Microbiota Modification with Probiotics Induces Hepatic Bile Acid Synthesis via Downregulation of the Fxr-Fgf15 Axis in Mice

Gut microbiota influences host health status by providing trophic, protective, and metabolic functions, including bile acid (BA) biotransformation. Microbial imprinting on BA signature modifies pool size and hydrophobicity, thus contributing to BA enterohepatic circulation. Microbiota-targeted therapies are now emerging as effective strategies for preventing and/or treating gut-related diseases. Here, we show that gut microbiota modulation induced by VSL#3 probiotics enhances BA deconjugation and fecal excretion in mice. These events are associated with changes in ileal BA absorption, repression of the enterohepatic farnesoid X receptor-fibroblast growth factor 15 (FXR-FGF15) axis, and increased hepatic BA neosynthesis. Treatment with a FXR agonist normalized fecal BA levels in probiotic-administered mice, whereas probiotic-induced alterations in BA metabolism are abolished upon FXR and FGF15 deficiency. Our data provide clear in vivo evidence that VSL#3 probiotics promote ileal BA deconjugation with subsequent fecal BA excretion and induce hepatic BA neosynthesis via downregulation of the gut-liver FXR-FGF15 axis

Polyunsaturated fatty acids omega 3: modulation of cholesterol metabolism

Le malattie cardiovascolari sono la prima causa di mortalità nel mondo ed elevati livelli plasmatici di colesterolo sono uno dei più importanti fattori di rischio cardiovascolare. Studi epidemiologici e trials clinici hanno dimostrato che cambiamenti nello stile di vita, in primis la dieta, possono contribuire positivamente alla prevenzione cardiovascolare. Le proprietà cardioprotettive degli acidi grassi omega 3, acidi eicosapentenoico e docosaesaenoico (EPA e DHA), sono ampiamente documentate sia nell’uomo che in modelli animali. Le fonti principali di EPA e DHA sono il pesce e l’olio di pesce; nei paesi industrializzati il consumo di pesce è tuttavia limitato e fonti alternative di EPA e DHA sono necessarie. L’acido alfa-linolenico (ALA) è il precursore metabolico di EPA e DHA ed è l’acido grasso omega 3 più abbondante nella dieta dei paesi industrializzati. Questo lavoro di tesi è stato mirato allo studio del potenziale ipocolesterolemico e ateroprotettivo dell’ALA in un modello murino di aterosclerosi. I nostri dati indicano che l'ALA è capace di limitare l’accumulo plasmatico ed epatico di colesterolo quando confrontato con acidi grassi monoinsaturi (acido oleico). L’ALA si accumula nei lipidi neutrali, quali colesterolo estere e trigliceridi, mentre il suo metabolita EPA nei fosfolipidi. Successivamente abbiamo valutato se il potenziale ipocolesterolemico dell’ALA e l’arricchimento dei fosfolipidi con EPA si traducessero in ateroprotezione. Diete ricche in ALA sono state confrontate con diete ricche in acido oleico e olio di pesce. L’ALA, pur arricchendo sia i lipidi plasmatici ed epatici con EPA e DHA, non ha alterato il metabolismo del colesterolo così efficacemente quanto l’EPA e il DHA provenienti dall’olio di pesce. In questo lavoro di ricerca abbiamo confrontato in modo diretto l’EPA e il DHA di origine esogena (olio di pesce) con quelli sintetizzati per via endogena dall’ALA e abbiamo dimostrato che non tutti gli acidi grassi omega 3 sono ugualmente protettivi in un modello murino di aterosclerosi

Targeted Deletion of Hepatocyte ABCA1 Leads to Very Low Density Lipoprotein Triglyceride Overproduction and Low Density Lipoprotein Hypercatabolism

Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism

Hepatic ACAT2 Knock Down Increases ABCA1 and Modifies HDL Metabolism in Mice

<div><p>Objectives</p><p>ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.</p><p>Design</p><p>WT and LXRα/β double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet.</p><p>Results</p><p>ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE.</p><p>Conclusions</p><p>The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.</p></div

Dietary n-3 LCPUFA from fish oil but not α-linolenic acid-derived LCPUFA confers atheroprotection in mice[S]

The atheroprotective potential of n-3 α-linolenic acid (ALA) has not yet been fully determined, even in murine models of atherosclerosis. We tested whether ALA-derived, n-3 long chain polyunsaturated fatty acids (LCPUFA) could offer atheroprotection in a dose-dependent manner. Apolipoprotein B (ApoB)100/100LDLr−/− mice were fed with diets containing two levels of ALA from flaxseed oil for 16 weeks. Fish oil- and cis-monounsaturated-fat-enriched diets were used as positive and negative controls, respectively. The mice fed cis-monounsaturated fat and ALA-enriched diets exhibited equivalent plasma total cholesterol (TPC) and LDL-cholesterol (LDL-c) levels; only mice fed the fish-oil diet had lower TPC and LDL-c concentrations. Plasma LDL-CE fatty acid composition analysis showed that ALA-enriched diets lowered the percentage of atherogenic cholesteryl oleate compared with cis-monounsaturated-fat diet (44% versus 55.6%) but not as efficiently as the fish-oil diet (32.4%). Although both ALA and fish-oil diets equally enriched hepatic phospholipids with eicosapentaenoic acid (EPA) and ALA-enriched diets lowered hepatic cholesteryl ester (CE) levels compared with cis-monounsaturated-fat diet, only fish oil strongly protected from atherosclerosis. These outcomes indicate that dietary n-3 LCPUFA from fish oil and n-3 LCPUFA (mostly EPA) synthesized endogenously from ALA were not equally atheroprotective in these mice

Inhibition of Stearoyl-CoA Desaturase 1 (SCD1) Dissociates Insulin Resistance and Obesity From Atherosclerosis

Background—Stearoyl-CoA Desaturase 1 (SCD1) is a well-known enhancer of the metabolic syndrome. The purpose of the present study was to investigate the role of SCD1 in lipoprotein metabolism and atherosclerosis progression. Methods and Results—Antisense oligonucleotides were used to inhibit SCD1 in a mouse model of hyperlipidemia and atherosclerosis (LDLr-/-Apob100/100). In agreement with previous reports, inhibition of SCD1 protected against diet-induced obesity, insulin resistance, and hepatic steatosis. However, unexpectedly SCD1 inhibition strongly promoted aortic atherosclerosis, which could not be reversed by dietary oleate. Further analyses revealed that SCD1 inhibition promoted accumulation of saturated fatty acids in plasma and tissues, reduced plasma triglyceride, yet had little impact on LDL cholesterol. Since dietary SFAs have been shown to promote inflammation through toll-like receptor 4 (TLR4), we examined macrophage TLR4 function. Interestingly, SCD1 inhibition resulted in alterations in macrophage membrane lipid composition and marked hypersensitivity to TLR4 agonists. Conclusions—This study demonstrates that atherosclerosis can occur independently of obesity and insulin resistance, and argues against SCD1 inhibition as a safe therapeutic target for the metabolic syndrome

Hepatic expression of genes involved in lipid metabolism.

<p>mRNA was quantified by real-time RT-PCR. Data were standardized for <i>Tfiib</i> mRNA expression, and normalized to the expression of each gene in WT Ctrl in each single experiment. mRNA data are expressed as average ± SEM (n = 6–8). Mann Whitney test, *p<0.05, † p<0.01, ‡ p<0.005.</p

Hepatic ACAT2 knock-down modifies HDL subclasses.

<p>HDL subclasses were separated by non-denaturing two-dimensional gel electrophoresis and transferred onto a nitrocellulose membrane, on which lipoproteins were detected with an anti-mouse apoA-I or apoE antibody; a representative animal in each group is shown. Preβ-HDL content was calculated as percentage of total apoA-I signal from 2D-electrophoresis developed against apoA-I. Error bars represent the median. Mann Whitney test, * p<0.05.</p

Lipid quantification in serum and HDL lipoproteins.

<p>Serum lipoproteins were separated by size exclusion chromatography, and the concentration of total (TC) and unesterified (UC) cholesterol, phospholipids (PL), and triglycerides (TG) was determined by a system for on-line detection. Data are expressed as average ± SEM (n = 6–8). Mann Whitney test, *p<0.05, † p<0.01, ‡ p<0.005.</p

Hepatic ACAT2 down regulation increases the ABCA1 protein independently of the mRNA expression.

<p>Liver membrane proteins were pooled group-wise, loaded, and separated on Tris-Acetate Gels. After transfer onto nitrocellulose membrane, samples were incubated with anti-mouse ABCA1 antibody. ABCA1 band (≈ 250 kD) was detected by chemiluminescence, and signals were plotted by μg-loaded protein. The slope of the curves was calculated by method of least square, and the slope of the ASOctrl group was set equal to 100%. Hepatic <i>Abca1</i> mRNA was quantified by real-time RT-PCR. Data were standardized for <i>Tfiib</i> mRNA expression, and normalized to WT Ctrl in each experiment. In the mRNA data error bars represent the median. Mann Whitney test, ** p< 0.01.</p