RESUMO
BACKGROUND/AIMS: Metabolic-associated fatty liver disease (MAFLD) is a common cause of chronic liver disease worldwide. However, there is currently no recognized effective drugs for treating it. MATERIALS AND METHODS: In this study, we investigated the efficacy of Honokiol (HNK) in vitro for mitigating MAFLD. Then, 0.4 mM palmitic acid (PA) and LO2 cells were used to establish the MAFLD model. The protective effect of HNK on MAFLD was confirmed by Oil Red O staining and cell counting kit (CCK-8) assay in LO2 cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were carried out to analyze the regulatory role of HNK on Nrf2 and RIPK3 signaling pathways. The effect of HNK and its downstream signaling pathways on oxidative stress were verified by the detection of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD). The concentration of IL-1ß, IL-6L, and TNF-α was assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The middle concentration of HNK (50 µmol/L) was selected as the best option for inhibiting lipidosis and oxidative stress in MAFLD models. Honokiol mitigates MAFLD via activation of nuclear factor E2-related factor 2 (Nrf2) signaling pathways in vitro. Honokiol suppressed MAFLD via activating the Nrf2 signaling pathway to play an antioxidant and anti-inflammatory role. Also, HNK regulates Nrf2 and RIPK3 signaling pathways to mitigate MAFLD. CONCLUSION: Our results showed that HNK may suppress the oxidative stress and inflammation in MAFLD via activation of Nrf2 signaling pathway.
Assuntos
Compostos de Bifenilo , Lignanas , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Proteína Serina-Treonina Quinases de Interação com Receptores , Transdução de Sinais , Lignanas/farmacologia , Lignanas/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Humanos , Compostos de Bifenilo/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Linhagem Celular , Espécies Reativas de Oxigênio/metabolismo , Ácido Palmítico/farmacologia , Compostos Alílicos , FenóisRESUMO
BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKß/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.
Assuntos
Proteínas Quinases Ativadas por AMP , Glucose , Resistência à Insulina , Fibras Musculares Esqueléticas , Phyllanthus emblica , Extratos Vegetais , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Animais , Camundongos , Glucose/metabolismo , Extratos Vegetais/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Frutas , Transportador de Glucose Tipo 4/metabolismo , Linhagem Celular , Palmitatos/farmacologia , Ácido Palmítico/farmacologiaAssuntos
Antioxidantes , Estresse Oxidativo , Quercetina , Quercetina/farmacologia , Quercetina/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Animais , Acetilação , Antioxidantes/farmacologia , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Anti-Inflamatórios/farmacologia , Ratos , Ácido Palmítico/toxicidade , Ácido Palmítico/farmacologiaRESUMO
BACKGROUND: Hepatic stellate cells (HSC) have numerous critical roles in liver function and homeostasis, while they are also known for their importance during liver injury and fibrosis. There is therefore a need for relevant in vitro human HSC models to fill current knowledge gaps. In particular, the roles of vitamin A (VA), lipid droplets (LDs), and energy metabolism in human HSC activation are poorly understood. METHODS: In this study, human pluripotent stem cell-derived HSCs (scHSCs), benchmarked to human primary HSC, were exposed to 48-hour starvation of retinol (ROL) and palmitic acid (PA) in the presence or absence of the potent HSC activator TGF-ß. The interventions were studied by an extensive set of phenotypic and functional analyses, including transcriptomic analysis, measurement of activation-related proteins and cytokines, VA- and LD storage, and cell energy metabolism. RESULTS: The results show that though the starvation of ROL and PA alone did not induce scHSC activation, the starvation amplified the TGF-ß-induced activation-related transcriptome. However, TGF-ß-induced activation alone did not lead to a reduction in VA or LD stores. Additionally, reduced glycolysis and increased mitochondrial fission were observed in response to TGF-ß. CONCLUSIONS: scHSCs are robust models for activation studies. The loss of VA and LDs is not sufficient for scHSC activation in vitro, but may amplify the TGF-ß-induced activation response. Collectively, our work provides an extensive framework for studying human HSCs in healthy and diseased conditions.
Assuntos
Células Estreladas do Fígado , Ácido Palmítico , Fator de Crescimento Transformador beta , Vitamina A , Humanos , Vitamina A/farmacologia , Vitamina A/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Ácido Palmítico/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Metabolismo Energético/efeitos dos fármacosRESUMO
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Hepatócitos , Isomerases de Dissulfetos de Proteínas , Transdução de Sinais , Tunicamicina , Chaperona BiP do Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Hepatócitos/metabolismo , Animais , Tunicamicina/farmacologia , Retículo Endoplasmático/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Linhagem Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Tapsigargina/farmacologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Sobrevivência Celular/efeitos dos fármacosRESUMO
Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1ß and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.
Assuntos
Hepatócitos , Inflamassomos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas de Ligação a Fosfato , Piroptose , Fatores de Transcrição , Animais , Humanos , Masculino , Camundongos , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Furanos , Gasderminas , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Indenos/farmacologia , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Nucleares , Ácido Palmítico/farmacologia , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Piroptose/efeitos dos fármacos , Sulfonamidas/farmacologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
The present study aimed to investigate the role of antidiabetic drug metformin on the cytoplasmic organization of oocytes. Germinal vesicle (GV) stage oocytes were collected from adult female Swiss albino mice and subjected to in vitro maturation (IVM) in various experimental groups- control, vehicle control (0.3% ethanol), metformin (50 µg/mL), high glucose and high lipid (HGHL, 10 mM glucose; 150 µM palmitic acid; 75 µM stearic acid and 200 µM oleic acid in ethanol), and HGHL supplemented with metformin. The metaphase II (MII) oocytes were analyzed for lipid accumulation, mitochondrial and endoplasmic reticulum (ER) distribution pattern, oxidative and ER stress, actin filament organization, cortical granule distribution pattern, spindle organization and chromosome alignment. An early polar body extrusion was observed in the HGHL group. However, the maturation rate at 24 h did not differ significantly among the experimental groups compared to the control. The HGHL conditions exhibited significantly higher levels of oxidative stress, ER stress, poor actin filament organization, increased lipid accumulation, altered mitochondrial distribution, spindle abnormalities, and chromosome misalignment compared to the control. Except for spindle organization, supplementation of metformin to the HGHL conditions improved all the parameters (non-significant for ER and actin distribution pattern). These results show that metformin exposure in the culture media helped to improve the hyperglycemia and hyperlipidemia-induced cytoplasmic anomalies except for spindle organization. Given the crucial role of spindle organization in proper chromosome segregation during oocyte maturation and meiotic resumption, the implications of metformin's limitations in this aspect warrant careful evaluation and further investigation.
Assuntos
Hiperglicemia , Hiperlipidemias , Metformina , Oócitos , Estresse Oxidativo , Fuso Acromático , Animais , Metformina/farmacologia , Feminino , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Camundongos , Fuso Acromático/efeitos dos fármacos , Hiperglicemia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hiperlipidemias/tratamento farmacológico , Hipoglicemiantes/farmacologia , Citoplasma/metabolismo , Citoplasma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Cultivadas , Ácido Palmítico/toxicidade , Ácido Palmítico/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodosRESUMO
OBJECTIVES: Obesity related glomerulopathy (ORG) is induced by obesity, but the pathogenesis remains unclear. This study aims to investigate the expression of early growth response protein 3 (EGR3) in the renal cortex tissues of ORG patients and high-fat diet-induced obese mice, and to further explore the molecular mechanism of EGR3 in inhibiting palmitic acid (PA) induced human podocyte inflammatory damage. METHODS: Renal cortex tissues were collected from ORG patients (n=6) who have been excluded from kidney damage caused by other diseases and confirmed by histopathology, and from obese mice induced by high-fat diet (n=10). Human and mouse podocytes were intervened with 150 µmol/L PA for 48 hours. EGR3 was overexpressed or silenced in human podocytes. Enzyme linked immunosorbent assay (ELISA) was used to detcet the levels of interleukin-6 (IL-6) and interleukin-1ß (IL-1ß). Real-time RT-PCR was used to detect the mRNA expressions of EGR3, podocytes molecular markers nephrosis 1 (NPHS1), nephrosis 2 (NPHS2), podocalyxin (PODXL), and podoplanin (PDPN). RNA-seq was performed to detect differentially expressed genes (DEGs) after human podocytes overexpressing EGR3 and treated with 150 µmol/L PA compared with the control group. Co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS) was used to detect potential interacting proteins of EGR3 and the intersected with the RNA-seq results. Co-IP confirmed the interaction between EGR3 and protein arginine methyltransferases 1 (PRMT1), after silencing EGR3 and PRMT1 inhibitor intervention, the secretion of IL-6 and IL-1ß in PA-induced podocytes was detected. Western blotting was used to detect the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) after overexpression or silencing of EGR3. RESULTS: EGR3 was significantly upregulated in renal cortex tissues of ORG patients and high-fat diet-induced obese mice (both P<0.01). In addition, after treating with 150 µmol/L PA for 48 hours, the expression of EGR3 in human and mouse podocytes was significantly upregulated (both P<0.05). Overexpression or silencing of EGR3 in human podocytes inhibited or promoted the secretion of IL-6 and IL-1ß in the cell culture supernatant after PA intervention, respectively, and upregulated or downregulated the expression of NPHS1, PODXL, NPHS2,and PDPN (all P<0.05). RNA-seq showed a total of 988 DEGs, and Co-IP+LC-MS identified a total of 238 proteins that may interact with EGR3. Co-IP confirmed that PRMT1 was an interacting protein with EGR3. Furthermore, PRMT1 inhibitors could partially reduce PA-induced IL-6 and IL-1ß secretion after EGR3 silencing in human podocytes (both P<0.05). Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3. CONCLUSIONS: EGR3 may reduce ORG podocyte inflammatory damage by inhibiting the PRMT1/p-STAT3 pathway.
Assuntos
Proteína 3 de Resposta de Crescimento Precoce , Obesidade , Podócitos , Proteína-Arginina N-Metiltransferases , Proteínas Repressoras , Fator de Transcrição STAT3 , Animais , Humanos , Masculino , Camundongos , Dieta Hiperlipídica/efeitos adversos , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/genética , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , Córtex Renal/metabolismo , Córtex Renal/patologia , Nefropatias/metabolismo , Nefropatias/etiologia , Nefropatias/patologia , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/metabolismo , Ácido Palmítico/farmacologia , Podócitos/metabolismo , Podócitos/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a prevalent condition characterized by hepatic fat accumulation, often progressing to severe liver injury, for which approved treatments are currently lacking. This study explores the potential therapeutic impact of alpha-lipoic acid (ALA), a natural compound crucial in lipid metabolism, on NAFLD using an in vitro model. METHODS: HepG2 cells were treated with a palmitic acid:oleic acid (PA:OA) mixture, representing a cellular model of steatosis. Subsequent treatment with ALA at concentrations of 1 µM and 5 µM aimed to evaluate its effects on lipid content and metabolism. Real-time polymerase chain reaction (PCR), BODIPY staining, cytofluorimetric analysis, and lipidomics were used to assess gene expression, lipid droplet accumulation, and fatty acid profiles. RESULTS: Our results showed that ALA significantly reduced lipid droplets in PA:OA-treated HepG2 cells, with a concentration-dependent effect. Analysis of fatty acid profiles demonstrated a decrease in palmitic acid levels with ALA treatment, while oleic acid reduction was observed only at the higher concentration. Moreover, ALA modulated the expression of genes involved in cholesterol biosynthesis and low-density lipoprotein (LDL) metabolism, indicating a potential role in lipid homeostasis. Further insights into molecular mechanisms revealed that ALA modulated peroxisome proliferator activated receptors (PPARs), specifically PPAR-alpha and PPAR-gamma, involved in fatty acid metabolism and insulin sensitivity. Finally, ALA counteracted the overexpression of thermogenic genes induced by exogenous fatty acids, suggesting a regulatory role in energy dissipation pathways. CONCLUSION: In conclusion, this study highlights ALA as a therapeutic agent in mitigating lipid accumulation and dysregulation in NAFLD.
Assuntos
Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica , Ácido Oleico , Ácido Palmítico , Ácido Tióctico , Humanos , Ácido Tióctico/farmacologia , Células Hep G2 , Metabolismo dos Lipídeos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Ácido Oleico/farmacologia , Ácido Oleico/metabolismo , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ácidos Graxos/metabolismo , PPAR gama/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR alfa/genética , Proteína Desacopladora 2/metabolismo , Proteína Desacopladora 2/genéticaRESUMO
Diffuse large B-cell lymphoma (DLBCL) is characterized by significant treatment resistance. Palmitic acid (PA) has shown promising antitumor properties. This study aims to elucidate the molecular mechanisms by which PA influences DLBCL progression. We quantified the expression levels of microRNAs (miRNAs), Forkhead box protein O1 (FOXO1), and DNA methyltransferase 3A (DNMT3A) in both untreated and PA-treated DLBCL tumors and cell lines. Assessments were made of cell viability, apoptosis, and autophagy-related protein expression following PA administration. Interaction analyses among miR-429, DNMT3A, and FOXO1 were conducted using luciferase reporter assays and methylation-specific (MSP) Polymerase chain reaction (PCR). After transfecting the miR-429 inhibitor, negative control (NC) inhibitor, shRNA against DNMT3A (sh-DNMT3A), shRNA negative control (sh-NC), overexpression vector for DNMT3A (oe-DNMT3A), or overexpression negative control (oe-NC), we evaluated the effects of miR-429 and DNMT3A on cell viability, mortality, and autophagy-related protein expression in PA-treated DLBCL cell lines. The efficacy of PA was also tested in vivo using DLBCL tumor-bearing mouse models. MiR-429 and FOXO1 expression levels were downregulated, whereas DNMT3A was upregulated in DLBCL compared to the control group. PA treatment was associated with enhanced autophagy, mediated by the upregulation of miR-429 and downregulation of DNMT3A. The luciferase reporter assay and MSP confirmed that miR-429 directly inhibits DNMT3A, thereby reducing FOXO1 methylation. Subsequent experiments demonstrated that PA promotes autophagy and inhibits DLBCL progression by upregulating miR-429 and modulating the DNMT3A/FOXO1 axis. In vivo PA significantly reduced the growth of xenografted tumors through its regulatory impact on the miR-429/DNMT3A/FOXO1 axis. Palmitic acid may modulate autophagy and inhibit DLBCL progression by targeting the miR-429/DNMT3A/FOXO1 signaling pathway, suggesting a novel therapeutic target for DLBCL management.
Assuntos
DNA Metiltransferase 3A , Proteína Forkhead Box O1 , Linfoma Difuso de Grandes Células B , MicroRNAs , Ácido Palmítico , MicroRNAs/genética , MicroRNAs/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Humanos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Animais , Camundongos , Ácido Palmítico/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Camundongos Nus , Masculino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Feminino , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
Pancreatic ductal adenocarcinoma (PDAC)'s resistance to therapies is mainly attributed to pancreatic cancer stem cells (PCSCs). Mitochondria-impairing agents can be used to hamper PCSC propagation and reduce PDAC progression. Therefore, to develop an efficient vector for delivering drugs to the mitochondria, we synthesized tris(3,5-dimethylphenyl)phosphonium-conjugated palmitic acid. Triphenylphosphonium (TPP) is a lipophilic cationic moiety that promotes the accumulation of conjugated agents in the mitochondrion. Palmitic acid (PA), the most common saturated fatty acid, has pro-apoptotic activity in different types of cancer cells. TPP-PA was prepared by the reaction of 16-bromopalmitic acid with TPP, and its structure was characterized by 1H and 13C NMR and HRMS. We compared the proteomes of TPP-PA-treated and untreated PDAC cells and PCSCs, identifying dysregulated proteins and pathways. Furthermore, assessments of mitochondrial membrane potential, intracellular ROS, cardiolipin content and lipid peroxidation, ER stress, and autophagy markers provided information on the mechanism of action of TPP-PA. The findings showed that TPP-PA reduces PDAC cell proliferation through mitochondrial disruption that leads to increased ROS, activation of ER stress, and autophagy. Hence, TPP-PA might offer a new approach for eliminating both the primary population of cancer cells and PCSCs, which highlights the promise of TPP-derived compounds as anticancer agents for PDAC.
Assuntos
Mitocôndrias , Compostos Organofosforados , Ácido Palmítico , Neoplasias Pancreáticas , Proteômica , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Ácido Palmítico/farmacologia , Ácido Palmítico/química , Compostos Organofosforados/farmacologia , Compostos Organofosforados/química , Proteômica/métodos , Linhagem Celular Tumoral , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Proteoma/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Autofagia/efeitos dos fármacosRESUMO
Stearoyl-CoA desaturase 1 (SCD1) is an attractive target for cancer therapy. However, the clinical efficacy of SCD1 inhibitor monotherapy is limited. There is thus a need to elucidate the mechanisms of resistance to SCD1 inhibition and develop new therapeutic strategies for combination therapy. In this study, we investigated the molecular mechanisms by which cancer cells acquire resistance to endoplasmic reticulum (ER) stress-dependent cancer cell death induced by SCD1 inhibition. SCD1 inhibitor-sensitive and -resistant cancer cells were treated with SCD1 inhibitors in vitro, and SCD1 inhibitor-sensitive cancer cells accumulated palmitic acid and underwent ER stress response-induced cell death. Conversely, SCD1-resistant cancer cells did not undergo ER stress response-induced cell death because fatty acid desaturase 2 (FADS2) eliminated the accumulation of palmitic acid. Furthermore, genetic depletion using siRNA showed that FADS2 is a key determinant of sensitivity/resistance of cancer cells to SCD1 inhibitor. A549 cells, an SCD1 inhibitor-resistant cancer cell line, underwent ER stress-dependent cancer cell death upon dual inhibition of SCD1 and FADS2. Thus, combination therapy with SCD1 inhibition and FADS2 inhibition is potentially a new cancer therapeutic strategy targeting fatty acid metabolism.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático , Ácidos Graxos Dessaturases , Estearoil-CoA Dessaturase , Estearoil-CoA Dessaturase/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/antagonistas & inibidores , Humanos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Linhagem Celular Tumoral , Células A549 , Ácido Palmítico/farmacologia , Morte Celular/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Neoplasias/tratamento farmacológicoRESUMO
STING (stimulator of interferon genes) is a critical immunoregulatory protein in sepsis and is regulated by various mechanisms, especially palmitoylation. FASN (fatty acid synthase) is the rate-limiting enzyme to generate cellular palmitic acid (PA) via acetyl-CoA and malonyl-CoA and participates in protein palmitoylation. However, the mechanisms underlying the interaction between STING and FASN have not been completely understood. In this study, STING-knockout mice were used to confirm the pivotal role of STING in sepsis-induced liver injury. Metabolomics confirmed the dyslipidemia in septic mice and patients. The compounds library was screened, revealing that FASN inhibitors exerted a significant inhibitory effect on the STING pathway. Mechanically, the regulatory effect of FASN on the STING pathway was dependent on palmitoylation. Further experiments indicated that the upstream of FASN, malonyl-CoA inhibited STING pathway possibly due to C91 (palmitoylated residue) of STING. Overall, this study reveals a novel paradigm of STING regulation and provides a new perspective on immunity and metabolism.
Assuntos
Ácido Graxo Sintase Tipo I , Lipoilação , Macrófagos , Malonil Coenzima A , Proteínas de Membrana , Sepse , Animais , Humanos , Masculino , Camundongos , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo I/genética , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Malonil Coenzima A/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido Palmítico/farmacologia , Sepse/metabolismo , Sepse/complicações , Sepse/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacosRESUMO
The functional differences between preadipocytes and fully differentiated mature adipocytes derived from stromal vascular fraction stem cells, as well as primary adipocytes have been analysed by evaluating their response to the obesogenic factor (a saturated fatty acid) and TNF-triggered inflammation. The analysis of single adipocytes shows that the saturated fatty acid (palmitic acid) accumulation is accompanied by inflammation and considerably dependent on the stage of the adipogenesis. In particular, preadipocytes show the exceptional potential for palmitic acid uptake resulting in their hypertrophy and the elevated cellular expression of the inflammation marker (ICAM-1). Our research provides new information on the impact of obesogenic factors on preadipocytes that is important in the light of childhood obesity prevention.
Assuntos
Adipócitos , Adipogenia , Ácido Palmítico , Adipogenia/efeitos dos fármacos , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Ácido Palmítico/efeitos adversos , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacos , Humanos , Obesidade/metabolismo , Obesidade/patologia , Animais , Diferenciação Celular , Fator de Necrose Tumoral alfa/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Molécula 1 de Adesão IntercelularRESUMO
SARS-CoV-2 S-protein-mediated fusion is thought to involve the interaction of the membrane-distal or N-terminal heptad repeat (NHR) ("HR1") of the cleaved S2 segment of the protein and the membrane-proximal or C-terminal heptad repeat (CHR) ("HR2") regions of the protein. We examined the fusion inhibitory activity of a PEGylated HR2-derived peptide and its palmitoylated derivative using a pseudovirus infection assay. The latter peptide caused a 76% reduction in fusion activity at 10 µM. Our results suggest that small variations in peptide derivatization and differences in the membrane composition of pseudovirus preparations may affect the inhibitory potency of HR2-derived peptides. We suggest that future studies on the inhibition of infectivity of SARS-CoV-2 in both in vitro and in vivo systems consider the need for higher concentrations of peptide inhibitors.
Assuntos
Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Peptídeos/farmacologia , Peptídeos/química , Ácido Palmítico/farmacologia , Ácido Palmítico/química , Internalização do Vírus/efeitos dos fármacos , COVID-19/virologia , COVID-19/metabolismo , Antivirais/farmacologia , Antivirais/químicaRESUMO
Nucleotides (NTs) act as pivotal regulatory factors in numerous biological processes, playing indispensable roles in growth, development, and metabolism across organisms. This study delves into the effects of exogenous NTs on hepatic insulin resistance using palmitic-acid-induced HepG2 cells, administering interventions at three distinct dosage levels of exogenous NTs. The findings underscore that exogenous NT intervention augments glucose consumption in HepG2 cells, modulates the expression of glycogen-synthesis-related enzymes (glycogen synthase kinase 3ß and glycogen synthase), and influences glycogen content. Additionally, it governs the expression levels of hepatic enzymes (hexokinase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase). Moreover, exogenous NT intervention orchestrates insulin signaling pathway (insulin receptor substrate-1, protein kinase B, and forkhead box protein O1) and AMP-activated protein kinase (AMPK) activity in HepG2 cells. Furthermore, exogenous NT intervention fine-tunes the expression levels of oxidative stress-related markers (malondialdehyde, glutathione peroxidase, and NADPH oxidase 4) and the expression of inflammation-related nuclear transcription factor (NF-κB). Lastly, exogenous NT intervention regulates the expression levels of glucose transporter proteins (GLUTs). Consequently, exogenous NTs ameliorate insulin resistance in HepG2 cells by modulating the IRS-1/AKT/FOXO1 pathways and regulate glucose consumption, glycogen content, insulin signaling pathways, AMPK activity, oxidative stress, and inflammatory status.
Assuntos
Proteína Forkhead Box O1 , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Ácido Palmítico , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Células Hep G2 , Ácido Palmítico/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteína Forkhead Box O1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Glucose/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Glicogênio/metabolismo , Insulina/metabolismoRESUMO
Individuals with type 2 diabetes mellitus frequently display heightened levels of palmitic acid (PA) in their serum, which may lead to ß-cell damage. The involvement of ferroptosis, a form of oxidative cell death in lipotoxic ß-cell injury remains uncertain. Here, we have shown that PA induces intracellular lipid peroxidation, increases intracellular Fe2+ content and decreases intracellular glutathione peroxidase 4 (GPX4) expression. Furthermore, PA causes distinct changes in pancreatic islets and INS-1 cells, such as mitochondrial atrophy and increased membrane density. Furthermore, the presence of the ferroptosis inhibitor has a significant mitigating effect on PA-induced ß-cell damage. Mechanistically, PA increased ceramide content and c-Jun N-terminal kinase (JNK) phosphorylation. The ceramide synthase inhibitor effectively attenuated PA-induced ß-cell damage and GPX4/Fe2+ abnormalities, while inhibiting JNK phosphorylation. Additionally, the JNK inhibitor SP600125 improved PA-induced cell damage. In conclusion, by promoting ceramide synthesis, PA inhibited GPX4 expression and increased intracellular Fe2+ to induce ß-cell ferroptosis. Moreover, JNK may be a downstream mechanism of ceramide-triggered lipotoxic ferroptosis in ß-cells.
Assuntos
Ceramidas , Ferroptose , Células Secretoras de Insulina , Ácido Palmítico , Transdução de Sinais , Ferroptose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ceramidas/metabolismo , Ácido Palmítico/farmacologia , Animais , Transdução de Sinais/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Ratos , Peroxidação de Lipídeos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ferro/metabolismoRESUMO
Diets rich in saturated fats are more detrimental to health than those containing mono- or unsaturated fats. Fatty acids are an important source of energy, but they also relay information regarding nutritional status to hypothalamic metabolic circuits and when in excess can be detrimental to these circuits. Astrocytes are the main site of central fatty acid ß-oxidation, and hypothalamic astrocytes participate in energy homeostasis, in part by modulating hormonal and nutritional signals reaching metabolic neurons, as well as in the inflammatory response to high-fat diets. Thus, we hypothesized that how hypothalamic astrocytes process-specific fatty acids participates in determining the differential metabolic response and that this is sex dependent as males and females respond differently to high-fat diets. Male and female primary hypothalamic astrocyte cultures were treated with oleic acid (OA) or palmitic acid (PA) for 24 h, and an untargeted metabolomics study was performed. A clear predictive model for PA exposure was obtained, while the metabolome after OA exposure was not different from controls. The observed modifications in metabolites, as well as the expression levels of key metabolic enzymes, indicate a reduction in the activity of the Krebs and glutamate/glutamine cycles in response to PA. In addition, there were specific differences between the response of astrocytes from male and female mice, as well as between hypothalamic and cerebral cortical astrocytes. Thus, the response of hypothalamic astrocytes to specific fatty acids could result in differential impacts on surrounding metabolic neurons and resulting in varied systemic metabolic outcomes.
Assuntos
Astrócitos , Hipotálamo , Ácido Oleico , Ácido Palmítico , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Ácido Oleico/farmacologia , Feminino , Ácido Palmítico/farmacologia , Hipotálamo/metabolismo , Hipotálamo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Caracteres Sexuais , Células CultivadasRESUMO
Epidemiological and clinical evidence have extensively documented the role of obesity in the development of endometrial cancer. However, the effect of fatty acids on cell growth in endometrial cancer has not been widely studied. Here, we reported that palmitic acid significantly inhibited cell proliferation of endometrial cancer cells and primary cultures of endometrial cancer and reduced tumor growth in a transgenic mouse model of endometrial cancer, in parallel with increased cellular stress and apoptosis and decreased cellular adhesion and invasion. Inhibition of cellular stress by N-acetyl-L-cysteine effectively reversed the effects of palmitic acid on cell proliferation, apoptosis, and invasive capacity in endometrial cancer cells. Palmitic acid increased the intracellular formation of lipid droplets in a time- and dose-dependent manner. Depletion of lipid droplets by blocking DGAT1 and DGAT2 effectively increased the ability of palmitic acid to inhibit cell proliferation and induce cleaved caspase 3 activity. Collectively, this study provides new insight into the effect of palmitic acid on cell proliferation and invasion and the formation of lipid droplets that may have potential clinical relevance in the treatment of obesity-driven endometrial cancer.
Assuntos
Apoptose , Proliferação de Células , Neoplasias do Endométrio , Gotículas Lipídicas , Ácido Palmítico , Feminino , Ácido Palmítico/farmacologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/patologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Diacilglicerol O-Aciltransferase/metabolismo , Camundongos TransgênicosRESUMO
Cardiac lipotoxicity is a prevalent consequence of lipid metabolism disorders occurring in cardiomyocytes, which in turn precipitates the onset of heart failure. Mimetics of brain-derived neurotrophic factor (BDNF), such as 7,8-dihydroxyflavone (DHF) and 7,8,3'-trihydroxyflavone (THF), have demonstrated significant cardioprotective effects. However, it remains unclear whether these mimetics can protect cardiomyocytes against lipotoxicity. The aim of this study was to examine the impact of DHF and THF on the lipotoxic effects induced by palmitic acid (PA), as well as the concurrent mitochondrial dysfunction. H9c2 cells were subjected to treatment with PA alone or in conjunction with DHF or THF. Various factors such as cell viability, lactate dehydrogenase (LDH) release, death ratio, and mitochondrial function including mitochondrial membrane potential (MMP), mitochondrial-derived reactive oxygen species (mito-SOX) production, and mitochondrial respiration were assessed. PA dose-dependently reduced cell viability, which was restored by DHF or THF. Additionally, both DHF and THF decreased LDH content, death ratio, and mito-SOX production, while increasing MMP and regulating mitochondrial oxidative phosphorylation in cardiomyocytes. Moreover, DHF and THF specifically activated Akt signaling. The protective effects of DHF and THF were abolished when an Akt inhibitor was used. In conclusion, BDNF mimetics attenuate PA-induced injury in cardiomyocytes by alleviating mitochondrial impairments through the activation of Akt signaling.