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1.
J Pharmacol Exp Ther ; 370(3): 390-398, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31262967

RESUMO

Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ-coactivator 1α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase-α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes.


Assuntos
Bebedeira/genética , Bebedeira/fisiopatologia , Epigênese Genética , Etanol/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/patologia , Fatores Sexuais , Animais , Bebedeira/metabolismo , Bebedeira/patologia , Citocromo P-450 CYP2E1/metabolismo , Diacilglicerol Quinase/metabolismo , Feminino , Glicerofosfolipídeos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transcrição Genética/efeitos dos fármacos
2.
Monoclon Antib Immunodiagn Immunother ; 38(3): 120-123, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31149888

RESUMO

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). Both DG and PA serve as lipidic second messengers; therefore, DGK plays a critical role in regulating the balance of two signaling pathways mediated by DG and PA in cellular functions. DGK gamma (DGKγ), 1 of the 10 mammalian DGK isozymes, has been reported to be involved in membrane traffic, mast cell function, and leukemic cell differentiation. We previously developed a novel anti-DGKγ monoclonal antibody (mAb), DgMab-6, which is very useful in immunocytochemistry of human cultured cells. In this study, we characterized the binding epitope of DgMab-6 using Western blotting, and Glu12 is important for facilitating the DgMab-6 binding to the DGKγ protein. These results might lead to further development for sensitive and specific mAbs against DGKγ.


Assuntos
Anticorpos Monoclonais/imunologia , Diacilglicerol Quinase/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Diacilglicerol Quinase/metabolismo , Humanos , Ligação Proteica , Transdução de Sinais
3.
PLoS One ; 14(6): e0217819, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31167004

RESUMO

Subsets of small-diameter dorsal root ganglia (DRG) neurons detect pruritogenic (itch-causing) and algogenic (pain-causing) stimuli and can be activated or sensitized by chemical mediators. Many of these chemical mediators activate receptors that are coupled to lipid hydrolysis and diacylglycerol (DAG) production. Diacylglycerol kinase iota (DGKI) can phosphorylate DAG and is expressed at high levels in small-diameter mouse DRG neurons. Given the importance of these neurons in sensing pruritogenic and algogenic chemicals, we sought to determine if loss of DGKI impaired responses to itch- or pain-producing stimuli. Using male and female Dgki-knockout mice, we found that in vivo sensitivity to histamine-but not other pruritogens-was enhanced. In contrast, baseline pain sensitivity and pain sensitization following inflammatory or neuropathic injury were equivalent between wild type and Dgki-/- mice. In vitro calcium responses in DRG neurons to histamine was enhanced, while responses to algogenic ligands were unaffected by Dgki deletion. These data suggest Dgki regulates sensory neuron and behavioral responses to histamine, without affecting responses to other pruritogenic or algogenic agents.


Assuntos
Diacilglicerol Quinase/deficiência , Histamina/efeitos adversos , Prurido/induzido quimicamente , Prurido/enzimologia , Animais , Comportamento Animal , Cálcio/farmacologia , Diacilglicerol Quinase/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptividade , Dor/enzimologia , Dor/patologia , Dor/fisiopatologia , Prurido/patologia , Prurido/fisiopatologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia
4.
Mol Biol Rep ; 46(4): 4105-4111, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31087244

RESUMO

Schizophrenia (SCZ) is a disabling and severe mental illness characterized by abnormal social behavior and disrupted emotions. Similar to other neuropsychological disorders, both genetics and environmental factors interplay so as to develop SCZ. It is acknowledged that genes such as DGKZ are involved in lipid signaling pathways that are the basis of neural activities, memory, and learning and are considered as candidate loci for SCZ. The aim of the present study was to evaluate the expression level and genotypes of DGKZ in patients with SCZ and controls. We used q-PCR to measure the relative expression of DGKZ in blood. To determine DGKZ-rs7951870 genotypes, tetra-ARMS PCR was used. Our results showed a significant difference in DGKZ mRNA ratio between SCZ patients and healthy controls (P = 2 × 10-4). Also, we showed that rs7951870-TT genotype was strongly associated with increased DGKZ expression level (P = 0.038). In conclusion, our findings revealed dysregulation of DGKZ in SCZ patients and a significant correction between the gene expression and DGKZ variant rs7951870.


Assuntos
Diacilglicerol Quinase/genética , Esquizofrenia/genética , Adulto , Diacilglicerol Quinase/metabolismo , Feminino , Expressão Gênica/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Transcriptoma/genética
5.
Monoclon Antib Immunodiagn Immunother ; 38(3): 124-128, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31112065

RESUMO

The diacylglycerol kinases (DGKs) are a family of proteins that catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), a cellular component that is important in lipid biochemistry and signal transduction, into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has previously been reported to be terminated in most cases by one of 10 DGK isoforms, DGKζ. In this study, we performed immunohistochemical analysis using a rabbit anti-DGKζ monoclonal antibody (mAb) (clone EPR22040-80) against tissues from the tonsils of a patient with oropharyngeal squamous cell carcinoma. We demonstrated that many DGKζ-expressing T cells are localized in the tonsils. We further characterized the binding epitope using an enzyme-linked immunosorbent assay and found that Pro790, Gln791, Gly792, and Leu795 residues of DGKζ are important for facilitating anti-DGKζ mAb binding to DGKζ. This anti-DGKζ mAb could be valuable in immunohistochemical analyses in determining the distribution of DGKζ-expressing T cells in pathophysiological tissues.


Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/metabolismo , Diacilglicerol Quinase/imunologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Neoplasias Orofaríngeas/metabolismo , Linfócitos T/imunologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Diacilglicerol Quinase/metabolismo , Humanos , Neoplasias Orofaríngeas/imunologia , Neoplasias Orofaríngeas/patologia , Fragmentos de Peptídeos/imunologia , Fosforilação , Ligação Proteica , Transdução de Sinais
6.
Histochem Cell Biol ; 151(6): 461-474, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963236

RESUMO

Cochlear hair cells are essential for the mechanotransduction of hearing. Sensorineural hearing loss can be irreversible because hair cells have a minimal ability to repair or regenerate themselves once damaged. In order to develop therapeutic interventions to prevent hair cell loss, it is necessary to understand the signaling pathway operating in cochlear hair cells and its alteration upon damage. Diacylglycerol kinase (DGK) regulates intracellular signal transduction through phosphorylation of lipidic second messenger diacylglycerol. We have previously reported characteristic expression and localization patterns of DGKs in various organs under pathophysiological conditions. Nevertheless, little is known about morphological and functional aspects of this enzyme family in the cochlea. First RT-PCR analysis reveals predominant mRNA expression of DGKα, DGKε and DGKζ. Immunohistochemical analysis shows that DGKζ localizes to the nuclei of inner hair cells (IHCs), outer hair cells (OHCs), supporting cells and spiral ganglion neurons in guinea pig cochlea under normal conditions. It is well known that loud noise exposure induces cochlear damage, thereby resulting in hair cell loss. In particular, OHCs are highly vulnerable to noise exposure than IHCs. We found that after 1 week of noise exposure DGKζ translocates from the nucleus to the cytoplasm in damage-sensitive OHCs and gradually disappears thereafter. In sharp contrast, DGKζ remains to the nucleus in damage-resistant IHCs. These results suggest that DGKζ cytoplasmic translocation is well correlated with cellular damage under noise-exposure stress conditions and is involved in delayed cell death in cochlear outer hair cells.


Assuntos
Cóclea/enzimologia , Diacilglicerol Quinase/análise , Ruído/efeitos adversos , Estresse Fisiológico , Animais , Cóclea/citologia , Cóclea/metabolismo , Diacilglicerol Quinase/metabolismo , Cobaias
7.
Biochem J ; 476(8): 1205-1219, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000624

RESUMO

The diacylglycerol kinases (DGK) are lipid kinases that transform diacylglycerol (DAG) into phosphatidic acid (PA) in a reaction that terminates DAG-based signals. DGK provide negative regulation to conventional and novel protein kinase C (PKC) enzymes, limiting local DAG availability in a tissue- and subcellular-restricted manner. Defects in the expression/activity of certain DGK isoforms contribute substantially to cognitive impairment and mental disorders. Abnormal DGK overexpression in tumors facilitates invasion and resistance to chemotherapy preventing tumor immune destruction by tumor-infiltrating lymphocytes. Effective translation of these findings into therapeutic approaches demands a better knowledge of the physical and functional interactions between the DGK and PKC families. DGKζ is abundantly expressed in the nervous and immune system, where physically and functionally interacts with PKCα. The latest discoveries suggest that PDZ-mediated interaction facilitates spatial restriction of PKCα by DGKζ at the cell-cell contact sites in a mechanism where the two enzymes regulate each other. In T lymphocytes, DGKζ interaction with Sorting Nexin 27 (SNX27) guarantees the basal control of PKCα activation. SNX27 is a trafficking component required for normal brain function whose deficit has been linked to Alzheimer's disease (AD) pathogenesis. The enhanced PKCα activation as the result of SNX27 silencing in T lymphocytes aligns with the recent correlation found between gain-of-function PKCα mutations and AD and suggests that disruption of the mechanisms that provides a correct spatial organization of DGKζ and PKCα may lie at the basis of immune and neuronal synapse impairment.


Assuntos
Diacilglicerol Quinase/metabolismo , Proteína Quinase C/metabolismo , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Isoenzimas/metabolismo , Domínios Proteicos , Transdução de Sinais/fisiologia
8.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1864(7): 1031-1038, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30980919

RESUMO

Decreased levels of the δ isozyme of diacylglycerol kinase (DGK) in skeletal muscle attenuate glucose uptake and, consequently, are critical for the pathogenesis of type 2 diabetes. We recently found that free myristic acid (14:0), but not free palmitic acid (16:0), increased the DGKδ protein levels and enhanced glucose uptake in C2C12 myotube cells. However, it has been unclear how myristic acid regulates the level of DGKδ2 protein. In the present study, we characterized the myristic acid-dependent increase of DGKδ protein. A cycloheximide chase assay demonstrated that myristic acid, but not palmitic acid, markedly stabilized DGKδ protein. Moreover, other DGK isozymes, DGKη and ζ, as well as glucose uptake-related proteins, such as protein kinase C (PKC) α, PKCζ, Akt and glycogen synthase kinase 3ß, failed to be stabilized by myristic acid. Furthermore, DGKδ was not stabilized in cultured hepatocellular carcinoma cells, pancreas carcinoma cells or neuroblastoma cells, and only a moderate stabilizing effect was observed in embryonic kidney cells. A proteasome inhibitor and a lysosome inhibitor, MG132 and chloroquine, respectively, partly inhibited DGKδ degradation, suggesting that myristic acid prevents, at least in part, the degradation of DGKδ by the ubiquitin-proteasome system and the autophagy-lysosome pathway. Overall, these results strongly suggest that myristic acid attenuates DGKδ protein degradation in skeletal muscle cells and that this attenuation is fatty acid-, protein- and cell line-specific. These new findings provide novel insights into the molecular mechanisms of the pathogenesis of type 2 diabetes mellitus.


Assuntos
Diacilglicerol Quinase/metabolismo , Músculo Esquelético/citologia , Ácido Mirístico/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/etiologia , Glucose/farmacocinética , Humanos , Isoenzimas/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Proteólise/efeitos dos fármacos
9.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889878

RESUMO

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.


Assuntos
Diacilglicerol Quinase/genética , Perfilação da Expressão Gênica , Genômica , Família Multigênica , Soja/enzimologia , Soja/genética , Estresse Fisiológico/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Especificidade de Órgãos/genética , Filogenia , Regiões Promotoras Genéticas/genética , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo
10.
J Biochem ; 165(6): 517-522, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715374

RESUMO

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) into phosphatidic acid (PA). DG and PA function as lipid messengers contributing to various signalling pathways. Thus, DGK plays a pivotal role in the signalling pathways by maintaining DG and PA levels. For example, DGKδ is involved in diabetes and DGKß is important for higher brain function including memory and emotion. Recently, we also revealed that the activation of DGKα ameliorated diabetic nephropathy (DN) in mice, suggesting that DGK can be therapeutic target. However, there is no commercially available DGK subtype-specific inhibitors or activators. Therefore, in a series of experiment to find DGK subtype-specific inhibitors or activators, we tried to screen novel DGKα activators from 9,600 randomly selected compounds by using high-throughput screening we had recently developed. Finally, we obtained two lead compounds for DGKα activators, KU-8 and KU-10. Focusing KU-8, we assessed the effect of KU-8 on all mammalian DGKs activities. Thus, KU-8 activates not only DGKα but also DGKθ by approximately 20%, and strongly inhibited DGKκ. In conclusion, KU-8 would be a good lead compound for DGKα and DGKθ activators, and useful as a DGKκ inhibitor.


Assuntos
Ciclopropanos/farmacologia , Diacilglicerol Quinase/antagonistas & inibidores , Diacilglicerol Quinase/metabolismo , Dioxinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Xilenos/farmacologia , Animais , Células COS , Células Cultivadas , Ciclopropanos/química , Dioxinas/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Xilenos/química
11.
Mol Metab ; 19: 13-23, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389349

RESUMO

OBJECTIVE: Obesity is a complex disorder involving many genetic and environmental factors that are required to maintain energy homeostasis. While studies in human populations have led to significant progress in the generation of an obesity gene map and broadened our understanding of the genetic basis of common obesity, there is still a large portion of heritability and etiology that remains unknown. Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance. METHODS: We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects. RESULTS: We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. Downregulation of neuronal Dgk levels increases TAG and carbohydrate levels and these phenotypes can be recapitulated by reducing Dgk levels specifically within the insulin-producing cells that secrete Drosophila insulin-like peptides (dILPs). Conversely, overexpression of kinase-dead Dgk, but not wild-type, decreased circulating dILP2 and dILP5 levels resulting in lower insulin signalling activity. Despite having higher circulating dILP levels, Dgk RNAi flies have decreased pathway activity suggesting that they are insulin-resistant. CONCLUSION: Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila.


Assuntos
Diacilglicerol Quinase/metabolismo , Homeostase/genética , Secreção de Insulina/genética , Animais , Diacilglicerol Quinase/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético/genética , Estudo de Associação Genômica Ampla , Insulina/metabolismo , Secreção de Insulina/fisiologia , Neurônios/metabolismo , Obesidade/genética , Fenótipo , Interferência de RNA , Reprodutibilidade dos Testes , Transdução de Sinais
12.
Exp Cell Res ; 373(1-2): 211-220, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30399372

RESUMO

Diacylglycerol kinases (DGK) are a family of enzymes catalyzing the transformation of diacylglycerol into phosphatidic acid, which have been recognized as key regulators in cell signaling pathways. The role of DGKγ in human malignancies has seldom been studied. In this study, we investigated the role of DGKγ in hepatocellular carcinoma (HCC). We found that DGKγ was down-regulated in HCC tumor tissues and cell lines as compared to that in non-tumor tissues. The prognostic value of DGKγ expression was evaluated by Cox regression and Kaplan-Meier analyses. Lower DGKγ expression in tumor tissues was an independent prognostic factor for poor post-surgical overall survival. By using HDACs inhibitors treatment and ChIP-PCR, we discovered that histone H3 and H4 deacetylation mainly contributed to the downregulation of DGKγ expression. Functional studies revealed that ectopic expression of DGKγ inhibited cell proliferation and cell migration in HCC cells. Mechanism studies showed that DGKγ overexpression led to down regulation of GLUT1 protein level and AMPK activity, which result in glucose uptake suppression as well as lactate and ATP production declination. The decrease of GLUT1 level could be partially rescued by treatments with either DGK inhibitor and lysosome inhibitor, indicating DGKγ may down-regulate GLUT1 through its kinase activity and lysosome degradation process. Together, this study demonstrated that DGKγ plays a tumor suppressor role in HCC by negatively regulating GLUT1. DGKγ could be a novel prognostic indicator and therapeutic target for HCC.


Assuntos
Carcinoma Hepatocelular/enzimologia , Diacilglicerol Quinase/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Neoplasias Hepáticas/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/cirurgia , Linhagem Celular Tumoral , Movimento Celular , Diacilglicerol Quinase/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal , Glucose/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/cirurgia , Prognóstico , Regiões Promotoras Genéticas , Proteínas Supressoras de Tumor/genética
13.
Dis Model Mech ; 11(9)2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30135067

RESUMO

Valproic acid (VPA) provides a common treatment for both epilepsy and bipolar disorder; however, common cellular mechanisms relating to both disorders have yet to be proposed. Here, we explore the possibility of a diacylglycerol kinase (DGK) playing a role in regulating the effect of VPA relating to the treatment of both disorders, using the biomedical model Dictyostelium discoideum DGK enzymes provide the first step in the phosphoinositide recycling pathway, implicated in seizure activity. They also regulate levels of diacylglycerol (DAG), thereby regulating the protein kinase C (PKC) activity that is linked to bipolar disorder-related signalling. Here, we show that ablation of the single Dictyostelium dgkA gene results in reduced sensitivity to the acute effects of VPA on cell behaviour. Loss of dgkA also provides reduced sensitivity to VPA in extended exposure during development. To differentiate a potential role for this DGKA-dependent mechanism in epilepsy and bipolar disorder treatment, we further show that the dgkA null mutant is resistant to the developmental effects of a range of structurally distinct branched medium-chain fatty acids with seizure control activity and to the bipolar disorder treatment lithium. Finally, we show that VPA, lithium and novel epilepsy treatments function through DAG regulation, and the presence of DGKA is necessary for compound-specific increases in DAG levels following treatment. Thus, these experiments suggest that, in Dictyostelium, loss of DGKA attenuates a common cellular effect of VPA relating to both epilepsy and bipolar disorder treatments, and that a range of new compounds with this effect should be investigated as alternative therapeutic agents.This article has an associated First Person interview with the first author of the paper.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/enzimologia , Diacilglicerol Quinase/metabolismo , Dictyostelium/enzimologia , Epilepsia/tratamento farmacológico , Epilepsia/enzimologia , Ácido Valproico/uso terapêutico , Sequência de Aminoácidos , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Transtorno Bipolar/patologia , Diacilglicerol Quinase/química , Dictyostelium/efeitos dos fármacos , Diglicerídeos/metabolismo , Epilepsia/patologia , Proteínas de Fluorescência Verde/metabolismo , Lítio/farmacologia , Lítio/uso terapêutico , Modelos Biológicos , Mutação/genética , Ácido Valproico/farmacologia
14.
Plant Physiol ; 177(3): 1303-1318, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29853600

RESUMO

Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (Arabidopsis thaliana). With or without cold acclimation, the dgat1 mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the dgat1 mutants showed reduced expression of C-REPEAT/DRE BINDING FACTOR2 and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of dgat1 mutant plants. During freezing stress, the accumulation of PA in dgat1 plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of DGK2, DGK3, and DGK5 were significantly more up-regulated in the dgat1 mutants than in the wild type during cold stress. Consistent with this observation, dgk2, dgk3, and dgk5 knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by DGAT1 is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Resposta ao Choque Frio/fisiologia , Diacilglicerol Quinase/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas de Arabidopsis/genética , Diacilglicerol Quinase/genética , Diacilglicerol O-Aciltransferase/genética , Diglicerídeos/genética , Diglicerídeos/metabolismo , Congelamento , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/metabolismo , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Ácido Salicílico/metabolismo , Transativadores/genética , Transativadores/metabolismo , Triglicerídeos/metabolismo
15.
Biochimie ; 151: 45-53, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29859210

RESUMO

Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKδ is highly expressed in the skeletal muscle, and a decrease in DGKδ expression increases the severity of type 2 diabetes. However, the role of DGKδ in myogenic differentiation is still unknown. The present study demonstrated that DGKδ expression was down-regulated in the early stage of C2C12 myogenic differentiation almost concurrently with a decrease in cyclin D1 expression. The knockdown of DGKδ by DGKδ-specific siRNAs significantly increased the levels of cyclin D1 expression at 48 h after C2C12 myogenic differentiation. In contrast, at the same time, the knockdown of DGKδ decreased the levels of myogenin expression and the number of myosin heavy chain (MHC)-positive cells. These results indicate that DGKδ regulates the early differentiation of C2C12 myoblasts via controlling the down-regulation of cyclin D1 expression. Moreover, the suppression of DGKδ expression increased the phosphorylation levels of conventional and novel protein kinase Cs (cnPKCs). Furthermore, DGKδ suppression increased the levels of cyclin D1 and phospho-cnPKCs even at the first 24 h of myogenic differentiation. These results suggest that DGKδ controls the down-regulation of cyclin D1 expression by attenuating the PKC signaling pathway for C2C12 myogenic differentiation.


Assuntos
Diferenciação Celular , Ciclina D1/metabolismo , Diacilglicerol Quinase/metabolismo , Regulação para Baixo , Músculo Esquelético/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Ciclina D3/metabolismo , Diacilglicerol Quinase/genética , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Miogenina/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
16.
Sci Signal ; 11(530)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29764991

RESUMO

Skeletal muscle rapidly remodels in response to various stresses, and the resulting changes in muscle mass profoundly influence our health and quality of life. We identified a diacylglycerol kinase ζ (DGKζ)-mediated pathway that regulated muscle mass during remodeling. During mechanical overload, DGKζ abundance was increased and required for effective hypertrophy. DGKζ not only augmented anabolic responses but also suppressed ubiquitin-proteasome system (UPS)-dependent proteolysis. We found that DGKζ inhibited the transcription factor FoxO that promotes the induction of the UPS. This function was mediated through a mechanism that was independent of kinase activity but dependent on the nuclear localization of DGKζ. During denervation, DGKζ abundance was also increased and was required for mitigating the activation of FoxO-UPS and the induction of atrophy. Conversely, overexpression of DGKζ prevented fasting-induced atrophy. Therefore, DGKζ is an inhibitor of the FoxO-UPS pathway, and interventions that increase its abundance could prevent muscle wasting.


Assuntos
Diacilglicerol Quinase/metabolismo , Diacilglicerol Quinase/fisiologia , Proteína Forkhead Box O3/metabolismo , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/patologia , Ubiquitina/metabolismo , Resposta a Proteínas não Dobradas , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Feminino , Regulação da Expressão Gênica , Hipertrofia/etiologia , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , NF-kappa B/metabolismo , Proteólise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
17.
PLoS One ; 13(5): e0197548, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29847571

RESUMO

We have previously reported that Ildr2 knockdown via adenovirally-delivered shRNA causes hepatic steatosis in mice. In the present study we investigated hepatic biochemical and anatomic phenotypes of Cre-mediated Ildr2 knock-out mice. Liver-specific Ildr2 knock-out mice were generated in C57BL/6J mice segregating for a floxed (exon 1) allele of Ildr2, using congenital and acute (10-13-week-old male mice) Cre expression. In addition, Ildr2 shRNA was administered to Ildr2 knock-out mice to test the effects of Ildr2 shRNA, per se, in the absence of Ildr2 expression. RNA sequencing was performed on livers of these knockdown and knockout mice. Congenital and acute liver-specific and hepatocyte-specific knockout mice did not develop hepatic steatosis. However, administration of Ildr2 shRNA to Ildr2 knock-out mice did cause hepatic steatosis, indicating that the Ildr2 shRNA had apparent "off-target" effects on gene(s) other than Ildr2. RNA sequencing and BLAST sequence alignment revealed Dgka as a candidate gene mediating these "off-target" effects. Ildr2 shRNA is 63% homologous to the Dgka gene, and Dgka expression decreased only in mice displaying hepatic steatosis. Dgka encodes diacylglycerol kinase (DGK) alpha, one of a family of DGKs which convert diacylglycerides to phosphatidic acid for second messenger signaling. Dgka knockdown mice would be expected to accumulate diacylglyceride, contributing to the observed hepatic steatosis. We conclude that ILDR2 plays a negligible role in hepatic steatosis. Rather, hepatic steatosis observed previously in Ildr2 knockdown mice was likely due to shRNA targeting of Dgka and/or other "off-target" genes. We propose that the gene candidates identified in this follow-up study may lead to identification of novel regulators of hepatic lipid metabolism.


Assuntos
Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , RNA Interferente Pequeno/genética , Análise de Sequência de RNA , Triglicerídeos/metabolismo
18.
FASEB J ; 32(8): 4121-4131, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29509511

RESUMO

Lipid metabolism is closely involved with signal transduction and energy homeostasis. Excess calorie intake causes abnormal lipid metabolism, promoting obesity and insulin resistance. Diacylglycerol (DG) represents not only a lipidic second messenger but also an intermediate metabolite for triglyceride metabolism in the endoplasmic reticulum (ER). However, it remains undetermined how the roles of DG in signaling and energy homeostasis is regulated within the cell. Of DG kinases (DGKs), which are enzymes that phosphorylate DG, DGKε resides in the ER. This study examined how DGKε is implicated in signal transduction and lipid homeostasis. DGKε-deficient mice were fed a high-fat diet (HFD) for 40 d. We observed that DGKε deficiency promotes fat accumulation in adipocytes and subsequently promotes insulin resistance in mice fed an HFD. This abnormal fat metabolism is mediated by down-regulation of lipolytic activities, such as adipose triglyceride lipase and hormone-sensitive lipase. In addition, activation of DG-sensitive PKC leads to insulin resistance in adipose tissue, which may be caused by delayed metabolism of DG. Our data suggest that DGKε links the second messenger signaling system to energy homeostasis in adipocytes and that its deficiency results in abnormal lipid metabolism such as obesity and insulin resistance.-Nakano, T., Seino, K., Wakabayashi, I., Stafforini, D. M., Topham, M. K., Goto, K. Deletion of diacylglycerol kinase ε confers susceptibility to obesity via reduced lipolytic activity in murine adipocytes.


Assuntos
Adipócitos/metabolismo , Diacilglicerol Quinase/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Regulação para Baixo/fisiologia , Homeostase/fisiologia , Resistência à Insulina/fisiologia , Lipase/metabolismo , Metabolismo dos Lipídeos/fisiologia , Camundongos , Transdução de Sinais/fisiologia
19.
Biochem Biophys Res Commun ; 497(4): 1031-1037, 2018 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-29486157

RESUMO

We previously reported that brain-specific diacylglycerol kinase (DGK) δ-knockout (KO) mice showed obsessive-compulsive disorder (OCD)-like behaviors, which were alleviated by a serotonin (5-HT) transporter (SERT) inhibitor. However, the molecular mechanisms causing the OCD-like abnormal behaviors remain unclear. In the present study, we found that DGKδ deficiency increased SERT protein levels in the mouse cerebral cortex. Moreover, DGKδ interacted and co-localized with SERT. Furthermore, DGKδ-KO decreased tryptophan hydroxylase-2 expression and increased monoamine oxidase-A expression. Indeed, the amount of 5-HT in the cerebral cortex was significantly decreased in DGKδ-KO mice. These data strongly suggest that OCD-like behaviors in the DGKδ-KO mice are caused by comprehensive and composite serotonergic hypofunction.


Assuntos
Encéfalo/enzimologia , Diacilglicerol Quinase/deficiência , Serotonina/metabolismo , Animais , Córtex Cerebral/metabolismo , Diacilglicerol Quinase/metabolismo , Camundongos , Camundongos Knockout , Transtorno Obsessivo-Compulsivo/etiologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
20.
Biochem Biophys Res Commun ; 496(2): 309-315, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29326040

RESUMO

Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Túbulos Renais/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Proteínas de Transporte Vesicular/genética , Animais , Bioensaio , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Cães , Inibidores Enzimáticos/farmacologia , Exocitose , Regulação da Expressão Gênica , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Quinazolinonas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas de Transporte Vesicular/agonistas , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismo , Cicatrização/fisiologia
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