RESUMO
Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.
Assuntos
Síndrome do Cromossomo X Frágil , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurogênese/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , RNA Mensageiro/genética , Camundongos KnockoutRESUMO
Protein kinase C (PKC) zeta has been implicated in insulin-induced glucose uptake in skeletal muscle cell, although the underlying mechanism remains unknown. In this study, we investigated the effect of PKCzeta on actin remodeling and glucose transport in differentiated rat L6 muscle cells expressing myc-tagged glucose transporter 4 (GLUT4). On insulin stimulation, PKCzeta translocated from low-density microsomes to plasma membrane accompanied by increase in GLUT4 translocation and glucose uptake. Z-scan confocal microscopy revealed a spatial colocalization of relocated PKCzeta with the small GTPase Rac-1, actin, and GLUT4 after insulin stimulation. The insulin-mediated colocalization, PKCzeta distribution, GLUT4 translocation, and glucose uptake were inhibited by wortmannin and cell-permeable PKCzeta pseudosubstrate peptide. In stable transfected cells, overexpression of PKCzeta caused an insulin-like effect on actin remodeling accompanied by a 2.1-fold increase in GLUT4 translocation and 1.7-fold increase in glucose uptake in the absence of insulin. The effects of PKCzeta overexpression were abolished by cell-permeable PKCzeta pseudosubstrate peptide, but not wortmannin. Transient transfection of constitutively active Rac-1 recruited PKCzeta to new structures resembling actin remodeling, whereas dominant negative Rac-1 prevented the insulin-mediated PKCzeta translocation. Together, these results suggest that PKCzeta mediates insulin effect on glucose transport through actin remodeling in muscle cells.
Assuntos
Actinas/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Células Musculares/metabolismo , Proteína Quinase C/metabolismo , Actinas/análise , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Transportador de Glucose Tipo 4/análise , Transportador de Glucose Tipo 4/genética , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células Musculares/química , Células Musculares/efeitos dos fármacos , Fosforilação , Proteína Quinase C/análise , Proteína Quinase C/genética , Transporte Proteico , Ratos , Proteínas rac1 de Ligação ao GTP/análise , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Analgesics, including opioids, steroidal and nonsteroidal anti-inflammatory drugs, aspirin, acetaminophen, antiepileptics, and serotonin-norepinephrine reuptake inhibitors, are medications commonly used to treat many forms of pain. However, all of these agents may have significant adverse side effects. Adverse effects may occasionally be inseparable from desired effects. Side effects are often dose dependent and time dependent. It is critical that the prescribing practitioner and the dispensing pharmacist provide a thorough, understandable review of the potential side effects to all patients before these drugs are administered. Proper monitoring and follow-up during therapy are crucial.
Assuntos
Dor Crônica/tratamento farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Manejo da Dor/efeitos adversos , Analgésicos/efeitos adversos , Analgésicos/uso terapêutico , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/uso terapêutico , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/uso terapêutico , Dor Crônica/diagnóstico , Aconselhamento/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/fisiopatologia , Feminino , Humanos , Incidência , Masculino , Monitorização Fisiológica , Manejo da Dor/métodos , Dor Intratável/diagnóstico , Dor Intratável/tratamento farmacológico , Prognóstico , Medição de Risco , Índice de Gravidade de DoençaRESUMO
Both microtubule and actin are required for insulin-induced glucose uptake. However, the roles of these two cytoskeletons and their relationship in insulin action still remain unclear. In this work, we examined the morphological change of microtubule/actin and their involvement in insulin signal transduction using rat skeletal muscle cells. Insulin rapidly led to microtubule clustering from ventral to dorsal surface of the cell. Microtubule filaments were rearranged to create space where new actin structures formed. Disruption of microtubule prevented insulin-induced actin remodeling and distal insulin signal transduction, with reduction in surface glucose transporter isoform 4 (GLUT4) and glucose uptake. Though microtubule mediated actin remodeling through PKCζ, reorganization of microtubule depended on tyrosine phosphorylation of insulin receptor, the mechanism is different from insulin-induced actin remodeling, which relied on the activity of PI3-kinase and PKCζ. We propose that microtubule network is required for insulin-induced signal transduction and actin remodeling in skeletal muscle cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Microtúbulos/metabolismo , Mioblastos Esqueléticos/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular , Glucose/metabolismo , Cinética , Mioblastos Esqueléticos/citologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , RatosRESUMO
Berberine has been shown to have insulin-sensitizing effect, but the molecular mechanism underlying remains elusive. In this work, we investigated the effect of berberine on insulin-induced signal transduction and glucose uptake in both insulin-sensitive and insulin-resistant rat skeletal muscle cells. Berberine increased the activity of AMPK and PKCzeta and AS160 phosphorylation in normal cells, but had little effect on PKB activation. In insulin-resistant state, berberine exhibited synergistic effect on insulin-induced glucose uptake and GLUT4 translocation. Berberine improved insulin-induced tyrosine-phosphorylation of IRS-1 and the recruitment of p85 to IRS-1. These changes were accompanied by enhancement in insulin-induced PKCzeta and PKB activity and actin remodeling. The ameliorated insulin signal transduction was related to the inhibition of mTOR by berberine, which attenuated serine-phosphorylation of IRS-1. These results suggest that berberine may overcome insulin resistance via modulating key molecules in insulin signaling pathway, leading to increased glucose uptake in insulin-resistant cells.
Assuntos
Berberina/farmacologia , Resistência à Insulina , Insulina/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Actinas , Animais , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Células Musculares/enzimologia , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , RatosRESUMO
Insulin and AMP-activated protein kinase (AMPK) signal pathways are involved in the regulation of glucose uptake. The integration of signals between these two pathways to maintain glucose homeostasis remains elusive. In this work, stimulation of insulin and berberine conferred a glucose uptake or surface glucose transporter 4 (GLUT4) translocation that was less than simple summation of their effects in insulin-sensitive muscle cells. Using specific inhibitors to key kinases of both pathways and PKCzeta small interference RNA, protein kinase C zeta (PKCzeta) was found to regulate insulin-stimulated protein kinase B (PKB) activation and inhibit AMPK activity on dorsal cell surface. In the presence of berberine, PKCzeta controlled AMPK activation and AMPK blocked PKB activity in perinuclear region. The inhibition effect of PKCzeta on AMPK activation or the arrestment of PKB activity by AMPK still existed in basal condition. These results suggest that there is antagonistic regulation between insulin and AMPK signal pathways, which is mediated by the switch roles of PKCzeta.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Fibras Musculares Esqueléticas/enzimologia , Proteína Quinase C/fisiologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Berberina/farmacologia , Transporte Biológico , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
In skeletal muscle cells, insulin stimulates cytoskeleton actin remodeling to facilitate the translocation of glucose transporter GLUT4 to plasma membrane. Defect of insulin-induced GLUT4 translocation and actin remodeling may cause insulin resistance. Free fatty acids cause insulin resistance in skeletal muscle. The aim of this study was to investigate the effects of fatty acids on glucose transport and actin remodeling. Differentiated L6 muscle cells expressing c-myc epitope-tagged GLUT4 were treated with palmitic acid, linoleic acid and oleic acid. Surface GLUT4 and 2-deoxyglucose uptake were measured in parallel with the morphological imaging of actin remodeling and GLUT4 immunoreactivity with fluorescence, confocal and transmission electron microscopy. Differentiated L6 cells showed concentration responses of insulin-induced actin remodeling and glucose uptake. The ultrastructure of insulin-induced actin remodeling was cell projections clustered with actin and GLUT4. Acute and chronic treatment with the 3 fatty acids had no effect on insulin-induced actin remodeling and GLUT4 immunoreactivity. However, insulin-mediated glucose uptake significantly decreased by palmitic acid (25, 50, 75, 100 µmol/L), oleic acid (180, 300 µmol/L) and linoleic acid (120, 180, 300 µmol/L). Oleic acid (120, 300 µmol/L) and linoleic acid (300 µmol/L), but not palmitic acid, significantly decreased insulin-mediated GLUT4 translocation. These data suggest that fatty acids inhibit insulin-induced glucose transport associated with actin remodeling in L6 muscle cells.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Ácidos Graxos/farmacologia , Glucose/metabolismo , Insulina/farmacologia , Células Musculares/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Glucose/farmacocinética , Transportador de Glucose Tipo 4/metabolismo , Células Musculares/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
BACKGROUND: Data on patch test findings in Hong Kong are scarce, with the last survey performed more than 10 years ago. A retrospective analysis of results from all patch tests performed on patients with suspected allergic contact dermatitis from January 1995 to December 1999 in the Social Hygiene Service, which provides a public dermatology service in Hong Kong, was undertaken. We aimed to explore the demographic data associated with positive reactions and the profile of contact sensitizing allergens in Hong Kong. METHODS: A total of 2585 patients were patch tested with the European standard series during the period. Most were Chinese, with a female-to-male ratio of 3 : 2. RESULTS: One or more positive responses were noted in 1415 patients (54.7%). The most common allergen was nickel sulfate (24.4%), followed by fragrance mix (13.7%), cobalt chloride (8.7%), p-phenylenediamine (6.0%), and balsam of Peru (5.7%). Nickel sensitivity was more common in female patients, and dichromate sensitivity was more common in male patients (P < 0.001). Female gender, an age of 40 years or below, truncal and upper limb involvement, absence of lower limb involvement, and a positive atopy history were significant risk factors for nickel sensitivity. CONCLUSIONS: This study provides a profile of allergens responsible for allergic contact dermatitis in the public dermatology service in Hong Kong. A prospective study, using a larger panel of allergens, involving patients from both the private and public sectors, would provide a more comprehensive profile of contact allergens in Hong Kong and contribute to the establishment of a local standard series.