RESUMO
Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans-tert-butylcyclohexanol revealed that the anti-adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro-adipogenic transcription factor peroxisome-proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu-let-7d-5p, which has been associated with decreased PPARγ levels.
Assuntos
Adipogenia/efeitos dos fármacos , Capsaicina/análogos & derivados , MicroRNAs/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Animais , Capsaicina/farmacologia , Camundongos , Canais de Cátion TRPV/metabolismoRESUMO
Red pepper and its major pungent component, capsaicin, have been associated with hypolipidemic effects in rats, although mechanistic studies on the effects of capsaicin and/or structurally related compounds on lipid metabolism are scarce. In this work, the effects of capsaicin and its structural analog nonivamide, the aliphatic alkamide trans-pellitorine and vanillin as the basic structural element of all vanilloids on the mechanisms of intestinal fatty acid uptake in differentiated intestinal Caco-2 cells were studied. Capsaicin and nonivamide were found to reduce fatty acid uptake, with IC50 values of 0.49 µM and 1.08 µM, respectively. trans-Pellitorine was shown to reduce fatty acid uptake by 14.0±2.14% at 100 µM, whereas vanillin was not effective, indicating a pivotal role of the alkyl chain with the acid amide group in fatty acid uptake by Caco-2 cells. This effect was associated neither with the activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or the epithelial sodium channel (ENaC) nor with effects on paracellular transport or glucose uptake. However, acetyl-coenzyme A synthetase activity increased (p<0.05) in the presence of 10 µM capsaicin, nonivamide or trans-pellitorine, pointing to an increased fatty acid biosynthesis that might counteract the decreased fatty acid uptake.
Assuntos
Acetato-CoA Ligase/metabolismo , Capsaicina/metabolismo , Enterócitos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fármacos Gastrointestinais/metabolismo , Hipolipemiantes/metabolismo , Absorção Intestinal , Acetato-CoA Ligase/química , Benzaldeídos/efeitos adversos , Benzaldeídos/metabolismo , Células CACO-2 , Capsaicina/efeitos adversos , Capsaicina/análogos & derivados , Sobrevivência Celular , Suplementos Nutricionais/efeitos adversos , Regulação para Baixo , Enterócitos/enzimologia , Canais Epiteliais de Sódio/metabolismo , Ácidos Graxos Insaturados/efeitos adversos , Ácidos Graxos Insaturados/metabolismo , Fármacos Gastrointestinais/efeitos adversos , Humanos , Hipolipemiantes/efeitos adversos , Cinética , Alcamidas Poli-Insaturadas/efeitos adversos , Alcamidas Poli-Insaturadas/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
SCOPE: Since bolus administration of capsaicin has been shown to reduce appetite and ad libitum energy intake, this study elucidated the satiating effect of the less pungent capsaicin analog, nonivamide, on subjective feelings of hunger, ad libitum food intake, and satiating hormones in moderately overweight male subjects. METHODS AND RESULTS: Following a randomized, crossover design, 24 male subjects (BMI 27.5 ± 1.53 kg/m(2) ) received either 75 g glucose in 300 mL water (control treatment, CT) or the same glucose solution supplemented with 0.15 mg nonivamide (nonivamide treatment, NT). Ratings of hunger were assessed before and 2 h after each intervention by means of visual analog scales. Ad libitum energy and macronutrient intakes from a standardized breakfast 2 h postintervention were calculated. Plasma glucose, insulin, peptide YY (3-36), glucagon-like peptide 1, and serotonin were quantified in blood samples drawn before and 15, 30, 60, 90, and 120 min after each intervention. NT reduced subjective feelings of hunger and ad libitum energy and carbohydrate intakes from a standardized breakfast compared to CT. Plasma analysis revealed higher mean plasma glucagon-like peptide 1 and serotonin concentrations after NT versus CT. CONCLUSION: Addition of 0.15 mg nonivamide to a glucose solution reduced ad libitum energy intake from a standardized breakfast in moderately overweight men.
Assuntos
Desjejum , Capsaicina/análogos & derivados , Ingestão de Energia/efeitos dos fármacos , Sobrepeso/sangue , Serotonina/sangue , Adulto , Apetite/efeitos dos fármacos , Glicemia , Índice de Massa Corporal , Capsaicina/administração & dosagem , Capsaicina/sangue , Estudos Cross-Over , Peptídeo 1 Semelhante ao Glucagon/sangue , Teste de Tolerância a Glucose , Humanos , Fome/efeitos dos fármacos , Insulina/sangue , Masculino , Fragmentos de Peptídeos/sangue , Peptídeo YY/sangue , Adulto JovemRESUMO
Pyridines are widely distributed in foods. Nicotinic acid (NA), a carboxylated pyridine derivative, inhibits lipolysis in adipocytes by activation of the orphan NA receptor (HM74A) and is applied to treat hyperlipidemia. However, knowledge on the impact of pyridine derivatives on intestinal lipid metabolism is scarce. This study was performed to identify the structural determinants of pyridines for their effects on fatty acid uptake in enterocyte-like Caco-2 cells and to elucidate the mechanisms of action. The impact of 17 pyridine derivatives on fatty acid uptake was tested. Multiple regression analysis revealed the presence of a methyl group to be the structural determinant at 0.1 mM, whereas at 1 mM, the presence of a carboxylic group and the N-methylation presented further structural characteristics to affect the fatty acid uptake. NA, showing a stimulating effect on FA uptake, and N-methyl-4-phenylpyridinium (MPP), inhibiting FA uptake, were selected for mechanistic studies. Gene expression of the fatty acid transporters CD36, FATP2 and FATP4, and the lipid metabolism regulating transcription factors peroxisome proliferator-activated receptor (PPAR) α and PPARγ was up-regulated upon NA treatment. Caco-2 cells were demonstrated to express the low-affinity NA receptor HM74 of which the gene expression was up-regulated upon NA treatment. We hypothesize that the NA-induced fatty acid uptake might result from NA receptor activation and related intracellular signaling cascades. In contrast, MPP increased transepithelial electrical resistance. We therefore conclude that NA and MPP, both sharing the pyridine motif core, exhibit their contrary effects on intestinal FA uptake by activation of different mechanisms.
Assuntos
Antígenos CD36/biossíntese , Ácidos Graxos/metabolismo , Niacina/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/biossíntese , 1-Metil-4-fenilpiridínio/farmacologia , Antígenos CD36/efeitos dos fármacos , Células CACO-2 , Impedância Elétrica , Proteínas de Transporte de Ácido Graxo/biossíntese , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , PPAR alfa/metabolismo , PPAR gama/metabolismo , Relação Estrutura-AtividadeRESUMO
The use of photolabile protecting groups is a versatile and well-established means of synthesizing high complexity microarrays of biopolymers, such as nucleic acids and peptides, for high-throughput analysis. The synthesis takes place in a photochemical reaction cell which positions the microarray substrate at the focus of the optical system delivering the light and which can be connected to a fluidics system which delivers appropriate reagents to the surface in synchrony with the light exposure. Here we describe a novel photochemical reaction cell which allows for the simultaneous synthesis of microarrays on two substrates. The reaction cell positions both substrates within the limited depth-of-focus of the optical system while maintaining the necessary reagent flow conditions. The resulting microarrays are mirror images of each other but otherwise essentially identical. The new reaction cell doubles the throughput of microarray synthesis without increasing the consumption of reagents. In addition, a secondary flow chamber behind the reaction cell can be filled with an absorbent and index-matching fluid to eliminate reflections from light exiting the reaction cell assembly, greatly reducing unintended light exposure that reduces the sequence fidelity of the microarray probes.
Assuntos
Hibridização In Situ/métodos , Luz , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Processos FotoquímicosRESUMO
SCOPE: Dietary intake of capsaicin has been shown to reduce body weight by increasing energy expenditure, and to enhance alertness and mood by stimulating the brain's reward system. Binding of capsaicin to the vanilloid receptor 1 (transient receptor potential cation channel subfamily V member 1 (TRPV1)) is one of the major cellular mechanisms responsible for these effects. However, strong TRPV1 agonists like capsaicin elicit a sharp, burning pain that limits their dietary intake. The present study aimed to investigate the effect of the less pungent capsaicin-analog nonivamide on dopamine and serotonin release in neural SH-SY5Y cells. METHODS AND RESULTS: Nonivamide (1 µM) stimulated the Ca(2+) -dependent release of serotonin (272 ± 115%) and dopamine (646 ± 48%) in SH-SY5Y cells compared to nontreated cells (100%) to a similar extent as capsaicin. qRT-PCR analysis of 1 µM nonivamide-treated SH-SY5Y cells revealed gene regulation of the receptors dopamine D1 and D2, serotonin HTR1A, 1B and 2A, cannabinoid 1, and TRPV1. Co-incubation experiments of SH-SY5Y cells with the TRPV1 inhibitors trans-tert-butylcyclohexanol and capsazepine demonstrated that capsaicin, but not nonivamide, induces serotonin and dopamine release through TRPV1 activation. CONCLUSION: The results indicate a TRPV1-independent signaling pathway for nonivamide that might allow dietary administration of higher doses of nonivamide compared to capsaicin.
Assuntos
Capsaicina/análogos & derivados , Dopamina/metabolismo , Fármacos do Sistema Sensorial/farmacologia , Serotonina/metabolismo , Canais de Cátion TRPV/metabolismo , Capsaicina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloexanóis/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Transdução de Sinais , Canais de Cátion TRPV/genéticaRESUMO
Maillard reaction products, which are formed in highly thermally treated foods, are commonly consumed in a Western diet. In this study, we investigated the impact of N(ε)-carboxymethyllysine (CML), a well-characterized product of the Maillard reaction, on the gene regulation of the human neuroblastoma cell line SH-SY5Y. Pathway analysis of data generated from customized DNA microarrays revealed 3 h incubation with 50 µM and 500 µM CML to affect serotonin receptor expression. Further experiments employing qRT-PCR showed an up-regulation of serotonin receptors 2A, 1A and 1B after 0.25 h and 3 h. In addition, 500 µM CML increased serotonin release, thus showing effects of CML not only at a genetic, but also at a functional level. Intracellular calcium mobilization, which mediates serotonin release, was increased by CML at concentrations of 0.05-500 µM. Since calcium mobilization has been linked to the activation of the receptor for advanced glycation end products (RAGE), we further investigated the effects of CML on RAGE expression. RAGE was found to be up-regulated after incubation with 500 µM CML for 0.25 h. Co-incubation with the calcium blocker neomycin for 0.25 h blocked the up-regulation of RAGE and the serotonin receptors 2A, 1A and 1B. These results indicate a possible link between a CML-induced calcium-mediated serotonin release and RAGE.
Assuntos
Lisina/análogos & derivados , Receptores Imunológicos/genética , Serotonina/metabolismo , Regulação para Cima , Cálcio/metabolismo , Linhagem Celular Tumoral , Humanos , Lisina/metabolismo , Reação de Maillard , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like ß-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.
Assuntos
Ácido Bongcréquico/biossíntese , Burkholderia gladioli/metabolismo , Ácido Bongcréquico/química , Conformação MolecularRESUMO
Caffeine has been hypothesised as a thermogenic agent that might help to maintain a healthy body weight. Since very little is known about its actions on cellular energy metabolism, we investigated the effect of caffeine on mitochondrial oxidative phosphorylation, cellular energy supply and thermogenesis in HepG2 cells, and studied its action on fatty acid uptake and lipid accumulation in 3T3-L1 adipocytes at concentrations ranging from 30-1500 µM. In HepG2 cells, caffeine induced a depolarisation of the inner mitochondrial membrane, a feature of mitochondrial thermogenesis, both directly and after 24 h incubation. Increased concentrations of uncoupling protein-2 (UCP-2) also indicated a thermogenic activity of caffeine. Energy generating pathways, such as mitochondrial respiration, fatty acid oxidation and anaerobic lactate production, were attenuated by caffeine treatment. Nevertheless, HepG2 cells demonstrated a higher energy charge potential after exposure to caffeine that might result from energy restoration through attenuation of energy consuming pathways, as typically found in hibernating animals. In 3T3-L1 cells, in contrast, caffeine increased fatty acid uptake, but did not affect lipid accumulation. We provide evidence that caffeine stimulates thermogenesis but concomitantly causes energy restoration that may compensate enhanced energy expenditure.
Assuntos
Trifosfato de Adenosina/metabolismo , Cafeína/farmacologia , Relação Dose-Resposta a Droga , Mitocôndrias/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/administração & dosagem , Ácidos Graxos/farmacocinética , Células Hep G2 , Humanos , Canais Iônicos/metabolismo , Ácido Láctico/metabolismo , Metabolismo dos Lipídeos , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Proteína Desacopladora 2RESUMO
Biosynthetic studies with (13)C-labelled acetates and methionine revealed that the infamous, food-related toxin bongkrekic acid from Burkholderia gladioli is a polyketide with acetate-derived beta-branches and a carboxylate terminus derived from the methyl group of an acetate.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Ácido Bongcréquico/química , Ácido Bongcréquico/metabolismo , Burkholderia gladioli/metabolismo , Translocases Mitocondriais de ADP e ATP/antagonistas & inibidores , Mitocôndrias/enzimologia , Estrutura MolecularRESUMO
Mould fungi are not only well known for food spoilage through toxin formation but also for the production of fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production with various Rhizopus strains is widespread. Here we report the finding of toxinogenic bacteria in a starter culture used for sufu production. By means of metabolic profiling of the fungus under standard conditions for tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts. Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our findings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting toxinogenic endofungal bacteria in starter cultures is proposed.