RESUMO
Metabolic dysfunction-associated steatohepatitis (MASH) is the severe form of non-alcoholic fatty liver disease which has a high potential to progress to cirrhosis and hepatocellular carcinoma, yet adequate effective therapies are lacking. Hypoadiponectinemia is causally involved in the pathogenesis of MASH. This study investigated the pharmacological effects of adiponectin replacement therapy with the adiponectin-derived peptide ALY688 (ALY688-SR) in a mouse model of MASH. Human induced pluripotent stem (iPS) cell-derived hepatocytes were used to test cytotoxicity and signaling of unmodified ALY688 in vitro. High-fat diet with low methionine and no added choline (CDAHF) was used to induce MASH and test the effects of ALY688-SR in vivo. Histological MASH activity score (NAS) and fibrosis score were determined to assess the effect of ALY688-SR. Transcriptional characterization of mice through RNA sequencing was performed to indicate potential molecular mechanisms involved. In cultured hepatocytes, ALY688 efficiently induced adiponectin-like signaling, including the AMP-activated protein kinase and p38 mitogen-activated protein kinase pathways, and did not elicit cytotoxicity. Administration of ALY688-SR in mice did not influence body weight but significantly ameliorated CDAHF-induced hepatic steatosis, inflammation, and fibrosis, therefore effectively preventing the development and progression of MASH. Mechanistically, ALY688-SR treatment markedly induced hepatic expression of genes involved in fatty acid oxidation, whereas it significantly suppressed the expression of pro-inflammatory and pro-fibrotic genes as demonstrated by transcriptomic analysis. ALY688-SR may represent an effective approach in MASH treatment. Its mode of action involves inhibition of hepatic steatosis, inflammation, and fibrosis, possibly via canonical adiponectin-mediated signaling.
Assuntos
Adiponectina , Modelos Animais de Doenças , Hepatócitos , Hepatopatia Gordurosa não Alcoólica , Animais , Adiponectina/metabolismo , Adiponectina/farmacologia , Adiponectina/deficiência , Camundongos , Humanos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/patologia , Doenças Metabólicas/tratamento farmacológico , Doenças Metabólicas/metabolismo , Doenças Metabólicas/prevenção & controle , Doenças Metabólicas/etiologia , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado Gorduroso/prevenção & controle , Fígado Gorduroso/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologiaRESUMO
The diverse beneficial effects of adiponectin-receptor signaling, including its impact on the regulation of inflammatory processes in vivo, have resulted in development of adiponectin receptor agonists as a treatment for metabolic disorders. However, there are no established non-invasive bioassays for detection of adiponectin target engagement in humans or animal models. Here, we designed an assay using small amounts of blood to assess adiponectin action. Specifically, we tested effects of the small 10-amino acid peptide adiponectin receptor agonist, ALY688, in a sublethal LPS endotoxemia model in mice. LPS-induced pro-inflammatory cytokine levels in serum were significantly reduced in mice treated with ALY688, assessed via multiplex ELISA in flow cytometry. Furthermore, ALY688 alone significantly induced TGF-ß release in serum 1 h after treatment and was elevated for up to 24 h. Additionally, using a flow-cytometry panel for detection of changes in circulating immune cell phenotypes, we observed a significant increase in absolute T cell counts in mice after ALY688 treatment. To assess changes in intracellular signaling effectors downstream of adiponectin, phospho-flow cytometry was conducted. There was a significant increase in phosphorylation of AMPK and p38-MAPK in mice after ALY688 treatment. We then used human donor immune cells (PBMCs) treated with ALY688 ex vivo and observed elevation of AMPK and p38-MAPK phosphorylation from baseline in response to ALY688. Together, these results indicate we can detect adiponectin action on immune cells in vivo by assessing adiponectin signaling pathway for AMPK and p38-MAPK, as well as pro-inflammatory cytokine levels. This new approach provides a blood-based bioassay for screening adiponectin action.
Assuntos
Adiponectina , Citocinas , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Animais , Adiponectina/sangue , Adiponectina/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Masculino , Citocinas/metabolismo , Citocinas/sangue , Bioensaio/métodos , Endotoxemia/imunologia , Endotoxemia/metabolismo , Receptores de Adiponectina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Modelos Animais de Doenças , FemininoRESUMO
The vagus nerve conveys gastrointestinal cues to the brain to control eating behavior. In obesity, vagally mediated gut-brain signaling is disrupted. Here, we show that the cocaine- and amphetamine-regulated transcript (CART) is a neuropeptide synthesized proportional to the food consumed in vagal afferent neurons (VANs) of chow-fed rats. CART injection into the nucleus tractus solitarii (NTS), the site of vagal afferent central termination, reduces food intake. Conversely, blocking endogenous CART action in the NTS increases food intake in chow-fed rats, and this requires intact VANs. Viral-mediated Cartpt knockdown in VANs increases weight gain and daily food intake via larger meals and faster ingestion rate. In obese rats fed a high-fat, high-sugar diet, meal-induced CART synthesis in VANs is blunted and CART antibody fails to increase food intake. However, CART injection into the NTS retains its anorexigenic effect in obese rats. Restoring disrupted VAN CART signaling in obesity could be a promising therapeutic approach.
Assuntos
Hiperfagia/genética , Proteínas do Tecido Nervoso/metabolismo , Nervo Vago/efeitos dos fármacos , Aumento de Peso/genética , Animais , Humanos , Masculino , RatosRESUMO
OBJECTIVE: Glucagon-like peptide-1 (GLP-1) neurons in the hindbrain densely innervate the dorsomedial hypothalamus (DMH), a nucleus strongly implicated in body weight regulation and the sympathetic control of brown adipose tissue (BAT) thermogenesis. Therefore, DMH GLP-1 receptors (GLP-1R) are well placed to regulate energy balance by controlling sympathetic outflow and BAT function. METHODS: We investigate this possibility in adult male rats by using direct administration of GLP-1 (0.5 ug) into the DMH, knocking down DMH GLP-1R mRNA with viral-mediated RNA interference, and by examining the neurochemical phenotype of GLP-1R expressing cells in the DMH using in situ hybridization. RESULTS: GLP-1 administered into the DMH increased BAT thermogenesis and hepatic triglyceride (TG) mobilization. On the other hand, Glp1r knockdown (KD) in the DMH increased body weight gain and adiposity, with a concomitant reduction in energy expenditure (EE), BAT temperature, and uncoupling protein 1 (UCP1) expression. Moreover, DMH Glp1r KD induced hepatic steatosis, increased plasma TG, and elevated liver specific de-novo lipogenesis, effects that collectively contributed to insulin resistance. Interestingly, DMH Glp1r KD increased neuropeptide Y (NPY) mRNA expression in the DMH. GLP-1R mRNA in the DMH, however, was found in GABAergic not NPY neurons, consistent with a GLP-1R-dependent inhibition of NPY neurons that is mediated by local GABAergic neurons. Finally, DMH Glp1r KD attenuated the anorexigenic effects of the GLP-1R agonist exendin-4, highlighting an important role of DMH GLP-1R signaling in GLP-1-based therapies. CONCLUSIONS: Collectively, our data show that DMH GLP-1R signaling plays a key role for BAT thermogenesis and adiposity.
Assuntos
Tecido Adiposo Marrom/metabolismo , Adiposidade , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hipotálamo/metabolismo , Termogênese , Animais , Exenatida/metabolismo , Neurônios GABAérgicos/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Resistência à Insulina , Lipogênese , Masculino , Neuropeptídeo Y/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Desacopladora 1/metabolismoRESUMO
Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24-39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.
Assuntos
Anticorpos Monoclonais/imunologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , Lisina/imunologia , Acetilação , Animais , Humanos , Lisina/química , Processamento de Proteína Pós-Traducional/imunologiaRESUMO
IL-17 is a proinflammatory cytokine that promotes the expression of different cytokines and chemokines via the induction of gene transcription and the posttranscriptional stabilization of mRNAs. In this study, we show that IL-17 increases the half-life of the Zc3h12a mRNA via interaction of the adaptor protein CIKS with the DEAD box protein DDX3X. IL-17 stimulation promotes the formation of a complex between CIKS and DDX3X, and this interaction requires the helicase domain of DDX3X but not its ATPase activity. DDX3X knockdown decreases the IL-17-induced stability of Zc3h12a without affecting the stability of other mRNAs. IKKε, TNFR-associated factor 2, and TNFR-associated factor 5 were also required to mediate the IL-17-induced Zc3h12a stabilization. DDX3X directly binds the Zc3h12a mRNA after IL-17 stimulation. Collectively, our findings define a novel, IL-17-dependent mechanism regulating the stabilization of a selected mRNA.
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Interleucina-17/metabolismo , Estabilidade de RNA , Ribonucleases/genética , Fatores de Transcrição/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Interleucina-17/farmacologia , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismoRESUMO
BACKGROUND: Mitochondria are dynamic organelles that frequently undergo fission and fusion processes, and imbalances in these processes may be involved in obesity and insulin resistance. AIMS: The present work had the following aims: (a) to evaluate whether the mitochondrial dysfunction present in the hepatic steatosis induced by a high-fat diet is associated with changes in mitochondrial dynamics and morphology; (b) to evaluate whether effects on the above parameters differ between high-lard and high-fish-oil diets, as it has been suggested that fish oil may have anti-obesity and anti-steatotic effects by stimulating fatty acids utilisation. METHODS: The development of hepatic steatosis and insulin resistance was monitored in rats fed a high-lard or high-fish-oil diet. Immunohistochemical and electronic microscopic observations were performed on liver sections. In isolated liver mitochondria, assessments of fatty acids oxidation rate, proton conductance and oxidative stress (by measuring H2O2 release and aconitase activity) were performed. Western blot and immunohistochemical analyses were performed to evaluate the presence of proteins involved in mitochondrial dynamics (i.e., fusion and fission processes). To investigate the fusion process, mitofusin 2 and autosomal dominant optic atrophy-1 (OPA1) were analysed. To investigate the fission process, the presence of dynamin-related protein 1 (Drp1) and fission 1 protein (Fis1) was assessed. RESULTS: High-lard feeding elicited greater hepatic lipid accumulation, insulin resistance with associated mitochondrial dysfunction, greater oxidative stress and a shift towards mitochondrial fission processes (versus high-fish-oil feeding, which had an anti-steatotic effect associated with increased mitochondrial fusion processes). CONCLUSIONS: Different types of high-fat diets differ in their effect on mitochondrial function and dynamic behaviour, leading to different cellular adaptations to over-feeding.
Assuntos
Gorduras na Dieta/farmacologia , Óleos de Peixe/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Animais , Masculino , Mitocôndrias Hepáticas/ultraestrutura , Ratos , Ratos WistarRESUMO
Dietary fat sources may differentially affect the development of inflammation in insulin-sensitive tissues during chronic overfeeding. Considering the anti-inflammatory properties of ω-3 fatty acids, this study aimed to compare the effects of chronic high-fish oil and high-lard diets on obesity-related inflammation by evaluating serum and tissue adipokine levels and histological features in insulin-sensitive tissues (white adipose tissue, skeletal muscle and liver). As expected, a high-lard diet induced systemic and peripheral inflammation and insulin resistance. Conversely, compared with a high-lard diet, a high-fish oil diet resulted in a lower degree of systemic inflammation and insulin resistance that were associated with a lower adipocyte diameter as well as lower immunoreactivity for transforming growth factor ß 1 (TGFß1) in white adipose tissue. A high-fish oil diet also resulted in a lower ectopic lipid depot, inflammation degree and insulin resistance in the skeletal muscle and liver. Moreover, a high-fish oil diet attenuated hepatic stellate cell activation and fibrogenesis in the liver, as indicated by the smooth muscle α-actin (α-SMA) and TGFß1 levels. The replacement of lard (saturated fatty acids) with fish oil (ω-3 fatty acids) in chronic high-fat feeding attenuated the development of systemic and tissue inflammation.
Assuntos
Dieta Hiperlipídica , Gorduras Insaturadas na Dieta , Actinas/metabolismo , Adipocinas/sangue , Adiponectina/sangue , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/análise , Quimiocina CCL2/sangue , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/patologia , Imuno-Histoquímica , Insulina/sangue , Leptina/sangue , Fígado/patologia , Masculino , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/sangueRESUMO
Various studies have shown that eicosapentaenoic acid (EPA) has beneficial effects on obesity and associated disorders. Apelin, the ligand of APJ receptor also exerts insulin-sensitizing effects especially by improving muscle metabolism. EPA has been shown to increase apelin production in adipose tissue but its effects in muscle have not been addressed. Thus, the effects of EPA supplementation (36 g/kg EPA) in high-fat diet (HFD) (45% fat, 20% protein, 35% carbohydrate) were studied in mice with focus on muscle lipid metabolism and apelin/APJ expression. Compared with HFD mice, HFD+EPA mice had significantly less weight gain, fat mass, lower blood glucose, insulinemia and hepatic steatosis after 10 weeks of diet. In addition, EPA prevented muscle metabolism alterations since intramuscular triglycerides were decreased and ß-oxidation increased. In soleus muscles of HFD+EPA mice, apelin and APJ expression were significantly increased compared to HFD mice. However, plasma apelin concentrations in HFD and HFD+EPA mice were similar. EPA-induced apelin expression was confirmed in differentiated C2C12 myocytes but in this model, apelin secretion was also increased in response to EPA treatment. In conclusion, EPA supplementation in HFD prevents obesity and metabolic alterations in mice, especially in skeletal muscle. Since EPA increases apelin/APJ expression in muscle, apelin may act in a paracrine/autocrine manner to contribute to these benefical effects.
Assuntos
Gorduras na Dieta/farmacologia , Suplementos Nutricionais , Ácido Eicosapentaenoico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Adipocinas , Animais , Apelina , Glicemia/metabolismo , Linhagem Celular , Gorduras na Dieta/efeitos adversos , Masculino , Camundongos , Músculo Esquelético/patologia , Obesidade/metabolismo , Obesidade/patologia , Obesidade/prevenção & controleRESUMO
Dietary PUFA, mainly those of the n-3 family, are known to play essential roles in the maintenance of energy balance and in the reduction of body fat deposition through the upregulation of mitochondrial uncoupling that is the main source of reactive oxygen species. We hypothesized that rat supplementation with raw donkey's milk (DM), characterized by low-fat content and higher n3:n6 ratio, may affect energy balance, lipid metabolism, and prooxidant status as compared to animals treated with cow's milk. In the present study, the effects of drinking raw DM (for 4 weeks) on energy balance, lipid metabolism, antiinflammatory, and antioxidant/detoxifying defences was compared to that produced by rat intake of an iso-energetic amount of raw cow's milk. The hypolipidemic effect produced by DM paralleled with the enhanced mitochondrial activity/proton leakage and with the increased activity or expression of mitochondrial markers namely, carnitine palmitoyl transferase and uncoupling protein 2. The association of decreased energy efficiency with reduced proinflammatory signs (TNF-α and LPS levels) with the significant increase antioxidant (total thiols) and detoxifying enzyme activities (glutathione-S-transferase NADH quinone oxidoreductase) in DM-treated animals, indicated that beneficial effects were attributable, at least in part, to the activation of nuclear factor 2 erythroid-related factor 2 pathway.