RESUMO
Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.
Assuntos
Doenças Cardiovasculares/sangue , Microbioma Gastrointestinal/genética , Glutamina/análogos & derivados , Trombose/metabolismo , Animais , Artérias/lesões , Artérias/metabolismo , Artérias/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plaquetas/metabolismo , Plaquetas/microbiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/microbiologia , Doenças Cardiovasculares/patologia , Morte Súbita Cardíaca/patologia , Glutamina/sangue , Glutamina/genética , Humanos , Masculino , Metaboloma/genética , Metabolômica/métodos , Camundongos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/microbiologia , Ativação Plaquetária/genética , Receptores Adrenérgicos alfa/sangue , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos beta/sangue , Receptores Adrenérgicos beta/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/microbiologia , Acidente Vascular Cerebral/patologia , Trombose/genética , Trombose/microbiologia , Trombose/patologiaRESUMO
BACKGROUND: Although artificial and non-nutritive sweeteners are widely used and generally recognized as safe by the US and European Union regulatory agencies, there have been no clinical trials to assess either long-term cardiovascular disease risks or short-term cardiovascular disease-relevant phenotypes. Recent studies report that fasting plasma levels of erythritol, a commonly used sweetener, are clinically associated with heightened incident cardiovascular disease risks and enhance thrombosis potential in vitro and in animal models. Effects of dietary erythritol on thrombosis phenotypes in humans have not been examined. METHODS: Using a prospective interventional study design, we tested the impact of erythritol or glucose consumption on multiple indices of stimulus-dependent platelet responsiveness in healthy volunteers (n=10 per group). Erythritol plasma levels were quantified with liquid chromatography tandem mass spectrometry. Platelet function at baseline and following erythritol or glucose ingestion was assessed via both aggregometry and analysis of granule markers released. RESULTS: Dietary erythritol (30 g), but not glucose (30 g), lead to a >1000-fold increase in erythritol plasma concentration (6480 [5930-7300] versus 3.75 [3.35-3.87] µmol/L; P<0.0001) and exhibited acute enhancement of stimulus-dependent aggregation responses in all subjects, agonists, and doses examined. Erythritol ingestion also enhanced stimulus-dependent release of the platelet dense granule marker serotonin (P<0.0001 for TRAP6 [thrombin activator peptide 6] and P=0.004 for ADP) and the platelet α-granule marker CXCL4 (C-X-C motif ligand-4; P<0.0001 for TRAP6 and P=0.06 for ADP). In contrast, glucose ingestion triggered no significant increases in stimulus-dependent release of either serotonin or CXCL4. CONCLUSIONS: Ingestion of a typical quantity of the non-nutritive sweetener erythritol, but not glucose, enhances platelet reactivity in healthy volunteers, raising concerns that erythritol consumption may enhance thrombosis potential. Combined with recent large-scale clinical observational studies and mechanistic cell-based and animal model studies, the present findings suggest that discussion of whether erythritol should be reevaluated as a food additive with the Generally Recognized as Safe designation is warranted. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04731363.
RESUMO
The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.
Assuntos
Fígado Gorduroso , Microbioma Gastrointestinal , Humanos , Camundongos , Animais , Polifenóis/farmacologia , Microbioma Gastrointestinal/fisiologia , Fígado Gorduroso/prevenção & controle , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Flavonoides/farmacologiaRESUMO
BACKGROUND AND AIMS: The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute. METHODS: Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10). RESULTS: In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects. CONCLUSIONS: Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.
Assuntos
Doenças Cardiovasculares , Xilitol , Humanos , Xilitol/farmacologia , Xilitol/efeitos adversos , Masculino , Feminino , Pessoa de Meia-Idade , Doenças Cardiovasculares/epidemiologia , Trombose , Edulcorantes/efeitos adversos , Edulcorantes/farmacologia , Idoso , Animais , Metabolômica , Espectrometria de Massas em Tandem , Adulto , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Fatores de Risco de Doenças CardíacasRESUMO
AIMS: Little is known about associations of trimethylamine N-oxide (TMAO), a novel gut microbiota-generated metabolite of dietary phosphatidylcholine and carnitine, and its changes over time with all-cause and cause-specific mortality in the general population or in different race/ethnicity groups. The study aimed to investigate associations of serially measured plasma TMAO levels and changes in TMAO over time with all-cause and cause-specific mortality in a multi-ethnic community-based cohort. METHODS AND RESULTS: The study included 6,785 adults from the Multi-Ethnic Study of Atherosclerosis. TMAO was measured at baseline and year 5 using mass spectrometry. Primary outcomes were adjudicated all-cause mortality and cardiovascular disease (CVD) mortality. Secondary outcomes were deaths due to kidney failure, cancer, or dementia obtained from death certificates. Cox proportional hazards models with time-varying TMAO and covariates assessed the associations with adjustment for sociodemographics, lifestyles, diet, metabolic factors, and comorbidities. During a median follow-up of 16.9 years, 1704 participants died and 411 from CVD. Higher TMAO levels associated with higher risk of all-cause mortality [hazard ratio (HR): 1.12, 95% confidence interval (CI): 1.08-1.17], CVD mortality (HR: 1.09, 95% CI: 1.00-1.09), and death due to kidney failure (HR: 1.44, 95% CI: 1.25-1.66) per inter-quintile range, but not deaths due to cancer or dementia. Annualized changes in TMAO levels associated with higher risk of all-cause mortality (HR: 1.10, 95% CI: 1.05-1.14) and death due to kidney failure (HR: 1.54, 95% CI: 1.26-1.89) but not other deaths. CONCLUSION: Plasma TMAO levels were positively associated with mortality, especially deaths due to cardiovascular and renal disease, in a multi-ethnic US cohort.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Demência , Neoplasias , Insuficiência Renal , Adulto , Humanos , Fatores de Risco , Biomarcadores , Metilaminas/metabolismo , Insuficiência Renal/etiologia , Aterosclerose/complicações , Neoplasias/complicaçõesRESUMO
AIMS: Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions. METHODS AND RESULTS: Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan). CONCLUSION: Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health.
Assuntos
Microbioma Gastrointestinal , Infarto do Miocárdio , Humanos , Camundongos , Animais , Aminoácidos Aromáticos/metabolismo , Triptofano , Glutamina , Glucuronídeos , Indóis/metabolismo , Progressão da Doença , TirosinaRESUMO
AIMS: Atherosclerotic cardiovascular disease (ACVD) is a major cause of mortality and morbidity worldwide, and increased low-density lipoproteins (LDLs) play a critical role in development and progression of atherosclerosis. Here, we examined for the first time gut immunomodulatory effects of the microbiota-derived metabolite propionic acid (PA) on intestinal cholesterol metabolism. METHODS AND RESULTS: Using both human and animal model studies, we demonstrate that treatment with PA reduces blood total and LDL cholesterol levels. In apolipoprotein E-/- (Apoe-/-) mice fed a high-fat diet (HFD), PA reduced intestinal cholesterol absorption and aortic atherosclerotic lesion area. Further, PA increased regulatory T-cell numbers and interleukin (IL)-10 levels in the intestinal microenvironment, which in turn suppressed the expression of Niemann-Pick C1-like 1 (Npc1l1), a major intestinal cholesterol transporter. Blockade of IL-10 receptor signalling attenuated the PA-related reduction in total and LDL cholesterol and augmented atherosclerotic lesion severity in the HFD-fed Apoe-/- mice. To translate these preclinical findings to humans, we conducted a randomized, double-blinded, placebo-controlled human study (clinical trial no. NCT03590496). Oral supplementation with 500 mg of PA twice daily over the course of 8 weeks significantly reduced LDL [-15.9 mg/dL (-8.1%) vs. -1.6 mg/dL (-0.5%), P = 0.016], total [-19.6 mg/dL (-7.3%) vs. -5.3 mg/dL (-1.7%), P = 0.014] and non-high-density lipoprotein cholesterol levels [PA vs. placebo: -18.9 mg/dL (-9.1%) vs. -0.6 mg/dL (-0.5%), P = 0.002] in subjects with elevated baseline LDL cholesterol levels. CONCLUSION: Our findings reveal a novel immune-mediated pathway linking the gut microbiota-derived metabolite PA with intestinal Npc1l1 expression and cholesterol homeostasis. The results highlight the gut immune system as a potential therapeutic target to control dyslipidaemia that may introduce a new avenue for prevention of ACVDs.
Assuntos
Aterosclerose , Propionatos , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/etiologia , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Humanos , Absorção Intestinal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Propionatos/farmacologia , Propionatos/uso terapêuticoRESUMO
We prospectively performed serial differential sugar absorption test in 29 consecutively consented patients with advanced decompensated heart failure admitted to the heart failure intensive care unit for hemodynamically-guided therapy. We observed that intestinal barrier function was significantly impaired in our study cohort, and increased intestinal permeability was associated with elevated right atrial pressure and poorer prognosis yet without any association with systemic levels of the gut microbial metabolite, trimethylamine N-oxide (TMAO) or intestinal fatty acid binding protein that were thought to be indicative of intestinal abnormalities.
Assuntos
Pressão Atrial , Insuficiência Cardíaca , Estudos de Coortes , Hospitalização , Humanos , Fatores de RiscoRESUMO
Bile acids (BAs) serve multiple biological functions, ranging from the absorption of lipids and fat-soluble vitamins to serving as signaling molecules through the direct activation of dedicated cellular receptors. Synthesized by both host and microbial pathways, BAs are increasingly understood as participating in the regulation of numerous pathways relevant to metabolic diseases, including lipid and glucose metabolism, energy expenditure, and inflammation. Quantitative analyses of BAs in biological matrices can be problematic due to their unusual and diverse physicochemical properties, making optimization of a method that shows good accuracy, precision, efficiency of extraction, and minimized matrix effects across structurally distinct human and murine BAs challenging. Herein we develop and clinically validate a stable-isotope-dilution LC/MS/MS method for the quantitative analysis of numerous primary and secondary BAs in both human and mouse biological matrices. We also utilize this tool to investigate gut microbiota participation in the generation of structurally specific BAs in both humans and mice. We examine circulating levels of specific BAs and in a clinical case-control study of age- and gender-matched type 2 diabetes mellitus (T2DM) versus nondiabetics. BAs whose circulating levels are associated with T2DM include numerous 12α-hydroxyl BAs (taurocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, deoxycholic acid, and 3-ketodeoxycholic acid), while taurohyodeoxycholic acid was negatively associated with diabetes. The LC/MS/MS-based platform described should serve as a robust, high-throughput investigative tool for studying the potential involvement of structurally specific BAs and the gut microbiome on both physiological and disease processes.
Assuntos
Ácidos e Sais Biliares/análise , Diabetes Mellitus Tipo 2/metabolismo , Microbioma Gastrointestinal , Animais , Ácidos e Sais Biliares/química , Estudos de Casos e Controles , Cromatografia Líquida , Diabetes Mellitus Tipo 2/microbiologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Controle de Qualidade , Espectrometria de Massas em TandemRESUMO
The gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) has recently been linked to cardiovascular disease (CVD) pathogenesis, prompting the development of therapeutic strategies to reduce TMAO. Previous work has shown that experimental alteration of circulating TMAO levels via dietary alterations or inhibition of the host TMAO producing enzyme flavin containing monooxygenase 3 (FMO3) is associated with reorganization of host cholesterol and bile acid metabolism in mice. In this work, we set out to understand whether recently developed nonlethal gut microbe-targeting small molecule choline trimethylamine (TMA) lyase inhibitors also alter host cholesterol and bile acid metabolism. Treatment of mice with the mechanism-based choline TMA lyase inhibitor, iodomethylcholine (IMC), increased fecal neutral sterol loss in the form of coprostanol, a bacteria metabolite of cholesterol. In parallel, IMC treatment resulted in marked reductions in the intestinal sterol transporter Niemann-pick C1-like 1 (NPC1L1) and reorganization of the gut microbial community, primarily reversing choline supplemented diet-induced changes. IMC also prevented diet-driven hepatic cholesterol accumulation, causing both upregulation of the host hepatic bile acid synthetic enzyme CYP7A1 and altering the expression of hepatic genes critical for bile acid feedback regulation. These studies suggest that the gut microbiota-driven TMAO pathway is closely linked to both microbe and host sterol and bile acid metabolism. Collectively, as gut microbe-targeting choline TMA lyase inhibitors move through the drug discovery pipeline from preclinical models to human studies, it will be important to understand how these drugs impact both microbe and host cholesterol and bile acid metabolism.NEW & NOTEWORTHY The gut microbe-dependent metabolite trimethylamine-N-oxide (TMAO) has been strongly associated with cardiovascular mortality, prompting drug discovery efforts to identify points of therapeutic intervention within the microbe host TMAO pathway. Recently, mechanism-based small molecule inhibitors of the major bacterial trimethylamine (TMA) lyase enzymes have been developed, and these drugs show efficacy as anti-atherothrombotic agents. The novel findings of this study are that small molecule TMA lyase inhibition results in beneficial reorganization of host cholesterol and bile acid metabolism. This study confirms previous observations that the gut microbial TMAO pathway is intimately linked to host cholesterol and bile acid metabolism and provides further rationale for the development of small molecule choline TMA lyase inhibitors for the treatment of cardiometabolic disorders.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/metabolismo , Animais , Colina/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , CamundongosRESUMO
Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.
Assuntos
Modelos Animais de Doenças , Proteínas de Membrana/antagonistas & inibidores , Pirazóis/farmacologia , Piridazinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Síndromes de Usher/tratamento farmacológico , Animais , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Pirazóis/uso terapêutico , Piridazinas/síntese química , Piridazinas/química , Piridazinas/uso terapêutico , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Síndromes de Usher/genéticaRESUMO
LW-1 is a collagen-linked blue fluorophore whose skin levels increase with age, diabetes and end-stage renal disease (ESRD), and correlate with the long-term progression of microvascular disease and indices of subclinical cardiovascular disease in type 1 diabetes. The chemical structure of LW-1 is still elusive, but earlier NMR analyses showed it has a lysine residue in an aromatic ring coupled to a sugar molecule reminiscent of advanced glycation end-products (AGEs). We hypothesized and demonstrate here that the unknown sugar is a N-linked glucuronic acid. LW-1 was extracted and highly purified from ~99 g insoluble skin collagen obtained at autopsy from patients with diabetes/ESRD using multiple rounds of proteolytic digestion and purification by liquid chromatography (LC). Advanced NMR techniques (1H-NMR, 13C-NMR, 1H-13C HSQC, 1H-1H TOCSY, 1H-13C HMBC) together with LC-mass spectrometry (MS) revealed a loss of 176 amu (atomic mass unit) unequivocally point to the presence of a glucuronic acid moiety in LW-1. To confirm this data, LW-1 was incubated with ß-glycosidases (glucosidase, galactosidase, glucuronidase) and products were analyzed by LC-MS. Only glucuronidase could cleave the sugar from the parent molecule. These results establish LW-1 as a glucuronide, now named glucuronidine, and for the first time raise the possible existence of a "glucuronidation pathway of diabetic complications". Future research is needed to rigorously probe this concept and elucidate the molecular origin and biological source of a circulating glucuronidine aglycone.
Assuntos
Colágeno/metabolismo , Complicações do Diabetes/metabolismo , Ácido Glucurônico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Colágeno/química , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Pessoa de Meia-Idade , Pele/metabolismoRESUMO
The photoreceptor outer segment (OS) is comprised of two compartments: plasma membrane (PM) and disk membranes. It is unknown how the PM renewal is coordinated with that of the disk membranes. Here we visualized the localization and trafficking process of rod cyclic nucleotide-gated channel α-subunit (CNGA1), a PM component essential for phototransduction. The localization was visualized by fusing CNGA1 to a fluorescent protein Dendra2 and expressing in Xenopus laevis rod photoreceptors. Dendra2 allowed us to label CNGA1 in a spatiotemporal manner and therefore discriminate between old and newly trafficked CNGA1-Dendra2 in the OS PM. Newly synthesized CNGA1 was preferentially trafficked to the basal region of the lateral OS PM where newly formed and matured disks are also added. Unique trafficking pattern and diffusion barrier excluded CNGA1 from the PM domains, which are the proposed site of disk membrane maturation. Such distinct compartmentalization allows the confinement of cyclic nucleotide-gated channel in the PM, while preventing the disk membrane incorporation. Cytochalasin D and latrunculin A treatments, which are known to disrupt F-actin-dependent disk membrane morphogenesis, prevented the entrance of newly synthesized CNGA1 to the OS PM, but did not prevent the entrance of rhodopsin and peripherin/rds to the membrane evaginations believed to be disk membrane precursors. Uptake of rhodopsin and peripherin/rds coincided with the overgrowth of the evaginations at the base of the OS. Thus F-actin is essential for the trafficking of CNGA1 to the ciliary PM, and coordinates the formations of disk membrane rim region and OS PM.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Morfogênese/fisiologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Animais Geneticamente Modificados , Membrana Celular/ultraestrutura , Quelantes/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endopeptidases/farmacologia , Técnicas In Vitro , Larva , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Morfogênese/genética , Fotodegradação , Transporte Proteico/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Rodopsina/genética , Rodopsina/metabolismo , XenopusRESUMO
It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway. When expressed in ciliated hTERT-RPE1 human cell line, P/rd is localized to cilia. Cilium trafficking of P/rds was sustained even when the Golgi functions, including trans-Golgi-mediated conventional secretion, were inhibited by the small molecules brefeldin A, 30N12, and monensin. The unconventional cilia targeting of P/rds is dependent on COPII-mediated exit from the ER, but appears to be independent of GRASP55-mediated secretion. The regions in the C-terminal tail of P/rds are essential for this unconventional trafficking. In the absence of the region required for cilia targeting, P/rds was prohibited from entering the secretory pathways and was retained in the Golgi apparatus. A region essential for this Golgi retention was also found in the C-terminal tail of P/rds and supported the cilia targeting of P/rds mediated by unconventional secretion. In ciliated cells, including bovine and Xenopus laevis rod photoreceptors, P/rds was robustly sensitive to endoglycosidase H, which is consistent with its bypassing the medial Golgi and traversing the unconventional secretory pathway. Because rhodopsin is known to traffic through conventional secretion, this study of P/rds suggests that both conventional secretion and unconventional secretion need to cooperate for the renewal of the photoreceptor sensory cilium.
Assuntos
Cílios/metabolismo , Periferinas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Via Secretória/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Bovinos , Linhagem Celular , Cílios/genética , Humanos , Periferinas/genética , Transporte Proteico/fisiologia , Xenopus laevisRESUMO
In the past few decades, fluorescent proteins have revolutionized the field of cell biology. Phototransformable fluorescent proteins are capable of changing their excitation and emission spectra after being exposed to specific wavelength(s) of light. The majority of phototransformable fluorescent proteins have originated from marine organisms. Genetic engineering of these proteins has made available many choices for different colors, modes of conversion, and other biophysical properties. Their phototransformative property has allowed the highlighting and tracking of subpopulations of cells, organelles, and proteins in living systems. Furthermore, phototransformable fluorescent proteins have offered new methods for superresolution fluorescence microscopy and optogenetics manipulation of proteins. One of the major advantages of phototransformable fluorescent proteins is their applicability for visualizing newly synthesized proteins that are en route to their final destinations. In this paper, we will discuss the biological applications of phototransformable fluorescent proteins with special emphasis on the application of tracking membrane proteins in vertebrate photoreceptor cells.
Assuntos
Células/citologia , Luz , Proteínas Luminescentes/metabolismo , Animais , Técnicas Biossensoriais , Células/metabolismo , Células/efeitos da radiação , Humanos , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiaçãoRESUMO
Rhodopsin is a cilia-specific GPCR essential for vision. Rhodopsin mislocalization is associated with blinding diseases called retinal ciliopathies. The mechanism by which rhodopsin mislocalizes in rod photoreceptor neurons is not well understood. Therefore, we investigated the roles of trafficking signals in rhodopsin mislocalization. Rhodopsin and its truncation mutants were fused to a photoconvertible fluorescent protein, Dendra2, and expressed in Xenopus laevis rod photoreceptors. Photoconversion of Dendra2 causes a color change from green to red, enabling visualization of the dynamic events associated with rhodopsin trafficking and renewal. We found that rhodopsin mislocalization is a facilitated process for which a signal located within 322-326 aa (CCGKN) is essential. An additional signal within 327-336 aa further facilitated the mislocalization. This collective mistrafficking signal confers toxicity to rhodopsin and causes mislocalization when the VXPX cilia-targeting motif is absent. We also determined that the VXPX motif neutralizes this mistrafficking signal, enhances ciliary targeting at least 10-fold, and accelerates trafficking of post-Golgi vesicular structures. In the absence of the VXPX motif, mislocalized rhodopsin is actively cleared through secretion of vesicles into the extracellular milieu. Therefore, this study unveiled the multiple roles of trafficking signals in rhodopsin localization and renewal.
Assuntos
Transporte Proteico/genética , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Anuros , Olho/anatomia & histologia , Feminino , Regulação da Expressão Gênica/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Modelos Moleculares , Mutação/genética , Técnicas de Cultura de Órgãos , Estimulação Luminosa , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Retina/citologia , Retina/metabolismo , Retina/ultraestrutura , Rodopsina/genética , Transdução de Sinais/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Xenopus laevisRESUMO
Advanced glycation end products (AGEs) represent a family of protein, peptide, amino acid, nucleic acid and lipid adducts formed by the reaction of carbonyl compounds derived directly or indirectly from glucose, ascorbic acid and other metabolites such as methylglyoxal. AGE formation in diabetes is of growing importance for their role as markers and potential culprits of diabetic complications, in particular retinopathy, nephropathy and neuropathy. Development of sensitive and specific assays utilizing liquid chromatography mass spectrometry with isotope dilution method has made it possible to detect and quantitate non-UV active AGEs such as carboxymethyl-lysine and glucosepane, the most prevalent AGE and protein crosslink of the extracellular matrix. Below we review studies on AGE formation in two skin biopsies obtained near the closeout of the Diabetes Control and Complications Trial (DCCT), one of which was processed in 2011 for assay of novel AGEs. The results of these analyses show that while several AGEs are associated and predict complication progression, the glucose/fructose-lysine/glucosepane AGE axis is one of the most robust markers for microvascular disease, especially retinopathy, in spite of adjustment for past or future average glycemia. Yet overall little biological and clinical information is available on glucosepane, making this review a call for data in a field of growing importance for diabetes and chronic metabolic diseases of aging.
Assuntos
Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Animais , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Complicações do Diabetes/etiologia , Complicações do Diabetes/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Produtos Finais de Glicação Avançada/química , Humanos , Fatores de Risco , Pele/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
Bile acids (BAs) are cholesterol-derived compounds that regulate glucose, lipid, and energy metabolism. Despite their significance in glucose homeostasis, the association between specific BA molecular species and their synthetic pathways with diabetes is unclear. Here, we used a recently validated, stable-isotope dilution, high-performance liquid chromatography with tandem mass spectrometry method to quantify a panel of BAs in fasting plasma from 2,145 study participants and explored structural and genetic determinants of BAs linked to diabetes, insulin resistance, and obesity. Multiple 12α-hydroxylated BAs were associated with diabetes (adjusted odds ratio [aOR] range, 1.3-1.9; P < 0.05 for all) and insulin resistance (aOR range, 1.3-2.2; P < 0.05 for all). Conversely, multiple 6α-hydroxylated BAs and isolithocholic acid (iso-LCA) were inversely associated with diabetes and obesity (aOR range, 0.3-0.9; P < 0.05 for all). Genome-wide association studies revealed multiple genome-wide significant loci linked with 9 of the 14 diabetes-associated BAs, including a locus for iso-LCA (rs11866815). Mendelian randomization analyses showed genetically elevated deoxycholic acid levels were causally associated with higher BMI, and iso-LCA levels were causally associated with reduced BMI and diabetes risk. In conclusion, comprehensive, large-scale, quantitative mass spectrometry and genetics analyses show circulating levels of multiple structurally specific BAs, especially DCA and iso-LCA, are clinically associated with and genetically linked to obesity and diabetes.
Assuntos
Ácidos e Sais Biliares , Estudo de Associação Genômica Ampla , Resistência à Insulina , Obesidade , Humanos , Ácidos e Sais Biliares/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Obesidade/genética , Obesidade/sangue , Resistência à Insulina/genética , Adulto , Diabetes Mellitus/sangue , Diabetes Mellitus/genética , Diabetes Mellitus/epidemiologia , Polimorfismo de Nucleotídeo Único , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/sangue , Idoso , Análise da Randomização MendelianaRESUMO
AIM: Phenylacetylglutamine (PAGln) is a phenylalanine-derived metabolite produced by gut microbiota with mechanistic links to heart failure (HF)-relevant phenotypes. We sought to investigate the prognostic value of PAGln in patients with stable HF. METHODS AND RESULTS: Fasting plasma PAGln levels were measured by stable-isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) in patients with stable HF from two large cohorts. All-cause mortality was assessed at 5-year follow-up in the Cleveland cohort, and HF, hospitalization, or mortality were assessed at 3-year follow-up in the Berlin cohort. Within the Cleveland cohort, median PAGln levels were 4.2 (interquartile range [IQR] 2.4-6.9) µM. Highest quartile of PAGln was associated with 3.09-fold increased mortality risk compared to lowest quartile. Following adjustments for traditional risk factors, as well as race, estimated glomerular filtration rate, amino-terminal pro-B-type natriuretic peptide, high-sensitivity C-reactive protein, left ventricular ejection fraction, ischaemic aetiology, and HF drug treatment, elevated PAGln levels remained predictive of 5-year mortality in quartile comparisons (adjusted hazard ratio [HR] [95% confidence interval, CI] for Q4 vs Q1: 1.64 [1.07-2.53]). In the Berlin cohort, a similar distribution of PAGln levels was observed (median 3.2 [IQR 2.0-4.8] µM), and PAGln levels were associated with a 1.92-fold increase in 3-year HF hospitalization or all-cause mortality risk (adjusted HR [95% CI] for Q4 vs Q1: 1.92 [1.02-3.61]). Prognostic value of PAGln appears to be independent of trimethylamine N-oxide levels. CONCLUSION: High levels of PAGln are associated with adverse outcomes independent of traditional cardiac risk factors and cardio-renal risk markers.