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1.
Mol Metab ; 74: 101750, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37302544

RESUMO

OBJECTIVE: Unexplained changes in regulation of branched chain amino acids (BCAA) during diabetes therapy with metformin have been known for years. Here we have investigated mechanisms underlying this effect. METHODS: We used cellular approaches, including single gene/protein measurements, as well as systems-level proteomics. Findings were then cross-validated with electronic health records and other data from human material. RESULTS: In cell studies, we observed diminished uptake/incorporation of amino acids following metformin treatment of liver cells and cardiac myocytes. Supplementation of media with amino acids attenuated known effects of the drug, including on glucose production, providing a possible explanation for discrepancies between effective doses in vivo and in vitro observed in most studies. Data-Independent Acquisition proteomics identified that SNAT2, which mediates tertiary control of BCAA uptake, was the most strongly suppressed amino acid transporter in liver cells following metformin treatment. Other transporters were affected to a lesser extent. In humans, metformin attenuated increased risk of left ventricular hypertrophy due to the AA allele of KLF15, which is an inducer of BCAA catabolism. In plasma from a double-blind placebo-controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin caused selective accumulation of plasma BCAA and glutamine, consistent with the effects in cells. CONCLUSIONS: Metformin restricts tertiary control of BCAA cellular uptake. We conclude that modulation of amino acid homeostasis contributes to therapeutic actions of the drug.


Assuntos
Metformina , Humanos , Metformina/farmacologia , Metformina/uso terapêutico , Aminoácidos de Cadeia Ramificada/metabolismo , Aminoácidos/metabolismo , Glucose , Homeostase
2.
J Nat Prod ; 79(11): 2856-2864, 2016 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-27792327

RESUMO

To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.


Assuntos
Produtos Biológicos/química , Trifolium/química , Proteínas Quinases Ativadas por AMP/metabolismo , Algoritmos , Cromatografia Líquida de Alta Pressão , Genisteína/química , Estrutura Molecular , Padrões de Referência , Suíça
3.
J Immunol ; 179(5): 2681-5, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17709478

RESUMO

Inflammatory mediators such as TNF-alpha, IL-6, and IL-1 are important in the pathogenesis of inflammatory bowel diseases and are regulated by the activation of NF-kappaB. The aim of the present study was to investigate whether the NF-kappaB essential modulator (NEMO)-binding domain (NBD) peptide, which has been shown to block the association of NEMO with the IkappaB kinasebeta subunit (IKKbeta) and inhibit NF-kappaB activity, reduces inflammatory injury in mice with colitis. Two colitis models were established by the following: 1) inclusion of dextran sulfate sodium salt (DSS) in the drinking water of the mice; and 2) a trinitrobenzene sulfonic acid enema. Marked NF-kappaB activation and expression of proinflammatory cytokines were observed in colonic tissues. The NBD peptide ameliorated colonic inflammatory injury through the down-regulation of proinflammatory cytokines mediated by NF-kappaB inhibition in both models. These results indicate that an IKKbeta-targeted NF-kappaB blockade using the NBD peptide could be an attractive therapeutic approach for inflammatory bowel disease.


Assuntos
Colite/tratamento farmacológico , Quinase I-kappa B/antagonistas & inibidores , Peptídeos/uso terapêutico , Animais , Colite/induzido quimicamente , Colite/patologia , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , Ácido Trinitrobenzenossulfônico/toxicidade
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