RESUMO
BACKGROUND & AIMS: Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. METHODS: We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. RESULTS: Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. CONCLUSIONS: Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.
Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/prevenção & controle , Metabolismo Energético/efeitos dos fármacos , Trealose/farmacologia , Animais , Proteínas de Bactérias/metabolismo , Células CACO-2 , Clostridioides difficile/enzimologia , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Dissacaridases/metabolismo , Modelos Animais de Doenças , Jejum/metabolismo , Fezes/microbiologia , Glucose/metabolismo , Células HEK293 , Hepatócitos , Humanos , Mucosa Intestinal/citologia , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Cultura Primária de Células , Trealose/análogos & derivados , Trealose/uso terapêuticoRESUMO
In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function.
Assuntos
Antibacterianos/urina , Proteínas de Transporte/urina , Catecóis/urina , Infecções por Escherichia coli/urina , Escherichia coli , Infecções Urinárias/urina , Adolescente , Adulto , Antibacterianos/imunologia , Proteínas de Transporte/imunologia , Catecóis/imunologia , Infecções por Escherichia coli/imunologia , Feminino , Humanos , Imunidade Inata , Lipocalina-2 , Metaboloma/imunologia , Pessoa de Meia-Idade , Infecções Urinárias/imunologiaRESUMO
During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.
Assuntos
Proteínas de Transporte/imunologia , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Infecções Urinárias/imunologia , Urina/química , Enterobactina/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Lipocalina-2 , Sideróforos/metabolismo , Infecções Urinárias/microbiologiaRESUMO
Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and is proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 5-[(13)C4]aminolevulinic acid, and this assay confirmed that de novo heme synthesis was ablated in FC knock-out parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
Assuntos
Anopheles/parasitologia , Eritrócitos/parasitologia , Heme/biossíntese , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , Animais , Feminino , Ferroquelatase/genética , Técnicas de Inativação de Genes , Heme/metabolismo , Humanos , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Espectrometria de Massas em TandemRESUMO
Bacterial pathogens secrete chemically diverse iron chelators called siderophores, which may exert additional distinctive functions in vivo. Among these, uropathogenic Escherichia coli often coexpress the virulence-associated siderophore yersiniabactin (Ybt) with catecholate siderophores. Here we used a new MS screening approach to reveal that Ybt is also a physiologically favorable Cu(II) ligand. Direct MS detection of the resulting Cu(II)-Ybt complex in mice and humans with E. coli urinary tract infections demonstrates copper binding to be a physiologically relevant in vivo interaction during infection. Ybt expression corresponded to higher copper resistance among human urinary tract isolates, suggesting a protective role for this interaction. Chemical and genetic characterization showed that Ybt helps bacteria resist copper toxicity by sequestering host-derived Cu(II) and preventing its catechol-mediated reduction to Cu(I). Together, these studies reveal a new virulence-associated function for Ybt that is distinct from iron binding.
Assuntos
Cobre/toxicidade , Fenóis/metabolismo , Tiazóis/metabolismo , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/metabolismo , Animais , Domínio Catalítico , Cromatografia Líquida , Infecções por Escherichia coli/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Fenóis/química , Ligação Proteica , Espectrometria de Massas em Tandem , Tiazóis/químicaRESUMO
Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.
Assuntos
Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Bilirrubina/metabolismo , Biliverdina/metabolismo , Cromatografia Líquida de Alta Pressão , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Heme Oxigenase (Desciclizante)/genética , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Ligação Proteica , Proteólise , Protoporfirinas/metabolismo , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by mitochondrial abnormalities, infantile or childhood onset of cardioskeletal myopathy, and high mortality rates. It is currently unknown if BTHS related mitochondrial dysfunction results in substrate metabolism abnormalities and thereby contributes to cardioskeletal myopathy in patients with BTHS. METHODS: Adolescents and young adults with BTHS (n = 5, 20 ± 4 yrs) and age and activity matched healthy controls (n = 5, 18 ± 4 yrs) underwent an hyperinsulinemic-euglycemic clamp procedure with stable isotopically labeled tracers for measurement of lipolysis, fatty acid oxidation, glucose disposal, and whole-body proteolysis rates; dual energy x-ray absorptiometry for measurement of body composition and 2-D and strain echocardiography for measurement of left ventricular function. RESULTS: Participants with BTHS had lower fat-free mass (FFM) (BTHS: 31.4 ± 6.9 vs. CONTROL: 46.7 ± 5.3 kg, p < 0.005), lower systolic function (strain, BTHS: -15.2 ± 2.4 vs. CONTROL: -19.0 ± 2.4 %, p < 0.05), greater insulin-stimulated glucose disposal rate per kg FFM (BTHS: 96.5 ± 16.3 vs. CONTROL: 67.4 ± 17.6 µmol/kgFFM/min, p < 0.05), lower basal (BTHS: 4.6 ± 2.7 vs. CONTROL: 11.9 ± 4.4 µmol/kgFM/min, p < 0.05) and hyperinsulinemic (BTHS: 1.6 ± 0.4 vs. CONTROL: 3.6 ± 1.6 µmol/kgFM/min, p < 0.05) lipolytic rate per kg fat mass (FM), and a trend towards higher basal leucine rate of appearance per kg FFM (BTHS: 271.4 ± 69.3 vs. CONTROL: 193.1 ± 28.7 µmol/kgFFM/hr, p = 0.07) compared to controls. Higher basal leucine rate of appearance per kg FFM (i.e. whole-body proteolytic rate) tended to be associated with lower left ventricular systolic strain (r = -0.57, p = 0.09). CONCLUSION: Whole-body fatty acid, glucose and amino acid metabolism kinetics when expressed per unit of body composition are altered and appear to be related to cardioskeletal myopathy in humans with BTHS. Further studies examining myocardial substrate metabolism and whole-body substrate metabolism during increased energy demands (e.g., exercise) and their relationships to skeletal and cardiac function are recommended.
Assuntos
Síndrome de Barth/metabolismo , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Adolescente , Adulto , Síndrome de Barth/sangue , Glicemia/metabolismo , Composição Corporal/fisiologia , Ecocardiografia/métodos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Insulina/sangue , Leucina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipólise/fisiologia , Masculino , Mitocôndrias/metabolismo , Oxirredução , Adulto JovemRESUMO
Studies in mice indicate that the gut microbiota promotes energy harvest and storage from components of the diet when these components are plentiful. Here we examine how the microbiota shapes host metabolic and physiologic adaptations to periods of nutrient deprivation. Germ-free (GF) mice and mice who had received a gut microbiota transplant from conventionally raised donors were compared in the fed and fasted states by using functional genomic, biochemical, and physiologic assays. A 24-h fast produces a marked change in gut microbial ecology. Short-chain fatty acids generated from microbial fermentation of available glycans are maintained at higher levels compared with GF controls. During fasting, a microbiota-dependent, Ppar alpha-regulated increase in hepatic ketogenesis occurs, and myocardial metabolism is directed to ketone body utilization. Analyses of heart rate, hydraulic work, and output, mitochondrial morphology, number, and respiration, plus ketone body, fatty acid, and glucose oxidation in isolated perfused working hearts from GF and colonized animals (combined with in vivo assessments of myocardial physiology) revealed that the fasted GF heart is able to sustain its performance by increasing glucose utilization, but heart weight, measured echocardiographically or as wet mass and normalized to tibial length or lean body weight, is significantly reduced in both fasted and fed mice. This myocardial-mass phenotype is completely reversed in GF mice by consumption of a ketogenic diet. Together, these results illustrate benefits provided by the gut microbiota during periods of nutrient deprivation, and emphasize the importance of further exploring the relationship between gut microbes and cardiovascular health.
Assuntos
Jejum/fisiologia , Trato Gastrointestinal/microbiologia , Corpos Cetônicos/metabolismo , Metagenoma , Miocárdio/metabolismo , Animais , Alimentos , Genômica , Vida Livre de Germes , Glucose/metabolismo , Hipertrofia , Técnicas In Vitro , Fígado/metabolismo , Camundongos , Miocárdio/patologia , Tamanho do Órgão , Oxirredução , Perfusão , Resistência FísicaRESUMO
Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.
Assuntos
Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metabolômica/métodos , Sideróforos/genética , Infecções Urinárias/metabolismo , Cromatografia Líquida , Epigênese Genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Feminino , Expressão Gênica , Humanos , Espectrometria de Massas , Mutação , Reto/microbiologia , Sideróforos/metabolismo , Estatísticas não Paramétricas , Infecções Urinárias/microbiologiaRESUMO
Familial hemiplegic migraine is an episodic neurological disorder characterized by transient sensory and motor symptoms and signs. Mutations of the ion pump α2-Na/K ATPase cause familial hemiplegic migraine, but the mechanisms by which α2-Na/K ATPase mutations lead to the migraine phenotype remain incompletely understood. Here, we show that mice in which α2-Na/K ATPase is conditionally deleted in astrocytes display episodic paralysis. Functional neuroimaging reveals that conditional α2-Na/K ATPase knockout triggers spontaneous cortical spreading depression events that are associated with EEG low voltage activity events, which correlate with transient motor impairment in these mice. Transcriptomic and metabolomic analyses show that α2-Na/K ATPase loss alters metabolic gene expression with consequent serine and glycine elevation in the brain. A serine- and glycine-free diet rescues the transient motor impairment in conditional α2-Na/K ATPase knockout mice. Together, our findings define a metabolic mechanism regulated by astrocytic α2-Na/K ATPase that triggers episodic motor paralysis in mice.
Assuntos
Astrócitos/metabolismo , Ataxia/genética , Metaboloma/genética , Enxaqueca com Aura/genética , ATPase Trocadora de Sódio-Potássio/genética , Transcriptoma , Animais , Astrócitos/patologia , Ataxia/metabolismo , Ataxia/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Neuroimagem Funcional , Glicina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Enxaqueca com Aura/metabolismo , Enxaqueca com Aura/patologia , Teste de Desempenho do Rota-Rod , Serina/metabolismo , ATPase Trocadora de Sódio-Potássio/deficiênciaRESUMO
High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.
Assuntos
Doenças Cardiovasculares/metabolismo , Dieta Rica em Proteínas , Macrófagos/metabolismo , Mitofagia/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Fatores de Risco de Doenças Cardíacas , Ativação de Macrófagos , Camundongos , Placa Aterosclerótica/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Clostridioides difficile infection (CDI) accounts for a substantial proportion of deaths attributable to antibiotic-resistant bacteria in the United States. Although C. difficile can be an asymptomatic colonizer, its pathogenic potential is most commonly manifested in patients with antibiotic-modified intestinal microbiomes. In a cohort of 186 hospitalized patients, we showed that host and microbe-associated shifts in fecal metabolomes had the potential to distinguish patients with CDI from those with non-C. difficile diarrhea and C. difficile colonization. Patients with CDI exhibited a chemical signature of Stickland amino acid fermentation that was distinct from those of uncolonized controls. This signature suggested that C. difficile preferentially catabolizes branched chain amino acids during CDI. Unexpectedly, we also identified a series of noncanonical, unsaturated bile acids that were depleted in patients with CDI. These bile acids may derive from an extended host-microbiome dehydroxylation network in uninfected patients. Bile acid composition and leucine fermentation defined a prototype metabolomic model with potential to distinguish clinical CDI from asymptomatic C. difficile colonization.
Assuntos
Ácidos e Sais Biliares/química , Infecções por Clostridium/microbiologia , Microbioma Gastrointestinal , Redes e Vias Metabólicas , Adulto , Idoso , Idoso de 80 Anos ou mais , Clostridioides difficile , Diarreia/microbiologia , Fezes/microbiologia , Feminino , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Análise dos Mínimos Quadrados , Leucina/química , Masculino , Metabolômica , Pessoa de Meia-Idade , Análise Multivariada , Análise de Componente PrincipalRESUMO
BACKGROUND: Ground corncob animal bedding and corn food products contain substances that disrupt endocrine function in rats. The disruptors were identified as isomeric mixtures of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid) and leukotoxindiols (LTX-diols; 9,10-dihydroxy-12-octadecenoic acid and 12,13-dihydroxy-9-octadecenoic acid). The authentic compounds blocked sexual behavior in male rats and estrous cyclicity in female rats at oral doses of 2 ppm. OBJECTIVES: To define the lowest observed adverse effect level (LOAEL) for the THF-diols and LTX-diols in rats, we examined the nature of their interaction (additive or synergistic) and quantified the concentration of THF-diols in rat tissues. METHODS: Adult male and female rats were provided drinking solutions containing various doses of THF-diols and/or LTX-diols, and we evaluated their effects on male sexual behavior and female estrous cyclicity. Tissues were collected for THF-diol determination by gas chromatography-mass spectrometry. RESULTS: The LOAEL for THF-diols and LTX-diols for blocking estrous cyclicity was 0.5-1.0 ppm and 0.2-0.5 ppm, respectively. Higher concentrations (1-2 ppm) of THF-diols were required to block male sexual behavior. Combination studies with subthreshold doses of 0.05 ppm THF-diols plus 0.05 ppm LTX-diols revealed that their effects on estrous cyclicity were not synergistic. We were unable to detect THF-diols in tissues from rats treated with 10 ppm of the compounds, suggesting that metabolism may be involved. DISCUSSION: THF-diols, LTX-diols, and/or their metabolites likely act additively to disrupt endocrine function in male and female rats at concentrations (0.5-1 ppm) that are 200-fold lower than those of classical phytoestrogen endocrine disruptors.
Assuntos
Disruptores Endócrinos/toxicidade , Ciclo Estral/efeitos dos fármacos , Comportamento Sexual Animal/efeitos dos fármacos , Ácidos Esteáricos/toxicidade , Zea mays/química , Animais , Relação Dose-Resposta a Droga , Disruptores Endócrinos/química , Feminino , Furanos , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Linoleicos , Masculino , Nível de Efeito Adverso não Observado , Ratos , Ácidos Esteáricos/químicaRESUMO
Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy-lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy-lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1ß levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy-lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease.
Assuntos
Aterosclerose/terapia , Autofagia , Macrófagos/fisiologia , Placa Aterosclerótica/patologia , Trealose/farmacologia , Animais , Aterosclerose/patologia , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Humanos , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Placa Aterosclerótica/terapia , Proteína Sequestossoma-1/metabolismoRESUMO
Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.
Assuntos
Autofagia , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Trealose/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Sequência de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Transporte Biológico , Linhagem Celular , Ácidos Graxos/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/química , Proteínas Facilitadoras de Transporte de Glucose/genética , Hepatócitos/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Fosforilação , Ligação Proteica , Transdução de Sinais , Trealose/química , Trealose/farmacologia , Triglicerídeos/metabolismoRESUMO
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a poorly understood syndrome affecting up to 6.5% of adult women in the U.S. The lack of broadly accepted objective laboratory markers for this condition hampers efforts to diagnose and treat this condition. To identify biochemical markers for IC/BPS, we applied mass spectrometry-based global metabolite profiling to urine specimens from a cohort of female IC/BPS subjects from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network. These analyses identified multiple metabolites capable of discriminating IC/BPS and control subjects. Of these candidate markers, etiocholan-3α-ol-17-one sulfate (Etio-S), a sulfoconjugated 5-ß reduced isomer of testosterone, distinguished female IC/BPS and control subjects with a sensitivity and specificity >90%. Among IC/BPS subjects, urinary Etio-S levels are correlated with elevated symptom scores (symptoms, pelvic pain, and number of painful body sites) and could resolve high- from low-symptom IC/BPS subgroups. Etio-S-associated biochemical changes persisted through 3-6months of longitudinal follow up. These results raise the possibility that an underlying biochemical abnormality contributes to symptoms in patients with severe IC/BPS.
Assuntos
Cistite Intersticial/urina , Metabolômica/métodos , Esteroides/urina , Sulfatos/urina , Adulto , Biomarcadores/urina , Estudos de Coortes , Cistite Intersticial/diagnóstico , Feminino , Humanos , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Medição da DorRESUMO
Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer.
Assuntos
Ácidos/metabolismo , Metabolismo Energético , Glucose/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Linhagem Celular Tumoral , Glicólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Metástase Neoplásica , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/patologia , Niclosamida/administração & dosagem , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.
Assuntos
Autofagia , Fígado Gorduroso/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Trealose/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Proteínas Facilitadoras de Transporte de Glucose/genética , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Camundongos KnockoutRESUMO
Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (iso-leukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.