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1.
Nutrients ; 13(7)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34371902

RESUMO

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.


Assuntos
Caquexia/tratamento farmacológico , Caproatos/farmacologia , Interferon gama/toxicidade , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Caquexia/induzido quimicamente , Caquexia/metabolismo , Caquexia/patologia , Linhagem Celular , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Metilistidinas/metabolismo , Camundongos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fosforilação , Biossíntese de Proteínas , Proteólise
2.
Am J Physiol Cell Physiol ; 317(2): C189-C199, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042421

RESUMO

Sepsis is a systemic inflammatory response syndrome resulting from infection. This study aimed at exploring the role of microRNA-140 (miR-140) in septic mice. Wnt family member 11 (WNT11) was verified to be a target gene of miR-140 after bioinformatic prediction and dual luciferase reporter gene assay. Importantly, miR-140 negatively regulated WNT11. We initially induced the model of sepsis by endotoxin, and then ectopic expression and knockdown experiments were performed to explore the functional role of miR-140 in sepsis. Additionally, cross-sectional areas of muscle fiber, lactic acid production, 3-methylhistidine (3-MH) and tyrosine (Tyr) production in extensor digitorium longus (EDL) muscles, and serum levels of inflammatory factors were examined. The effect of miR-140 on the expression of WNT signaling pathway-related and apoptosis-related factors in skeletal muscle tissue was determined. The experimental results indicated that upregulated miR-140 or silenced WNT11 increased cross-sectional areas of muscle fiber while decreasing lactic acid production, skeletal muscle cell apoptosis [corresponding to downregulated B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3 and upregulated Bcl-2], and the proteolytic rate of Tyr and 3-MH. Also, overexpressed miR-140 or silenced WNT11 reduced inflammation as reflected by decreased serum levels of IL-6, IL-10, and TNF-α. Furthermore, overexpression of miR-140 was shown to suppress the activation of the WNT signaling pathway, accompanied by decreased expression of WNT11, ß-catenin, and GSK-3ß. Taken together, upregulation of miR-140 could potentially inhibit skeletal muscle lactate release, an indirect measure of glycolysis, and atrophy in septic mice through suppressing the WNT signaling pathway via inhibiting WNT11 expression.


Assuntos
Glicólise , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Sepse/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Citocinas/sangue , Modelos Animais de Doenças , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Mediadores da Inflamação/sangue , Ácido Láctico/metabolismo , Lipopolissacarídeos , Masculino , Metilistidinas/metabolismo , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Músculo Esquelético/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/genética , Atrofia Muscular/patologia , Sepse/induzido quimicamente , Sepse/genética , Sepse/patologia , Tirosina/metabolismo , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
3.
Org Lett ; 20(17): 5427-5430, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30141637

RESUMO

Ovothiols are thiolhistidine derivatives. The first step of ovothiol biosynthesis is OvoA-catalyzed oxidative coupling between histidine and cysteine. In this report, the remaining steps of ovothiol A biosynthesis were reconstituted in vitro. ETA_14770 (OvoB) was reported as a PLP-dependent sulfoxide lyase, responsible for mercaptohistidine production. OvoA was found to be a bifunctional enzyme, which mediates both oxidative C-S bond formation and methylation of mercaptohistidine to afford ovothiol A. Besides reconstituting the whole biosynthetic pathway, two unique features proposed in the literature were also examined: a potential cysteine-recycling mechanism of the C-S lyase (OvoB) and the selectivity of the π- N methyltransferase.


Assuntos
Liases/metabolismo , Metilistidinas/metabolismo , Metiltransferases/metabolismo , Liases/química , Metilistidinas/química , Metiltransferases/química , Modelos Moleculares , Conformação Proteica
4.
PLoS One ; 12(3): e0174894, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28358856

RESUMO

Dexmedetomidine is generally used for sedaton in critically ill, it could shorten duration of mechanical ventilation, ICU stay and lower basic metabolism. However, the exact mechanism of these positive effects remains unkown. Here we investigated the hypothesis that dexmedetomidine could ameliorate muscle wasting in endotoxemic rats and whether it was related to hypothalamic neuropeptides alteration and inflammation. Fourty-eight adult male Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (5 mg/kg) or saline, followed by 50 µg/kg dexmedetomidine or saline administration via the femoral vein catheter (infusion at 5 µg·kg-1·hr-1). Twenty-four hours after injection, hypothalamus tissues and skeletal muscle were obtained. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF-1) as well as 3-methylhistidine (3-MH) and tyrosine release. Hypothalamic inflammatory markers and neuropeptides expression were also detected in all four groups. Results showed that LPS administration led to significant increase in hypothalamic inflammation together with muscle wasting. Increased hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine and amphetamine-related transcript (CART) and neuropeptides Y (NPY) and decreased agouti-related protein (AgRP) were also observed. Meanwhile dexmedetomidine administration ameliorated muscle wasting, hypothalamic inflammation and modulated the alteration of neuropeptides, POMC, CART and AgRP, in endotoxemic rats. In conclusion, dexmedetomidine could alleviate muscle wasting in endotoxemic rats, and it could also attenuate the alteration of hypothalamic neuropeptides and reduce hypothalamic inflammation.


Assuntos
Dexmedetomidina/uso terapêutico , Endotoxemia/tratamento farmacológico , Hipotálamo/metabolismo , Inflamação/tratamento farmacológico , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Neuropeptídeos/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Endotoxemia/metabolismo , Hipotálamo/efeitos dos fármacos , Inflamação/metabolismo , Interleucina-1/metabolismo , Masculino , Metilistidinas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeo Y/metabolismo , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
5.
Mol Cell Biochem ; 410(1-2): 93-100, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26307368

RESUMO

Muscle wasting impairs physical function and leads people to a bedridden state. We previously demonstrated that lysine (Lys) suppresses autophagic-proteolysis through the Akt pathway. However, the effect of metabolites of Lys on proteolysis is unclear. In this study, we investigated the effect of saccharopine (Sac), a metabolite of Lys, on proteolysis in C2C12 cells. When C2C12 myotubes were incubated in serum-free medium containing Sac, the rate of proteolysis, which was evaluated by 3-methylhistidine released from C2C12 myotubes, and autophagy activity, which was assessed by amount of light chain 3-II, were suppressed. Sac stimulated Akt and mammalian target of rapamycin signaling, which was evaluated from eIF4E-binding protein 1 phosphorylation. The suppressive effects of Sac on proteolysis and autophagy were completely abolished by an Akt inhibitor. Therefore, we concluded that Sac suppresses autophagic-proteolysis through Akt as with Lys.


Assuntos
Autofagia/efeitos dos fármacos , Lisina/análogos & derivados , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Fatores de Iniciação em Eucariotos , Cinética , Lisina/farmacologia , Metilistidinas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/enzimologia , Atrofia Muscular/patologia , Fosfoproteínas/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
6.
Int J Neuropsychopharmacol ; 18(10): pyv045, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25899065

RESUMO

BACKGROUND: The neurobiological changes underlying depression resistant to treatments remain poorly understood, and failure to respond to selective serotonin reuptake inhibitors may result from abnormalities of neurotransmitter systems that excite serotonergic neurons, such as histamine. METHODS: Using behavioral (tail suspension test) and neurochemical (in vivo microdialysis, Western-blot analysis) approaches, here we report that antidepressant responses to selective serotonin reuptake inhibitors (citalopram or paroxetine) are abolished in mice unable to synthesize histamine due to either targeted disruption of histidine decarboxylase gene (HDC(-/-)) or injection of alpha-fluoromethylhistidine, a suicide inhibitor of this enzyme. RESULTS: In the tail suspension test, all classes of antidepressants tested reduced the immobility time of controls. Systemic reboxetine or imipramine reduced the immobility time of histamine-deprived mice as well, whereas selective serotonin reuptake inhibitors did not even though their serotonergic system is functional. In in vivo microdialysis experiments, citalopram significantly increased histamine extraneuronal levels in the cortex of freely moving mice, and methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, abolished this effect, thus suggesting the involvement of endogenous serotonin. CREB phosphorylation, which is implicated in the molecular mechanisms of antidepressant treatment, was abolished in histamine-deficient mice treated with citalopram. The CREB pathway is not impaired in HDC(-/-) mice, as administration of 8-bromoadenosine 3', 5'-cyclic monophosphate increased CREB phosphorylation, and in the tail suspension test it significantly reduced the time spent immobile by mice of both genotypes. CONCLUSIONS: Our results demonstrate that selective serotonin reuptake inhibitors selectively require the integrity of the brain histamine system to exert their preclinical responses.


Assuntos
Encéfalo/efeitos dos fármacos , Citalopram/farmacologia , Transtorno Depressivo/tratamento farmacológico , Histamina/metabolismo , Paroxetina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Antidepressivos/farmacologia , Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transtorno Depressivo/metabolismo , Transtorno Depressivo Resistente a Tratamento/metabolismo , Modelos Animais de Doenças , Feminino , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Masculino , Metilistidinas/metabolismo , Metisergida/farmacologia , Camundongos Knockout , Antagonistas da Serotonina/farmacologia
7.
JPEN J Parenter Enteral Nutr ; 39(7): 847-54, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24906686

RESUMO

BACKGROUND: Enhanced glutamine (GLN) intake may affect the catabolism of branched-chain amino acids (BCAAs; valine, leucine, and isoleucine), which play a regulatory role in protein turnover. We examined the effects of enhanced GLN availability on leucine oxidation, amino acid concentrations, and protein metabolism in muscles from healthy and septic rats. METHODS: Cecal ligation and puncture were used as a model of sepsis. Twenty-four hours after surgery, the soleus (SOL, red muscle) and the extensor digitorum longus (EDL, white muscle) were incubated in medium containing 0.5 or 2.0 mM GLN. Protein breakdown, protein synthesis, and leucine oxidation were determined via 3-methylhistidine release, muscle L-[1-(14)C]leucine radioactivity, and the radioactivity of released (14)CO2, respectively. RESULTS: In muscles from septic animals, increased proteolysis and leucine oxidation and decreased protein synthesis were detected. These effects were more pronounced in the EDL. In septic muscles, the addition of GLN decreased leucine oxidation in both muscles and increased protein synthesis in the EDL. In muscles from untreated animals, decreased leucine oxidation after the addition of GLN to the medium was associated with decreased protein synthesis in the SOL and decreased concentrations of serine, glycine, histidine, alanine, arginine, proline, and lysine in both muscles. CONCLUSIONS: White muscle fibers are more sensitive to septic stimuli than red fibers are. In sepsis, enhanced GLN intake may ameliorate GLN deficiency, inhibit BCAA catabolism, and stimulate protein synthesis. In the healthy state, surplus of GLN may lead to severe alterations in the intramuscular concentration of several amino acids and impair protein synthesis.


Assuntos
Aminoácidos/metabolismo , Glutamina/farmacocinética , Músculo Esquelético/efeitos dos fármacos , Proteínas/metabolismo , Animais , Suplementos Nutricionais , Glutamina/administração & dosagem , Glutamina/deficiência , Leucina/metabolismo , Masculino , Metilistidinas/metabolismo , Músculo Esquelético/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Wistar
8.
Org Lett ; 16(8): 2122-5, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24684381

RESUMO

OvoA in ovothiol biosynthesis is a mononuclear non-heme iron enzyme catalyzing the oxidative coupling between histidine and cysteine. It can also catalyze the oxidative coupling between hercynine and cysteine, yet with a different regio-selectivity. Due to the potential application of this reaction for industrial ergothioneine production, in this study, we systematically characterized OvoA by a combination of three different assays. Our studies revealed that OvoA can also catalyze the oxidation of cysteine to either cysteine sulfinic acid or cystine. Remarkably, these OvoA-catalyzed reactions can be systematically modulated by a slight modification of one of its substrates, histidine.


Assuntos
Cisteína/análogos & derivados , Metilistidinas/metabolismo , Mycobacterium smegmatis/metabolismo , Betaína/análogos & derivados , Betaína/metabolismo , Catálise , Cisteína/química , Cisteína/metabolismo , Cisteína Dioxigenase/metabolismo , Heme/metabolismo , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Modelos Biológicos , Estrutura Molecular , Mycobacterium smegmatis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Oxirredução
9.
Org Lett ; 15(18): 4854-7, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-24016264

RESUMO

Ergothioneine (5) and ovothiol (8) are two novel thiol-containing natural products. Their C-S bonds are formed by oxidative coupling reactions catalyzed by EgtB and OvoA enzymes, respectively. In this work, it was discovered that in addition to catalyzing the oxidative coupling between histidine and cysteine (1 → 6 conversion), OvoA can also catalyze a direct oxidative coupling between hercynine (2) and cysteine (2 → 4 conversion), which can shorten the ergothioneine biosynthetic pathway by two steps.


Assuntos
Carbono-Enxofre Ligases/metabolismo , Ergotioneína/biossíntese , Metilistidinas/síntese química , Betaína/análogos & derivados , Betaína/química , Catálise , Cisteína/química , Ergotioneína/química , Ergotioneína/metabolismo , Histidina/análogos & derivados , Histidina/biossíntese , Histidina/química , Histidina/metabolismo , Metilistidinas/química , Metilistidinas/metabolismo , Estrutura Molecular , Oxirredução , Estereoisomerismo , Compostos de Sulfidrila/química
10.
Brain Res ; 1442: 15-24, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22306030

RESUMO

Combined amylin+leptin (AMN+LEP) can reduce diet induced obesity and is very effective in combating LEP resistance. The purpose of this study was to evaluate the effect of AMN+LEP on central histaminergic signaling in lean and obese rats. Male rats were administered LEP (300 µg/kg/d), AMN (100 µg/kg/d), AMN+LEP or vehicle (SAL, 0.9% normal saline), via a subcutaneous mini-osmotic pump or single injection (LEP, 300 µg/kg and AMN, 100 µg/kg) for acute studies. AMN+LEP administration increased expression of histamine H1 receptor (HIR) and histidine decarboxylase (HDC) mRNA in the hypothalamus. Increased levels of H1R were seen in arcuate (Arc) and ventromedial hypothalamus (VMH) as well as the area postrema (APOS) and nucleus of solitary tract (NTS) following AMN+LEP administration. APOS and NTS also showed expression of immediate early gene c-FOS in the hindbrain in AMN+LEP-treated rats. We confirmed previous evidence indicating that AMN+LEP increased STAT-3 protein phosphorylation in Arc and VMH. Finally, by in vivo microdialysis, we observed an increase in methyl HIS levels in the VMH of AMN, LEP and AMN+LEP-treated rats. Taken together, these observations are consistent with an important role that neuronal HIS may play in mediating the potent effects of AMN+LEP on food intake and body weight.


Assuntos
Histamina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/administração & dosagem , Leptina/administração & dosagem , Transdução de Sinais , Animais , Peso Corporal , Ingestão de Alimentos , Genes fos , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Hipotálamo/metabolismo , Masculino , Metilistidinas/metabolismo , Obesidade/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Rombencéfalo/metabolismo , Regulação para Cima
11.
J Biol Chem ; 285(48): 37598-606, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-20864530

RESUMO

We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven ß-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.


Assuntos
Metilistidinas/metabolismo , Metiltransferases/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
12.
Anal Chem ; 82(7): 2959-68, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20192244

RESUMO

Despite several decades of active research, the success of large-scale clinical trials involving antioxidants remains equivocal given the complex biological interactions of reactive oxygen/nitrogen species in human health. Herein, we outline a differential metabolomics strategy by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) to assess the efficacy of nutritional intervention to attenuate oxidative stress induced by strenuous exercise. A healthy volunteer was recruited to perform a submaximal prolonged ergometer cycling trial until volitional exhaustion with frequent blood collection over a 6 h time interval, which included pre-, during, and postexercise periods while at rest. A follow-up study was subsequently performed by the same subject after high-dose oral intake of N-acetyl-L-cysteine (NAC) prior to performing the same exercise protocol under standardized conditions. Time-dependent changes in global metabolism of filtered red blood cell lysates by CE-ESI-MS were measured to reveal a significant attenuation of cellular oxidation associated with high-dose oral NAC intake relative to a control. Untargeted metabolite profiling allowed for the identification and quantification of several putative early- and late-stage biomarkers that reflected oxidative stress inhibition due to nutritional intervention, including oxidized glutathione (GSSG), reduced glutathione (GSH), 3-methylhistidine (3-MeHis), L-carnitine (C0), O-acetyl-L-carnitine (C2), and creatine (Cre). Our work demonstrates the proof-of-principle that NAC pretreatment is effective at dampening acute episodes of oxidative stress by reversible perturbations in global metabolism that can provide deeper insight into the mechanisms of thiol-specific protein inhibition relevant to its successful translation as a prophylaxis in clinical medicine.


Assuntos
Acetilcisteína/farmacologia , Eletroforese Capilar/métodos , Exercício Físico , Metabolômica/métodos , Estresse Oxidativo , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetilcarnitina/metabolismo , Administração Oral , Carnitina/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Masculino , Metilistidinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
13.
J Physiol Pharmacol ; 59 Suppl 9: 19-27, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19261968

RESUMO

Our previous results show that leptin, as well as nitric oxide (NO) and some antioxidants (histidine dipeptides - HDP) change the secretion of gonadotrophins from ovine adenohypophysis cells in vitro. NO and HDP are produced by pituitary and can modulate gonadotropin secretion by autocrine action. It is possible that these compounds mediate leptin influence on gonadotropin secretion. Therefore, the objective of the present study was to analyse leptin effect on NO and HDP (3-metyl-L-histidine, carnosine and anserine) release from ovine pituitary in vitro. Adenohypophysis cells were cultured in McCoy 5A medium with GnRH (4 x 10(-9) M) and 10(-10)-10(-5) M/l of leptin, respectively. Next, the media for analysis of NO (Griess method) and HDP (HPLC) were collected. Leptin in concentration of 10(-8)-10(-6) M/l caused a significant augmentation in NO in the culture medium, whereas in the dose of 10(-5) M/l reduced (P< or =0.05) NO release. The level of 3-metyl-L-histidine and anserine, but not carnosine, was significantly lower in the culture with 10(-8)-10(-7) M/l of leptin. Taking into account that 10(-8)-10(-7) M/l leptin stimulates LH and FSH secretion, as show in our previous study, it is possible that this effect in ewes is mediated by augmented release of NO and reduction of HDP level.


Assuntos
Anserina/metabolismo , Leptina/farmacologia , Metilistidinas/metabolismo , Óxido Nítrico/metabolismo , Animais , Carnosina/efeitos dos fármacos , Carnosina/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Leptina/administração & dosagem , Adeno-Hipófise/citologia , Adeno-Hipófise/efeitos dos fármacos , Adeno-Hipófise/metabolismo , Ovinos
14.
Clin Nutr ; 26(4): 483-90, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17513024

RESUMO

BACKGROUND & AIMS: Since both cancer and surgery are known to alter protein turnover, we investigated how the presence of tumour affects post-operative protein breakdown. METHODS: Controls and tumour-bearing non-cachectic mice were studied, both with and without laparotomy (n=8 per group). One day after laparotomy, stable isotopes of phenylalanine and 3-methylhistidine were used in a steady-state protocol to assess total protein breakdown on whole-body level and in muscle, intestines, liver and kidney, in addition to whole-body myofibrillar protein breakdown and body composition. Proteasomal chymotrypsin-like activity was measured to determine activity of the ATP-dependent ubiquitin pathway. RESULTS: On whole-body level, the presence of tumour increased total protein breakdown from 47+/-6 to 58+/-4 nmol/10 g/min (p<0.05) and myofibrillar protein breakdown from 0.70+/-0.04 to 1.22+/-0.14 nmol/10 g/min (p<0.05) without affecting protein breakdown in organs, body composition or proteasomal activity. Laparotomy increased myofibrillar protein breakdown in controls (from 0.70+/-0.04 to 0.98+/-0.12 nmol/10 g/min, p<0.05) and tumour-bearing mice (from 1.22+/-0.14 to 1.54+/-0.22 nmol/10 g/min, p=0.15) to a similar extent. CONCLUSIONS: Whole-body total protein breakdown, total protein breakdown across organs, body composition or proteasomal activity were not affected by laparotomy. Tumour-bearing mice had increased total and myofibrillar protein breakdown on whole-body level even before weight loss was obvious. However, this did not affect the post-operative response in protein breakdown or body composition.


Assuntos
Composição Corporal , Metabolismo Energético/fisiologia , Músculo Esquelético/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas/metabolismo , Animais , Mucosa Intestinal/metabolismo , Rim/metabolismo , Laparotomia , Fígado/metabolismo , Masculino , Metilistidinas/metabolismo , Camundongos , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Especificidade de Órgãos , Período Pós-Operatório , Distribuição Aleatória
15.
J Trauma ; 62(1): 125-31; discussion 131-2, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17215743

RESUMO

BACKGROUND: Negative nitrogen balance and skeletal muscle loss are common in critically injured patients and may contribute to morbidity, mortality and resource utilization. Juven, an enteral supplement which is a combination of beta-hydroxy-beta-methylbutyrate (HMB), arginine (ARG), and glutamine (GLN) has been shown to restore muscle in cachetic acquired immunodeficiency syndrome (AIDS) and cancer patients. More recently HMB has been shown to attenuate cancer-induced muscle loss by decreasing muscle proteolysis. The purpose of this study was to analyze whether HMB alone or in combination with ARG and GLN would have a similar effect on critically injured trauma patients. We hypothesized that nitrogen balance would be improved and muscle proteolysis decreased with HMB and HMB/ARG/GLN supplementation. METHODS: There were 100 adult trauma patients with Injury Severity Score (ISS) >18 were enrolled in this prospective, randomized, blinded study. All patients received standard tube feeds and one of three iso-nitrogenous supplements; HMB, HMB/ARG/ GLN, or placebo (PLAC) for 28 days. Urine, serum, and clinical data were collected for 72 patients receiving at least 7 days of supplementation during the first 14 days of treatment. Urinary 3-methylhistidine (3-MH) was used as a proxy for muscle proteolysis. RESULTS: The three groups were similar in age, gender, mechanism, and severity of injury, with the average ISS being 31.9. Utilizing covariant (ISS) repeated measure (days 1-14) mixed model (SAS) analysis, there was a significant treatment effect (p = 0.05) on nitrogen balance (g/d). Change in nitrogen balance from the first 7 days to the last 7 days was -4.3 for the HMB and -5.6 g/d HMB/ARG/GLN groups compared with -8.9 g/d for the PLAC group. 3-MH to creatinine ratios were not different in the PLAC group as compared with the HMB/ARG/GLN and HMB groups (Treatment Effect, p = 0.80). CONCLUSIONS: These data suggest that supplementation with HMB alone may improve nitrogen balance in critically injured adult patients and that this effect is not a result of lowered muscle protein turnover as originally hypothesized.


Assuntos
Ácido 3-Hidroxibutírico/uso terapêutico , Nutrição Enteral , Atrofia Muscular/prevenção & controle , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Ferimentos e Lesões/terapia , Adulto , Arginina/uso terapêutico , Biomarcadores/metabolismo , Feminino , Glutamina/uso terapêutico , Humanos , Masculino , Metilistidinas/metabolismo , Músculo Esquelético/metabolismo , Nitrogênio/metabolismo , Análise de Sobrevida
16.
Free Radic Biol Med ; 40(1): 156-64, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16337889

RESUMO

Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 micromol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystin-pretreated ischemic hearts was significantly (P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly (P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss (-6.5%) of 3-methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome.


Assuntos
Coração/fisiopatologia , Precondicionamento Isquêmico Miocárdico , Isquemia Miocárdica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Actinas/metabolismo , Animais , Inibidores de Cisteína Proteinase/farmacologia , Imunoprecipitação , Masculino , Metilistidinas/metabolismo , Isquemia Miocárdica/patologia , Oxirredução , Inibidores de Proteassoma , Ratos , Ratos Sprague-Dawley , Reperfusão , Ubiquitina/metabolismo
17.
Zhonghua Yi Xue Za Zhi ; 85(41): 2895-8, 2005 Nov 02.
Artigo em Chinês | MEDLINE | ID: mdl-16324361

RESUMO

OBJECTIVE: To study the changes of skeletal muscle proteolysis in severely burned patients with sepsis and analyze its possible mechanism. METHODS: Blood samples were collected from 20 burned patients with sepsis, aged 42 +/- 10 (burn sepsis group), and 12 patients undergoing plastic operation after burn, aged 43 +/- 8 (control group) to determine the plasma concentrations of cortisol and tumor necrosis factor (TNF-alpha) by radioimmunoassay. 24 h urine was retained to determine the level of 3-methylhistidine (3-MH) by high performance liquid chromatography. An appropriate amount of quadriceps femoris muscle sample was collected during operation to determine the concentration of 3-methylhistidine with high performance liquid chromatography, the expression of ubiquitin mRNA, E(2)-14k mRNA and C(2) subunit mRNA by Northern blot analysis, and the protein expression of C(2) subunit by Western blot analysis. RESULTS: The plasma concentrations of cortisol and TNF-alpha in the burn sepsis group were 478 +/- 56 microg/L and 22.4 +/- 3.8 microg/L respectively, both significantly higher than the those in the normal control group, 72 +/- 12 microg/L and 0.5 +/- 0.1 microg/L respectively (both P < 0.01). The amount of 3-MH in the quadriceps femoris muscle of the burn sepsis group was 4.3 +/- 0.6 micromol/g, significantly higher than that of the control group (2.5 +/- 0.4 micromol/g, P < 0.01). The 24 h urinary output of 3-MH of the burn sepsis group was 456 +/- 25 micromol, significantly higher than that of the control group (202 +/- 29 micromol, P < 0.091). The expressions of ubiquitin mRNA 2.4 Kilobase pair (kb) and 1.2 kb, E(2)-14k mRNA 1.2 kb and C(2) subunit mRNA in quadriceps femoris muscles of the burn sepsis group were increased with 51%, 32%, 75%, and 39% respectively, as compared with those of the control group (all P < 0.01). The protein expression of C(2) subunit in quadriceps femoris muscles of the burn sepsis group was increased with 130% than that of the control group (P < 0.01). CONCLUSION: The proteolytic rate in skeletal muscle is increased during sepsis after severe burn and the mechanism is related to the enhanced function of the ubiquitin system at both gene and protein levels with the hypersecretion of glucocorticoid and TNF-alpha.


Assuntos
Queimaduras/complicações , Metilistidinas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Sepse/metabolismo , Adulto , Queimaduras/metabolismo , Feminino , Humanos , Hidrocortisona/sangue , Masculino , Pessoa de Meia-Idade , Proteoglicanas , Sepse/etiologia , Fator de Necrose Tumoral alfa/sangue , Ubiquitina/metabolismo
18.
Am J Physiol Endocrinol Metab ; 288(3): E493-501, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15522995

RESUMO

Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-alpha can stimulate muscle proteolysis; therefore, a potential relationship between TNF-alpha and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 (P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated (P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-alpha levels were increased and correlated positively with free and conjugated ubiquitin (P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-alpha might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.


Assuntos
Cirrose Hepática Biliar/metabolismo , Músculo Esquelético/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Ubiquitina/metabolismo , Animais , Western Blotting , Peso Corporal , Calpaína/genética , Catepsinas/genética , Modelos Animais de Doenças , Expressão Gênica/genética , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Masculino , Metilistidinas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Músculo Esquelético/patologia , Miofibrilas/química , Miofibrilas/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/metabolismo , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
19.
Biosci Biotechnol Biochem ; 68(11): 2326-31, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15564672

RESUMO

The effects of cysteine as an antioxidant nutrient on change in protein modification and myofibrillar proteolysis in chick myotubes by induction of oxidative stress by H(2)O(2) treatment were investigated. Myotubes were treated for 1 h with H(2)O(2) (1 mM). After this treatment, the H(2)O(2) was removed and the cells were cultured in cysteine (0.1 and 1 mM) containing serum-free medium for 24 h. Protein carbonyl content as an index of protein modification and N(tau)-methylhistidine release as an index of myofibrillar proteolysis were increased at 24 h after H(2)O(2) treatment, and the increment was reduced by cysteine. Calpain, proteasome and cathepsin (B+L and D) activities were increased at 24 h after H(2)O(2) treatment, and the increment was also reduced by cysteine. These results indicate that cysteine suppresses protein modification by oxidative stress, resulting in a decrease of protease acitivities, finally resulting in a decrease in myofibrillar proteolysis in chick myotubes.


Assuntos
Antioxidantes/farmacologia , Cisteína/farmacologia , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Estresse Oxidativo/fisiologia , Peptídeo Hidrolases/metabolismo , Animais , Catepsina B/metabolismo , Catepsina L , Catepsinas/metabolismo , Células Cultivadas , Embrião de Galinha , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/metabolismo , Peróxido de Hidrogênio/farmacologia , Metilistidinas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos
20.
J Nutr Sci Vitaminol (Tokyo) ; 50(1): 45-9, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15228217

RESUMO

Changes in protein conformation and proteolysis in chick myotubes in response to the induction of oxidative stress by H2O2 treatment were studied. Myotubes were treated for 1 h with H2O2. After this treatment, the H2O2 was removed and the cells were cultured in serum-free medium for 6 and 24 h. Protein carbonyl content, as an index of protein modification, was increased at 6 and 24 h after H2O2 treatment. N(tau)-methylhistidine release, as an index of myofibrillar proteolysis, was also increased at 6 and 24 h after H2O2 treatment. Calpain and cathepsin (B+L and D) activities were increased at 24 but not 6 h after H2O2 treatment. Proteasome activity was increased at 6 and 24 h after H2O2 treatment. These results indicate that oxidative stress increased proteasome activity and caused an increase in myofibrillar proteolysis during short-term incubation, whereas it increased calpain, proteasome and cathepsin activities during long-term incubation, finally resulting in an increase of myofibrillar proteolysis in chick myotubes.


Assuntos
Calpaína/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Fibras Musculares Esqueléticas/enzimologia , Miofibrilas/metabolismo , Estresse Oxidativo , Animais , Galinhas , Peróxido de Hidrogênio/farmacologia , Cinética , Metilistidinas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Substâncias Reativas com Ácido Tiobarbitúrico/análise
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