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1.
Nat Commun ; 11(1): 2695, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483258

RESUMO

Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors that are characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority are located in non-coding DNA regions. This suggests that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Elementos Facilitadores Genéticos , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Obesidade/patologia , Ácido Palmítico/farmacologia , Fatores de Iniciação de Peptídeos/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/farmacologia
2.
Am J Physiol Endocrinol Metab ; 319(2): E265-E275, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459525

RESUMO

Saturated fatty acids (SFAs) are implicated in muscle inflammation/cell stress and insulin resistance, but the catalog of factors involved is incomplete. SFA derivatives that accumulate with mismatched FA availability and FA oxidation (FAO) are likely involved, and evidence has emerged that select acylcarnitines should be considered. To understand if excessive long-chain acylcarnitine accumulation and limited FAO associate with lipotoxicity, carnitine palmitoyltransferase 2 knockout C2C12 cells were generated (CPT2 KO). CPT2 KO was confirmed by Western blot, increased palmitoylcarnitine accumulation, and loss of FAO capacity. There was no effect of CPT2 KO on palmitic acid (PA) concentration-dependent increases in media IL-6 or adenylate kinase. PA at 200 and 500 µM did not trigger cell stress responses (phospho-Erk, -JNK, or -p38) above that of vehicle in WT or CPT2 KO cells. In contrast, loss of CPT2 exacerbated PA-induced insulin resistance (acute phospho-Akt; 10 or 100 nM insulin) by as much as ~50-96% compared with WT. Growing cells in carnitine-free media abolished differences between WT and CPT2 KO, but this did not fully rescue PA-induced insulin resistance. The results suggest that PA-induced insulin resistance stems in part from palmitoylcarnitine accumulation, further supporting the hypothesis that select acylcarnitines participate in cell signaling and, when in excess, can compromise cell function. Since carnitine-free conditions could not fully rescue insulin signaling, and CPT2 KO did not alter cell stress responses, the majority of PA-induced "lipotoxicity" in C2C12 myotubes cannot be attributed to palmitoylcarnitine alone.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/fisiologia , Técnicas de Inativação de Genes , Resistência à Insulina/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Ácido Palmítico/farmacologia , Animais , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Palmitoilcarnitina/metabolismo , Transdução de Sinais/fisiologia
3.
J Dairy Sci ; 103(6): 5131-5142, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32253043

RESUMO

We evaluated the effects of commercially available fatty acid (FA) supplements containing palmitic (C16:0) and stearic acid (C18:0) on nutrient digestibility and production responses of dairy cows. Thirty-six mid-lactation (146 ± 55 d in milk) multiparous Holstein cows were randomly assigned to twelve 3 × 3 balanced truncated Latin squares, with 3 treatments and 2 consecutive 35-d periods, with the final 5 d used for sample and data collection. Treatments were (1) a control diet containing no supplemental FA (CON), (2) a control diet supplemented with a commercially available C16:0 supplement (PA), and (3) a control diet supplemented with a commercially available C16:0 and C18:0 supplement (MIX). Supplements were fed at 1.5% dry matter and replaced soyhulls in CON. The statistical model included the random effect of cow nested within square and the fixed effects of treatment, period, square, and their interactions. Preplanned contrasts were (1) overall effect of FA treatments [CON vs. the average of the FA treatments (FAT); 1/2 (PA + MIX)], and (2) effect of FA supplement (PA vs. MIX). Treatment had no effects on dry matter intake, body weight, or body weight change. Compared with CON, FAT decreased digestibilities of total FA and 18-carbon FA but did not affect dry matter and neutral detergent fiber digestibility. Compared with MIX, PA increased dry matter and neutral detergent fiber digestibilities by 3.6 and 4.8 percentage units, respectively. The PA also increased total FA and 18-carbon FA digestibilities but did not alter 16-carbon FA digestibility compared with MIX. Using a Lucas test, we estimated apparent digestibility coefficients of 0.768 and 0.553 for the PA and MIX supplements, respectively. Compared with CON, FAT increased milk yield and tended to increase energy-corrected milk, but did not affect yield of milk fat or milk protein. The PA increased energy-corrected milk and milk fat yield but had no effect on milk protein yield compared with MIX. Our results indicate that dairy cows producing around 45 kg of milk respond better to a FA supplement enriched in C16:0 compared with a supplement containing both C16:0 and C18:0, which is likely due in part to PA increasing FA and neutral detergent fiber digestibility compared with MIX.


Assuntos
Bovinos/fisiologia , Suplementos Nutricionais/análise , Lactação/efeitos dos fármacos , Leite/metabolismo , Ácido Palmítico/farmacologia , Ácidos Esteáricos/farmacologia , Ração Animal/análise , Animais , Peso Corporal/efeitos dos fármacos , Dieta/veterinária , Fibras na Dieta/metabolismo , Digestão/efeitos dos fármacos , Feminino , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Leite/química , Proteínas do Leite/metabolismo , Nutrientes/metabolismo , Distribuição Aleatória
4.
Chem Biol Interact ; 329: 109094, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32278738

RESUMO

BACKGROUND: Oxidative stress in cardiac myocytes is an important pathogenesis of cardiac lipotoxicity. Autophagy is a cellular self-digestion process that can selectively remove damaged organelles under oxidative stress, and thus presents a potential therapeutic target against cardiac lipotoxicity. Globular CTRP9 (gCTRP9) is a newly identified adiponectin paralog with established metabolic regulatory properties. The aim of this work is to investigate whether autophagy participates the protection effects of gCTRP9 in neonatal rat cardiac myocytes (NRCMs) under oxidative stress and the underlying mechanism. RESULTS: NRCMs were treated with PA of various concentrations for indicated time period. Our results showed that PA enhanced intracellular ROS accumulation, decreased mitochondrial membrane potential (Δψm) and increased activation of caspases 3. These changes suggested lipotoxicity due to excessive PA. In addition, PA was observed to impair autophagic flux in NRCMs and impaired autophagosome clearance induced by PA contributes to cardiomyocyte death. Besides, we found that gCTRP9 increased the ratio of LC3II/I and the expression of ATG5 which was vital to the formation of autophagosomes and decreased the level of P62, suggesting enhanced autophagic flux in the absence or presence of PA. The result was further confirmed by the methods of infection with LC3-mRFP-GFP lentivirus and blockage of autophagosome-lysosome fusion by BafA1. Moreover, gCTRP9 reestablished the loss of mitochondrial membrane potential, suppressed ROS generation, and reduced PA -induced myocyte death. However, the protective effect of gCTRP9 on the cardiac lipotoxicity was partly abolished by blockade of autophagy by autophagy-related 5 (ATG5) siRNA, indicating that the effect of gCTRP9 on cell survival is critically mediated through regulation of autophagy. CONCLUSION: Autophagy induction by gCTRP9 could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.


Assuntos
Adiponectina/metabolismo , Autofagia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Proteína 5 Relacionada à Autofagia/antagonistas & inibidores , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo
5.
Biochim Biophys Acta Mol Basis Dis ; 1866(6): 165763, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32169502

RESUMO

Excess circulating fatty acids contribute to endothelial dysfunction that subsequently aggravates the metabolic conditions such as fatty liver diseases. However, the exact mechanism of this event is not fully understood, and the investigation on the effect of a direct exposure to fatty acids together with their subsequent fate is of interest. In this work we employed a chemically specific and label-free techniques such as Raman and CARS microscopies, to investigate the process of lipid droplets (LDs) formation in endothelial cells and hepatocytes after exposure to oleic and palmitic acid. We aimed to observe the changes in the composition of LDs associated with metabolism and degradation of lipids. We were able to characterize the diversity in the formation of LDs in endothelium as compared to hepatocytes, as well as the differences in the formation of LDs and degradation manner with respect to the used fatty acid. Thus, for the first time the spectral characteristics of LDs formed in endothelial cells after incubation with oleic and palmitic acid is presented, including the time-dependent changes in their chemical composition.


Assuntos
Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Ácido Oleico/farmacologia , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Análise Espectral Raman
6.
Am J Physiol Endocrinol Metab ; 318(5): E791-E805, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32182124

RESUMO

Irisin, a newly identified myokine, is critical to modulating body metabolism and biological homeostasis. However, whether irisin protects the skeletal muscles against metabolic stresses remains unknown. In this study, we determine the effect of irisin on high glucose and fatty acid-induced damages using irisin-overexpressed mouse C2C12 (irisin-C2C12) myoblasts and skeletal muscle from irisin-injected mice. Compared with empty vector-transfected control C2C12 cells, irisin overexpression resulted in a marked increase in cell viability and decrease in apoptosis under high-glucose stress. Progression of the cell cycle into the G2/M phase in the proliferative condition was also observed with irisin overexpression. Furthermore, glucose uptake, glycogen accumulation, and phosphorylation of AMPKα/insulin receptor (IR) ß-subunit/Erk1/2 in response to insulin stimulation were enhanced by irisin overexpression. In irisin-C2C12 myoblasts, these responses of phosphorylation were preserved under palmitate treatment, which induced insulin resistance in the control cells. These effects of irisin were reversed by inhibiting AMPK with compound C. In addition, high glucose-induced suppression of the mitochondrial membrane potential was also prevented by irisin. Moreover, suppression of IR in irisin-C2C12 myoblasts by cotransfection of shRNA against IR also mitigated the effects of irisin while not affecting AMPKα phosphorylation. As an in vivo study, soleus muscles from irisin-injected mice showed elevated phosphorylation of AMPKα and Erk1/2 and glycogen contents. Our results indicate that irisin counteracts the stresses generated by high glucose and fatty acid levels and irisin overexpression serves as a novel approach to elicit cellular protection. Furthermore, AMPK activation is a crucial factor that regulates insulin action as a downstream target.


Assuntos
Adenilato Quinase/metabolismo , Fibronectinas/farmacologia , Glucose/farmacologia , Mioblastos/efeitos dos fármacos , Ácido Palmítico/farmacologia , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Fibronectinas/genética , Fibronectinas/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Mioblastos/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/fisiologia
7.
Am J Physiol Cell Physiol ; 318(1): C48-C62, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31618077

RESUMO

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.


Assuntos
Centrossomo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas 14-3-3/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Glicemia/metabolismo , Centrossomo/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Exorribonucleases/metabolismo , Feminino , Receptores Frizzled/genética , Produtos Finais de Glicação Avançada/farmacologia , Células HCT116 , Humanos , Insulina/sangue , Camundongos Endogâmicos ICR , Ácido Palmítico/farmacologia , Ligação Proteica , Ratos , Proteínas Wnt/genética , Quinases Associadas a rho/metabolismo
8.
Am J Physiol Endocrinol Metab ; 318(1): E1-E10, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31613643

RESUMO

The molecular circadian clock plays a role in metabolic homeostasis. We tested the hypothesis obesity and systemic factors associated with insulin resistance affect skeletal muscle clock gene expression. We determined clock gene expression in skeletal muscle of obese women (n = 5) and men (n = 18) before and 6 mo after Roux-en-Y gastric bypass (RYGB) surgery and normal-weight controls (women n = 6, men n = 8). Skeletal muscle clock gene expression was affected by obesity and weight loss. CRY1 mRNA (P = 0.05) was increased and DBP mRNA (P < 0.05) was decreased in obese vs. normal weight women and restored to control levels after RYGB-induced weight loss. CLOCK, CRY1, CRY2, and DBP mRNA (P < 0.05) was decreased in obese men compared with normal weight men. Expression of all other clock genes was unaltered by obesity or weight loss in both cohorts. We correlated clock gene expression with clinical characteristics of the participants. Among the genes studied, DBP and PER3 expression was inversely correlated with plasma lipids in both cohorts. Circadian time-course studies revealed that core clock genes oscillate over time (P < 0.05), with BMAL1, CIART, CRY2, DBP, PER1, and PER3 expression profiles altered by palmitate treatment. In conclusion, skeletal muscle clock gene expression and function is altered by obesity, coincident with changes in plasma lipid levels. Palmitate exposure disrupts clock gene expression in myotubes, indicating that dyslipidemia directly alters the circadian program. Strategies to reduce lipid overload and prevent elevations in nonesterified fatty acid and cholesterol levels may sustain circadian clock signals in skeletal muscle.


Assuntos
Músculo Esquelético/metabolismo , Obesidade/genética , RNA Mensageiro/metabolismo , Perda de Peso , Fatores de Transcrição ARNTL/genética , Adulto , Proteínas CLOCK/genética , Estudos de Casos e Controles , Criptocromos/genética , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/metabolismo , Feminino , Derivação Gástrica , Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Obesidade/metabolismo , Obesidade/cirurgia , Ácido Palmítico/farmacologia , Proteínas Circadianas Period/genética , Cultura Primária de Células , Fatores de Transcrição/genética
9.
Redox Biol ; 28: 101314, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514051

RESUMO

Nuclear factor-erythroid 2 related factor 2 (Nrf2)-mediated signaling plays a central role in maintaining cellular redox homeostasis of hepatic cells. Carbon monoxide releasing molecule-A1 (CORM-A1) has been reported to stimulate up-regulation and nuclear translocation of Nrf2 in hepatocytes. However, the role of CORM-A1 in improving lipid metabolism, antioxidant signaling and mitochondrial functions in nonalcoholic steatohepatitis (NASH) is unknown. In this study, we report that CORM-A1 prevents hepatic steatosis in high fat high fructose (HFHF) diet fed C57BL/6J mice, used as model of NASH. The beneficial effects of CORM-A1 in HFHF fed mice was associated with improved lipid homeostasis, Nrf2 activation, upregulation of antioxidant responsive (ARE) genes and increased ATP production. As, mitochondria are intracellular source of reactive oxygen species (ROS) and important sites of lipid metabolism, we further investigated the mechanisms of action of CORM-A1-mediated improvement in mitochondrial function in palmitic acid (PA) treated HepG2 cells. Cellular oxidative stress and cell viability were found to be improved in PA + CORM-A1 treated cells via Nrf2 translocation and activation of cytoprotective genes. Furthermore, in PA treated cells, CORM-A1 improved mitochondrial oxidative stress, membrane potential and rescued mitochondrial biogenesis thru upregulation of Drp1, TFAM, PGC-1α and NRF-1 genes. CORM-A1 treatment improved cellular status by lowering glycolytic respiration and maximizing OCR. Improvement in mitochondrial respiration and increment in ATP production in PA + CORM-A1 treated cells further corroborate our findings. In summary, our data demonstrate for the first time that CORM-A1 ameliorates tissue damage in steatotic liver via Nrf2 activation and improved mitochondrial function, thus, suggesting the anti-NASH potential of CORM-A1.


Assuntos
Boranos/administração & dosagem , Carbonatos/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Xarope de Milho Rico em Frutose/efeitos adversos , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Boranos/farmacologia , Carbonatos/farmacologia , Sobrevivência Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Transdução de Sinais/efeitos dos fármacos
10.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 72-83, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844893

RESUMO

Type 2 diabetes increases the risk for cancer. Centrosome amplification can initiate tumorigenesis. We have described that type 2 diabetes increases the centrosome amplification of peripheral blood mononuclear cells, with high glucose, insulin, and palmitic acid as the triggers, which suggests that centrosome amplification is a candidate biological mechanism linking diabetes to cancer. In this study, we aimed to further investigate the signaling pathways of the diabetes-associated centrosome amplification and to examine whether and how resveratrol inhibits the centrosome amplification. The results showed that treatment with high glucose, insulin, and palmitic acid, alone or in combination, could increase the protein levels of phospho-protein kinase C alpha (p-PKCα), phospho-p38 mitogen-activated protein kinases (p-p38), c-myc, and c-jun, as well as the mRNA levels of c-myc and c-jun. PKCα inhibitor could inhibit the treatment-induced increase in the protein levels of p-p38, c-myc, and c-jun. Inhibitor or siRNA of p38 was also able to inhibit the treatment-induced increase in the levels of p-p38, c-myc, and c-jun. Meanwhile, knockdown of c-myc or c-jun did not alter the treatment-induced increase in the phosphorylation of PKCα or p38. Importantly, inhibition of the phosphorylation of PKCα or p38 and knockdown of c-myc or c-jun could attenuate the centrosome amplification. In diabetic mice, the levels of p-PKCα, p-p38, c-myc, and c-jun were all increased in the colon tissues. Interestingly, resveratrol, but not metformin, was able to attenuate the treatment-induced increase in the levels of p-PKCα, p-p38, c-myc, and c-jun, as well as the centrosome amplification. In conclusion, our results suggest that PKCα-p38 to c-myc/c-jun is the signaling pathway of the diabetes-associated centrosome amplification, and resveratrol attenuates the centrosome amplification by inhibiting this signaling pathway.


Assuntos
Centrossomo/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Centrossomo/metabolismo , Colo/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Técnicas de Silenciamento de Genes , Glucose/farmacologia , Células HCT116 , Humanos , Insulina/farmacologia , Camundongos , Ácido Palmítico/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Estreptozocina/efeitos adversos , Estreptozocina/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genética
11.
Int J Mol Sci ; 20(23)2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810180

RESUMO

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 µM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Lipogênese/efeitos dos fármacos , Ácido Palmítico/farmacologia , Pele Artificial , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/biossíntese , Lipídeos/química , Morfogênese/efeitos dos fármacos , Ácido Palmítico/química , Pele/química , Pele/efeitos dos fármacos , Pele/metabolismo
12.
Biomed Res Int ; 2019: 8171989, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828133

RESUMO

Obesity-related insulin resistance and high fatty acid concentrations occur during the development of type 2 diabetes mellitus. The role of high concentrations of plasma-free fatty acids is not fully understood. In this study, palmitic acid (PA, 0.8 mM for 24 h) induced the expression of miR-221 that bound to phosphoinositide 3-kinases (PI3K) mRNA to inhibit glucose uptake by HepG2 cells. Compared with controls, PA significantly decreased glucose uptake, increased insulin receptor substrate-2 (IRS-2) and miR-221 expression, and decreased phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and glucose transporter type 4 (GLUT4) mRNA expression. Luciferase reporter assay revealed that miR-221 binding inhibited PI3K expression. Transfection of HepG2 cells with an miR-221 mimic induced miR-221 expression and inhibited the PI3K/AKT pathway. PA decreased glucose uptake in HepG2 cells by inducing the expression of miR-221, which bound to PI3K mRNA and suppressed PI3K/AKT signaling. miR-221 may be a novel target for preventing and treating obesity-induced insulin resistance.


Assuntos
Diabetes Mellitus Tipo 2/genética , Transportador de Glucose Tipo 4/genética , Resistência à Insulina/genética , MicroRNAs/genética , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Humanos , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , Proteína Oncogênica v-akt/genética , Ácido Palmítico/farmacologia , Fosfatidilinositol 3-Quinases/genética , RNA Mensageiro/genética , Transdução de Sinais
13.
Int J Mol Sci ; 20(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842349

RESUMO

Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic ß-cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead to cell cycle arrest, DNA repair, senescence, or apoptosis, depending on the severity/type of stress. Nonetheless, the precise role of p53 in lipotoxicity induced by palmitic acid is not clear. Here, our results show that palmitic acid induces p53 activation in a dose- and time-dependent manner. Furthermore, loss of p53 makes cells sensitive to palmitic acid-induced apoptosis. These results were demonstrated in human colon carcinoma cells (HCT116) and primary mouse embryo fibroblasts (MEF) through analysis of DNA fragmentation, flow cytometry, colony formation, and Western blots. In the HCT116 p53-/- cell line, palmitic acid induced greater reactive oxygen species formation compared to the p53+/+ cell line. The reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and reduced glutathione (GSH) partially attenuated apoptosis in the HCT116 p53-/- cell line but had no obvious effect on the p53+/+ cell line. Furthermore, p53 induced the expression of its downstream target genes, p21 and Sesn2, in response to ROS induced by palmitic acid. Loss of p21 also leads to more palmitic acid-induced cell apoptosis in the HCT116 cell line compared with HCT116 p53+/+ and HCT116 p53-/-. In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53-/- mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53-/- and p53+/+ mice that received a regular diet. We conclude that p53 may provide some protection against palmitic acid- induced apoptosis in cells by targeting its downstream genes in response to this stress.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Resistência a Medicamentos/genética , Ácido Palmítico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Fibroblastos , Deleção de Genes , Células HCT116 , Humanos , Camundongos
14.
PLoS One ; 14(12): e0226696, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31860682

RESUMO

Elevated levels of glucose and fatty acids are the main characteristics of diabetes, obesity and other metabolic disorders, associated with increased oxidative stress, mitochondrial dysfunction and inflammation. Once the primary pathogenesis of diabetes is established, which is potentially linked to both genetic and environmental factors, hyperglycemia and hyperlipidemia exert further destructive and/or toxic effects on ß-cells. The concept of glucolipotoxicity has arisen from the combination of deleterious effects of chronic elevation of glucose and fatty acid levels on pancreatic ß- cell function and/or survival. Though numerous studies have been conducted in this field, the exact molecular mechanisms and causative factors still need to be established. The aim of the present work was to elucidate the molecular mechanisms of oxidative stress, and inflammatory/antioxidant responses in the presence of high concentrations of glucose/fatty acids in a cell-culture system using an insulin-secreting pancreatic ß-cell line (Rin-5F) and to study the effects of the antioxidant, N-acetyl cysteine (NAC) on ß-cell toxicity. In our study, we investigated the molecular mechanism of cytotoxicity in the presence of high glucose (up to 25 mM) and high palmitic acid (up to 0.3 mM) on Rin-5F cells. Our results suggest that the cellular and molecular mechanisms underlying ß-cell toxicity are mediated by increased oxidative stress, imbalance of redox homeostasis, glutathione (GSH) metabolism and alterations in inflammatory responses. Pre-treatment with NAC attenuated oxidative stress and alterations in GSH metabolism associated with ß-cells cytotoxicity.


Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Glucose/farmacologia , Glutationa/metabolismo , Células Secretoras de Insulina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/farmacologia , Animais , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo
15.
Food Funct ; 10(12): 7634-7644, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31728459

RESUMO

Chemotherapy is currently used to treat colorectal cancer (CRC), the most common cancer worldwide. However, chemotherapeutic drugs are limited by severe side effects or drug resistance. In this study, bioactive compound(s), a mixture of palmitic acid, stearic acid, and glyceryl 1,3-dipalmitate (PSG), in Lactobacillus paracasei subsp. paracasei NTU 101-fermented reconstituted skimmed milk ethanol extract (NTU 101-FMEE) were isolated and identified. PSG (1 : 1.5 : 6.3) at 125 µg mL-1 could significantly decrease CRC cell viability at dosages that were not cytotoxic to healthy colon epithelial cells or macrophages. Moreover, the inhibitory effect of the combination of 62.5 µg mL-1 PSG (1 : 1.5 : 6.3) and 5-fluorouracil (5-FU) was significantly higher than that of 5-FU alone (p < 0.05). PSG up-regulated the activities of apoptosis-related proteins and down-regulated the nuclear factor-κB signaling pathway compared to the levels in the control group. Overall, PSG purified from NTU 101-FMEE possesses the potential to ameliorate CRC by improving the effects of adjuvant chemotherapy drugs and reducing side effects.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Produtos Fermentados do Leite/análise , Fluoruracila/farmacologia , Lactobacillus paracasei/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/fisiopatologia , Produtos Fermentados do Leite/microbiologia , Fermentação , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Ácidos Esteáricos/metabolismo , Ácidos Esteáricos/farmacologia
16.
Med Sci Monit ; 25: 8544-8553, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31719515

RESUMO

BACKGROUND Gegen qinlian decoction (GGQLD) is a form of traditional Chinese medicine used for hundreds of years for its efficacy in treating diabetes. However, the mechanisms underlying the therapeutic effects of GGQLD on diabetes are still not clear. We aimed to evaluate the effect of GGQLD on hepatic insulin resistance (IR) through silent information regulator1 (SIRT1)/forkhead box O1 (FOXO1) in an IR mouse model. MATERIAL AND METHODS A high-fat diet (HFD) mouse model was established and GGQLD was administrated by oral gavage. Metabolic parameters were detected, including body weights, triglyceride, fasting glucose, fasting insulin and HOMA-IR index, glucose intolerance, and insulin resistance. HE-stained sections were used to observe the histopathology of liver tissue. For in vitro study, GGQLD-medicated serum was used to treat palmitic acid-stimulated HepG2 cells. The glycogen synthesis and downstream SIRT1/FOXO1 signaling pathways were examined. Specific siRNAs were used to knock down SIRT1 in HepG2 cells. RESULTS GGQLD administration significantly decreased body weights, triglyceride level, fasting glucose level, fasting insulin level, and HOMA-IR index, and improved IR in HFD mice. GGQLD enhanced SIRT1 expression and suppressed the expression of Ac-FOXO1 in liver tissues. Further, GGQLD-medicated serum promoted SIRT1 upregulation and suppressed Ac-FOXO1 levels in palmitate-stimulated HepG2 cells. GGQLD-medicated serum also increased the protein expression of PPARγ and reduced the expression of FABP4 in palmitate-stimulated HepG2 cells. CONCLUSIONS We found that GGQLD alleviates insulin resistance through SIRT1-dependent deacetylation of FOXO1.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Proteína Forkhead Box O1/metabolismo , Resistência à Insulina/fisiologia , Sirtuína 1/metabolismo , Animais , China , Dieta Hiperlipídica , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/metabolismo , Proteína Forkhead Box O1/genética , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Células Hep G2 , Humanos , Insulina/metabolismo , Fígado/metabolismo , Masculino , Medicina Tradicional Chinesa/métodos , Camundongos , Camundongos Endogâmicos C57BL , Ácido Palmítico/farmacologia , Transdução de Sinais
17.
Pharmacol Rep ; 71(6): 1160-1167, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31675670

RESUMO

BACKGROUND: Hepatic insulin resistance can be induced by excess dietary intake of saturated fat. Ginsenoside Rg1 (GRg1), the major active ginsenoside enriched in tonic food ginseng, was reported to help alleviate liver diseases. In the present study, GRg1 was evaluated for its impact on palmitic acid (PA)-induced hepatic insulin resistance model in vitro. METHODS: Insulin resistance in HepG2 cells was induced by 0.5 mM PA exposure for 24 h and then the effect of GRg1 on cellular glucose consumption was measured. Expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphate (G6Pase) were analyzed by Western blot and quantitative real-time polymerase chain reaction. Activation of protein kinases and transcript factor was analyzed by measuring protein phosphorylation. The influence of GRg1 on reactive oxygen species (ROS) production in HepG2 was also examined. RESULTS: GRg1 reversed PA-induced decrease in glucose consumption of HepG2 cells by downregulating gluconeogenesis genes G6pase and PEPCK. GRg1 increased Akt activation but inhibited JNK activation in PA-challenged HepG2 cells. Cellular ROS level was elevated in insulin-resistant HepG2 cells but was reduced by GRg1. CONCLUSIONS: Together these findings indicate that GRg1 protects against hepatic insulin resistance via preserving insulin signaling sensitivity and is a promising alternative medicine.


Assuntos
Ginsenosídeos/farmacologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , MAP Quinase Quinase 4/efeitos dos fármacos , Ácido Palmítico/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
18.
Fish Shellfish Immunol ; 95: 595-605, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31676430

RESUMO

Fatty acids (FAs) are key elements that affect not only growth but also different immune functions, and therefore, nutrition is important for growing healthy fish. Zebrafish (Danio rerio) is a good model for assessing the beneficial effects of immunostimulants, including FAs, before applying them in aquaculture. Accordingly, this study evaluated the effects of palmitic acid (PA) treatment on different immune parameters of zebrafish and on the mortality caused by the spring viremia of carp virus (SVCV). The results suggest that PA modulates the infection outcome in vivo, which benefits zebrafish and results in reduced mortality and viral titres. The antiviral protection elicited by this FA seems to be associated with the inhibition of autophagy and is independent of other immune processes, such as neutrophil proliferation or type I interferon (IFN) activity. The use of PA as an immunostimulant at low concentrations showed great potential in the prevention of SVCV infections; therefore, this FA could help to prevent the mortality and morbidity caused by viral agents in aquacultured fish. Nevertheless, the potentially detrimental effects of suppressing autophagy in the organism should be taken into account.


Assuntos
Antivirais/farmacologia , Autofagia/efeitos dos fármacos , Imunidade Inata , Ácido Palmítico/farmacologia , Peixe-Zebra/imunologia , Animais , Aquicultura , Linhagem Celular , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Rhabdoviridae , Infecções por Rhabdoviridae/imunologia , Peixe-Zebra/virologia
19.
Int J Mol Sci ; 20(20)2019 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-31614951

RESUMO

Obesity is closely associated with neuroinflammation in the hypothalamus, which is characterized by over-activated microglia and excessive production of pro-inflammatory cytokines. The present study was aimed at elucidating the effects of (-)-epigallocatechin gallate (EGCG) on palmitic acid-stimulated BV-2 microglia and high-fat-diet-induced obese mice. The results indicated the suppressive effect of EGCG on lipid accumulation, pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) release, and microglial activation in both cellular and high-fat-diet rodent models. These results were associated with lower phosphorylated levels of the janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway. In conclusion, EGCG can attenuate high-fat-induced hypothalamic inflammation via inhibiting the JAK2/STAT3 signaling pathways in microglia.


Assuntos
Fármacos Antiobesidade/farmacologia , Catequina/análogos & derivados , Microglia/efeitos dos fármacos , Obesidade/tratamento farmacológico , Animais , Fármacos Antiobesidade/uso terapêutico , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Hipotálamo/efeitos dos fármacos , Hipotálamo/imunologia , Hipotálamo/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Janus Quinase 2/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Microglia/metabolismo , Obesidade/metabolismo , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Polifenóis/farmacologia , Fator de Transcrição STAT3/metabolismo , Chá/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Lipids Health Dis ; 18(1): 179, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31639005

RESUMO

BACKGROUND: Ceramide plays pathogenic roles in nonalcoholic fatty liver disease (NAFLD) via multiple mechanisms, and as such inhibition of ceramide de novo synthesis in the liver may be of therapeutically beneficial in patients with NAFLD. In this study, we aimed to explore whether inhibition of ceramide signaling by myriocin is beneficial in animal model of NAFLD via regulating autophagy. METHODS: Sprague Dawley rats were randomly divided into three groups: standard chow (n = 10), high-fat diet (HFD) (n = 10) or HFD combined with oral administration of myriocin (0.3 mg/kg on alternate days for 8 weeks) (n = 10). Liver histology and autophagy function were measured. HepG2 cells were incubated with fatty acid with or without myriocin treatment. Lipid accumulation and autophagy markers in the HepG2 cells were analyzed. Serum ceramide changes were studied in 104 subjects consisting healthy adults, liver biopsy-proven patients with NAFLD and liver biopsy-proven patients with chronic hepatitis B (CHB). RESULTS: Myriocin reversed the elevated body weight and serum transaminases and alleviated dyslipidemia in HFD fed rats. Myriocin treatment significantly attenuated liver pathology including steatosis, lobular inflammation and ballooning. By qPCR analysis, it was revealed that myriocin corrected the expression pattern of fatty acid metabolism associated genes including Fabp1, Pparα, Cpt-1α and Acox-2. Further, myriocin also restored the impaired hepatic autophagy function in rats with HFD-induced NASH, and this has been verified in HepG2 cells. Among the sphingolipid species that we screened in lipidomic profiles, significantly increased ceramide was observed in NASH patients as compared to the controls and non-NASH patients, regardless of whether or not they have active CHB. CONCLUSIONS: Ceramide may play an important regulatory role in the autophagy function in the pathogenesis of NASH. Hence, blockade of ceramide signaling by myriocin may be of therapeutically beneficial in NASH. TRIAL REGISTRATION: Registration ID: ChiCTR-DDT-13003983 . Data of registration: 13 May, 2013, retrospectively registered.


Assuntos
Autofagia/efeitos dos fármacos , Ceramidas/metabolismo , Dislipidemias/tratamento farmacológico , Ácidos Graxos Monoinsaturados/farmacologia , Hipolipemiantes/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Adulto , Animais , Autofagia/genética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Estudos de Casos e Controles , Ceramidas/antagonistas & inibidores , Dieta Hiperlipídica/efeitos adversos , Dislipidemias/etiologia , Dislipidemias/genética , Dislipidemias/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatite B Crônica/genética , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácido Oleico/antagonistas & inibidores , Ácido Oleico/farmacologia , Oxirredutases/genética , Oxirredutases/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Ácido Palmítico/antagonistas & inibidores , Ácido Palmítico/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
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