RESUMEN
High blood concentrations of nonesterified fatty acids (NEFAs) provoke various metabolic disorders and are associated with mammary tissue injury and decreased milk production in dairy cows. Nuciferine, an alkaloid found in Nelumbo nucifera leaves, has great potential for correcting lipid metabolism derangements and lipotoxicity. In this study, we evaluated the lipotoxicity induced by excessive NEFA in bovine mammary epithelial cells (bMECs) and investigated whether nuciferine alleviates NEFA-induced lipotoxicity and the underlying molecular mechanisms. We found that excessive NEFA (1.2 and 2.4 mM) induced lipid accumulation, apoptosis, and migration ability impairment in bMECs, whereas nuciferine could ameliorate these disarrangements, as indicated by decreasing triglyceride content, protein abundance of SREBP-1c, cytoplasmic cytochrome c, and cleaved caspase-3 and increasing protein abundance of PPARα and migration ability. Moreover, nuciferine could reverse NEFA-induced LKB1/AMPK signaling inhibition, and the protective effect of nuciferine on lipotoxicity caused by NEFA was abrogated by AMPK inhibitor dorsomorphin. Furthermore, transfection with LKB1 siRNA (si-LKB1) largely abolished the activation effect of nuciferine on AMPK. Overall, nuciferine can protect bMECs from excessive NEFA-induced lipid accumulation, apoptosis, and impaired migration by activating LKB1/AMPK signaling pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Ácidos Grasos no Esterificados , Animales , Bovinos , Femenino , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis , Células Epiteliales/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Metabolismo de los Lípidos , Transducción de Señal , Quinasas de la Proteína-Quinasa Activada por el AMP/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD), characterized with oxidative stress and hepatic steatosis, is a serious threat to human health. As a specific activator of nuclear factor E2-related factor 2 (Nrf2), the 4-octyl itaconate (4-OI) has the beneficial effects in antioxidant and anti-inflammation; however, whether 4-OI can alleviate hepatic steatosis and its mechanism is still unknown. The present study was aimed at investigating the protective effects of 4-OI on free fat acid- (FFA-) induced lipid metabolism disorder and its potential molecular mechanism in hepatocytes. The results showed that 4-OI treatment markedly alleviated FFA-induced oxidative stress and excessive lipid accumulation in hepatocytes. Mechanistically, 4-OI significantly suppressed the overproduction of reactive oxygen species (ROS) through activation of Nrf2; the downregulation of ROS level induced a downregulation of AMP-dependent protein kinase (AMPK) phosphorylation level which finally ameliorated excessive lipid accumulation in FFA-stimulated hepatocytes. In general, our data demonstrated that 4-OI relieves the oxidative stress and lipid metabolism disorder in FFA-stimulated hepatocytes; and these beneficial effects were achieved by activating the Nrf2-AMPK signaling pathway. These data not only expand the new biological function of 4-OI but also provide a theoretical basis for 4-OI to protect against lipid metabolism disorders and related diseases, such as NAFLD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Hepatocitos/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Succinatos/farmacología , Animales , Línea Celular , Hepatocitos/metabolismo , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action leading to insulin resistance and Type 2 diabetes mellitus. Several serine/threonine kinases including JNK, mTOR, and p70 S6K cause serine phosphorylation of the insulin receptor substrate (IRS) and have been implicated in insulin resistance. Activation of AMP-activated protein kinase (AMPK) increases glucose uptake, and in recent years, AMPK has been viewed as an important target to counteract insulin resistance. We reported previously that carnosic acid (CA) found in rosemary extract (RE) and RE increased glucose uptake and activated AMPK in muscle cells. In the present study, we examined the effects of CA on palmitate-induced insulin-resistant L6 myotubes and 3T3L1 adipocytes. Exposure of cells to palmitate reduced the insulin-stimulated glucose uptake, GLUT4 transporter levels on the plasma membrane, and Akt activation. Importantly, CA attenuated the deleterious effect of palmitate and restored the insulin-stimulated glucose uptake, the activation of Akt, and GLUT4 levels. Additionally, CA markedly attenuated the palmitate-induced phosphorylation/activation of JNK, mTOR, and p70S6K and activated AMPK. Our data indicate that CA has the potential to counteract the palmitate-induced muscle and fat cell insulin resistance.
Asunto(s)
Abietanos/farmacología , Adipocitos/patología , Ácidos Grasos no Esterificados/toxicidad , Resistencia a la Insulina , Células Musculares/patología , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos/efectos de los fármacos , Animales , Línea Celular , Glucosa/metabolismo , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Modelos Biológicos , Células Musculares/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Palmitatos/toxicidad , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Background: Inflammation and apoptosis play a crucial role in the progression of nonalcoholic steatohepatitis (NASH). Suppressor of cytokine signaling 2 (SOCS2) is one of classic negative regulators of cytokine signaling, which has recently been described as anti-inflammatory mediators. However, the role of SOCS2 in macrophages during NASH progression and the relationship among SOCS2, inflammation, apoptosis and NASH is largely unknown. Herein, we aimed to study the function of SOCS2 in NASH progression. Methods: We detected SOCS2 expression in macrophages in human subjects without steatosis, with simple steatosis and with NASH to confirm the relationship between SOCS2 and NASH. Free fatty acids was used to establish stress environment in RAW 264.7 cell lines stably overexpressing or knockdown SOCS2. In vitro and vivo assays also performed to study the molecular function of SOCS2 in NASH progression. Findings: Our human samples illustrated that SOCS2 was decreased in macrophages during NASH progression and was negatively correlated to NASH level. Meanwhile, In vitro assays showed SOCS2 overexpression in macrophages suppressed inflammation and apoptosis via inhibiting NF-κB signaling pathway, while SOCS2 knock-down in macrophages caused an increased activation of NF-κB, which could be blocked by ammonium 1-pyrrolidinedithiocarbamate (PDTC). In addition, SOCS2 in macrophages also suppressed inflammation via limiting the activation of inflammasomes. Consistent with these, our BMT model also confirmed the SOCS2 function in macrophages during NASH. Interpretation: Our data strongly indicate that SOCS2 plays a role in inhibiting inflammation and apoptosis via NF-κB and inflammasome signaling pathway in macrophages during NASH. Further studies are required to explore the potential preventive and therapeutic strategies of SOCS2 for this common liver disease.
Asunto(s)
Apoptosis/fisiología , Inflamación/metabolismo , Macrófagos/metabolismo , FN-kappa B/metabolismo , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Trasplante de Médula Ósea , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos no Esterificados/toxicidad , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/genética , Macrófagos/efectos de los fármacos , Ratones , FN-kappa B/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Células RAW 264.7 , Transducción de Señal , Estrés Fisiológico , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
In the perinatal period of dairy cows, negative energy balance (NEB) is likely to occur, which increases the level of non-esterified fatty acids (NEFA) in the follicular fluid, hinders the proliferation of granulosa cells (GCs), and thus endangers the development of oocytes and the fecundity of dairy cows. We found that there were oxidative stress and inflammatory response in the serum of cows with perinatal ketosis. Whether the oxidative stress induced by NEFA is involved in the pyroptosis and inflammation of GCs remains unclear. After NEFA treatment, the expression of NLRP3 and caspase-1 and the release of inflammatory cytokines IL-1ß were increased in a dose-dependent manner, indicating that NEFA may contribute to pyroptosis. Besides, NEFA stimulation induced oxidative stress, resulting in the phosphorylation of NF-κB, and increased the production of interleukin (IL)-6 and nitric oxide (NO), indicating that NEFA may induce inflammation in GCs. However, the NEFA-mediated effects were observably reversed when the GCs were pre-treated with antioxidant and radical scavenger, N-acetylcysteine (NAC). Taken together, our results reveal that NEFA can induce pyroptosis and inflammation through NLRP3 inflammasome and TLR4/NF-κB pathway, respectively, and NAC can alleviate these conditions.
Asunto(s)
Acetilcisteína/farmacología , Ácidos Grasos no Esterificados/toxicidad , Células de la Granulosa/patología , Inflamación/patología , Piroptosis/efectos de los fármacos , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Femenino , Células de la Granulosa/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Cetosis/patología , Modelos Biológicos , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Insulin resistance, a main characteristic of type 2 diabetes mellitus (T2DM), is linked to obesity and excessive levels of plasma free fatty acids (FFA). Studies indicated that significantly elevated levels of FFAs lead to skeletal muscle insulin resistance, by dysregulating the steps in the insulin signaling cascade. The polyphenol resveratrol (RSV) was shown to have antidiabetic properties but the exact mechanism(s) involved are not clearly understood. In the present study, we examined the effect of RSV on FFA-induced insulin resistance in skeletal muscle cells in vitro and investigated the mechanisms involved. Parental and GLUT4myc-overexpressing L6 rat skeletal myotubes were used. [3H]2-deoxyglucose (2DG) uptake was measured, and total and phosphorylated levels of specific proteins were examined by immunoblotting. Exposure of L6 cells to FFA palmitate decreased the insulin-stimulated glucose uptake, indicating insulin resistance. Palmitate increased ser307 (131% ± 1.84% of control, p < 0.001) and ser636/639 (148% ± 10.1% of control, p < 0.01) phosphorylation of IRS-1, and increased the phosphorylation levels of mTOR (174% ± 15.4% of control, p < 0.01) and p70 S6K (162% ± 20.2% of control, p < 0.05). Treatment with RSV completely abolished these palmitate-induced responses. In addition, RSV increased the activation of AMPK and restored the insulin-mediated increase in (a) plasma membrane GLUT4 glucose transporter levels and (b) glucose uptake. These data suggest that RSV has the potential to counteract the FFA-induced muscle insulin resistance.
Asunto(s)
Adenilato Quinasa/fisiología , Ácidos Grasos no Esterificados/toxicidad , Resistencia a la Insulina/fisiología , Músculo Esquelético/efectos de los fármacos , Resveratrol/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Serina-Treonina Quinasas TOR/fisiología , Animales , Línea Celular , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Palmitatos/farmacología , Palmitatos/toxicidad , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Although glucagon-like peptide-1 (GLP-1) analogue has been reported to suppress oxidative stress in non-alcoholic fatty liver disease (NAFLD), an effective therapeutic agent for NAFLD is currently unavailable. Therefore, in this study, we aimed to investigate the protective effects of the GLP-1 analogue liraglutide against lipotoxicity-induced oxidative stress in HepG2 cells and to elucidate the underlying mechanisms. HepG2 cells were cultured for 48 hours and treated with a free fatty acid (FFA) mixture: FFA mixture and liraglutide or FFA mixture, liraglutide, and exendin (9-39). Lipid accumulation was examined by oil red O staining. Oxidative stress was assessed by measuring the levels of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate and thiobarbituric acid-reactive substances, whereas antioxidant capacity was assessed by measuring the activity of superoxide dismutase and catalase. Expression of the nuclear factor erythroid-2-related factor 2 (NRF2) gene and the genes encoding antioxidant enzymes was analyzed using quantitative RT-PCR. Cellular and nuclear NRF2 expression levels were assessed using immunofluorescence cell staining and western blotting. Liraglutide treatment reduced high fat-induced lipid formation and the levels of oxidative stress markers and increased antioxidant enzyme activity in HepG2 cells. Liraglutide treatment increased the mRNA expression of NRF2 target genes, induced NRF2 nuclear translocation, and increased nuclear NRF2 levels without altering NRF2 mRNA expression. Collectively, these results indicate that liraglutide exhibits a protective effect against lipotoxicity-induced oxidative stress, possibly via modulation of NRF2 and expression of antioxidant enzymes in liver cells.
Asunto(s)
Ácidos Grasos no Esterificados/toxicidad , Liraglutida/farmacología , Hígado/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Citoprotección/efectos de los fármacos , Células Hep G2 , Humanos , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
An inappropriate diet, particularly excessive consumption of dietary fats and oils, may have a major negative impact on beta-cell function and cause type 2 diabetes mellitus. To investigate this issue, we examined the toxicity of free fatty acid (FFA) compositions mirroring the FFA profiles of various popular edible oils in human EndoC-ßH1 beta-cells and in rat islets. For this purpose, we made compositions consisting exclusively of various FFAs in different volumetric percentages mimicking these oils and additionally mixtures of these compositions. Human EndoC-ßH1 beta-cells were incubated with different oil compositions and the toxicity, lipid droplet formation, ER-stress, and H2O2 production were analyzed. Compositions with prominent content of saturated as well as unsaturated long-chain FFAs showed moderate but significant toxicity both in human EndoC-ßH1 beta-cells and rat islets, however, without further measurable metabolic impairments. On the other hand compositions with high content of medium-chain FFAs revealed no toxicity. A composition with 50% of the very long-chain unsaturated FFA erucic acid caused high toxicity with concomitant peroxisomal H2O2 production. The toxicity of FFAs to human EndoC-ßH1 beta-cells was dampened in mixtures of FFA compositions with a significant content of medium-chain FFAs, but not with a significant proportion of unsaturated FFAs.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Células Secretoras de Insulina/efectos de los fármacos , Aceites de Plantas/toxicidad , Animales , Mantequilla/toxicidad , Línea Celular , Estrés del Retículo Endoplásmico , Ácidos Grasos no Esterificados/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Células Secretoras de Insulina/metabolismo , Aceites de Plantas/metabolismo , RatasRESUMEN
Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120-126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.
Asunto(s)
Blastocisto/efectos de los fármacos , Ácidos Grasos no Esterificados/toxicidad , Fertilidad/efectos de los fármacos , Lipólisis , Mórula/efectos de los fármacos , Oviductos/metabolismo , Cigoto/efectos de los fármacos , Animales , Blastocisto/metabolismo , Blastocisto/patología , Bovinos , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Ácidos Grasos no Esterificados/metabolismo , Femenino , Fertilización In Vitro , Mórula/metabolismo , Mórula/patología , Embarazo , Cigoto/metabolismo , Cigoto/patologíaRESUMEN
In Japan and other Asian countries, increased fat uptake induced by a westernized diet is thought to be associated with an increased incidence of inflammatory bowel disease, colorectal cancer and food allergies; however, the mechanism for this remains unclear. High-fat diet (HFD)-fed mice are common animal models used to examine the effect of fat intake in vivo. HFDs are reported to exacerbate DSS-induced colitis and intestinal tumorigenesis, but the effect of HFDs on the intestines before disease induction is often overlooked. We found that the intestinal and gut-associated lymphoid tissue (GALT) morphology of HFD-fed mice differed from that of standard diet (SD)-fed mice. To clarify the mechanism by which fat intake increases intestinal diseases, we analyzed the morphological and immunological aspects of the intestines of HFD-fed mice as well as the molecular mechanisms and physiology. Feeding an HFD for 3 weeks induced atrophy of the small intestine, colon and GALT and reduced the number of small intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). Feeding an HFD for only one day reduced the number of small intestinal (SI)-IELs and SI-LPLs. The effect of feeding a 3-week HFD continued for 2 weeks after returning to the SD. The effect of the HFD on the intestinal immune system was independent of the gut microbes. We hypothesized that the cytotoxicity of the abundant HFD-derived free fatty acids in the intestinal lumen impairs the intestinal immune system. Both saturated and unsaturated free fatty acids were toxic to intestinal T-cells in vitro. Orally administering free fatty acids reduced the number of SI-IELs and LPLs. Using a lipase inhibitor to reduce the luminal free fatty acids attenuated the HFD-induced changes in the intestinal immune system, while using a statin to reduce the serum free fatty acids did not. Thus, HFD-induced free fatty acids damaged the intestines; this effect was termed "intestinal lipotoxicity". Because sustained reduction of SI-LPLs after HFD feeding exacerbated indomethacin-induced small intestinal damage, lipotoxicity to the human intestines incurred by consuming a westernized diet in Japan may increase intestinal diseases such as IBD, colorectal cancer or food allergies.
Asunto(s)
Dieta Alta en Grasa , Ácidos Grasos no Esterificados/toxicidad , Sistema Inmunológico/patología , Mucosa Intestinal/patología , Animales , Atrofia , Colon/patología , Ácidos Grasos no Esterificados/sangre , Conducta Alimentaria , Microbioma Gastrointestinal/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Indometacina , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/patología , Recuento de Linfocitos , Linfocitos/efectos de los fármacos , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/patología , Masculino , Ratones Endogámicos C57BLRESUMEN
The deleterious effects of chronically elevated free fatty acid (FFA) levels on glucose homeostasis are referred to as lipotoxicity, and the concurrent exposure to high glucose may cause synergistic glucolipotoxicity. Lipo- and glucolipotoxicity have been studied for over 25 years. Here, we review the current evidence supporting the role of pancreatic ß-cell lipo- and glucolipotoxicity in type 2 diabetes (T2D), including lipid-based interventions in humans, prospective epidemiological studies, and human genetic findings. In addition to total FFA quantity, the quality of FFAs (saturation and chain length) is a key determinant of lipotoxicity. We discuss in vitro and in vivo experimental models to investigate lipo- and glucolipotoxicity in ß-cells and describe experimental pitfalls. Lipo- and glucolipotoxicity adversely affect many steps of the insulin production and secretion process. The molecular mechanisms underpinning lipo- and glucolipotoxic ß-cell dysfunction and death comprise endoplasmic reticulum stress, oxidative stress and mitochondrial dysfunction, impaired autophagy, and inflammation. Crosstalk between these stress pathways exists at multiple levels and may aggravate ß-cell lipo- and glucolipotoxicity. Lipo- and glucolipotoxicity are therapeutic targets as several drugs impact the underlying stress responses in ß-cells, potentially contributing to their glucose-lowering effects in T2D.
Asunto(s)
Diabetes Mellitus Tipo 2 , Ácidos Grasos no Esterificados/toxicidad , Glucosa/toxicidad , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/metabolismo , Animales , Autofagia , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/metabolismo , Estrés del Retículo Endoplásmico , Ácidos Grasos no Esterificados/metabolismo , Receptor del Péptido 1 Similar al Glucagón/análisis , Receptor del Péptido 1 Similar al Glucagón/uso terapéutico , Glucosa/metabolismo , Humanos , Inflamación , Insulina/biosíntesis , Insulina/metabolismo , Metformina/farmacología , Mitocondrias/patología , Estrés Oxidativo , Transducción de Señal , Tiazolidinedionas/farmacologíaRESUMEN
Transient Receptor Potential Melastatin-2 (TRPM2) is a nonselective cation channel mediating Ca2+ influx in response to oxidative stress. Given that insulin resistance-related endothelial dysfunction in obesity attributes to fatty-acid-induced reactive oxygen species (ROS) overproduction, in this study, we addressed the possible role of TRPM2 in obesity-related endothelial insulin resistance and the underlying mechanisms. Whole-cell patch clamp technique, intracellular Ca2+ concentration measurement, western blot, vasorelaxation assay, and high-fat diet (HFD)-induced obese model were employed to assess the relationship between TRPM2 and endothelial insulin response. We found that both the expression and activity of TRPM2 were higher in endothelial cells of obese mice. Palmitate rose a cationic current in endothelial cells which was inhibited or enlarged by TRPM2 knockdown or overexpression. Silencing of TRPM2 remarkably improved insulin-induced endothelial Akt activation, nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production, while TRPM2 overexpression resulted in the opposite effects. Furthermore, TRPM2-mediated Ca2+ entry, CaMKII activation and the following activation of PERK/ATF4/TRB3 cascade were involved in the mechanism of obesity or palmitate-induced endothelial insulin resistance. Notably, in vivo study, knockdown of TRPM2 with adeno-associated virus harboring short-hairpin RNA (shRNA) against TRPM2 alleviated endothelial insulin resistance and ameliorated endothelium-dependent vasodilatation in obese mice. Thus, these results suggest that TRPM2-activated Ca2+ signaling is necessary to induce insulin resistance-related endothelial dysfunction in obesity. Downregulation or pharmacological inhibition of TRPM2 channels may lead to the development of effective drugs for treatment of endothelial dysfunction associated with oxidative stress state.
Asunto(s)
Calcio/metabolismo , Endotelio Vascular/patología , Ácidos Grasos no Esterificados/toxicidad , Peróxido de Hidrógeno/toxicidad , Resistencia a la Insulina , Obesidad/fisiopatología , Canales Catiónicos TRPM/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Oxidantes/toxicidad , Estrés Oxidativo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Canales Catiónicos TRPM/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Elevated free fatty acids (FFAs) are a risk factor for type 2 diabetes. Endothelial dysfunction induced by high levels of FFAs is one of the mechanisms related to the progression of diabetes. In clinical diabetes care, DPP-4 inhibitors have been shown to be effective in reducing glucose levels. In this study, we investigated the molecular mechanism of the clinically available DPP-4 inhibitor vildagliptin in the protection of FFA-induced endothelial dysfunction. Treatment of endothelial cells with vildagliptin inhibits FFA-induced cellular LDH release and generation of ROS. Vildagliptin also reverses FFA-induced reduced levels of GSH and elevated expression of the FFA-associated NAPHD oxidase protein NOX-4. Moreover, vildagliptin ameliorates the reduction in mitochondrial potential triggered by FFAs. Mechanistically, we show that vildagliptin suppresses FFA-induced expression of proteins of the NLRP3 inflammasome complex, including NLRP3, ASC, p20 and HMGB-1, and mitigates FFA-induced inactivation of the AMPK pathway. Consequently, vildagliptin inhibits production of two cytokines that are favored by NLRP3 inflammasome machinery: IL-1ß and IL-18. Finally, we demonstrate that vildagliptin ameliorates FFA-induced reduced eNOS, indicating its protective role against endothelial dysfunction. Collectively, we conclude that the protective role of vildagliptin in endothelial cells is mediated via suppression of the AMPK-NLRP3 inflammasome-HMGB-1 axis pathway. These findings imply that the anti-diabetic drug vildagliptin possesses dual therapeutic applications in lowering glucose and improving vascular function.
Asunto(s)
Ácidos Grasos no Esterificados/toxicidad , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Vildagliptina/farmacología , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteína HMGB1/genética , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Interleucina-18/biosíntesis , Interleucina-1beta/biosíntesis , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NADPH Oxidasa 4/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Elevated blood free fatty acids (FFAs), as seen in obesity, impair muscle insulin action leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) is linked to insulin resistance and a number of serine/threonine kinases including JNK, mTOR and p70 S6K have been implicated in this process. Activation of the energy sensor AMP-activated protein kinase (AMPK) increases muscle glucose uptake, and in recent years AMPK has been viewed as an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased muscle cell glucose uptake and activated AMPK. However, the effect of RE on FFA-induced muscle insulin resistance has never been examined. In the current study, we investigated the effect of RE in palmitate-induced insulin resistant L6 myotubes. Exposure of myotubes to palmitate reduced the insulin-stimulated glucose uptake, increased serine phosphorylation of IRS-1, and decreased the insulin-stimulated phosphorylation of Akt. Importantly, exposure to RE abolished these effects and the insulin-stimulated glucose uptake was restored. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 S6K whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Our data indicate that rosemary extract has the potential to counteract the palmitate-induced muscle cell insulin resistance and further studies are required to explore its antidiabetic properties.
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Ácidos Grasos no Esterificados/toxicidad , Resistencia a la Insulina/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Ácido Palmítico/toxicidad , Extractos Vegetales/farmacología , Rosmarinus/química , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Fosforilación/efectos de los fármacos , Extractos Vegetales/química , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Procyanidin B2, a naturally occurring phenolic compound, has been reported to exert multiple beneficial functions. However, the effect of procyanidin B2 on free fatty acids (FFAs)-induced hepatic steatosis remains obscure. The present study is therefore aimed to elucidate the protective effect of procyanidin B2 against hepatic steatosis and its underlying mechanism. Herein, we reported that procyanidin B2 attenuated FFAs-induced lipid accumulation and its associated oxidative stress by scavenging excessive ROS and superoxide anion radicals, blocking loss of mitochondrial membrane potential, restoring glutathione content, and increasing activity of antioxidant enzymes (GPx, SOD and CAT) in hepatocytes. Procyanidin B2 mechanistically promoted lipid degradation via modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathway. Molecular docking analysis indicated a possible ligand-binding position of procyanidin B2 with TFEB. In addition, administration of procyanidin B2 resulted in a significant reduction of hepatic fat accumulation in high-fat diet (HFD)-induced obese mice, and also ameliorated HFD-induced metabolic abnormalities, including hyperlipidemia and hyperglycemia. It was confirmed that procyanidin B2 prevented HFD-induced hepatic fat accumulation through down-regulating lipogenesis-related gene expressions (PPARγ, C/EBPα and SREBP-1c), inhibiting pro-inflammatory cytokines production (IL-6 and TNF-α) and increasing antioxidant enzymes activity (GPx, SOD and CAT). Moreover, hepatic fatty acids analysis indicated that procyanidin B2 caused a significant increase in the levels of palmitic acid, oleic acid and linoleic acid. Intriguingly, procyanidin B2 restored the decreased nuclear TFEB expression in HFD-induced liver steatosis and up-regulated its target genes involved in lysosomal pathway (Lamp1, Mcoln, Uvrag), which suggested a previously unrecognized mechanism of procyanidin B2 on ameliorating HFD-induced hepatic steatosis. Taken together, our results demonstrated that procyanidin B2 attenuated FFAs-induced hepatic steatosis through regulating TFEB-mediated lysosomal pathway and redox state, which had important implications that modulation of TFEB might be a potential therapeutic strategy for hepatic steatosis and procyanidin B2 could represent a promising novel agent in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD).
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Biflavonoides/administración & dosificación , Catequina/administración & dosificación , Hígado Graso/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Estrés Oxidativo/genética , Proantocianidinas/administración & dosificación , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/metabolismo , Hígado/patología , Lisosomas/genética , Lisosomas/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Ratones , Simulación del Acoplamiento Molecular , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Ácido Oléico/metabolismo , Oxidación-Reducción/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the role of Toll-like receptor-4 (TLR4) in the free fatty acids (FFAs) induced human umbilical vein endothelial cells (HUVECs) inflammation and to explore the underlying mechanisms. MATERIALS AND METHODS: HUVECs and HEK293 cell lines were obtained from Shanghai Type Culture Collection. Cell counting kit-8 (CCK8) and flow cytometry (FCM) were performed to examine the cell viability and apoptosis rate of HUVECs induced by FFAs treatments with or without infection of toll-like receptor-4 interference (TLR4i) adenovirus. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the inflammatory cytokines release. Quantitative polymerase chain reaction (qPCR) and Western Blot (WB) were used to test the molecular mechanisms of inflammation. RESULTS: FFAs induced inflammatory responses in HUVECs via modulating the TLR4 receptor complex. TLR4i adenovirus interference increased cell viability and decreased cell apoptosis rate. FFAs treatments significantly increased the expressions of inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5) and CXC chemokine ligand 10 (CXCL10), while TLR4i adenovirus interference significantly reduced these cytokines levels. TLR4-mediated myeloid differential protein-88 (MyD88) expression activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inhabiting kappa B kinase-beta (IKK-ß). TLR4i adenovirus interference decreased the expressions of these genes at both mRNA level and protein level. CONCLUSIONS: TLR4 mediates FFAs induced inflammatory responses in HUVECs. TLR4 interference in HUVECs significantly reduces the inflammatory cytokines expression, decreases the cell apoptosis rate and increases cell viability.
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Ácidos Grasos no Esterificados/toxicidad , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Mediadores de Inflamación/metabolismo , Receptor Toll-Like 4/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Receptor Toll-Like 4/antagonistas & inhibidores , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
Insulin resistance, a pathophysiology of type 2 diabetes, is associated with obesity. Lipotoxicity in obesity leads to the dysfunction and death of pancreatic ß-cells and inadequate insulin production, thereby aggravating type 2 diabetes. The present study was conducted to determine the effect of Aloe vera polysaccharides (APs) as an anti-hyperglycemic agent and their mechanisms of action. Gel polysaccharides from Aloe extracts were separated using ultrafiltration devices with molecular weight-cutoff membranes, and the protective effect of APs on pancreatic ß-cells in response to free fatty acids (FFAs) was determined. Hamster pancreatic ß-cell line HIT-T15 was treated with palmitate and APs to analyze cellular responses. We observed a large number of apoptotic ß-cell death after treatment with high levels of palmitate, but this was efficiently prevented by the addition of APs in a dose-dependent manner. It was found that the anti-apoptotic properties of APs were largely due to the relief of endoplasmic reticulum (ER) stress signaling. APs were effective in interfering with the FFA-induced activation of the PERK and IRE1 pathways as well as ROS generation, thereby protecting pancreatic ß-cells from lipotoxicity. Although variation in the chain length of APs can influence the activity of FFA-mediated ER stress signaling in different ways, polysaccharide mixtures with molecular weights higher than 50â¯kDa showed greater antiapoptotic and antioxidant activity in ß-cells. After oral administration of APs, markedly lowering fasting blood glucose levels were observed in db/db mice, providing evidence of the potential of APs as an alternative insulin sensitizer. Therefore, it was concluded that APs have a protective effect against type 2 diabetes by modulating obesity-induced ER stress in pancreatic ß-cells.
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Aloe/química , Citoprotección/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácidos Grasos no Esterificados/toxicidad , Células Secretoras de Insulina/patología , Polisacáridos/farmacología , Sustancias Protectoras/farmacología , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Línea Celular , Cricetinae , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Regulación hacia Abajo/efectos de los fármacos , Hiperglucemia/complicaciones , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/patología , Hiperlipidemias/complicaciones , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/patología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos C57BL , Palmitatos/toxicidad , Polisacáridos/uso terapéutico , Sustancias Protectoras/uso terapéuticoRESUMEN
In recent years, bio-based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L-1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L-1 . Next, an isolated oleate-resistant strain produced 382.8 mg L-1 of free fatty acids. Finely, acyl-CoA carboxylase gene (ACC1) over-expression resulted to production of 1436.7 mg L-1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L-1 during test tube cultivation.
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Ácidos Grasos no Esterificados/metabolismo , Glicerol/metabolismo , Ingeniería Metabólica/métodos , Yarrowia/metabolismo , Proliferación Celular/efectos de los fármacos , Ácidos Grasos no Esterificados/análisis , Ácidos Grasos no Esterificados/toxicidad , Glucosa/metabolismo , Glicerol/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Ácido Oléico/metabolismo , Ácido Oléico/toxicidad , Yarrowia/efectos de los fármacos , Yarrowia/genéticaRESUMEN
We investigated the effects of a Leonurus japonicus ethanol extract (LJE) on nonalcoholic fatty liver disease (NAFLD). An in vitro model of hepatic steatosis was treated with 1 mM free fatty acid (FFA) in HepG2 cells. An in vivo NAFLD model was established using C57BL/6 mice fed a high-fat diet (HFD) and administered LJE (100 or 200 mg/kg) orally for 14 weeks. LJE treatment suppressed lipid accumulation and intracellular triglyceride levels significantly in a concentration-dependent manner in HepG2 cells. Moreover, LJE significantly reduced the expression of sterol regulatory element binding protein 1-c, and its downstream genes, which are associated with lipogenesis, in HepG2 cells. In HFD-fed mice, LJE treatment decreased body weight significantly and decreased serum alanine transaminase levels to normal values, concurrent with a decrease in hepatic lipid accumulation. Furthermore, LJE supplementation ameliorated insulin sensitivity by decreasing serum glucose and insulin levels. LJE improved hepatic steatosis by increasing the expression of phosphorylated AMP-activated protein kinase and peroxisome proliferator-activated receptor-α in HFD-fed mice and FFA-treated HepG2 cells. The results suggested that LJE might be a potential therapeutic agent to treat NAFLD.
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Dieta Alta en Grasa , Ácidos Grasos no Esterificados/toxicidad , Hepatocitos/efectos de los fármacos , Leonurus , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Extractos Vegetales/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Supervivencia Celular/efectos de los fármacos , Citoprotección , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Insulina/sangre , Resistencia a la Insulina , Leonurus/química , Lipogénesis/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , PPAR alfa/metabolismo , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Tiempo , Triglicéridos/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
BACKGROUND: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. RESULTS: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. CONCLUSIONS: Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.