RESUMEN
Insufficient expression of steroidogenic acute regulatory-related lipid transfer protein 5 (StarD5) on liver cholesterol/lipid homeostasis is not clearly defined. The ablation of StarD5 was analyzed in mice on a normal or Western diet (WD) to determine its importance in hepatic lipid accumulation and fibrosis compared with wild-type (WT) mice. Rescue experiments in StarD5-/- mice and hepatocytes were performed. In addition to increased hepatic triglyceride (TG)-cholesterol levels, global StarD5-/- mice fed a normal diet displayed reduced plasma triglycerides and liver very low-density lipoprotein (VLDL) secretion as compared with WT counterparts. Insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) scoring were elevated, demonstrating developing insulin resistance (IR). WD-fed StarD5-/- mice upregulated WW domain containing transcription regulator 1 (TAZ or WWTR1) expression with accelerated liver fibrosis when compared with WD-fed WT mice. Suppression of oxysterol 7α-hydroxylase (CYP7B1) coupled with chronic accumulation of toxic oxysterol levels correlated with presentation of fibrosis. "Hepatocyte-selective" StarD5 overexpression in StarD5-/- mice restored expression, reduced hepatic triglycerides, and improved HOMA-IR. Observations in two additional mouse and one human metabolic dysfunction-associated steatotic liver disease (MASLD) model were supportive. The downregulation of StarD5 with hepatic lipid excess is a previously unappreciated physiological function appearing to promote lipid storage for future needs. Conversely, lingering downregulation of StarD5 with prolonged lipid-cholesterol excess accelerates fatty liver's transition to fibrosis; mediated via dysregulation in the oxysterol signaling pathway.NEW & NOTEWORTHY We have found that deletion of the cholesterol transport protein StarD5 in mice leads to an increase in insulin resistance and lipid accumulation due to the upregulation of lipid synthesis and decrease VLDL secretion from the liver. In addition, deletion of StarD5 increased fibrosis when mice were fed a Western diet. This represents a novel pathway of fibrosis development in the liver.
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Resistencia a la Insulina , Cirrosis Hepática , Hígado , Ratones Noqueados , Animales , Humanos , Masculino , Ratones , Colesterol/metabolismo , Colesterol/sangre , Dieta Occidental/efectos adversos , Progresión de la Enfermedad , Hígado Graso/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Metabolismo de los Lípidos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones Endogámicos C57BL , Triglicéridos/metabolismoRESUMEN
CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3ß-hydroxy-5-cholesten-(25R)26-oic acid (3ßHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3ßHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3ßHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1-/- mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3ßHCA levels were maintained at basal levels in ND-fed Cyp7b1-/- mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1-/- mice developed insulin resistance (IR) with subsequent 26HC/3ßHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1-/- mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3ßHCA accumulation. The results suggest 26HC/3ßHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3ßHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.
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Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Colesterol/metabolismo , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Dieta Alta en Grasa , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: To examine whether it is possible to screen for bile acid synthesis disorders (BASDs) including peroxisome biogenesis disorder 1a (PBD1A) and Niemann-Pick type C1 (NPC1) at the time of newborn mass screening by measuring the intermediary metabolites of bile acid (BA) synthesis. METHODS: Patients with 3ß-hydroxy-ΔSuchy et al. (2021)5-C27-steroid dehydrogenase/isomerase (HSD3B7) deficiency (n = 2), 3-oxo-ΔPandak and Kakiyama (n.d.)4-steroid 5ß-reductase (SRD5B1) deficiency (n = 1), oxysterol 7α-hydroxylase (CYP7B1) deficiency (n = 1), PBD1A (n = 1), and NPC1 (n = 2) with available dried blood spot (DBS) samples collected in the neonatal period were included. DBSs from healthy neonates at 4 days of age (n = 1055) were also collected for the control. Disease specific BAs were measured by newly optimized liquid chromatography-tandem mass spectrometry with short run cycle (5-min/run). The results were validated by comparing with those obtained by the conventional condition with longer run cycle (76-min/run). RESULTS: In healthy specimens, taurocholic acid and cholic acid were the two major BAs which constituted approximately 80% in the measured BAs. The disease marker BAs presented <10%. In BASDs, the following BAs were determined for the disease specific markers: Glyco/tauro 3ß,7α,12α-trihydroxy-5-cholenoic acid 3-sulfate for HSD3B7 deficiency (>70%); glyco/tauro 7α,12α-dihydroxy-3-oxo-4-cholenoic acid for SRD5B1 deficiency (54%); tauro 3ß-hydroxy-5-cholenoic acid 3-sulfate for CYP7B1 deficiency (94%); 3α,7α,12α-trihydroxy-5ß-cholestanoic acid for PBD1A (78%); and tauro 3ß,7ß-dihydroxy-5-cholenoic acid 3-sulfate for NPC1 (26%). *The % in the parenthesis indicates the portion found in the patient's specimen. CONCLUSIONS: Early postnatal screening for BASDs, PBD1A and NPC1 is feasible with the described DBS-based method by measuring disease specific BAs. The present method is a quick and affordable test for screening for these inherited diseases.
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Hepatopatías , Síndrome de Zellweger , Recién Nacido , Humanos , Ácidos y Sales Biliares , Tamizaje Neonatal , Esteroides , SulfatosRESUMEN
[Figure: see text].
Asunto(s)
Fosfatasa Alcalina/sangre , Aterosclerosis/metabolismo , Absorción Intestinal , Lipopolisacáridos/sangre , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Antígenos CD36/metabolismo , Proteínas Portadoras/metabolismo , Colon/anatomía & histología , Colon/metabolismo , Dieta Aterogénica/efectos adversos , Proteínas de Transporte de Ácidos Grasos/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas Ligadas a GPI/sangre , Humanos , Mucosa Intestinal , Metabolismo de los Lípidos , Lípidos/análisis , Lípidos/sangre , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Pérdida de PesoRESUMEN
Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1-67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified. Precursor ion scan data from LC/MS suggested the presence of long-chain FA-linked BAs. We chemically synthesized a series of fatty acid 3ß-acyl conjugates of isoDCA and isoLCA as analytical standards and analyzed their fecal profiles from newborns to adults (n = 64) by LC/MS. FA-conjugated isobile acids (FA-isoBAs) were constantly present from 2 years of age to adulthood. C16- and C18-chain FA-isoBA esters were predominantly found regardless of age, but small amounts of acetic acid esters were also found. FA-isoBA concentrations were not correlated to fecal FA concentrations. Interestingly, there were some adults who did not have FA-isoBAs. Gut bacteria involved in the production of FA-isoBAs have not been identified yet. The present study provides insight into the establishment of early gut microbiota and the interactive development of esterified BAs.The contribution of FA-isoBAs to gut physiology and their role in pathophysiologic conditions such as inflammatory bowel disease are currently under investigation.
Asunto(s)
Ácidos y Sales Biliares , Hidroxiácidos , Recién Nacido , Adulto , Humanos , Ácidos y Sales Biliares/análisis , Hidroxiácidos/análisis , Heces/química , Ácidos Grasos , Ácido Litocólico/análisis , EtanolRESUMEN
Oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic pathway" of cholesterol metabolism. Previously, we demonstrated that an inability to upregulate CYP7B1 in the setting of insulin resistance leads to the accumulation of cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) that initiate and promote hepatocyte injury; followed by an inflammatory response. The current study demonstrates that dietary coffee improves insulin resistance and restores Cyp7b1 levels in a well-characterized Western diet (WD)-induced nonalcoholic fatty liver disease (NAFLD) mouse model. Ingestion of a WD containing caffeinated (regular) coffee or decaffeinated coffee markedly reduced the serum ALT level and improved insulin resistance. Cyp7b1 mRNA and protein levels were preserved at normal levels in mice fed the coffee containing WD. Additionally, coffee led to upregulated steroid sulfotransferase 2b1 (Sult2b1) mRNA expression. In accordance with the response in these oxysterol metabolic genes, hepatocellular 26HC levels were maintained at physiologically low levels. Moreover, the current study provided evidence that hepatic Cyp7b1 and Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, hepatocyte nuclear factor (HNF)-4α. We conclude coffee achieves its beneficial effects through the modulation of insulin resistance. Both decaffeinated and caffeinated coffee had beneficial effects, demonstrating caffeine is not fundamental to this effect. The effects of coffee feeding on the insulin-HNF4α-Cyp7b1 signaling pathway, whose dysregulation initiates and contributes to the onset and progression of NASH as triggered by insulin resistance, offer mechanistic insight into approaches for the treatment of NAFLD.NEW & NOTEWORTHY This study demonstrated dietary coffee prevented the accumulation of hepatic oxysterols by maintaining Cyp7b1/Sult2b1 expression in a diet-induced NAFLD mice model. Lowering liver oxysterols markedly reduced inflammation in the coffee-ingested mice. Caffeine is not fundamental to this effect. In addition, this study showed Cyp7b1/Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, HNF4α. The insulin-HNF4α-Cyp7b1/Sult2b1 signaling pathway, which directly correlates to the onset of NASH triggered by insulin resistance, offers insight into approaches for NAFLD treatment.
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Hepatitis , Resistencia a la Insulina , Insulinas , Enfermedad del Hígado Graso no Alcohólico , Oxiesteroles , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Oxiesteroles/metabolismo , Café/metabolismo , Cafeína/farmacología , Cafeína/metabolismo , Hígado/metabolismo , Modelos Animales de Enfermedad , Colesterol/metabolismo , Hepatitis/metabolismo , Factores Nucleares del Hepatocito/metabolismo , ARN Mensajero/metabolismo , Insulinas/metabolismo , Familia 7 del Citocromo P450/metabolismo , Esteroide Hidroxilasas/metabolismoRESUMEN
BACKGROUND: Berberine (BBR) is a plant-based nutraceutical that has been used for millennia to treat diarrheal infections and in contemporary medicine to improve patient lipid profiles. Reduction in lipids, particularly cholesterol, is achieved partly through up-regulation of bile acid synthesis and excretion into the gastrointestinal tract (GI). The efficacy of BBR is also thought to be dependent on structural and functional alterations of the gut microbiome. However, knowledge of the effects of BBR on gut microbiome communities is currently lacking. Distinguishing indirect effects of BBR on bacteria through altered bile acid profiles is particularly important in understanding how dietary nutraceuticals alter the microbiome. RESULTS: Germfree mice were colonized with a defined minimal gut bacterial consortium capable of functional bile acid metabolism (Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides distasonis, Bilophila wadsworthia, Clostridium hylemonae, Clostridium hiranonis, Blautia producta; B4PC2). Multi-omics (bile acid metabolomics, 16S rDNA sequencing, cecal metatranscriptomics) were performed in order to provide a simple in vivo model from which to identify network-based correlations between bile acids and bacterial transcripts in the presence and absence of dietary BBR. Significant alterations in network topology and connectivity in function were observed, despite similarity in gut microbial alpha diversity (P = 0.30) and beta-diversity (P = 0.123) between control and BBR treatment. BBR increased cecal bile acid concentrations, (P < 0.05), most notably deoxycholic acid (DCA) (P < 0.001). Overall, analysis of transcriptomes and correlation networks indicates both bacterial species-specific responses to BBR, as well as functional commonalities among species, such as up-regulation of Na+/H+ antiporter, cell wall synthesis/repair, carbohydrate metabolism and amino acid metabolism. Bile acid concentrations in the GI tract increased significantly during BBR treatment and developed extensive correlation networks with expressed genes in the B4PC2 community. CONCLUSIONS: This work has important implications for interpreting the effects of BBR on structure and function of the complex gut microbiome, which may lead to targeted pharmaceutical interventions aimed to achieve the positive physiological effects previously observed with BBR supplementation.
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Bacterias/clasificación , Proteínas Bacterianas/genética , Berberina/administración & dosificación , Ácidos y Sales Biliares/metabolismo , ARN Ribosómico 16S/genética , Animales , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , Berberina/farmacología , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Masculino , Metabolómica , Ratones , Análisis de Secuencia de ARN , Especificidad de la EspecieRESUMEN
INTRODUCTION: The prevalence of coronary artery disease (CAD) is high among patients with cirrhosis; however, the impact of it on cardiovascular disease (CVD) is not known. The aim of the current study was to evaluate CVD events in patients with cirrhosis and impact of cirrhosis on biomarkers of atherogenesis. METHODS: The study included 682 patients with decompensated cirrhosis referred for liver transplantation (LT) evaluation between 2010 and 2017. All patients were followed until they experienced a CVD event, non-cardiac death, liver transplantation or last follow-up. To evaluate mechanistic link, patients with NASH cirrhosis were propensity matched 1:2 to non-cirrhosis NASH patients and biomarkers of atherogenic risk were compared. RESULTS: The composite CVD outcome occurred in 23(3.4%) patients after a median follow-up period of 585 days (IQR 139, 747). A strong association between presence of any CAD and CVD event was noted (HR = 6.8, 95% CI 2.9, 15.9) that was independent of age, gender, BMI, and MELD score. In competing risk model, the combined rate of LT and non-cardiac was significantly higher when compared to the rate of CVD events. Marker of insulin resistance and inflammation-related markers were similar in patients with and without cirrhosis. Patients with cirrhosis were more likely to have reduced VLDL, sdLDL-C, LDL-C, and triglycerides. Interestingly, patients with cirrhosis had an increase in serum HDL-2, the anti-atherogenic lipoprotein, and adiponectin, a protective serum adipokine. CONCLUSION: The risk of CVD events in patients with cirrhosis is low and may potentially be due to improvement in markers of atherogenic risk.
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Enfermedades Cardiovasculares/sangre , Progresión de la Enfermedad , Mediadores de Inflamación/sangre , Trasplante de Hígado/tendencias , Enfermedad del Hígado Graso no Alcohólico/sangre , Aterosclerosis/sangre , Aterosclerosis/diagnóstico , Enfermedades Cardiovasculares/diagnóstico , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/cirugía , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/cirugía , Estudios RetrospectivosRESUMEN
NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.
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Familia 7 del Citocromo P450/metabolismo , Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Esteroide Hidroxilasas/metabolismo , Animales , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/patología , Oxiesteroles/metabolismoRESUMEN
How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Macrófagos Peritoneales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Células CHO , Células Cultivadas , Colesterol/metabolismo , Cricetulus , Retículo Endoplásmico/metabolismo , Femenino , Homeostasis/genética , Homeostasis/fisiología , Immunoblotting , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , ARN Mensajero , Triglicéridos/metabolismoRESUMEN
Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)-associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B-containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR-/- mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet-induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet-induced dyslipidemia and atherosclerosis.
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Aterosclerosis/etiología , Aterosclerosis/metabolismo , Proteínas Portadoras/metabolismo , Dieta Occidental/efectos adversos , Dislipidemias/etiología , Dislipidemias/metabolismo , Animales , Aterosclerosis/sangre , Aterosclerosis/genética , Proteínas Portadoras/genética , Colesterol/sangre , Colesterol/metabolismo , Dislipidemias/sangre , Dislipidemias/genética , Femenino , Eliminación de Gen , Absorción Intestinal , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Masculino , Ratones , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
BACKGROUND & AIMS: Patients with cirrhosis have intestinal dysbiosis and are prone to itching and skin or soft-tissue infections. The skin microbiome, and its relationship with intestinal microbiome, have not been characterized. We investigated alterations in skin microbiota of patients with cirrhosis and their association with intestinal microbiota and modulators of itch. METHODS: We collected skin swabs at 7 sites and blood and stool samples from 20 healthy individuals (control subjects; mean age, 59 years) and 50 patients with cirrhosis (mean age, 61 years; mean model for end-stage disease score, 12; 20 with decompensation). Skin and stool samples were analyzed by 16s rRNA sequencing and serum samples were analyzed by liquid chromatography and mass spectrometry for levels of bile acids (BAs) and by an ELISA for autotaxin (an itch modulator). Participants were analyzed by the visual analog itch scale (VAS, 0-10,10 = maximum intensity). Data were compared between groups (cirrhosis vs control subjects, with vs without decompensation, VAS 5 or higher vs less than 5). Correlation networks between serum levels of BAs and skin microbiomes were compared between patients with cirrhosis with vs without itching. RESULTS: The composition of microbiomes at all skin sites differed between control subjects and patients with cirrhosis and between patients with compensated vs decompensated cirrhosis. Skin microbiomes of patients with cirrhosis (especially those with decompensation) contained a higher relative abundance of Gammaproteobacteria, Streptococaceae, and Staphylococcaceae, and fecal microbiomes contained a higher relative abundance of Gammaproteobacteria, than control subjects. These bacterial taxa were associated with serum levels of autotaxin and BAs, which were higher in patients with VAS scores ≥5. Based on network statistics, microbial and BA interactions at all sites were more complex in patients with greater levels of itching in the shin, the most common site of itch. CONCLUSIONS: We identified alterations in skin microbiome of patients with cirrhosis (in Gammaproteobacteria, Streptococcaceae, and Staphylococcaceae)-especially in patients with decompensation; fecal microbiomes of patients with cirrhosis had a higher relative abundance of Gammaproteobacteria than control subjects. These specific microbial taxa are associated with itching intensity and itch modulators, such as serum levels of BAs and autotaxin.
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Cirrosis Hepática/complicaciones , Microbiota , Prurito/etiología , Piel/microbiología , Ácidos y Sales Biliares/sangre , Estudios de Casos y Controles , Heces/microbiología , Femenino , Gammaproteobacteria/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Hidrolasas Diéster Fosfóricas/sangre , Staphylococcaceae/aislamiento & purificación , Streptococcaceae/aislamiento & purificación , Escala Visual AnalógicaRESUMEN
Patients with cirrhosis are often exposed to antibiotics that can lead to resistance and fungal overgrowth. The role of fecal microbial transplant (FMT) in restoring gut microbial function is unclear in cirrhosis. In a Food and Drug Administration-monitored phase 1 clinical safety trial, patients with decompensated cirrhosis on standard therapies (lactulose and rifaximin) were randomized to standard-of-care (SOC, no antibiotics/FMT) or 5 days of broad-spectrum antibiotics followed by FMT from a donor enriched in Lachnospiraceae and Ruminococcaceae. Microbial composition (diversity, family-level relative abundances), function (fecal bile acid [BA] deconjugation, 7α-dehydroxylation, short-chain fatty acids [SCFAs]), and correlations between Lachnospiraceae, Ruminococcaceae, and clinical variables were analyzed at baseline, postantibiotics, and 15 days post-FMT. FMT was well tolerated. Postantibiotics, there was a reduced microbial diversity and autochthonous taxa relative abundance. This was associated with an altered fecal SCFA and BA profile. Correlation linkage changes from beneficial at baseline to negative after antibiotics. All of these parameters became statistically similar post-FMT to baseline levels. No changes were seen in the SOC group. CONCLUSION: In patients with advanced cirrhosis on lactulose and rifaximin, FMT restored antibiotic-associated disruption in microbial diversity and function. (Hepatology 2018; 00:000-000).
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Antibacterianos/efectos adversos , Farmacorresistencia Microbiana , Trasplante de Microbiota Fecal/métodos , Microbioma Gastrointestinal/efectos de los fármacos , Cirrosis Hepática/terapia , Anciano , Antibacterianos/administración & dosificación , Humanos , Cirrosis Hepática/patología , Persona de Mediana Edad , Valores de Referencia , Rifaximina/uso terapéutico , Nivel de Atención , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
Liver transplantation (LT) improves daily function and ameliorates gut microbial composition. However, the effect of LT on microbial functionality, which can be related to overall patient benefit, is unclear and could affect the post-LT course. The aims were to determine the effect of LT on gut microbial functionality focusing on endotoxemia, bile acid (BA), ammonia metabolism, and lipidomics. We enrolled outpatient patients with cirrhosis on the LT list and followed them until 6 months after LT. Microbiota composition (Shannon diversity and individual taxa) and function analysis (serum endotoxin, urinary metabolomics and serum lipidomics, and stool BA profile) and cognitive tests were performed at both visits. We enrolled 40 patients (age, 56 ± 7 years; mean Model for End-Stage Liver Disease score, 22.6). They received LT 6 ± 3 months after enrollment and were re-evaluated 7 ± 3 months after LT with a stable course. A significant improvement in cognition with increase in microbial diversity, increase in autochthonous and decrease in potentially pathogenic taxa, and reduced endotoxemia were seen after LT compared with baseline. Stool BAs increased significantly after LT, and there was evidence of greater bacterial action (higher secondary, oxo and iso-BAs) after LT although the levels of conjugated BAs remained similar. There was a reduced serum ammonia and corresponding rise in urinary phenylacetylglutamine after LT. There was an increase in urinary trimethylamine-N-oxide, which was correlated with specific changes in serum lipids related to cell membrane products. The ultimate post-LT lipidomic profile appeared beneficial compared with the profile before LT. In conclusion, LT improves gut microbiota diversity and dysbiosis, which is accompanied by favorable changes in gut microbial functionality corresponding to BAs, ammonia, endotoxemia, lipidomic, and metabolomic profiles. Liver Transplantation 24 752-761 2018 AASLD.
Asunto(s)
Disbiosis/microbiología , Enfermedad Hepática en Estado Terminal/cirugía , Microbioma Gastrointestinal/fisiología , Cirrosis Hepática/cirugía , Trasplante de Hígado , Ácidos y Sales Biliares/sangre , Cognición/fisiología , Disbiosis/sangre , Disbiosis/fisiopatología , Enfermedad Hepática en Estado Terminal/sangre , Enfermedad Hepática en Estado Terminal/microbiología , Endotoxemia/diagnóstico , Endotoxemia/microbiología , Endotoxemia/fisiopatología , Heces/microbiología , Femenino , Humanos , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Hígado/cirugía , Cirrosis Hepática/sangre , Cirrosis Hepática/microbiología , Pruebas de Función Hepática , Masculino , Metaboloma/fisiología , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
BACKGROUND: Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS: Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS: Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS: Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.
Asunto(s)
Alcoholismo/fisiopatología , Disbiosis/fisiopatología , Tracto Gastrointestinal/fisiopatología , Cirrosis Hepática/fisiopatología , Alcoholismo/diagnóstico , Alcoholismo/epidemiología , Disbiosis/diagnóstico , Disbiosis/epidemiología , Endoscopía del Sistema Digestivo/métodos , Femenino , Humanos , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/epidemiología , Masculino , Microbiota/fisiología , Persona de Mediana EdadRESUMEN
The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR(-/-) background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.
Asunto(s)
Aterosclerosis/enzimología , Hígado/enzimología , Esterol Esterasa/genética , Animales , Aterosclerosis/sangre , Ácidos y Sales Biliares/metabolismo , Colesterol/sangre , Femenino , Expresión Génica , Intolerancia a la Glucosa , Humanos , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Esterol Esterasa/biosíntesis , Transgenes , Triglicéridos/metabolismoRESUMEN
We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.
Asunto(s)
Ácidos y Sales Biliares/análisis , Cromatografía Líquida de Alta Presión/métodos , Heces/química , Fibrosis , Cromatografía de Gases y Espectrometría de Masas , Ácidos y Sales Biliares/química , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1ß, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1ß, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.
Asunto(s)
Alcoholismo/complicaciones , Ácidos y Sales Biliares/metabolismo , Enfermedades del Colon/inducido químicamente , Inflamación/etiología , Cirrosis Hepática/complicaciones , Ácidos y Sales Biliares/química , Enfermedades del Colon/patología , Heces/química , Humanos , Inflamación/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Persona de Mediana EdadRESUMEN
OBJECTIVE: Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. APPROACH AND RESULTS: Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. CONCLUSIONS: Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.
Asunto(s)
Aterosclerosis/genética , Aterosclerosis/metabolismo , Hidrolasas de Éster Carboxílico/genética , Receptores de Lipoproteína/genética , Esteroles/metabolismo , Alimentación Animal , Animales , Ácidos y Sales Biliares/metabolismo , Hidrolasas de Éster Carboxílico/deficiencia , Hidrolasas de Éster Carboxílico/metabolismo , HDL-Colesterol/sangre , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Heces/enzimología , Femenino , Homeostasis/fisiología , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Receptores de Lipoproteína/metabolismoRESUMEN
Sterol regulatory element-binding protein-1c (SREBP-1c) increases lipogenesis at the transcriptional level, and its expression is upregulated by liver X receptor α (LXRα). The LXRα/SREBP-1c signaling may play a crucial role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We previously reported that a cholesterol metabolite, 5-cholesten-3ß,25-diol 3-sulfate (25HC3S), inhibits the LXRα signaling and reduces lipogenesis by decreasing SREBP-1c expression in primary hepatocytes. The present study aims to investigate the effects of 25HC3S on lipid homeostasis in diet-induced NAFLD mouse models. NAFLD was induced by feeding a high-fat diet (HFD) in C57BL/6J mice. The effects of 25HC3S on lipid homeostasis, inflammatory responses, and insulin sensitivity were evaluated after acute treatments or long-term treatments. Acute treatments with 25HC3S decreased serum lipid levels, and long-term treatments decreased hepatic lipid accumulation in the NAFLD mice. Gene expression analysis showed that 25HC3S significantly suppressed the SREBP-1c signaling pathway that was associated with the suppression of the key enzymes involved in lipogenesis: fatty acid synthase, acetyl-CoA carboxylase 1, and glycerol-3-phosphate acyltransferase. In addition, 25HC3S significantly reduced HFD-induced hepatic inflammation as evidenced by decreasing tumor necrosis factor and interleukin 1 α/ß mRNA levels. A glucose tolerance test and insulin tolerance test showed that 25HC3S administration improved HFD-induced insulin resistance. The present results indicate that 25HC3S as a potent endogenous regulator decreases lipogenesis, and oxysterol sulfation can be a key protective regulatory pathway against lipid accumulation and lipid-induced inflammation in vivo.