RESUMEN
We previously proposed that LYR motif containing 1 (LYRM1)-induced mitochondrial reactive oxygen species (ROS) production contributes to obesity-related insulin resistance. Metformin inhibits ROS production and promotes mitochondrial biogenesis in specific tissues. We assessed the effects of metformin on insulin resistance in LYRM1-over-expressing 3T3-L1 adipocytes. Metformin enhanced basal and insulin-stimulated glucose uptake and GLUT4 translocation, reduced IRS-1 and Akt phosphorylation and ROS levels, and affected the expression of regulators of mitochondrial biogenesis in LYRM1-over-expressing adipocytes. Metformin may ameliorate LYRM1-induced insulin resistance and mitochondrial dysfunction in part via a direct antioxidant effect and in part by activating the adenosine monophosphate-activated protein kinase (AMPK)-PGC1/NRFs pathway.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Hipoglucemiantes/metabolismo , Resistencia a la Insulina , Metformina/metabolismo , Mitocondrias/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Ratones , Especies Reactivas de Oxígeno/análisisRESUMEN
LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.
Asunto(s)
Adipocitos/citología , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Técnicas de Silenciamiento del Gen , Resistencia a la Insulina/genética , Mitocondrias/efectos de los fármacos , Células 3T3-L1 , Adenosina Trifosfato/biosíntesis , Animales , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ionóforos de Protónes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
NYGGF4 (also called PID1) is a recently discovered gene that is involved in obesity-related insulin resistance (IR). We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using metformin treatment in 3T3-L1 adipocytes. Our data showed that the metformin pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with metformin strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the metformin pretreatment. Our data also showed that metformin increased the expressions of PGC1-α, NRF-1, and TFAM, which were reduced in the NYGGF4 overexpression adipocytes. These results suggest that NYGGF4 plays a role in IR and its effects on IR could be reversed by metformin through activating IRS-1/PI3K/Akt and AMPK-PGC1-α pathways.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Metformina/farmacología , Transducción de Señal/efectos de los fármacos , Células 3T3-L1 , Adipocitos/patología , Animales , Proteínas Portadoras/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Factor Nuclear 1 de Respiración/genética , Factor Nuclear 1 de Respiración/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de TranscripciónRESUMEN
Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.
Asunto(s)
Antioxidantes/farmacología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Tióctico/farmacología , Células 3T3-L1 , Animales , Proteínas Reguladoras de la Apoptosis/genética , Expresión Génica , Glucosa/genética , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Resistencia a la Insulina/genética , Ratones , Fosforilación/efectos de los fármacos , Fosforilación/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Especies Reactivas de Oxígeno , Transducción de Señal/genéticaRESUMEN
NYGGF4 (also called PID1) was demonstrated that it may be related to the development of obesity-related IR. We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using α-Lipoic acid (LA) treatment, which could facilitate glucose transport and utilization in fully differentiated adipocytes. Our data showed that the LA pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the LA pretreatment. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with LA strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. These results suggest that LA protects 3T3-L1 adipocytes from NYGGF4-induced IR partially through increasing phosphorylation of IRS-1 and Akt and provide evidence that NYGGF4 may be a potential target for the treatment of obesity and obesity-related IR.
Asunto(s)
Adipocitos/efectos de los fármacos , Proteínas Portadoras/biosíntesis , Resistencia a la Insulina/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ácido Tióctico/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Ratones , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , TransfecciónRESUMEN
Polychlorinated biphenyls (PCBs) are persistent organic pollutants that affect embryonic development. The purpose of this study was to examine the effects of embryonic exposure to PCBs on early skeletal development in zebraï¬sh (Danio rerio). Zebraï¬sh embryos were immediately exposed to various concentrations (0, 0.125, 0.25, 0.5 and 1.0 mg/l) of PCBs (Aroclor 1254) after fertilization. Embryos were assessed at 24, 48, 72, 96 and 120 h post-fertilization (hpf) for changes in embryonic survival and malformation rates. Calcium content and vitamin D receptor (VDR), parathyroid hormone (PTH) and TRVP6 mRNA expressions were assessed at 120 hpf. The results showed that PCBs exposure decreased the survival rate of the embryos in a time-and dose-dependent manner. The embryos exposed to the higher concentrations of PCBs (0.5 and 1.0 mg/l) displayed obvious skeletal morphological deformities. At 120 hpf, the calcium content of the zebraï¬sh was downregulated in all the PCB-treated groups. VDR, PTH and TRVP6 mRNA expressions were all affected by PCBs. By 120 hpf, the mRNA expressions of VDR, PTH and TRVP6 from the PCB-treated larvae were all upregulated. The expressions of PTH and TRVP6 positively correlated with the level of PCBs to which the embryos were exposed. These results suggest that embryonic exposure to PCBs induces developmental deï¬cits in the zebraï¬sh skeleton.
Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Bifenilos Policlorados/efectos adversos , Contaminantes Químicos del Agua/efectos adversos , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Relación Dosis-Respuesta a Droga , Hormona Paratiroidea/biosíntesis , Bifenilos Policlorados/farmacología , Receptores de Calcitriol/biosíntesis , Canales Catiónicos TRPV/biosíntesis , Contaminantes Químicos del Agua/farmacologíaRESUMEN
To explore the effects of Lyrm1 knockdown on the mitochondrial function of 3 T3-L1 adipocytes using small interfering RNA (siRNA). 3 T3-L1 preadipocytes were infected with either a negative control (NC) expression lentivirus or a Lyrm1-shRNA expression lentivirus and induced to differentiate. The knockdown efficiency of Lrym1-specific shRNA in 3 T3-L1 cells was evaluated by real-time PCR. The ultrastructure of the mitochondria in adipocytes was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers and Ucp2 mRNA were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The mitochondrial membrane potential and ROS levels of cells were analyzed with a FACScan flow cytometer using Cell Quest software. Cells transfected with lentiviral-Lyrm1-shRNA showed a significantly reduced transcription of Lyrm1 mRNA compared with NC cells. The size and ultrastructure of mitochondria in Lyrm1 knockdown adipocytes was similar to those of the NC cells. There was no significant difference in mtDNA copy number between the two groups. The total level of ATP production, mitochondrial membrane potential and Ucp2 mRNA expression levels were dramatically increased in adipocytes transfected with Lyrm1 RNAi. Furthermore, the level of ROS was dramatically decreased in Lyrm1 knockdown adipocytes. Knockdown of the Lyrm1 gene in adipocytes resulted in dramatically increased cellular ATP production, mitochondrial membrane potentials and levels Ucp2 mRNA, while ROS levels were significantly decreased. These results imply that mitochondrial function is improved in adipocytes after the knockdown of Lyrm1.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Adipocitos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Mitocondrias/metabolismo , Obesidad/metabolismo , Células 3T3-L1 , Animales , Proteínas Reguladoras de la Apoptosis/genética , Citometría de Flujo , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Lentivirus , Potencial de la Membrana Mitocondrial , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mitocondrias/ultraestructura , Plásmidos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Obesity is a multifactorial disease resulting from interactions between susceptibility genes, psychosocial, and environmental factors. However, it is becoming evident that interindividual differences in obesity susceptibility depend also on epigenetic factors, although the mechanisms have not been fully elucidated. We have undertaken a genome-wide analysis of DNA methylation of human preadipocytes and mature adipocytes to examine the differences in methylation between them. We found hypomethylation occurring in 2,701 genes and hypermethylation in 1,070 genes after differentiation. Meanwhile, Gene Ontology analysis and Ingenuity Pathway Analysis showed many significant gene functions and pathways with altered methylation status after adipocyte differentiation. In addition, Signal-Net analysis showed that tumor necrosis factor-α, mitogen-activated protein kinase, and interleukin-8 were important to the formation of this network. Our results suggest that DNA methylation mechanisms may be involved in regulating the differentiation process of human preadipocytes.
Asunto(s)
Adipocitos/metabolismo , Metilación de ADN , Adipocitos/citología , Diferenciación Celular , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Obesidad/metabolismo , Obesidad/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002 mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Resistencia a la Insulina/fisiología , Insulina/farmacología , Proteínas de la Membrana/inmunología , Mitocondrias/metabolismo , Oxidorreductasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenosina Trifosfato/biosíntesis , Adipocitos/efectos de los fármacos , Adipocitos/inmunología , Adipocitos/metabolismo , Anticuerpos Monoclonales/inmunología , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/inmunología , Potencial de la Membrana Mitocondrial , Mitocondrias/efectos de los fármacos , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We previously identified the six-transmembrane epithelial antigen of prostate (STEAP) 4 as a novel plasma membrane protein that is up-regulated in obese patients and may play a significant role in the development of human obesity. In this study, a STEAP4-specific antibody was used to characterize the biological functions of the STEAP4 protein in human adipocytes. Cell viability assays (Trypan Blue exclusion), CCK-8 assays and cell cycle analysis showed that the STEAP4 antibody inhibited pre-adipocyte proliferation. Morphological observations by electron microscopy and confocal laser microscopy, annexin V-FITC labeling, caspase-3 and caspase-8 activity assays as well as data from quantitative real-time RT-PCR (qPCR) further determined that the STEAP4 antibody could promote apoptosis in pre-adipocytes. Based on quantitative Oil Red O staining and the expression profiles of specific markers, we demonstrated that the STEAP4 antibody did not affect adipogenesis, but the 2-deoxy-d-[3H]-glucose uptake tests showed that it induced the insulin-stimulated glucose uptake in mature human adipocytes. In conclusion, our results demonstrated that the STEAP4 antibody does not influence human adipocyte differentiation, but it is likely that the STEAP4 protein regulates proliferation and apoptosis and plays an important role in modulating the insulin sensitivity of human adipocytes.
Asunto(s)
Adipocitos/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Glucosa/metabolismo , Proteínas de la Membrana/inmunología , Oxidorreductasas/inmunología , Adipocitos/citología , Adipocitos/metabolismo , Análisis de Varianza , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Células Cultivadas , Humanos , Inmunohistoquímica , Sincalida/metabolismoRESUMEN
Previous studies have determined that lin-4, which was the first miRNA to be discovered, controls the timing of cell fate determination and life span in Caenorhabditis elegans. However, the mechanism of lin-4 involvement in these processes remains poorly understood. Fat storage is an essential aspect of the life cycle of organisms, and the function of lin-4 in fat accumulation is not clear. In this study, we showed that the fat content is reduced remarkably in C. elegans lin-4 mutants. Quantitative RT-PCR analysis revealed a considerable decrease in the levels of SBP-1 and OGA-1 mRNA in lin-4 mutants. We also showed that lin-4 mutants have a significantly shorter life span than wild-type worms. DCF assay experiments showed that the reactive oxygen species (ROS) levels increased and mitochondrial DNA (mtDNA) copy number decreased in loss-of-function lin-4 mutants. These mutants also showed attenuation of locomotion. Taken together, our findings suggest that lin-4 may play an important role in regulating fat accumulation and locomotion and that lin-4 may control the life span of C. elegans by mediating ROS production.