RESUMO
Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.
Assuntos
Chironomidae , Dessecação , Animais , Trealose/metabolismo , Larva/metabolismo , Chironomidae/genética , Insetos/metabolismo , Linhagem CelularRESUMO
Phospholipids are asymmetrically distributed between the lipid bilayer of plasma membranes in which phosphatidylserine (PtdSer) is confined to the inner leaflet. ATP11A and ATP11C, type IV P-Type ATPases in plasma membranes, flip PtdSer from the outer to the inner leaflet, but involvement of other P4-ATPases is unclear. We herein demonstrated that once PtdSer was exposed on the cell surface of ATP11A-/-ATP11C-/- mouse T cell line (W3), its internalization to the inner leaflet of plasma membranes was negligible at 15 °C. However, ATP11A-/-ATP11C-/- cells internalized the exposed PtdSer at 37 °C, a temperature at which trafficking of intracellular membranes was active. In addition to ATP11A and 11C, W3 cells expressed ATP8A1, 8B2, 8B4, 9A, 9B, and 11B, with ATP8A1 and ATP11B being present at recycling endosomes. Cells deficient in four P4-ATPases (ATP8A1, 11A, 11B, and 11C) (QKO) did not constitutively expose PtdSer on the cell surface but lost the ability to re-establish PtdSer asymmetry within 1 hour, even at 37 °C. The expression of ATP11A or ATP11C conferred QKO cells with the ability to rapidly re-establish PtdSer asymmetry at 15 °C and 37 °C, while cells expressing ATP8A1 or ATP11B required a temperature of 37 °C to achieve this function, and a dynamin inhibitor blocked this process. These results revealed that mammalian cells are equipped with two independent mechanisms to re-establish its asymmetry: the first is a rapid process involving plasma membrane flippases, ATP11A and ATP11C, while the other is mediated by ATP8A1 and ATP11B, which require an endocytosis process.
Assuntos
Transportador 1 de Cassete de Ligação de ATP , ATPases do Tipo-P , Fosfatidilserinas , Proteínas de Transferência de Fosfolipídeos , Animais , Camundongos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , ATPases do Tipo-P/genética , ATPases do Tipo-P/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Técnicas de Inativação de Genes , Linfócitos TRESUMO
Polypedilum vanderplanki is a striking and unique example of an insect that can survive almost complete desiccation. Its genome and a set of dehydration-rehydration transcriptomes, together with the genome of Polypedilum nubifer (a congeneric desiccation-sensitive midge), were recently released. Here, using published and newly generated datasets reflecting detailed transcriptome changes during anhydrobiosis, as well as a developmental series, we show that the TCTAGAA DNA motif, which closely resembles the binding motif of the Drosophila melanogaster heat shock transcription activator (Hsf), is significantly enriched in the promoter regions of desiccation-induced genes in P. vanderplanki, such as genes encoding late embryogenesis abundant (LEA) proteins, thioredoxins, or trehalose metabolism-related genes, but not in P. nubifer Unlike P. nubifer, P. vanderplanki has double TCTAGAA sites upstream of the Hsf gene itself, which is probably responsible for the stronger activation of Hsf in P. vanderplanki during desiccation compared with P. nubifer To confirm the role of Hsf in desiccation-induced gene activation, we used the Pv11 cell line, derived from P. vanderplanki embryo. After preincubation with trehalose, Pv11 cells can enter anhydrobiosis and survive desiccation. We showed that Hsf knockdown suppresses trehalose-induced activation of multiple predicted Hsf targets (including P. vanderplanki-specific LEA protein genes) and reduces the desiccation survival rate of Pv11 cells fivefold. Thus, cooption of the heat shock regulatory system has been an important evolutionary mechanism for adaptation to desiccation in P. vanderplanki.
Assuntos
Chironomidae/fisiologia , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Insetos/metabolismo , Animais , Evolução Biológica , Chironomidae/genética , Desidratação , Feminino , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico , Proteínas de Insetos/genética , Masculino , Estresse FisiológicoRESUMO
The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.
Assuntos
Adaptação Fisiológica/genética , Chironomidae/genética , Dessecação , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla/métodos , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Insetos/genética , Animais , Linhagem Celular , Chironomidae/citologia , Análise por Conglomerados , Perfilação da Expressão Gênica/métodosRESUMO
Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.
Assuntos
Chironomidae/genética , Dessecação , Técnicas de Transferência de Genes , Interferência de RNA , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Chironomidae/citologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Insetos/genética , Larva/citologia , Estresse Fisiológico/genéticaRESUMO
Cushing's disease (CD) and subclinical Cushing's disease (subCD) are both diseases caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. However, ACTH autonomy in subCD is weaker than in CD and there are no Cushingoid features in subCD. The differences of molecular mechanisms in ACTH autonomy between CD and subCD have not yet been reported. Therefore, we aimed to investigate the differences in molecular mechanisms of ACTH-secretion autonomy between CD and subCD. The study included 23 patients [7 CD, 6 subCD, and 10 non-functioning pituitary tumors (NFTs)] who underwent transsphenoidal surgery at the Osaka University Hospital between December 2009 and October 2013. Using quantitative real-time PCR, various ACTH-related gene expressions in tumor tissues from CD, subCD, and NFT were measured such as pro-opiomelanocortin (POMC), POMC transcription factor (Tpit, Pitx1, NeuroD1, and Nur77), POMC peptide processing enzymes (prohormone convertase: PC1/3 and PC2), and ACTH secretion-related factors (corticotropin-releasing hormone receptor 1: CRHR1 and glucocorticoid receptor α: GRα). Only Nur77 mRNA levels were significantly higher in CD than in subCD. Furthermore, we stained 6 CD and 6 subCD with anti-Nur77 antibody. All tumor samples from CD had Nur77 protein positive cells. On the other hand, Nur77 protein was expressed in only one tumor sample from subCD. This sample showed high expression of Nur77 mRNA. Nur77 is an important to regulate POMC transcription and negative-feedback by glucocorticoids. Nur77 gene expression levels might involve different autonomy of ACTH production between CD and subCD.
Assuntos
Adenoma Hipofisário Secretor de ACT/genética , Adenoma/genética , Hormônio Adrenocorticotrópico/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Hipersecreção Hipofisária de ACTH/genética , Adenoma Hipofisário Secretor de ACT/metabolismo , Adenoma/metabolismo , Adulto , Idoso , Doenças Assintomáticas , Estudos de Casos e Controles , Retroalimentação Fisiológica , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Hipersecreção Hipofisária de ACTH/metabolismo , Via Secretória/genética , Adulto JovemRESUMO
Obesity and diabetes are rapidly reaching epidemic proportions in many parts of world and are becoming one of the major public health problems. Many studies have been performed to develop treatments for obesity and diabetes. In clinical aspect, for example, vitamin D was assumed to be a causal factor of obesity and diabetes, and the effect of vitamin D supplementation on the patients was assessed. In addition to clinical study, basic researchers have tried to elucidate the mechanisms of obesity and diabetes. Recent studies show novel techniques for finding etiologic factors in obesity and diabetes. These effort will accelerate progress toward total eradication of obesity and diabetes.
Assuntos
Complicações do Diabetes/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Obesidade/tratamento farmacológico , Tecnologia , Vitamina D/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Descoberta de Drogas/tendências , Humanos , Obesidade/metabolismoRESUMO
Type 1 diabetes, one of two major forms of diabetes, results from the complete destruction of pancreatic beta cells. Viral infection has been suggested to be a trigger of beta cell destruction, the pathogenesis of type 1 diabetes. The aim of this study was to clarify the role of the protein encoded by intherferon stimulated gene (ISG) 15, an antiviral effector, in the development of this clinical entity. We used the mouse beta cell line MIN6 to investigate the role of ISG15 and paid special attention to apoptosis. Although not detected in native MIN6 cells, free ISG15 and ISG15 conjugated proteins were both present in dose-dependently increased amounts following stimulation with interferon alpha. As assessed both by caspase 3/7 activity and an annexin V assay, the percentage of apoptotic MIN6 cells (after exposure to the inflammatory cytokines of interleukin-1beta plus interferon gamma or tumor necrosis factor alpha) was decreased by pretreatment with adenovirus-expressing ISG15 and increased by expressing a short hairpin RNA directed against ISG15. In conclusion, ISG15 has an anti-apoptotic effect on MIN6 cells. Thus, promoting ISG15 expression in the pancreatic beta cells could be a potential therapeutic approach for patients with type 1 diabetes.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/fisiologia , Células Secretoras de Insulina/patologia , Animais , Linhagem Celular , Citocinas/biossíntese , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , Ubiquitinas/biossíntese , Ubiquitinas/fisiologiaRESUMO
The plasma membrane containing cholesterol exhibits phospholipid asymmetry, with phosphatidylcholine and sphingomyelin enriched in its outer leaflet and phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) on the cytoplasmic side. We herein describe steps for bacterial expression of recombinant proteins that bind to membrane lipids, followed by affinity purification. Using fluorescence-labeled phospholipid analogs, we further detail the assay to detect flippase activity, which maintains the single-sided distribution of PtdSer and PtdEtn, in mammalian cells. For complete details on the use and execution of this protocol, please refer to Segawa et al. (2021).1.
Assuntos
Fosfatidilcolinas , Fosfolipídeos , Animais , Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Fosfatidilcolinas/metabolismo , Transporte Biológico , Mamíferos/metabolismoRESUMO
Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.
RESUMO
Genomic safe harbors (GSHs) provide ideal integration sites for generating transgenic organisms and cells and can be of great benefit in advancing the basic and applied biology of a particular species. Here we report the identification of GSHs in a dry-preservable insect cell line, Pv11, which derives from the sleeping chironomid, Polypedilum vanderplanki, and similar to the larvae of its progenitor species exhibits extreme desiccation tolerance. To identify GSHs, we carried out genome analysis of transgenic cell lines established by random integration of exogenous genes and found four candidate loci. Targeted knock-in was performed into these sites and the phenotypes of the resulting transgenic cell lines were examined. Precise integration was achieved for three candidate GSHs, and in all three cases integration did not alter the anhydrobiotic ability or the proliferation rate of the cell lines. We therefore suggest these genomic loci represent GSHs in Pv11 cells. Indeed, we successfully constructed a knock-in system and introduced an expression unit into one of these GSHs. We therefore identified several GSHs in Pv11 cells and developed a new technique for producing transgenic Pv11 cells without affecting the phenotype.
Assuntos
Chironomidae , Animais , Linhagem Celular , Chironomidae/genética , Genômica , Insetos , LarvaRESUMO
Non-biting midges (Chironomidae) are known to inhabit a wide range of environments, and certain species can tolerate extreme conditions, where the rest of insects cannot survive. In particular, the sleeping chironomid Polypedilum vanderplanki is known for the remarkable ability of its larvae to withstand almost complete desiccation by entering a state called anhydrobiosis. Chromosome numbers in chironomids are higher than in other dipterans and this extra genomic resource might facilitate rapid adaptation to novel environments. We used improved sequencing strategies to assemble a chromosome-level genome sequence for P. vanderplanki for deep comparative analysis of genomic location of genes associated with desiccation tolerance. Using whole genome-based cross-species and intra-species analysis, we provide evidence for the unique functional specialization of Chromosome 4 through extensive acquisition of novel genes. In contrast to other insect genomes, in the sleeping chironomid a uniquely high degree of subfunctionalization in paralogous anhydrobiosis genes occurs in this chromosome, as well as pseudogenization in a highly duplicated gene family. Our findings suggest that the Chromosome 4 in Polypedilum is a site of high genetic turnover, allowing it to act as a 'sandbox' for evolutionary experiments, thus facilitating the rapid adaptation of midges to harsh environments.
RESUMO
Pv11 is an insect cell line established from the midge Polypedilum vanderplanki, whose larval form exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 itself is also capable of anhydrobiosis, which is induced by trehalose treatment. Here we report the successful construction of a genome editing system for Pv11 cells and its application to the identification of signaling pathways involved in anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established Pv11 cells that stably expressed GCaMP3 to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase in cytosolic Ca2+ concentration, and further experiments revealed that the calmodulin-calcineurin-NFAT pathway contributes to tolerance of trehalose treatment as well as desiccation tolerance, while the calmodulin-calmodulin kinase-CREB pathway conferred only desiccation tolerance on Pv11 cells. Thus, our results show a critical contribution of the trehalose-induced Ca2+ surge to anhydrobiosis and demonstrate temporally different roles for each signaling pathway.
Assuntos
Sistemas CRISPR-Cas , Sinalização do Cálcio , Desidratação , Edição de Genes , Animais , Cálcio/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Técnicas de Introdução de Genes , Ontologia Genética , Insetos , Larva , RNA Guia de Cinetoplastídeos , Estresse Fisiológico , Trealose/metabolismo , Trealose/farmacologiaRESUMO
Oxidative stress has been implicated as a causal role in atherosclerosis, microvascular complications of diabetes as well as in beta cell failure in type 2 diabetes. PPARgamma agonists not only improve insulin sensitivity but also eliminate oxidative stress. In mouse, catalase, a major antioxidant enzyme, is directly regulated by PPARgamma through two PPARgamma binding elements in its promoter. This study examined the regulatory mechanisms of catalase expression in human. Expression of catalase was significantly upregulated in human primary adipocytes upon treatment with a PPARgamma agonist. However, the mouse PPARgamma response elements are not functionally conserved in human catalase promoter. In luciferase reporter assay containing human catalase promoter, PPARgamma /RXRalpha, in combination of a PPARgamma agonist significantly transactivated 19 kb of promoter and this was mediated via a novel PPARgamma response element (PPRE) at -12 kb from transcription initiation site of human catalase gene. Electrophoretic mobility shift assay showed direct binding of PPARgamma to this PPRE. Together, our results indicate that PPARgamma regulates the expression of catalase gene in human through a PPRE distinct from that of mouse, and could explain, at least in part, the observed inhibitory effects of PPARgamma on oxidative stress in human.
Assuntos
Catalase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , PPAR gama/fisiologia , Elementos de Resposta/fisiologia , Adipócitos/enzimologia , Animais , Sítios de Ligação , DNA/química , DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Estresse Oxidativo/fisiologia , PPAR gama/agonistas , PPAR gama/genética , Pioglitazona , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Tiazolidinedionas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.
RESUMO
Larvae of the sleeping chironomid Polypedilum vanderplanki are known for their extraordinary ability to survive complete desiccation in an ametabolic state called "anhydrobiosis". The unique feature of P. vanderplanki genome is the presence of expanded gene clusters associated with anhydrobiosis. While several such clusters represent orthologues of known genes, there is a distinct set of genes unique for P. vanderplanki. These include Lea-Island-Located (LIL) genes with no known orthologues except two of LEA genes of P. vanderplanki, PvLea1 and PvLea3. However, PvLIL proteins lack typical features of LEA such as the state of intrinsic disorder, hydrophilicity and characteristic LEA_4 motif. They possess four to five transmembrane domains each and we confirmed membrane targeting for three PvLILs. Conserved amino acids in PvLIL are located in transmembrane domains or nearby. PvLEA1 and PvLEA3 proteins are chimeras combining LEA-like parts and transmembrane domains, shared with PvLIL proteins. We have found that PvLil genes are highly upregulated during anhydrobiosis induction both in larvae of P. vanderplanki and P. vanderplanki-derived cultured cell line, Pv11. Thus, PvLil are a new intriguing group of genes that are likely to be associated with anhydrobiosis due to their common origin with some LEA genes and their induction during anhydrobiosis.
Assuntos
Membrana Celular/metabolismo , Chironomidae/fisiologia , Desidratação , Proteínas de Insetos/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Análise por Conglomerados , Simulação por Computador , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Larva/fisiologia , Família Multigênica , Filogenia , Domínios Proteicos , RNA-SeqRESUMO
Obesity is associated with insulin resistance and a mild chronic inflammation in adipose tissues. Recent studies suggested that GM3 ganglioside mediates dysfunction in insulin signaling. However, it has not been determined the ganglioside profiling in adipose tissues of obese animals. Here, we for the first time examined semi-quantitative ganglioside profiles in the adipose tissues of high fat- and high sucrose-induced obese, diabetic C57BL/6J mice by TLC and HPLC/mass spectrometry. In control adipose tissues GM3 dominated with traces of GM1 and GD1a; obesity led to a dramatic increase in GM2, GM1, and GD1a with the GM3 content unchanged. Similar results were obtained in KK and KKAy mice. Adipocytes separated from stromal vascular cells including macrophages contained more of those gangliosides in KKAy mice than in KK mice. These results underscore those gangliosides in the pathophysiology of obesity-related diseases.
Assuntos
Tecido Adiposo/metabolismo , Gangliosídeos/metabolismo , Obesidade/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/química , Animais , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Gangliosídeo G(M2)/análise , Gangliosídeo G(M2)/genética , Gangliosídeo G(M2)/metabolismo , Gangliosídeo G(M3)/análise , Gangliosídeo G(M3)/genética , Gangliosídeo G(M3)/metabolismo , Gangliosídeos/análise , Gangliosídeos/genética , Expressão Gênica , Macrófagos/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos , N-Acetilgalactosaminiltransferases/biossíntese , Obesidade/complicações , RNA Mensageiro/biossínteseRESUMO
Rho GTPase regulates actin cytoskeleton organization and assembly in many cell types, however, its significance in adipose tissue is not well characterized. Here, we demonstrate high RhoA activity in adipose tissues of C57BL/6J mice. To determine the effect of RhoA activation on 3T3-L1 cells, stable cell lines overexpressing G14VRhoA fused to destabilizing domain of FKBP12 (DD-G14VRhoA-L1) were generated. Treatment of DD-G14VRhoA-L1 cells with Shield1 following their differentiation into adipocytes, resulted in the appearance of thick cortical actin filaments, and increased the mRNA expression levels of plasminogen activator inhibitor type-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1). The induction of PAI-1 and MCP-1 was inhibited by treatment with a Rho-associated kinase (ROCK) inhibitor, Y-27632. In 3T3-L1 adipocytes, tumor necrosis factor-alpha activated RhoA and increased mRNA expression of PAI-1 and MCP-1, and their treatment with Y-27632 partially inhibited these changes. The results indicate that RhoA-ROCK pathway induces inflammatory cytokine expression in adipocytes.
Assuntos
Adipócitos/enzimologia , Citocinas/biossíntese , Proteína rhoA de Ligação ao GTP/metabolismo , Células 3T3 , Amidas/farmacologia , Animais , Quimiocina CCL2/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Serpina E2 , Serpinas/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
OBJECTIVE: Macrophage-mediated chronic inflammation of adipose tissue is causally linked to insulin resistance in obesity. The beneficial effects of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase inhibitors (statins) on glucose metabolism have been suggested, but the effects of these agents on adipose tissue inflammation are unclear. The aim of the present study is to define the effects of statins on adipose tissue inflammation and macrophages. METHODS AND RESULTS: Pravastatin or pitavastatin treatment of obese mice attenuated an increase in mRNA expressions of proinflammatory genes, including MCP1 and IL6, in adipose tissue. The supernatant of TLR4-stimulated RAW264 macrophages strongly induced the expression of these genes in 3T3-L1 adipocytes, which was inhibited by pretreatment of macrophages with either statin. Statins inhibited TLR4-mediated activation of interferon (IFN) regulatory factor (IRF)3 by either lipopolysaccharide (LPS) or palmitic acid, resulting in suppression of IFN-beta expression, but not that of NF-kappaB or JNK. Moreover, statins strongly downregulated TLR3-mediated gene expressions by poly(I:C), but not TLR2-stimulation by zymosan A. Neutralization of IFN-beta attenuated proinflammatory activities of the macrophage supernatant. CONCLUSIONS: Statins partially attenuated the development of adipose tissue inflammation in obese mice, which might be associated with an inhibitory effect of statins on TLR4-triggered expression of IFN-beta via MyD88-independent signaling pathway in macrophages.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamação/prevenção & controle , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Macrófagos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Células 3T3-L1 , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Feminino , Inflamação/metabolismo , Inflamação/patologia , Fator Regulador 3 de Interferon/efeitos dos fármacos , Interferon beta/efeitos dos fármacos , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Obesos , Fator 88 de Diferenciação Mieloide/efeitos dos fármacos , Obesidade/metabolismo , Obesidade/patologia , Pravastatina/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismoRESUMO
Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes. Using a luciferase reporter assay, this strong transcriptional activity was shown to be conserved in cell lines from various insect species, including S2 (Drosophila melanogaster, Diptera), SaPe-4 (Sarcophaga peregrina, Diptera), Sf9 (Spodoptera frugiperda, Lepidoptera) and Tc81 (Tribolium castaneum, Coleoptera) cells. In conjunction with an appropriate selection maker gene, the 121 promoter was able to confer zeocin resistance on SaPe-4 cells and allowed the establishment of stable SaPe-4 cell lines expressing the fluorescent protein AcGFP1; this is the first report of heterologous gene expression in this cell line. These results show the 121 promoter to be a versatile tool for exogenous gene expression in a wide range of insect cell lines, particularly useful to those from non-model insect species.