Effects of changing from a diet with saturated fat to a diet with n-6 polyunsaturated fat on the serum metabolome in relation to cardiovascular disease risk factors

Purpose Replacing saturated fatty acids (SFA) with polyunsaturated fatty acids (PUFA) is associated with a reduced risk of cardiovascular disease. Yet, the changes in the serum metabolome after this replacement is not well known. Therefore, the present study aims to identify the metabolites differentiating diets where six energy percentage SFA is replaced with PUFA and to elucidate the association of dietary metabolites with cardiometabolic risk markers. Methods In an 8-week, double-blind, randomized, controlled trial, 99 moderately hyper-cholesterolemic adults (25–70 years) were assigned to a control diet (C-diet) or an experimental diet (Ex-diet). Both groups received commercially available food items with different fatty acid compositions. In the Ex-diet group, products were given where SFA was replaced mostly with n-6 PUFA. Fasting serum samples were analysed by untargeted ultra-performance liquid chromatography high-resolution mass spectrometry (UPLC-HRMS). Pre-processed data were analysed by double cross-validated Partial Least-Squares Discriminant Analysis (PLS-DA) to detect features differentiating the two diet groups. Results PLS-DA differentiated the metabolic profiles of the Ex-diet and the C-diet groups with an area under the curve of 0.83. The Ex-diet group showed higher levels of unsaturated phosphatidylcholine plasmalogens, an unsaturated acylcarnitine, and a secondary bile acid. The C-diet group was characterized by odd-numbered phospholipids and a saturated acylcarnitine. The Principal Component analysis scores of the serum metabolic profiles characterizing the diets were significantly associated with low-density lipoprotein cholesterol, total cholesterol, and triglyceride levels but not with glycaemia. Conclusion The serum metabolic profiles confirmed the compliance of the participants based on their diet-specific metabolome after replacing SFA with mostly n-6 PUFA. The participants' metabolic profiles in response to the change in diet were associated with cardiovascular disease risk markers. This study was registered at clinicaltrials.gov as NCT 01679496 on September 6th 2012.

Metabolic Soft Spot and Pharmacokinetics: Functionalization of C-3 Position of an Eph–Ephrin Antagonist Featuring a Bile Acid Core as an Effective Strategy to Obtain Oral Bioavailability in Mice

UniPR129, an L-β-homotryptophan conjugate of the secondary bile acid lithocholic acid (LCA), acts as an effective protein-protein interaction (PPI) inhibitor of the Eph–ephrin system but suffers from a poor oral bioavailability in mice. To improve UniPR129 bioavailability, a metabolic soft spot, i.e., the 3α-hydroxyl group on the LCA steroidal ring, was functionalized to 3-hydroxyimine. In vitro metabolism of UniPR129 and 3-hydroxyimine derivative UniPR500 was compared in mouse liver subcellular fractions, and main metabolites were profiled by high resolution (HR-MS) and tandem (MS/MS) mass spectrometry. In mouse liver microsomes (MLM), UniPR129 was converted into several metabolites: M1 derived from the oxidation of the 3-hydroxy group to 3-oxo, M2–M7, mono-hydroxylated metabolites, M8–M10, di-hydroxylated metabolites, and M11, a mono-hydroxylated metabolite of M1. Phase II reactions were only minor routes of in vitro biotransformation. UniPR500 shared several metabolic pathways with parent UniPR129, but it showed higher stability in MLM, with a half-life (t1/2) of 60.4 min, if compared to a t1/2 = 16.8 min for UniPR129. When orally administered to mice at the same dose, UniPR500 showed an increased systemic exposure, maintaining an in vitro valuable pharmacological profile as an EphA2 receptor antagonist and an overall improvement in its physico-chemical profile (solubility, lipophilicity), if compared to UniPR129. The present work highlights an effective strategy for the pharmacokinetic optimization of aminoacid conjugates of bile acids as small molecule Eph–ephrin antagonists.

Duodenal mucosal resurfacing with GLP-1 receptor agonism increases postprandial unconjugated bile acid in patients with insulin-dependent type 2 diabetes

Introduction: Duodenal Mucosal Resurfacing (DMR) is a new endoscopic ablation technique aimed at improving glycemia and metabolic control in patients with type 2 diabetes mellitus (T2DM). DMR appears to improve insulin resistance, which is the root cause of T2DM, but its mechanism of action is largely unknown. Bile acids function as intestinal signalling molecules in glucose and energy metabolism via the activation of farnesoid X receptor and secondary signalling (e.g. via fibroblast growth factor 19[FGF19]), and are linked to metabolic health. Methods: We investigated the effect of DMR and GLP-1 on postprandial bile acid responses in 16 patients with insulin-dependent T2DM, using mixed meal tests performed at baseline and six months after the DMR procedure. Results: The combination treatment allowed discontinuation of insulin treatment in 11/16 (69%) of patients while improving glycaemic and metabolic health. We found increased postprandial unconjugated bile acid responses (all p<0.05), an overall increased secondary bile acid response (p=0.036) and a higher 12α-hydroxylated:non12α-hydroxylated ratio (p<0.001). Total bile acid concentrations were unaffected by the intervention. Postprandial FGF19 and 7-alpha-hydroxy-4-cholesten-3-one (C4) concentrations decreased post-intervention (both p<0.01). Conclusion and discussion: Our study demonstrates that DMR with GLP-1 modulates the postprandial bile acid response. The alterations in postprandial bile acid responses may be the result of changes in the microbiome, ileal bile acid uptake and improved insulin sensitivity. Controlled studies are needed to elucidate the mechanism linking the combination treatment to metabolic health and bile acids.

The Potential Utility of Prebiotics to Modulate Alzheimer’s Disease: A Review of the Evidence

The gut microbiome has recently emerged as a critical modulator of brain function, with the so-called gut-brain axis having multiple links with a variety of neurodegenerative and mental health conditions, including Alzheimer’s Disease (AD). Various approaches for modulating the gut microbiome toward compositional and functional states that are consistent with improved cognitive health outcomes have been documented, including probiotics and prebiotics. While probiotics are live microorganisms that directly confer beneficial health effects, prebiotics are oligosaccharide and polysaccharide structures that can beneficially modulate the gut microbiome by enhancing the growth, survival, and/or function of gut microbes that in turn have beneficial effects on the human host. In this review, we discuss evidence showing the potential link between gut microbiome composition and AD onset or development, provide an overview of prebiotic types and their roles in altering gut microbial composition, discuss the effectiveness of prebiotics in regulating gut microbiome composition and microbially derived metabolites, and discuss the current evidence linking prebiotics with health outcomes related to AD in both animal models and human trials. Though there is a paucity of human clinical trials demonstrating the effectiveness of prebiotics in altering gut microbiome-mediated health outcomes in AD, current evidence highlights the potential of various prebiotic approaches for beneficially altering the gut microbiota or gut physiology by promoting the production of butyrate, indoles, and secondary bile acid profiles that further regulate gut immunity and mucosal homeostasis, which are associated with beneficial effects on the central immune system and brain functionality.

Circulating Metabolomics Suggest Neutropenic Fever As a Metabolic Derangement Related to Intestinal Tissue Damage and Gut Dysbiosis

Introduction: Despite antibiotic prophylaxis, most patients with acute myeloid leukemia (AML) develop neutropenic fever (NF) during intensive chemotherapy, suggesting a non-infectious etiology in many cases. In addition, escalated antibiotics used to treat NF increase the risk of Clostridioides difficile infection, promote pathogen colonization, prolong hospitalization, and increase healthcare costs. More effective prevention of NF, preferably using non-antibiotic approaches, is needed. We hypothesized that a longitudinal analysis of the circulating metabolome may reveal novel aspects of NF pathogenesis and identify potential targets for new preventative interventions. Methods: We analyzed 128 longitudinal serum samples from 17 intensively treated adult patients with AML between hospital admission and day 28 of chemotherapy. Samples were collected between 6-8 AM every Mon and Thu. Samples were subjected to ultrahigh performance liquid chromatography-tandem mass spectrometry. Results: All patients developed NF. A total of 1,031 metabolites were identified. Principal components analysis of the circulating metabolome could not resolve individual patients (Fig. 1a). In contrast, pre- vs. post-NF samples were partially clustered (Fig. 1b), suggesting a metabolomic shift associated with NF. After correcting for false discovery, 26 and 27 metabolites were higher in pre- and post-NF samples, respectively (q&lt;0.05, |log fold-change| &gt;1; Fig. 1c). The most significant metabolite that was different between post- and pre-NF samples was citrulline, with a mean concentration ratio of 0.65 between the two groups (q&lt;10-5, Fig. 1c). Citrulline is a known biomarker for total enterocyte mass and its lower levels in post-NF samples indicate intestinal tissue damage as a potential etiology for NF. Another notable metabolite was 3-indoxyl sulfate (3-IS), a tryptophan metabolite and biomarker of gut microbiota diversity and clostridia abundance. 3-IS levels also decreased in post-NF (post vs. pre ratio: 0.45, q=0.02; Fig. 1c), suggesting a protective role for commensal microbiota against NF. Indoles act via the aryl hydrocarbon receptor to repair the intestinal epithelial barrier. Sparse partial least squares discriminant analysis (sPLS-DA) further improved group separation (Fig. 1d). Significantly altered metabolites in the first analysis along with the top 50 metabolites in sPLS-DA were fed into a random forest which generated the final list of 47 metabolites with largest contributions to group separation, including 3-IS and several citrulline metabolites (top 10 metabolites in Table 1). The most frequent metabolites on this list were those in amino acid (n = 17) and lipid (n = 14, including a secondary bile acid) pathways. Conclusions: This first-time analysis of the circulating metabolome in AML patients with NF suggests NF as a metabolic derangement rather than an infectious event in many patients. Augmenting the intestinal epithelium and maintaining a commensal clostridia-rich gut microbiome may help prevent NF. In addition, our list of altered metabolites introduces an unexplored niche for the development of novel, non-antibiotic-based approaches to prevent NF. Figure 1 Figure 1. Disclosures Holtan: Incyte: Consultancy, Research Funding; Generon: Consultancy. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding.

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently shifts the gut microbiome consistent with the progression of steatosis to steatohepatitis with fibrosis

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids including taurolithocholic acid and deoxycholic acid, microbial modified bile acids involved in host bile acid regulation signaling pathways. To investigate the effects of TCDD on the gut microbiota, cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 µg/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and menaquinone biosynthesis genes. Analysis of gut microbiomes from cirrhosis patients identified increased abundance of these pathways as identified in the mouse cecum metagenomic analysis. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.

SARS-CoV-2 infection complicated by intrahepatic cholestasis of pregnancy

This article reports a case of SARS-CoV-2 infection complicated by intrahepatic cholestasis of pregnancy in the second trimester in a South Asian woman. She presented with itchiness and grossly elevated bile acid following SARS-CoV-2 virus infection. Her cholestasis resolved in parallel with her infection while she was being treated with ursodeoxycholic acid. Bile acid is considered harmful to a developing fetus and is associated with adverse pregnancy outcomes, particularly stillbirth. Ursodeoxycholic acid is still commonly used in the treatment of these patients despite controversy about its value. It has anti-inflammatory properties and there have been suggestions that this could benefit patients with SARS-CoV-2. Elevated levels of primary bile acids (cholic and chenodeoxycholic acid) are considered harmful to a developing fetus, and are associated with adverse pregnancy outcomes, in particular, stillbirth. The secondary bile acid, ursodeoxycholic acid, is thought to be beneficial by reducing cholesterol production in the liver. This clinical case study highlights a diagnostic enigma in the management of this unique clinical case presentation.

Lithocholic Acid Induces miR21, Promoting PTEN Inhibition via STAT3 and ERK-1/2 Signaling in Colorectal Cancer Cells

Micro-RNA-21 (miR-21) is a vital regulator of colorectal cancer (CRC) progression and has emerged as a potential therapeutic target in CRC treatment. Our study using real-time PCR assay found that a secondary bile acid, lithocholic acid (LCA), stimulated the expression of miR21 in the CRC cell lines. Promoter activity assay showed that LCA strongly stimulated miR21 promoter activity in HCT116 cells in a time- and dose-dependent manner. Studies of chemical inhibitors and miR21 promoter mutants indicated that Erk1/2 signaling, AP-1 transcription factor, and STAT3 are major signals involved in the mechanism of LCA-induced miR21 in HCT116 cells. The elevation of miR21 expression was upstream of the phosphatase and tensin homolog (PTEN) inhibition, and CRC cell proliferation enhancement that was shown to be possibly mediated by PI3K/AKT signaling activation. This study is the first to report that LCA affects miR21 expression in CRC cells, providing us with a better understanding of the cancer-promoting mechanism of bile acids that have been described as the very first promoters of CRC progression.

Prophylactic inhibition of colonization by Streptococcus pneumoniae with the secondary bile acid metabolite deoxycholic acid

Streptococcus pneumoniae (Spn) colonizes the nasopharynx of children and the elderly but also kills millions worldwide yearly. The secondary bile acid metabolite, deoxycholic acid (DoC), affects the viability of human pathogens but also plays multiple roles in host physiology. We assessed in vitro the antimicrobial activity of DoC and investigated its potential to eradicate Spn colonization using a model of human nasopharyngeal colonization and an in vivo mouse model of colonization. At a physiological concentration DoC (0.5 mg/ml; 1.27 mM) killed all tested Spn strains (N=48) two hours post-inoculation. The model of nasopharyngeal colonization showed that DoC eradicated colonization by Spn strains as soon as 10 min post-exposure. The mechanism of action did not involve activation of autolysis since the autolysis-defective double mutants Δ lytA Δ lytC and ΔspxBΔlctO were as susceptible to DoC as was the wild-type (WT). Oral streptococcal species (N=20), however, were not susceptible to DoC (0.5 mg/ml). Unlike trimethoprim, whose spontaneous resistance frequency (srF) for TIGR4 or EF3030 was ≥1x10 −9 , no spontaneous resistance was observed with DoC (srF≥1x10- 12 ). Finally, the efficacy of DoC to eradicate Spn colonization was assessed in vivo using a topical route via intranasal (i.n.) administration and as a prophylactic treatment. Mice challenged with Spn EF3030 carried a median of 4.05x10 5 cfu/ml four days post-inoculation compared to 6.67x10 4 cfu/ml for mice treated with DoC. Mice in the prophylactic group had a ∼99% reduction of the pneumococcal density (median, 2.61 x10 3 cfu/ml). Thus, DoC, an endogenous human bile salt, has therapeutic potential against Spn.