RESUMO
Increased levels of serum free fatty acids (FFAs) are closely associated with microvascular dysfunction. In our previous study, a coronary microvascular dysfunction (CMD) model was successfully established via lipid infusion to increase the levels of serum FFAs in mice. However, the underlying mechanisms remained poorly understood. Therefore, the aim of the present study was to explore the mechanism underlying FFAinduced CMD. A CMD mouse model was established via lipid combined with heparin infusion for 6 h to increase the concentration of serum FFAs. Following the establishment of the model, the coronary flow reserve (CFR), extent of leukocyte activation and cardiac microvascular structures were assessed in the mice. Cardiac microvascular endothelial cells (CMECs) were treated with different concentrations of palmitic acid and cell viability was evaluated. Changes in the expression levels of AMPactivated protein kinase (AMPK), Krüppellike factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) were identified by immunohistochemical and western blot analyses. Experiments using AMPK activator, KLF2 overexpression plasmid, small interfering RNAs and nicorandil were subsequently designed to investigate the potential involvement of the AMPK/KLF2/eNOS signaling pathway. These experiments revealed that FFAs could induce CMD in mice, which was characterized by reduced CFR (1.89±0.37 vs. 2.74±0.30) and increased leukocyte adhesion (4,350±1,057.5 vs. 11.8±5.4 cells/mm2) compared with the control mice. CD11b expression and intracellular reactive oxygen species (ROS) levels were increased in CMD model mice compared with control mice. Serum TNFα and IL6 levels were higher in the model group than in the control group. Transmission electron microscopy revealed that CMECs in heart tissues of model mice were severely swollen. In addition, palmitic acid decreased CMEC viability and increased ROS production in a dosedependent manner. Notably, the AMPK/KLF2/eNOS signaling pathway was demonstrated to be suppressed by FFAs both in vivo and in vitro. Activation of this axis with AMPK activator, KLF2 overexpression plasmid or nicorandil restored the CFR in CMD model mice, inhibited oxidative stress and increased CMEC viability. Taken together, the results of the present study demonstrated that FFAs could induce CMD via inhibition of the AMPK/KLF2/eNOS signaling pathway, whereas activation of this pathway led to the alleviation of FFAinduced CMD, which may be a therapeutic option for CMD.
Assuntos
Células Endoteliais , Ácidos Graxos não Esterificados , Microcirculação , Miocárdio , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos não Esterificados/efeitos adversos , Ácidos Graxos não Esterificados/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Nicorandil , Óxido Nítrico Sintase Tipo III/metabolismo , Ácidos Palmíticos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Microcirculação/fisiologia , Miocárdio/patologiaRESUMO
BACKGROUND: There are hypersensitivity reactions (HRs) to foods in which nonsteroidal anti-inflammatory drugs (NSAIDs) act as aggravating factors (NSAID-exacerbated food allergy [NEFA]) or cofactors (NSAID-induced food allergy [NIFA]), often misdiagnosed as HRs to NSAIDs. Urticarial/angioedematous and/or anaphylactic reactions to two or more chemically unrelated NSAIDs do not meet current classification criteria. However, they may be considered part of a cross-reactive type of acute HR, which is NSAID-induced urticaria/angioedema with or without respiratory or systemic symptoms of anaphylaxis. OBJECTIVE: To evaluate patients reporting acute HRs to NSAIDs and classify them according to updated criteria. METHODS: We prospectively studied 414 patients with suspected HRs to NSAIDs. For all whom met these criteria, NEFA/NIFA was diagnosed: (1) mild reactions to (NEFA) or tolerance of (NIFA) the suspected foods without taking NSAIDs; (2) cutaneous and/or anaphylactic reactions to the combination foods plus NSAIDs; (3) positive allergy tests to the suspected foods; and (4) negative drug challenges (DCs) with the NSAIDs involved. RESULTS: A total of 252 patients were given the diagnosis of NSAID hypersensitivity (60.9%), 108 of whom had NSAID-induced urticaria/angioedema with or without respiratory or systemic symptoms of anaphylaxis. We excluded NSAID hypersensitivity in 162 patients (39.1%) who tolerated DCs with the suspected NSAIDs, nine of whom received a diagnosis of NEFA, and 66 of NIFA. Pru p 3 was implicated in 67 of those 75 patients who received a diagnosis of NEFA or NIFA. CONCLUSIONS: NEFA and NIFA account for about 18% of patients reporting HRs to NSAIDs, in which Pru p 3 is the main responsible food allergen. Therefore, patients with cutaneous and/or anaphylactic reactions to NSAIDs should be carefully questioned about all foods ingested within 4 hours before or after NSAID exposure, and targeted food allergy tests should be considered in the diagnostic workup of these patients. If testing is positive, DCs with the suspected NSAIDs should also be considered.
Assuntos
Anafilaxia , Angioedema , Hipersensibilidade a Drogas , Hipersensibilidade Alimentar , Urticária , Humanos , Anti-Inflamatórios não Esteroides/efeitos adversos , Anafilaxia/diagnóstico , Anafilaxia/induzido quimicamente , Ácidos Graxos não Esterificados/efeitos adversos , Hipersensibilidade a Drogas/diagnóstico , Angioedema/diagnóstico , Angioedema/induzido quimicamente , Hipersensibilidade Alimentar/diagnóstico , Alérgenos/efeitos adversos , Urticária/diagnóstico , Urticária/induzido quimicamenteRESUMO
Mitochondrial dysfunction and oxidative stress-mediated inflammasome activation play critical roles in the pathogenesis of the non-alcoholic fatty liver disease (NAFLD). Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1), or growth differentiation factor-15 (GDF15), is associated with many biological processes and diseases, including NAFLD. However, the role of NAG-1/GDF15 in regulating oxidative stress and whether this process is associated with absent in melanoma 2 (AIM2) inflammasome activation in NAFLD are unknown. In this study, we revealed that NAG-1/GDF15 is significantly downregulated in liver tissues of patients with steatosis compared to normal livers using the Gene Expression Omnibus (GEO) database, and in free fatty acids (FFA, oleic acid/palmitic acid, 2:1)-induced HepG2 and Huh-7 cellular steatosis models. Overexpression of NAG-1/GDF15 in transgenic (Tg) mice significantly alleviated HFD-induced obesity and hepatic steatosis, improved lipid homeostasis, enhanced fatty acid ß-oxidation and lipolysis, inhibited fatty acid synthesis and uptake, and inhibited AIM2 inflammasome activation and the secretion of IL-18 and IL-1ß, as compared to their wild-type (WT) littermates without reducing food intake. Furthermore, NAG-1/GDF15 overexpression attenuated FFA-induced triglyceride (TG) accumulation, lipid metabolism deregulation, and AIM2 inflammasome activation in hepatic steatotic cells, while knockdown of NAG-1/GDF15 demonstrated opposite effects. Moreover, NAG-1/GDF15 overexpression inhibited HFD- and FFA-induced oxidative stress and mitochondrial damage which in turn reduced double-strand DNA (dsDNA) release into the cytosol, while NAG-1/GDF15 siRNA showed opposite effects. The reduced ROS production and dsDNA release may be responsible for attenuated AIM2 activation by NAG-1/GDF15 upon fatty acid overload. In conclusion, our results provide evidence that other than regulating lipid homeostasis, NAG-1/GDF15 protects against hepatic steatosis through a novel mechanism via suppressing oxidative stress, mitochondrial damage, dsDNA release, and AIM2 inflammasome activation.
Assuntos
Fator 15 de Diferenciação de Crescimento/metabolismo , Melanoma , Hepatopatia Gordurosa não Alcoólica , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/efeitos adversos , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Melanoma/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Estresse OxidativoRESUMO
Pink lotus essential oil (PLEO) is the volatile components extracted from lotus flowers and there are few relevant research. The purpose of this study was to observe the effect of PLEO on NAFLD in vitro model and its possible mechanism. The ingredients of PLEO were determined by gas chromatography-mass spectrometry (GS-MS) and its lipid-lowering and hepatoprotective activities were investigated. HepG2 cells were treated with free fatty acid (FFA) to establish a cell model of NAFLD. Cell viability was evaluated by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Total cholesterol (TC), triglyceride (TG), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were determined by Enzyme-Linked Immune Sorbent Assay (ELISA). Oil red O staining was performed to observe the lipid accumulation in the HepG2 cells. Lipid metabolism enzymes including fatty acid synthase (FAS), acetyl-coA carboxylase (ACC), stearoyl-CoA desaturase 1 (SCD-1), and carnitine palmitoyltransferase-1 (CPT-1), insulin signaling pathways including phosphatidylinositol 3 kinase (PI3K) and protein kinase B Akt, inflammatory signaling pathways such as nuclear factor kappa-B (NF-κB), were determined by Western blotting. There were 46 components determined in PLEO with many terpenoids compounds. PLEO decreased TC and TG contents in the FFA-treated HepG2 cells. Furthermore, PLEO inhibited TNF-α, IL-6 and IL-1ß excretion, decreased NF-κB, FAS, ACC and SCD-1 while increased phosphorylation of NF-κB, PI3K, Akt, and CPT-1 expression. It is the first time to reveal that PLEO alleviates FFA-induced steatosis in HepG2 cells by regulating lipid metabolism, inhibiting inflammatory response, and improving insulin sensitivity.
Assuntos
Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Lotus/química , NF-kappa B/metabolismo , Óleos Voláteis/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/patologia , Células Hep G2 , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Óleos Voláteis/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.
Assuntos
Compostos Benzidrílicos/administração & dosagem , Antígenos CD36/metabolismo , Ácidos Graxos não Esterificados/efeitos adversos , Glucosídeos/administração & dosagem , Túbulos Renais Proximais/citologia , PPAR gama/metabolismo , Substâncias Protetoras/administração & dosagem , Insuficiência Renal/induzido quimicamente , Insuficiência Renal/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/administração & dosagem , Animais , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ácido Palmítico/farmacologia , Insuficiência Renal/metabolismo , Resultado do TratamentoRESUMO
Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common type of chronic liver disease in developed nations, affecting around 25% of the population. Elucidating the factors causing NAFLD in individual patients to progress in different rates and to different degrees of severity, is a matter of active medical research. Here, we aim to provide evidence that the intra-hepatic heterogeneity of rheological, metabolic and tissue-regenerating capacities plays a central role in disease progression. We developed a generic mathematical model that constitutes the liver as ensemble of small liver units differing in their capacities to metabolize potentially cytotoxic free fatty acids (FFAs) and to repair FFA-induced cell damage. Transition from simple steatosis to more severe forms of NAFLD is described as self-amplifying process of cascading liver failure, which, to stop, depends essentially on the distribution of functional capacities across the liver. Model simulations provided the following insights: (1) A persistently high plasma level of FFAs is sufficient to drive the liver through different stages of NAFLD; (2) Presence of NAFLD amplifies the deleterious impact of additional tissue-damaging hits; and (3) Coexistence of non-steatotic and highly steatotic regions is indicative for the later occurrence of severe NAFLD stages.
Assuntos
Ácidos Graxos não Esterificados/efeitos adversos , Metabolismo dos Lipídeos/fisiologia , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/fisiopatologiaRESUMO
Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and ß-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1ß, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase ß activity, and the abundance of NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation.
Assuntos
Doenças dos Bovinos/prevenção & controle , Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/veterinária , GTP Fosfo-Hidrolases/antagonistas & inibidores , Inflamação/veterinária , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Animais , Estudos de Casos e Controles , Bovinos , Ácidos Graxos não Esterificados/sangue , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/prevenção & controle , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Lactação/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The aim of this study was to investigate the potential protective effects of the hot water extract of Eriobotrya japonica (EJW) on EtOH- or free fatty acid (FFA)-induced fatty liver injury in vitro. HepG2/2E1 cells were exposed to EtOH and HepG2 cells were exposed to a mixture of FFAs (oleic acid:palmitic acid, 2:1) to stimulate oxidative stress and to induce lipid accumulation, respectively. Antioxidant activity was significantly increased and lipid accumulation was inhibited in cells pretreated with EJW compared to those in cells exposed to EtOH or FFA only. Also, 5'adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylations were considerably increased, indicating activation of AMPK. Furthermore, EJW reduced the messenger RNA (mRNA) expression of lipogenesis-associated factors such as ACC, sterol regulatory element binding protein-1c (SREBP-1c), and fatty acid synthase (FAS), and increased mRNA expression related to components of the fatty acid ß-oxidation pathway, such as AMPK, carnitine palmitoyltransferase 1 (CPT-1), and peroxisome proliferator-activated receptor alpha (PPARα). These results suggest that EJW possessed potential preventive effects against both EtOH- and FFA-induced fatty liver disease by alleviation of oxidative stress and lipid accumulation in hepatocytes.
Assuntos
Eriobotrya/química , Fígado Gorduroso Alcoólico/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Extratos Vegetais/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Etanol/efeitos adversos , Ácido Graxo Sintases/metabolismo , Ácidos Graxos não Esterificados/efeitos adversos , Células Hep G2/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Produto da Acumulação Lipídica , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Ácido Oleico/efeitos adversos , Estresse Oxidativo , PPAR alfa/genética , Ácido Palmítico/efeitos adversos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , ÁguaRESUMO
Twist-related protein 2 (TWIST2) is identified as a basic helix-loop-helix (b-HLH) transcription repressor by dimerizing with other b-HLH proteins. The significance of TWIST2 has been emphasized in various tumors; however, few studies report its functions in metabolism and metabolic diseases. Here we aimed to explore the novel role and regulation mechanism of TWIST2 in hepatic steatosis. Our results showed that Twist2 knockdown caused mice obesity, insulin resistance, and hepatic steatosis, which were accompanied with inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction. In vitro, TWIST2 overexpression ameliorated hepatocellular steatosis, inhibited inflammation, and improved mitochondrial content and function with a fibroblast growth factor 21 (FGF21)-dependent pattern. NF-κB negatively regulated FGF21 transcription by directly binding to FGF21 promoter DNA, which was eliminated by TWIST2 overexpression by inhibiting NF-κB expression and translocation to nucleus. TWIST2 overexpression decreased intracellular reactive oxygen species level, increased mitochondrial DNA and biogenesis, and enhanced ATP production and antioxidation ability. Additionally, TWIST2 expression was repressed by insulin-targeting sterol regulatory element-binding protein 1c (SREBP1c) and forkhead box protein O1 and was enhanced by dexamethasone targeting Krüppel-like factor 15, which directly interacted with Twist2 promoter DNA. Together, our studies identify an important role and regulation mechanism of TWIST2 in maintaining hepatic homeostasis by ameliorating steatosis, inflammation, and oxidative stress via the NF-κB-FGF21 or SREBP1c-FGF21 pathway, which may provide a new therapeutic scheme for nonalcoholic fatty liver disease.-Zhou, L., Li, Q., Chen, A., Liu, N., Chen, N., Chen, X., Zhu, L., Xia, B., Gong, Y., Chen, X. KLF15-activating Twist2 ameliorated hepatic steatosis by inhibiting inflammation and improving mitochondrial dysfunction via NF-κB-FGF21 or SREBP1c-FGF21 pathway.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Fatores de Transcrição Kruppel-Like/metabolismo , NF-kappa B/metabolismo , Proteínas Repressoras/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , DNA/genética , Gorduras na Dieta/efeitos adversos , Ácidos Graxos não Esterificados/efeitos adversos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Inflamação/metabolismo , Resistência à Insulina , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , NF-kappa B/genética , Células NIH 3T3 , Regiões Promotoras Genéticas , Ligação Proteica , Células RAW 264.7 , Proteínas Repressoras/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Cardiovascular disease is recognized as a leading cause of death worldwide, but the risk of death is 2-3 times higher for individuals with diabetes. NLRP3 inflammasome activation is a leading pathway of vascular damage, and new treatment methods are needed to reduce NLRP3 inflammasome expression, along with a detailed understanding of how those treatments work. In a series of assays on human vascular endothelial cells that were exposed to high concentrations of free fatty acids (FFA) to induce a diabetes-like environment, we found a significant impact of cilostazol, a vasodilator widely used to treat blood flow problems and well-tolerated medication. To our knowledge, this study is the first to demonstrate the effects of cilostazol in primary human aortic endothelial cells. We found that cilostazol significantly reduced NLRP3 inflammasome activation, as well as the activity of other related and harmful factors, including oxidative stress, expression of NADPH oxidase 4 (NOX-4), thioredoxin-interacting protein (TxNIP), high mobility group box 1 (HMGB-1), interleukin 1ß (IL-1ß) and IL-18. Cilostazol also protected the functionality of sirtuin 1 (SIRT1), which serves to restrict NLRP3 inflammasome activity, when exposure to FFAs would have otherwise impaired its function. Thus, it appears that cilostazol's mechanism of action in reducing NLRP3 inflammasome activation is an indirect one; it protects SIRT1, which then allows SIRT1 to perform its regulatory job. Cilostazol has potential as an already-available, well-tolerated preventive medication that may alleviate some of the adverse vascular effects of living with diabetes. The findings of the present study lay the groundwork for further research on the potential of cilostazol as a safe and effective treatment against diabetic endothelial dysfunction and vacular disease.
Assuntos
Cilostazol/farmacologia , Ácidos Graxos não Esterificados/efeitos adversos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Relação Dose-Resposta a Droga , Humanos , Interleucina-18/biossíntese , Interleucina-1beta/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is closely related to metabolic diseases such as obesity and insulin resistance. PURPOSE: We studied whether an ethanol extract of Lycopus lucidus Turcz. ex Benth (LLE) exhibited effects on lipid metabolism in NAFLD. STUDY DESIGN: An in vitro modelwas established by treatment of HepG2 cells with a 1â¯mM free fatty acid (FFA) mixture (oleic acid/palmitic acid, 2:1). C57BL/6 mice were fed a high-fat diet (HFD; 60 kcal% fat) for 14 weeks to induce obesity and were treated with or without LLE (100 or 200⯠mg/kg daily by oral gavage). METHODS: HepG2 cells were exposed to 1â¯mM FFA, with or without LLE (250 - 1000⯠mg/ml). Intracellular lipid contents were measured by Oil Red O staining and a Nile Red assay. The body weight, relative liver weight, hepatic lipids, triglycerides (TGs), and total cholesterol (TC) were measured in the mice. Serum alanine aminotransferase (ALT), TG, TC, glucose, insulin, leptin, and tumor necrosis factor-alpha (TNF-α) levels were determined by biochemical or enzyme-linked immunosorbent assays. Histologic analysis was performed in the liver. Western blotting and quantitative real-time polymerase chain reaction were used to analyze the expression of key enzymes of hepatic lipid metabolism. RESULTS: LLE significantly decreased the intracellular lipid accumulation in FFA-treated HepG2 cells. LLE not only remarkably decreased the expression of lipogenesis genes but also increased ß-oxidation in FFA-induced HepG2 cells. In the in vivo study, LLE treatment significantly decreased the body weight, relative liver weight, serum ALT, TC, and low-density lipoprotein cholesterol, as well as the serum glucose, insulin, leptin, and TNF-α levels in HFD-fed mice. The hepatic TG and TC contents were significantly reduced in the LLE-treated groups. Western blot analysis showed that the expression of sterol-regulatory element-binding protein 1 decreased, while that of phosphorylated AMP-activated protein kinase and peroxisome proliferator-activated receptor α increased in the LLE-treated mice. CONCLUSION: These results suggest that LLE may exert protective effects against NAFLD-related obesity and metabolic disease.
Assuntos
Fármacos Antiobesidade/farmacologia , Dieta Hiperlipídica/efeitos adversos , Lycopus/química , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Ácidos Graxos não Esterificados/efeitos adversos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/tratamento farmacológico , Obesidade/etiologia , Extratos Vegetais/química , Triglicerídeos/sangueRESUMO
Aims: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver diseases. However, there are no approved pharmacotherapies for the treatment of NAFLD other than managing life style and controlling diets. Extensive studies have demonstrated that multiple mechanisms are involved in free fatty acid (FFA)- and high fat diet (HFD)-induced hepatic injury, including mitochondrial dysfunction, activation of oxidative stress and endoplasmic reticulum (ER) stress, and lysosome dysfunction. A previous study reported that Isosteviol (ISV), a derivative of stevioside, prevents HFD-induced hepatic injury. However, the underlying mechanisms remain unclear. Results: In this study, we examined the potential cellular/molecular mechanisms underlying ISV-mediated protective effect against FFA-/HFD-induced hepatic lipotoxicity by using both in vitro primary rat hepatocytes and the in vivo rat NAFLD model. The results indicated that ISV inhibits FFA-/HFD-induced hepatic injury via reducing oxidative and ER stress. Specifically, ISV inhibited the expression, activation, and mitochondrial translocation of Src-homology-2-domain-containing transforming protein 1 (p66Shc), an adapter protein that mediates oxidative stress-induced injury and is a substrate of protein kinase C-ß (PKC-ß), via inhibition of PKC-ß activity. However, ISV had no effect on the expression and activity of peptidyl-prolyl cis-trans isomerase and serine/threonine protein phosphatase 2A, isomerase and phosphorylase of p66Shc. In addition, ISV also inhibited FFA-induced ER stress and decreased ER-mitochondrial interaction. Innovation and Conclusion: We first identified that ISV prevents FFA-/HFD-induced hepatic injury through modulating PKC-ß/p66Shc/oxidative and ER stress pathways. ISV represents a promising therapeutic agent for NAFLD in the future. Antioxid. Redox Signal. 30, 1949-1968.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Diterpenos do Tipo Caurano/farmacologia , Ácidos Graxos não Esterificados/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína Quinase C beta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Animais , Apoptose , Biópsia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo , RatosRESUMO
BACKGROUND: Insulin resistance has been independently related to heart failure. However, the specific mechanisms of high FFA levels in the pathophysiology of heart failure in insulin-resistant states are remain largely unclear. This study investigated whether elevated circulating free fatty acids (FFA) levels result in impaired cardiac structure and function in vivo via insulin-related signaling pathways in myocardium. METHODS: Male Wistar rats were randomly divided into the intralipid group (20% intralipid plus heparin infusion) and the control group (glycerol infusion). Blood samples were collected before and after 6-, 12-, and 24-h infusions. Cardiac structure and function were measured using echocardiography. Maximum velocity of myocardial contraction (+dP/dt max) and diastole (-dP/dt max) were measured using a physiological polygraph in vivo. Heart tissues were collected for western blotting. RESULTS: Compared with the control group, plasma FFA, plasma glucose, and serum insulin levels increased significantly in the intralipid group. With increasing infusion time, cardiac function in the intralipid group decreased gradually compared with the control group. After a 24-h infusion, early (E', cm/s) diastolic peak velocities and (-dP/dt max) decreased significantly. Protein expression of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and phosphorylated Akt in myocardium increased after a 6-h infusion and decreased significantly after a 24-h infusion in the intralipid group. Protein expression of glucose transporter type 4 (GLUT4), Adenosine 5'-monophosphate -activated protein kinase (AMPK), phosphorylated AMPK(p-AMPK), and endothelial nitric oxide synthase (eNOS) in myocardium gradually decreased in the intralipid group. CONCLUSIONS: Elevated FFA levels may impair cardiac function and cardiac dysfunction might result from myocardial insulin resistance with significant changes to PI3K-Akt-GLUT4 and AMPK-eNOS signaling pathways with increasing FFA levels.
Assuntos
Ácidos Graxos não Esterificados/efeitos adversos , Coração/fisiopatologia , Insulina/metabolismo , Transdução de Sinais , Adenilato Quinase/metabolismo , Animais , Glicemia/metabolismo , Ácidos Graxos não Esterificados/sangue , Transportador de Glucose Tipo 4/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos WistarRESUMO
Elevated levels of free fatty acids (FFAs) in the liver, resulting from either increased lipolysis or imbalanced FFAs flux, is a key pathogenic factor of hepatic steatosis. This study was conducted to examine the therapeutic effect of tetrahydrocurcumin (THC), a naturally occurring curcuminoid and a metabolite of curcumin, on oleic acid (OA)-induced steatosis in human hepatocellular carcinoma cells and to elucidate the underlying mechanism. HepG2 cells were incubated with OA to induce steatosis, and then treated with various concentrations of THC. The results showed that THC treatment significantly decreased lipid accumulation in OA-treated HepG2 cells, possibly, by inhibiting the expression of the lipogenic proteins, sterol regulatory element-binding protein 1 (SREBP-1c), peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4). Moreover, THC attenuated OA-induced hepatic lipogenesis in an adenosine monophosphate-activated protein kinase (AMPK)-dependent manner, which was reversed by pretreatment with an AMPK inhibitor. THC promoted lipolysis and upregulated the expression of genes involved in ß-oxidation. Glucose uptake and insulin signaling impaired in HepG2 cells incubated with OA were abated by THC treatment, including phosphorylation of the insulin receptor substrate 1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Akt and downstream signaling pathways, forkhead box protein O1 (FOXO1) and glycogen synthase kinase 3 ß (GSK3ß), which are involved in gluconeogenesis and glycogen synthesis, respectively. Altogether, these results demonstrated the novel therapeutic benefit of THC against hepatic steatosis and, consequently, a potential treatment for non-alcoholic fatty liver disease (NAFLD).
Assuntos
Curcumina/análogos & derivados , Ácidos Graxos não Esterificados/efeitos adversos , Fígado Gorduroso/metabolismo , Resistência à Insulina , Curcumina/farmacologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/fisiopatologia , Glucose/metabolismo , Células Hep G2 , Humanos , Lipogênese/efeitos dos fármacos , Ácido Oleico/efeitos adversos , PPAR alfa/genética , PPAR alfa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
Tolllike receptor 4 (TLR4)mediated immune and inflammatory signaling serves a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Our previous study demonstrated that celastrol treatment was able to improve hepatic steatosis and inhibit the TLR4 signaling cascade pathway in type 2 diabetic rats. The present study aimed to investigate the effects of celastrol on triglyceride accumulation and inflammation in steatotic HepG2 cells, and the possible mechanisms responsible for the regulation of cellular responses following TLR4 gene knockdown by small interfering RNA (siRNA) in vitro. A cell model of hepatic steatosis was prepared by exposing the HepG2 cells to free fatty acid (FFA) in the absence or presence of celastrol. Intracellular triglycerides were visualized by Oil red O staining, and the TLR4/myeloid differentiation primary response 88 (MyD88)/nuclear factorκB (NFκB) signaling cascade pathway were investigated. To directly elucidate whether TLR4 was the blocking target of celastrol upon FFA exposure, the cellular response to inflammation was determined upon transfection with TLR4 siRNA. The results revealed that celastrol significantly reduced triglyceride accumulation in the steatotic HepG2 cells, and downregulated the expression levels of TLR4, MyD88 and phosphoNFκBp65, as well as of the downstream inflammatory cytokines interleukin1ß and tumor necrosis factor α. Knockdown of TLR4 also alleviated FFAinduced inflammatory response. In addition, cotreatment with TLR4 siRNA and celastrol further attenuated the expression of inflammatory mediators. These results suggest that celastrol exerts its protective effect partly via inhibiting the TLR4mediated immune and inflammatory response in steatotic HepG2 cells.
Assuntos
Ácidos Graxos não Esterificados/efeitos adversos , Hepatopatia Gordurosa não Alcoólica/imunologia , Receptor 4 Toll-Like/imunologia , Triglicerídeos/imunologia , Triterpenos/farmacocinética , Animais , Ácidos Graxos não Esterificados/farmacologia , Células Hep G2 , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Triterpenos Pentacíclicos , Ratos , Receptor 4 Toll-Like/genética , Triglicerídeos/genéticaRESUMO
The aim of this study was to investigate the changes in hepatic oxidative phosphorylation (OXPHOS) complexes (COs) in patients and cows with non-alcoholic steatohepatitis (NASH) and to investigate the mechanism that links mitochondrial dysfunction and hepatic insulin resistance induced by non-esterified fatty acids (NEFAs). Patients and cows with NASH displayed high blood NEFAs, TNF-α and IL-6 concentrations, mitochondrial dysfunction and insulin resistance. The protein levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitofusin-2 (Mfn-2) and OXPHOS complexes (human: COI and COIII; cow: COI-IV) were significantly decreased in patients and cows with NASH. NEFA treatment significantly impaired mitochondrial function and, increased reactive oxygen species (ROS) production, and excessive ROS overactivated the JNK and p38MAPK pathways and induced insulin resistance in cow hepatocytes. PGC-1α and Mfn-2 overexpression significantly decreased the NEFA-induced ROS production and TNF-α and IL-6 mRNA expressions, reversed the inhibitory effect of NEFAs on mitochondrial function and attenuated the overactivation of the ROS-JNK/p38MAPK pathway, alleviated insulin resistance induced by NEFAs in cow hepatocytes and HepG2 cells. These findings indicate that NEFAs induce mitochondrial dysfunction and insulin resistance mediated by the ROS-JNK/p38MAPK pathway. PGC-1α or Mfn-2 overexpression reversed the lipotoxicity of NEFAs on mitochondrial dysfunction and insulin resistance. Our study clarified the mechanism that links hepatic mitochondrial dysfunction and insulin resistance in NASH.
Assuntos
Ácidos Graxos não Esterificados/efeitos adversos , Insulina/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Estudos de Casos e Controles , Bovinos , Células Cultivadas , Feminino , GTP Fosfo-Hidrolases/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Resistência à Insulina , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A dietary influence on cancer progression has been evident for many decades, and dietary fatty acids, particularly long chain mono- and polyunsaturated fatty acids, have been shown to play significant roles in influencing growth of a variety of human cancers. The discovery of the family of cell-surface free-fatty acid receptors, which include the long-chain fatty acid receptors FFA1 and FFA4, suggest that many of the effects of dietary fats could be receptor-mediated. FFA4 is ubiquitously expressed and has recently been shown to modulate a variety of important anti-inflammatory and metabolic processes. Since FFA4 is currently an attractive drug target for treatment of metabolic disorders such as diabetes and obesity, understanding its role in cancer progression is critical towards the drug discovery process. In this research update, the current body of knowledge on the role of this receptor in regulating cancer cell proliferation, migration, and invasion, as well as in vivo tumorigenesis is reviewed.
Assuntos
Gorduras na Dieta/efeitos adversos , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Gorduras na Dieta/administração & dosagem , Ácidos Graxos não Esterificados/administração & dosagem , Ácidos Graxos não Esterificados/efeitos adversos , Ácidos Graxos não Esterificados/fisiologia , Humanos , Neoplasias/induzido quimicamente , Neoplasias/tratamento farmacológico , Receptores Acoplados a Proteínas G/administração & dosagem , Receptores Acoplados a Proteínas G/antagonistas & inibidoresRESUMO
Isosteviol (ISV), a diterpene molecule, is an isomer of the backbone structure of a group of substances with proven antidiabetic capabilities. The aim of this study was to investigate if ISV elicits dynamic insulin release from pancreatic islets and concomitantly is able to ameliorate gluco-, lipo-, and aminoacidotoxicity in clonal ß-cell line (INS-1E) in relation to cell viability and insulin secretion. Isolated mice islets placed into perifusion chambers were perifused with 3.3 mM and 16.7 mM glucose with/without 10-7 M ISV. INS-1E cells were incubated for 72 h with either 30 mM glucose, 1 mM palmitate or 10 mM leucine with or without 10-7 M ISV. Cell viability was evaluated with a Cytotoxic Fluoro-test and insulin secretion was measured in Krebs-Ringer Buffer at 3.3 mM and 16.7 mM glucose. In the presence of 3.3 mM glucose, 10-7 M ISV did not change basal insulin secretion from perifused islets. However, at a high glucose level of 16.7 mM, 10-7 M ISV elicited a 2.5-fold increase (-ISV: 109.92 ± 18.64 ng/mL vs. +ISV: 280.15 ± 34.97 ng/mL; p < 0.01). After 72 h gluco-, lipo-, or aminoacidotoxicity in INS-1E cells, ISV treatment did not significantly affect cell viability (glucotoxicity, -ISV: 19.23 ± 0.83%, +ISV: 18.41 ± 0.90%; lipotoxicity, -ISV: 70.46 ± 3.15%, +ISV: 65.38 ± 2.81%; aminoacidotoxicity: -ISV: 8.12 ± 0.63%; +ISV: 7.75 ± 0.38%, all nonsignificant). ISV did not improve impaired insulin secretion (glucotoxicity, -ISV: 52.22 ± 2.90 ng/mL, +ISV: 47.24 ± 3.61 ng/mL; lipotoxicity, -ISV: 19.94 ± 4.10 ng/mL, +ISV: 22.12 ± 3.94 ng/mL; aminoacidotoxicity: -ISV: 32.13 ± 1.00 ng/mL; +ISV: 30.61 ± 1.54 ng/mL, all nonsignificant). In conclusion, ISV acutely stimulates insulin secretion at high but not at low glucose concentrations. However, ISV did not counteract cell viability or cell dysfunction during gluco-, lipo-, or aminoacidotoxicity in INS-1E cells.
Assuntos
Diterpenos do Tipo Caurano/farmacologia , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Insulina/metabolismo , Animais , Carbacol/efeitos adversos , Carbacol/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Agonistas Colinérgicos/efeitos adversos , Agonistas Colinérgicos/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diterpenos do Tipo Caurano/efeitos adversos , Ácidos Graxos não Esterificados/efeitos adversos , Ácidos Graxos não Esterificados/metabolismo , Feminino , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/efeitos adversos , Glucose/metabolismo , Hipoglicemiantes/efeitos adversos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Leucina/efeitos adversos , Leucina/metabolismo , Camundongos , Concentração Osmolar , Ácido Palmítico/efeitos adversos , Ácido Palmítico/metabolismo , Substâncias Protetoras/efeitos adversos , Substâncias Protetoras/farmacologia , Técnicas de Cultura de TecidosRESUMO
3-Acetyl-oleanolic acid (3Ac-OA) is a derivative of oleanolic acid (OA), which has shown therapeutic beneficial effects on diabetes and metabolic syndrome. In this study we investigated whether 3Ac-OA exerted beneficial effect on non-alcoholic fatty liver disease (NAFLD) in rats and its potential underlying mechanisms. Treatment with 3Ac-OA (1-100 µmol/L) dose-dependently decreased the intracellular levels of total cholesterol (TC) and triglyceride (TG) in FFA-treated primary rat hepatocytes and human HepG2 cell lines in vitro. Furthermore, oil red staining studies showed that 3Ac-OA caused dose-dependent decrease in the number of lipid droplets in FFA-treated primary rat hepatocytes. SD rats were fed a high fat diet (HFD) for 6 weeks and subsequently treated with 3Ac-OA (60, 30, 15 mg·kg-1·d-1) for 4 weeks. 3Ac-OA administration significantly decreased the body weight, liver weight and serum TC, TG, LDL-C levels in HFD rats. Furthermore, 3AcOA administration ameliorated lipid accumulation and cell apoptosis in the liver of HFD rats. Using adipokine array analyses, we found that the levels of 11 adipokines (HGF, ICAM, IGF-1, IGFBP-3, IGFBP-5, IGFBP-6, lipocalin-2, MCP-1, M-CSF, Pref-1 and RAGE) were increased by more than twofold in the serum of 3Ac-OA-treated rats, whereas ICAM, IGF-1 and lipocalin-2 had levels increased by more than 20-fold. Moreover, 3Ac-OA administration significantly increased the expression of glucose transporter type 2 (GLUT-2) and low-density lipoprotein receptor (LDLR), as well as the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase B (AKT) and glycogen synthase kinase 3ß (GSK-3ß) in the liver tissues of HFD rats. In conclusion, this study demonstrates that 3Ac-OA exerts a protective effect against hyperlipidemia in NAFLD rats through AMPK-related pathways.
Assuntos
Hiperlipidemias/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Triterpenos/uso terapêutico , Adipocinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos não Esterificados/efeitos adversos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Masculino , Ratos Sprague-DawleyRESUMO
We hypothesised that skeletal muscles of healthy young people have a large variation in oxidative capacity and fibre-type composition, and aimed therefore to investigate glucose metabolism in biopsies and myotubes isolated from musculus vastus lateralis from healthy males with varying degrees of maximal oxygen uptake. Trained and intermediary trained subjects showed higher carbohydrate oxidation in vivo. Fibre-type distribution in biopsies and myotubes did not differ between groups. There was no correlation between fibre-type I expression in biopsies and myotubes. Myotubes from trained had higher deoxyglucose accumulation and fractional glucose oxidation (glucose oxidation relative to glucose uptake), and were also more sensitive to the suppressive action of acutely added oleic acid to the cells. Despite lack of correlation of fibre types between skeletal muscle biopsies and cultured cells, myotubes from trained subjects retained some of their phenotypes in vitro with respect to enhanced glucose metabolism and metabolic flexibility.