RESUMO
Given worldwide increases in the incidence of obesity and type 2 diabetes, new strategies for preventing and treating metabolic diseases are needed. The nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma) plays a central role in lipid and glucose metabolism; however, current PPARγ-targeting drugs are characterized by undesirable side effects. Natural products from edible biomaterial provide a structurally diverse resource to alleviate complex disorders via tailored nutritional intervention. We identified a family of natural products, the amorfrutins, from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa, as structurally new and powerful antidiabetics with unprecedented effects for a dietary molecule. Amorfrutins bind to and activate PPARγ, which results in selective gene expression and physiological profiles markedly different from activation by current synthetic PPARγ drugs. In diet-induced obese and db/db mice, amorfrutin treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity. These results show that selective PPARγ-activation by diet-derived ligands may constitute a promising approach to combat metabolic disease.
Assuntos
Produtos Biológicos/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fabaceae/química , Hipoglicemiantes/farmacologia , Salicilatos/farmacologia , Células 3T3-L1 , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Expressão Gênica/efeitos dos fármacos , Glycyrrhiza/química , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/etiologia , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salicilatos/química , Salicilatos/metabolismoRESUMO
Cellular communication is a fundamental process in biology. The interaction of adipocytes with macrophages is a key event in the development of common diseases such as type 2 diabetes. We applied an established bilayer cell co-culture system and comprehensive mass spectrometry analysis to detect proteome-wide the paracrine interaction of murine adipocytes and macrophages. Altogether, we identified 4486 proteins with at least two unique peptides of which 2392 proteins were informative for 3T3-L1 adipocytes and 2957 proteins for RAW 264.7 macrophages. Further, we observed over 12,000 phosphorylation sites of which we could assign 3,200 informative phosphopeptides with a single phosphosite for adipocytes and 4,514 for macrophages. Using protein set enrichment and phosphosite analyses, we deciphered regulatory protein pathways involved in cellular stress and inflammation, which can contribute to metabolic impairment of cells including insulin resistance and other disorders. The generated data sets provide a holistic, molecular pathway-centric view on the interplay of adipocytes and macrophages in disease processes and a resource for further studies.
Assuntos
Adipócitos/metabolismo , Comunicação Celular , Macrófagos/metabolismo , Proteoma/metabolismo , Animais , Linhagem Celular , Técnicas de Cocultura , Camundongos , Fosfopeptídeos/metabolismo , Fosforilação , Transdução de Sinais , TranscriptomaRESUMO
TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10µM), and all three metal ions activated the ATPase at a concentration of 0.3µM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJâ mol(-1)) over the whole concentration range while it increased from 17 to 42kJâ mol(-1) with rising copper concentration and decreased from 39 to 23kJâ mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.
Assuntos
Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/isolamento & purificação , Biofísica , Eletroforese em Gel de Poliacrilamida , Espectrofotometria AtômicaRESUMO
The antimycotic drug clotrimazole inhibits the function of the gastric H,K-ATPase in a manner similar to that observed for the Na,K-ATPase. Because of the high hydrophobicity of the compound, the interaction between clotrimazole and the ion pump occurs at the membrane domain in the apolar core of the membrane. The enzymatic activity was inhibited with a half-saturating concentration of 5.2 microM. Various partial reactions of the pump cycle were analyzed with the electrochromic styryl dye RH421 that has been widely used to study the transport mechanism of P-type ATPases. We discovered that the interaction of clotrimazole with the H,K-ATPase introduces a single "dead-end" branch added to the Post-Albers scheme in the E(1) state of the pump. In this inhibiting state, the ion binding sites have a significantly enhanced affinity for protons and bind up to two protons even at pH 8.5. Inhibition of the pump can be reversed by a decreased pH or increased K(+) concentrations. The mechanistic proposal that allows an explanation of all experiments presented is similar to that published for the Na,K-ATPase.
Assuntos
Adenosina Trifosfatases/metabolismo , Clotrimazol/metabolismo , Íons/metabolismo , Prótons , Estômago/enzimologia , Animais , Sítios de Ligação , Mucosa Gástrica/metabolismo , SuínosRESUMO
OBJECTIVE: The liver X receptor α (LXRα) is a ligand-dependent nuclear receptor and the major regulator of reverse cholesterol transport in macrophages. This makes it an interesting target for mechanistic study and treatment of atherosclerosis. METHODS AND RESULTS: We optimized a promising stilbenoid structure (STX4) in order to reach nanomolar effective concentrations in LXRα reporter-gene assays. STX4 displayed the unique property to activate LXRα effectively but not its subtype LXRß. The potential of STX4 to increase transcriptional activity as an LXRα ligand was tested with gene expression analyses in THP1-derived human macrophages and oxLDL-loaded human foam cells. Only in foam cells but not in macrophage cells STX4 treatment showed athero-protective effects with similar potency as the synthetic LXR ligand T0901317 (T09). Surprisingly, combinatorial treatment with STX4 and T09 resulted in an additive effect on reporter-gene activation and target gene expression. In physiological tests the cellular content of total and esterified cholesterol was significantly reduced by STX4 without the undesirable increase in triglyceride levels as observed for T09. CONCLUSIONS: STX4 is a new LXRα-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development.