RESUMO
Aminopeptidases, such as dipeptidyl peptidase-4 (DPP-4, CD26), are potent therapeutic targets for pharmacological interventions because they play key roles in many important pathological pathways. To analyze aminopeptidase activity in vitro (including high-throughput screening [HTS]), in vivo, and ex vivo, we developed a highly sensitive and quantitative bioluminescence-based readout method. We successfully applied this method to screening drugs with potential DPP-4 inhibitory activity. Using this method, we found that cancer drug mitoxantrone possesses significant DPP-4 inhibitory activity both in vitro and in vivo. The pharmacophore of mitoxantrone was further investigated by testing a variety of its structural analogues.
Assuntos
Antineoplásicos/farmacologia , Dipeptidil Peptidase 4/análise , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Descoberta de Drogas , Mitoxantrona/farmacologia , Humanos , Medições LuminescentesRESUMO
Faecalibacterium prausnitzii (F. prausnitzii) is a bacterial taxon in the human gut with anti-inflammatory properties, and this may contribute to the beneficial effects of healthy eating habits. However, little is known about the nutrients that enhance the growth of F. prausnitzii other than simple sugars and fibers. Here, we combined dietary and microbiome data from the American Gut Project (AGP) to identify nutrients that may be linked to the relative abundance of F. prausnitzii. Using a machine learning approach in combination with univariate analyses, we identified that sugar alcohols, carbocyclic sugar, and vitamins may contribute to F. prausnitzii growth. We next explored the effects of these nutrients on the growth of two F. prausnitzii strains in vitro and observed robust and strain-dependent growth patterns on sorbitol and inositol, respectively. In the context of a complex community using in vitro fermentation, neither inositol alone nor in combinations with vitamin B exerted a significant growth-promoting effect on F. prausnitzii, partly due to high variability among the fecal microbiota community from four healthy donors. However, the fecal communities that showed an increase in F. prausnitzii on inulin also responded with at least 60% more F. prausnitzii on any of inositol containing media than control. Future nutritional studies aiming to increase the relative abundance of F. prausnitzii should explore a personalized approach accounting for strain-level genetic variations and community-level microbiome composition.
Assuntos
Microbiota , Complexo Vitamínico B , Humanos , Faecalibacterium prausnitzii , Inositol , InulinaRESUMO
The microbiome-produced enzyme bile salt hydrolase (BSH) plays a central role in human health, but its function remains unclear due to the lack of suitable methods for measuring its activity. Here, we have developed a novel optical tool based on ultrasensitive bioluminescent imaging and demonstrated that this assay can be used for quick and cost-effective quantification of BSH activity across a broad range of biological settings including pure enzymes and bacteria, intact fecal slurries, and noninvasive imaging in live animals, as well as for the assessment of BSH activity in the entire gastrointestinal tract of mice and humans. Using this assay, we showed that certain types of prebiotics are capable of increasing BSH activity of the gut microbiota in vivo and successfully demonstrated potential application of this assay as a noninvasive diagnostic test to predict the clinical status of inflammatory bowel disease (IBD) patients.
Assuntos
Amidoidrolases , Microbioma Gastrointestinal , Amidoidrolases/análise , Amidoidrolases/química , Animais , Bactérias , Ácidos e Sais Biliares , Microbioma Gastrointestinal/fisiologia , Humanos , Medições Luminescentes/métodos , Camundongos , PrebióticosRESUMO
BACKGROUND: In observational studies, coffee consumption has been consistently associated with a lower risk of type 2 diabetes mellitus. Trials examining the effect of coffee consumption on glucose metabolism have been limited by the use of surrogate insulin sensitivity indices, small sample sizes, lack of blinding, and short follow-up duration. OBJECTIVES: We aimed to overcome limitations of previously conducted coffee trials in a randomized placebo-controlled trial of the effect of coffee consumption on insulin sensitivity. METHODS: We conducted a 24-wk randomized placebo-controlled trial in 126 overweight, non-insulin sensitive (HOMA-IR ≥1.30), Chinese, Malay, and Asian-Indian males and females aged 35-69 y. Participants were randomly assigned to receive 4 cups of instant regular coffee (n = 62) or 4 cups of a coffee-like placebo beverage (n = 64) per day. The primary outcome was the amount of glucose metabolized per kilogram of body weight per minute (Mbw) assessed during steady-state conditions with a hyperinsulinemic euglycemic clamp. Secondary outcomes included other clamp-based insulin sensitivity measures, biological mediators of insulin sensitivity, and measures of fasting glucose metabolism. RESULTS: Coffee consumption did not significantly change insulin sensitivity compared with placebo (percentage mean difference in Mbw = 4.0%; 95% CI: -8.3, 18.0%; P = 0.53). Furthermore, no significant differences in fasting plasma glucose (2.9%; 95% CI: -0.4, 6.3%; P = 0.09) or biological mediators of insulin resistance, such as plasma adiponectin (2.3%; 95% CI: -1.4, 6.2%; P = 0.22), were observed between coffee and placebo groups over 24 wk of intervention. Participants in the coffee arm experienced a loss of fat mass (FM) (-3.7%; 95% CI: -6.3, -1.1%; P = 0.006) and reduction in urinary creatinine concentrations (-21.2%; 95% CI: -31.4, -9.5%; P = 0.001) compared with participants in the placebo arm over 24 wk of intervention. CONCLUSIONS: Consuming 4 cups/d of caffeinated coffee for 24 wk had no significant effect on insulin sensitivity or biological mediators of insulin resistance but was associated with a modest loss of FM and reduction in urinary creatinine concentrations.This trial was registered at clinicaltrials.gov as NCT01738399. Registered on November 28, 2012. Trial sponsor: Nestlé Research, Lausanne, Switzerland. Trial site: National University of Singapore.
Assuntos
Café , Diabetes Mellitus Tipo 2 , Resistência à Insulina , Adulto , Idoso , Feminino , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Recent data suggest that the gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type 2 diabetes. To assess this issue, we modulated gut microbiota via antibiotics administration in two different mouse models with insulin resistance. Results from dose-determination studies showed that a combination of norfloxacin and ampicillin, at a dose of 1 g/L, maximally suppressed the numbers of cecal aerobic and anaerobic bacteria in ob/ob mice. After a 2-wk intervention with the antibiotic combination, both ob/ob and diet-induced obese and insulin-resistant mice showed a significant improvement in fasting glycemia and oral glucose tolerance. The improved glycemic control was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the control ob/ob mice. Reduced liver triglycerides and increased liver glycogen correlated with improved glucose tolerance in the treated mice. Concomitant reduction of plasma lipopolysaccharides and increase of adiponectin further supported the antidiabetic effects of the antibiotic treatment in ob/ob mice. In summary, modulation of gut microbiota ameliorated glucose tolerance of mice by altering the expression of hepatic and intestinal genes involved in inflammation and metabolism, and by changing the hormonal, inflammatory, and metabolic status of the host.
Assuntos
Ampicilina/farmacologia , Bacteroides/efeitos dos fármacos , Bifidobacterium/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Norfloxacino/farmacologia , Animais , Antibacterianos/farmacologia , Bacteroides/fisiologia , Bifidobacterium/fisiologia , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Enterobacteriaceae/fisiologia , Lactobacillus/fisiologia , Camundongos , Camundongos Obesos , Testes de Sensibilidade Microbiana , Obesidade/microbiologia , Obesidade/fisiopatologiaRESUMO
The circadian clock and associated feeding rhythms have a profound impact on metabolism and the gut microbiome. To what extent microbiota reciprocally affect daily rhythms of physiology in the host remains elusive. Here, we analyzed transcriptome and metabolome profiles of male and female germ-free mice. While mRNA expression of circadian clock genes revealed subtle changes in liver, intestine, and white adipose tissue, germ-free mice showed considerably altered expression of genes associated with rhythmic physiology. Strikingly, the absence of the microbiome attenuated liver sexual dimorphism and sex-specific rhythmicity. The resulting feminization of male and masculinization of female germ-free animals is likely caused by altered sexual development and growth hormone secretion, associated with differential activation of xenobiotic receptors. This defines a novel mechanism by which the microbiome regulates host metabolism.
Assuntos
Tecido Adiposo Branco/metabolismo , Relógios Circadianos , Grelina/metabolismo , Intestinos/microbiologia , Fígado/metabolismo , Transcriptoma , Animais , Ritmo Circadiano , Feminino , Microbioma Gastrointestinal , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Caracteres SexuaisRESUMO
In this study we investigated the structural requirements for inhibition of human salivary alpha-amylase by flavonoids. Four flavonols and three flavones, out of the 19 flavonoids tested, exhibited IC50 values less than 100 microM against human salivary alpha-amylase activity. Structure-activity relationships of these inhibitors by computational ligand docking showed that the inhibitory activity of flavonols and flavones depends on (i) hydrogen bonds between the hydroxyl groups of the polyphenol ligands and the catalytic residues of the binding site and (ii) formation of a conjugated pi-system that stabilizes the interaction with the active site. Our findings show that certain naturally occurring flavonoids act as inhibitors of human alpha-amylase, which makes them promising candidates for controlling the digestion of starch and postprandial glycemia.
Assuntos
Flavonas/química , Flavonóis/química , Modelos Moleculares , Amido/metabolismo , alfa-Amilases/antagonistas & inibidores , Domínio Catalítico , Digestão , Humanos , Ligação de Hidrogênio , Ligantes , Conformação Proteica , Saliva/enzimologia , Relação Estrutura-Atividade , alfa-Amilases/químicaRESUMO
Escherichia coli is one of the common inhabitants of the mammalian gastrointestinal track. We isolated a strain from an ob/ob mouse and performed whole-genome sequencing, which yielded a chromosome of ~5.1 Mb and three plasmids of ~160 kb, ~6 kb, and ~4 kb.
RESUMO
The gut microbiome and lipid metabolism are both recognized as essential components in the maintenance of metabolic health. The mechanisms involved are multifactorial and (especially for microbiome) poorly defined. A strategic approach to investigate the complexity of the microbial influence on lipid metabolism would facilitate determination of relevant molecular mechanisms for microbiome-targeted therapeutics. E. coli is associated with obesity and metabolic syndrome and we used this association in conjunction with gnotobiotic models to investigate the impact of E. coli on lipid metabolism. To address the complexities of the integration of the microbiome and lipid metabolism, we developed transcriptomics-driven lipidomics (TDL) to predict the impact of E. coli colonization on lipid metabolism and established mediators of inflammation and insulin resistance including arachidonic acid metabolism, alterations in bile acids and dietary lipid absorption. A microbiome-related therapeutic approach targeting these mechanisms may therefore provide a therapeutic avenue supporting maintenance of metabolic health.
RESUMO
The gut microbiota is involved in many aspects of host physiology but its role in body weight and glucose metabolism remains unclear. Here we studied the compositional changes of gut microbiota in diet-induced obesity mice that were conventionally raised or received microbiota transplantation. In conventional mice, the diversity of the faecal microbiota was weakly associated with 1(st) week weight gain but transferring the microbiota of mice with contrasting weight gain to germfree mice did not change obesity development or feed efficiency of recipients regardless whether the microbiota was taken before or after 10 weeks high fat (HF) feeding. Interestingly, HF-induced glucose intolerance was influenced by microbiota inoculation and improved glucose tolerance was associated with a low Firmicutes to Bacteroidetes ratio. Transplantation of Bacteroidetes rich microbiota compared to a control microbiota ameliorated glucose intolerance caused by HF feeding. Altogether, our results demonstrate that gut microbiota is involved in the regulation of glucose metabolism and the abundance of Bacteroidetes significantly modulates HF-induced glucose intolerance but has limited impact on obesity in mice. Our results suggest that gut microbiota is a part of complex aetiology of insulin resistance syndrome, individual microbiota composition may cause phenotypic variation associated with HF feeding in mice.
Assuntos
Dieta Hiperlipídica , Gorduras na Dieta/efeitos adversos , Intolerância à Glucose/metabolismo , Obesidade/metabolismo , Animais , Bacteroidetes/classificação , Bacteroidetes/crescimento & desenvolvimento , Transplante de Microbiota Fecal , Firmicutes/classificação , Firmicutes/crescimento & desenvolvimento , Microbioma Gastrointestinal/fisiologia , Intolerância à Glucose/etiologia , Intolerância à Glucose/microbiologia , Intolerância à Glucose/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/microbiologia , Obesidade/patologia , Proteobactérias/classificação , Proteobactérias/crescimento & desenvolvimentoRESUMO
Development of NGS has revolutionized the analysis in microbial ecology contributing to our deeper understanding of microbiota in health and disease. However, the quality, quantity and confidence of summarized taxonomic abundances are in need of further scrutiny due to sample dependent and independent effects. In this article we introduce 'AVIT (Abundance and Variability In Taxonomy), an unbiased method to enrich for assigned members of microbial communities. As opposed to using a priori thresholds, 'AVIT uses inherent abundance and variability of taxa in a dataset to determine the inclusion or rejection of each taxa for further downstream analysis. Using in-vitro and in-vivo studies, we benchmarked performance and parameterized 'AVIT to establish a framework for investigating the dynamic range of microbial community membership in clinically relevant scenarios.
Assuntos
Microbiota , Algoritmos , Animais , Vida Livre de Germes , Humanos , CamundongosRESUMO
BACKGROUND: Coffee consumption has been consistently associated with a lower risk of type 2 diabetes mellitus in cohort studies. In addition, coffee components increased insulin sensitivity in animal models. However, data from intervention studies on the effect of coffee consumption on glucose metabolism have been limited by small sample sizes, lack of blinding, short follow-up duration and the use of surrogate indices of insulin sensitivity. We designed the Coffee for Metabolic Health (COMETH) study to evaluate the effect of coffee consumption on insulin sensitivity. METHODOLOGY: The COMETH study is a double-blind randomized placebo-controlled 24-week trial. Participants were overweight, male and female habitual coffee consumers who were of Chinese, Malay and Asian-Indian ethnicity. We excluded smokers, persons with diabetes, and persons with low insulin resistance (HOMA-IR < 1.30). Participants were randomly assigned to receive daily 4 cups of instant regular coffee or 4 cups of a coffee-like placebo beverage. The hyperinsulinemic euglycemic clamp was performed at baseline and at the end of 24 weeks to determine changes in the bodyweight standardized M-value. Secondary outcomes included changes in fasting glucose and insulin sensitivity mediators such as adiponectin, markers of inflammation, liver function, and oxidative stress.We enrolled 128 participants, 126 (57.1% males; aged 35-67 years) of whom completed baseline assessments. DISCUSSION: If improvement in insulin sensitivity in the coffee group is significantly greater than that of the placebo group, this would support the hypothesis that coffee consumption reduced risk of type 2 diabetes through biological pathways involving insulin sensitivity. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01738399. Registered on 28 November 2012. Trial Sponsor: Nestlé Research Center, Lausanne, Switzerland. Trial Site: National University of Singapore.
RESUMO
Proteins are needed for the development and sustainability of life. They are the molecular machines and building blocks in the human body that drive or exert most biological functions and confer structure and function to cell and tissue architecture. Dietary proteins provide essential amino acids and complement lipid and carbohydrate as a major source of energy. Therefore, humans must consume a sufficient amount and quality of proteins to stay healthy and avoid deficiencies. Even with a reasonable amount of intake, variability in protein consumption can result in measurable health consequences in specific conditions. This said, dietary protein delivers more than energy and building blocks to the human body: the pools of body, tissue, and cell proteins, peptides, and amino acids are under complex metabolic control, resulting in a highly dynamic protein turnover, that is, the interplay between synthesis and degradation. Proteins also contain peptide sequences that can be interpreted as bioactive precursors which can be liberated upon digestion to exert biological functions locally (e.g., in the gut) or systemically (i.e., via the bloodstream). In this chapter, we will first review holistic readouts of protein intake assessed by omics technologies such as gene expression, proteomics, and metabolite profiling. Second, we will look at protein benefits beyond macronutrient supply and describe how to generate, analyze, and leverage bioactive peptides. In the third part, we will discuss protein turnover as tackled by proteomics tools that allow single-protein resolution at proteome-wide scale.
Assuntos
Proteínas Alimentares/administração & dosagem , Nutrigenômica , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteínas Alimentares/metabolismo , Humanos , CamundongosRESUMO
Consumption of low-carbohydrate, high-protein, high-fat diets lead to rapid weight loss but the cardioprotective effects of these diets have been questioned. We examined the impact of high-protein and high-fat diets on cholesterol metabolism by comparing the plasma cholesterol and the expression of cholesterol biosynthesis genes in the liver of mice fed a high-fat (HF) diet that has a high (H) or a low (L) protein-to-carbohydrate (P/C) ratio. H-P/C-HF feeding, compared with L-P/C-HF feeding, decreased plasma total cholesterol and increased HDL cholesterol concentrations at 4-wk. Interestingly, the expression of genes involved in hepatic steroid biosynthesis responded to an increased dietary P/C ratio by first down-regulation (2-d) followed by later up-regulation at 4-wk, and the temporal gene expression patterns were connected to the putative activity of SREBF1 and 2. In contrast, Cyp7a1, the gene responsible for the conversion of cholesterol to bile acids, was consistently up-regulated in the H-P/C-HF liver regardless of feeding duration. Over expression of Cyp7a1 after 2-d and 4-wk H-P/C-HF feeding was connected to two unique sets of transcription regulators. At both time points, up-regulation of the Cyp7a1 gene could be explained by enhanced activations and reduced suppressions of multiple transcription regulators. In conclusion, we demonstrated that the hypocholesterolemic effect of H-P/C-HF feeding coincided with orchestrated changes of gene expressions in lipid metabolic pathways in the liver of mice. Based on these results, we hypothesize that the cholesterol lowering effect of high-protein feeding is associated with enhanced bile acid production but clinical validation is warranted. (246 words).
Assuntos
Colesterol/metabolismo , Dieta Hiperlipídica , Carboidratos da Dieta/farmacologia , Proteínas Alimentares/farmacologia , Animais , Colesterol 7-alfa-Hidroxilase/metabolismo , Dislipidemias/complicações , Dislipidemias/metabolismo , Dislipidemias/patologia , Ingestão de Energia/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Teste de Tolerância a Glucose , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Modelos Biológicos , Obesidade/complicações , Obesidade/metabolismo , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esteroides/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Fatores de Tempo , Aumento de Peso/efeitos dos fármacosRESUMO
Recent data suggest that gut microbiota plays a significant role in fat accumulation. However, it is not clear whether gut microbiota is involved in the pathophysiology of type-2 diabetes. To address this issue, we modulated gut microbiota with two combinations of antibiotics in two different mouse models with insulin resistance. Treatment with norfloxacin and ampicillin for 2 weeks reduced the cecal bacterial DNA below the level of detection in ob/ob, diet-induced obese and insulin resistance (DIO) mice, and significantly improved fasting glycemia and oral glucose tolerance of the treated animals. The enhanced insulin sensitivity was independent of food intake or adiposity because pair-fed ob/ob mice were as glucose intolerant as the untreated ob/ob mice. The reduced liver triglycerides, increased liver glycogen and improved glucose tolerance in the treated mice indicate broad impacts on metabolism by gut decontamination. The treatment with non-absorbable antibiotics polymyxin B and neomycin significantly modified cecal microbiota profile in the DIO mice, and the modified intestinal microbiota was associated with a gradual reduction in glycemia during a washout period. In summary, modulation of gut microbiota ameliorated glucose intolerance in mice and altered the hormonal, inflammatory and metabolic status of the host.