearing loss. We herein investigated the adverse effect of proteasome inhibitor on auditory hair cells. Treatment of a proteasome inhibitor destroys stereocilia bundles of hair cells resulting in the disarray of stereocilia in the organ of Corti explants. Since proteasome activity may be potentially important for biogenesis and function of the peroxisome, we tested whether proteasome activity is necessary for maintaining functional peroxisomes. Our results showed that treatment of a proteasome inhibitor significantly decreases both the number of peroxisomes and expression of peroxisomal proteins such as PMP70 and Catalase. In addition, we also found that proteasome inhibitor impairs the import pathway of PTS1-peroxisome matrix proteins. Taken together, our findings support recent clinical reports of hearing loss associated with proteasome inhibition. Mechanistically, peroxisome dysfunction may contribute to hair cell damage and hearing loss in response to the treatment of a proteasome inhibitor.
Assuntos
Células Ciliadas Auditivas/efeitos dos fármacos , Perda Auditiva/induzido quimicamente , Perda Auditiva/metabolismo , Peroxissomos/metabolismo , Inibidores de Proteassoma/efeitos adversos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/efeitos adversos , Ácidos Borônicos/efeitos adversos , Bortezomib , Catalase/metabolismo , Linhagem Celular , Sobrevivência Celular , Humanos , Lipídeos/química , Órgão Espiral/efeitos dos fármacos , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/efeitos adversos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de OxigênioRESUMO
Cisplatin has many adverse effects, which are a major limitation to its use, including ototoxicity, neurotoxicity, and nephrotoxicity. This study aims to elucidate the protective mechanisms of erdosteine against cisplatin in HEI-OC1 cells. Pretreatment with erdosteine protects HEI-OC1 cells from cisplatin-medicated apoptosis, which is characterized by increase in nuclear fragmentation, DNA laddering, sub-G0/G1 phase, H2AX phosphorylation, PARP cleavage, and caspase-3 activity. Erdosteine significantly suppressed the production of reactive nitrogen/oxygen species and pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in cisplatin-treated cells. Studies using pharmacologic inhibitors demonstrated that phosphatidylinositol-3-kinases (PI3K) and protein kinase B (Akt) have protective roles in the action of erdosteine against cisplatin in HEI-OC1 cells. In addition, pretreatment with erdosteine clearly suppressed the phosphorylation of p53 (Ser15) and expression of p53-upregulated modulator of apoptosis. Erdosteine markedly induces expression of NF-E2-related factor 2 (Nrf2), which may contribute to the increase in expression of glutathione redox genes γ-l-glutamate-l-cysteine-ligase catalytic and γ-l-glutamate-l-cysteine-ligase modifier subunits, as well as in the antioxidant genes HO-1 and SOD2 in cisplatin-treated HEI-OC1 cells. Furthermore, the increase in expression of phosphorylated p53 induced by cisplatin is markedly attenuated by pretreatment with erdosteine in the mitochondrial fraction. This increased expression may inhibit the cytosolic expression of the apoptosis-inducing factor, cytochrome c, and Bax/Bcl-xL ratio. Thus, our results suggest that treatment with erdosteine is significantly attenuated cisplatin-induced damage through the activation of Nrf2-dependent antioxidant genes, inhibition of pro-inflammatory cytokines, activation of the PI3K/Akt signaling, and mitochondrial-related inhibition of pro-apoptotic protein expression in HEI-OC1 auditory cells.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cisplatino/toxicidade , Citocinas/metabolismo , Orelha Interna/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Tioglicolatos/farmacologia , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Citocinas/imunologia , Citoproteção , Relação Dose-Resposta a Droga , Orelha Interna/imunologia , Orelha Interna/metabolismo , Orelha Interna/patologia , Regulação da Expressão Gênica , Mediadores da Inflamação/imunologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ubxd8, a multidomain protein sensor for long-chain unsaturated fatty acids (FAs), plays a crucial role to maintain cellular homeostasis of FAs. Ubxd8 polymerizes upon interaction with long-chain unsaturated FAs, but the molecular mechanism involved in this polymerization remains unclear. Here we report that the UAS domain of Ubxd8 mediates this polymerization. We show that a positively charged surface area in the domain is required for the reaction. Mutations changing the positively charged residues in this area to glutamates prevented long-chain unsaturated FAs from inducing oligomerization of Ubxd8. Consequently, the mutant protein no longer responded to regulation by long-chain unsaturated FAs in cultured cells. Long-chain unsaturated FAs also induced polymerization of Fas-associated factor 1 (FAF1), the only other mammalian protein that contains a UAS domain homologous to that of Ubxd8. These results provide further insights into protein-FA interactions by identifying the UAS domain as a motif interacting with long-chain unsaturated FAs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Sanguíneas/química , Ácidos Graxos Insaturados/química , Proteínas de Membrana/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Sanguíneas/metabolismo , Células CHO , Células Cultivadas , Cricetulus , Ácidos Graxos Insaturados/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Polimerização , Estrutura Terciária de ProteínaRESUMO
Endotoxin tolerance allows macrophages to produce large quantities of proinflammatory cytokines immediately after their contact with lipopolysaccharides (LPSs), but prevents their further production after repeated exposure to LPSs. While this response is known to prevent overproduction of proinflammatory cytokines, the mechanism through which endotoxin tolerance is established has not been identified. In the current study, we demonstrate that sufficient production of geranylgeraniol (GGOH) in macrophages is required to maintain endotoxin tolerance. We show that increased synthesis of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) protein following LPS treatment is required to produce enough GGOH to inhibit expression of Malt1, a protein known to stimulate expression of proinflammatory cytokines, in macrophages repeatedly exposed to LPSs. Depletion of GGOH caused by inhibition of HMGCR led to increased Malt1 expression in macrophages subjected to repeated exposure to LPSs. Consequently, endotoxin tolerance was impaired, and production of interleukin 1-ß and other proinflammatory cytokines was markedly elevated in these cells. These results suggest that insufficient production of GGOH in macrophages may cause autoinflammatory diseases.
Assuntos
Diterpenos/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is an anti-hyperlipidemic agent that has been widely used in the treatment of dyslipidemia. In this study, we examined the effect of fenofibrate on liver damage caused by refeeding a high-fat diet (HFD) in mice after 24h fasting. Here, we showed that refeeding HFD after fasting causes liver damage in mice determined by liver morphology and liver cell death. A detailed analysis revealed that hepatic lipid droplet formation is enhanced and triglyceride levels in liver are increased by refeeding HFD after starvation for 24h. Also, NF-κB is activated and consequently induces the expression of TNF-α, IL1-ß, COX-2, and NOS2. However, treating with fenofibrate attenuates the liver damage and triglyceride accumulation caused by the fasting-refeeding HFD process. Fenofibrate reduces the expression of NF-κB target genes but induces genes for peroxisomal fatty acid oxidation, peroxisome biogenesis and mitochondrial fatty acid oxidation. These results strongly suggest that the treatment of fenofibrate ameliorates the liver damage induced by fasting-refeeding HFD, possibly through the activation of fatty acid oxidation.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Jejum/efeitos adversos , Fenofibrato/administração & dosagem , Hipolipemiantes/administração & dosagem , Fígado/efeitos dos fármacos , PPAR alfa/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Ácidos Graxos/metabolismo , Ligantes , Fígado/metabolismo , Fígado/patologia , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Oxirredução , Triglicerídeos/metabolismoRESUMO
Fatty acids (FAs) are essential for cell survival, yet their overaccumulation causes lipotoxicity. To prevent lipotoxicity, cells store excess FAs as triglycerides (TGs). In cultured cells TG synthesis is activated by excess unsaturated but not saturated FAs. Here, we identify Ubxd8 as a sensor for unsaturated FAs and regulator of TG synthesis. In cultured cells depleted of FAs, Ubxd8 inhibits TG synthesis by blocking conversion of diacylglycerols (DAGs) to TGs. Excess unsaturated but not saturated FAs relieve this inhibition. As a result, unsaturated FAs are incorporated into TGs, whereas saturated FAs are incorporated into DAGs. In vitro, unsaturated but not saturated FAs alter the structure of purified recombinant Ubxd8 as monitored by changes in its thermal stability, trypsin cleavage pattern, and oligomerization. These results suggest that Ubxd8 acts as a brake that limits TG synthesis, and this brake is released when its structure is altered by exposure to unsaturated FAs.
Assuntos
Proteínas Sanguíneas/metabolismo , Ácidos Graxos Insaturados/metabolismo , Proteínas de Membrana/metabolismo , Triglicerídeos/biossíntese , Proteínas Sanguíneas/genética , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos Insaturados/química , Humanos , Proteínas de Membrana/genética , Desnaturação ProteicaRESUMO
This paper describes a convergent mechanism for the feedback control of cholesterol synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for cholesterol synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.
Assuntos
Colesterol/biossíntese , Retroalimentação Fisiológica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Esteróis/metabolismo , Ubiquitina/metabolismo , Animais , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Humanos , Hidroxicolesteróis/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
Peroxisomes are dynamic organelles that participate in a diverse array of cellular processes, including ß-oxidation, which produces a considerable amount of reactive oxygen species (ROS). Although we showed that catalase depletion induces ROS-mediated pexophagy in cells, the effect of catalase deficiency during conditions that favor ROS generation remains elusive in mice. In this study, we reported that prolonged fasting in catalase-knockout (KO) mice drastically increased ROS production, which induced liver-specific pexophagy, an autophagic degradation of peroxisomes. In addition, increased ROS generation induced the production of pro-inflammatory cytokines in the liver tissues of catalase-KO mice. Furthermore, there was a significant increase in the levels of aspartate transaminase and alanine transaminase as well as apparent cell death in the liver of catalase-KO mice during prolonged fasting. However, an intra-peritoneal injection of the antioxidant N-acetyl-l-cysteine (NAC) and autophagy inhibitor chloroquine inhibited the inflammatory response, liver damage, and pexophagy in the liver of catalase-KO mice during prolonged fasting. Consistently, genetic ablation of autophagy, Atg5 led to suppression of pexophagy during catalase inhibition by 3-aminotriazole (3AT). Moreover, treatment with chloroquine also ameliorated the inflammatory response and cell death in embryonic fibroblast cells from catalase-KO mice. Taken together, our data suggest that ROS-mediated liver-specific pexophagy observed during prolonged fasting in catalase-KO mice may be responsible for the process associated with hepatic cell death.
Assuntos
Catalase/fisiologia , Fígado/patologia , Macroautofagia , Peroxissomos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/uso terapêutico , Animais , Catalase/genética , Células Cultivadas , Privação de Alimentos , Hepatite/tratamento farmacológico , Hepatite/etiologia , Hepatite/metabolismo , Hepatite/patologia , Fígado/metabolismo , Camundongos KnockoutRESUMO
BACKGROUND: Fatty acids (FA) derived from adipose tissue and liver serve as the main fuel in thermogenesis of brown adipose tissue (BAT). Catalase, a peroxisomal enzyme, plays an important role in maintaining intracellular redox homeostasis by decomposing hydrogen peroxide to either water or oxygen that oxidize and provide fuel for cellular metabolism. Although the antioxidant enzymatic activity of catalase is well known, its role in the metabolism and maintenance of energy homeostasis has not yet been revealed. The present study investigated the role of catalase in lipid metabolism and thermogenesis during nutrient deprivation in catalase-knockout (KO) mice. RESULTS: We found that hepatic triglyceride accumulation in KO mice decreased during sustained fasting due to lipolysis through reactive oxygen species (ROS) generation in adipocytes. Furthermore, the free FA released from lipolysis were shuttled to BAT through the activation of CD36 and catabolized by lipoprotein lipase in KO mice during sustained fasting. Although the exact mechanism for the activation of the FA receptor enzyme, CD36 in BAT is still unclear, we found that ROS generation in adipocytes mediated the shuttling of FA to BAT. CONCLUSIONS: Taken together, our findings uncover the novel role of catalase in lipid metabolism and thermogenesis in BAT, which may be useful in understanding metabolic dysfunction.
RESUMO
Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , Receptores do Fator Autócrino de Motilidade , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in ppara-/- cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in ppara-/- mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while ppara-/- mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in ppara-/- mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.