Statin therapy causes gut dysbiosis in mice through a PXR-dependent mechanism.

BACKGROUND: Statins are a class of therapeutics used to regulate serum cholesterol and reduce the risk of heart disease. Although statins are highly effective in removing cholesterol from the blood, their consumption has been linked to potential adverse effects in some individuals. The most common events associated with statin intolerance are myopathy and increased risk of developing type 2 diabetes mellitus. However, the pathological mechanism through which statins cause these adverse effects is not well understood. RESULTS: Using a murine model, we describe for the first time profound changes in the microbial composition of the gut following statin treatment. This remodelling affected the diversity and metabolic profile of the gut microbiota and was associated with reduced production of butyrate. Statins altered both the size and composition of the bile acid pool in the intestine, tentatively explaining the observed gut dysbiosis. As also observed in patients, statin-treated mice trended towards increased fasting blood glucose levels and weight gain compared to controls. Statin treatment affected the hepatic expression of genes involved in lipid and glucose metabolism. Using gene knockout mice, we demonstrated that the observed effects were mediated through pregnane X receptor (PXR). CONCLUSION: This study demonstrates that statin therapy drives a profound remodelling of the gut microbiota, hepatic gene deregulation and metabolic alterations in mice through a PXR-dependent mechanism. Since the demonstrated importance of the intestinal microbial community in host health, this work provides new perspectives to help prevent the statin-associated unintended metabolic effects.

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides.

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.

Spectroscopic characterization of genotoxic chromium(V) peptide complexes: Oxidation of Chromium(III) triglycine, tetraglycine and pentaglycine complexes.

Evidence is growing that metabolites of Cr(III) dietary supplements are partially oxidized to carcinogenic Cr(VI) and Cr(V) in vivo. Hence, we examined oxidations of Cr(III) peptide (triglycine, tetraglycine and pentaglycine) complexes to Cr(VI) and Cr(V) by PbO2 at 37°C and physiological pH values between 3.85 and 7.4. The products were characterized by EPR and UV/Vis spectroscopies and electrospray mass spectrometry. At pH3.85, the monomeric Cr(V) complexes produced were relatively unstable and degraded over min to hr under the acidic conditions. The triglycine and tetraglycine Cr(V) complexes had five-line 14N-superhyperfine-coupled EPR signals; giso, (AN) values 1.9824 (2.44×10-4cm-1) and 1.9825 (2.43×10-4cm-1), respectively. The pentaglycine Cr(V) complex had a seven-line 14N-superhyperfine-coupled EPR signal: giso=1.9844; AN=2.27×10-4cm-1. In phosphate buffer (pH7.4 and 5.85), several Cr(V) intermediates were produced, but Cr(VI) was the end product. For the triglycine, tetraglycine and pentaglycine Cr(V) complexes, the giso (AN, 10-4cm-1) values were 1.9831 (2.17), 1.9843 (2.27) and 1.9844 (2.30), respectively. A second EPR signal with unresolved superhyperfine structure was observed at giso~1.966. At 1min, the tetraglycine and pentaglycine Cr(V) complexes, had another signal at giso~1.978, which decayed relative to the other signals with time. This chemistry has relevance to: (i) certain types of DNA damage produced by Cr carcinogens; (ii) the intracellular oxidation of Cr(III) to Cr(VI); and (iii) redox recycling of Cr(III) metabolites formed from both the intracellular reduction of carcinogenic Cr(VI) and from Cr(III) supplements.

Beta-scission of side-chain alkoxyl radicals on peptides and proteins results in the loss of side-chains as aldehydes and ketones.

Exposure of proteins to radicals in the presence of O(2) results in side-chain oxidation and backbone fragmentation; the interrelationship between these processes is not fully understood. Recently, initial attack on Ala side-chains was shown to give alpha-carbon radicals (and hence backbone cleavage) and formaldehyde, via the formation and subsequent beta-scission, of C-3 alkoxyl radicals. We now show that this side-chain to backbone damage transfer, is a general mechanism for aliphatic side-chains. Oxidation of Val, Leu, and Asp residues by HO(*)/O(2) results in the release of a family of carbonyls (including formaldehyde, acetone, isobutyraldehyde, and glyoxylic acid) via the formation, and subsequent beta-scission of alkoxyl radicals. The concentration of these products increases with the HO(*) flux. The release of multiple carbonyls confirms the occurrence of oxidation at C-3 and C-4 for Val, and these sites, plus C-5, for Leu. The detection of glyoxylic acid and CO(2)(-*) from Asp demonstrates the occurrence of competing beta-scission processes for the Asp C-3 alkoxyl radical. The yield of hydroperoxides and released carbonyls account for 10-145% of the initial HO(*). The greater than 100% yields confirm the occurrence of chain reactions in peptide/protein oxidation, with more than one residue being damaged per initiating radical.

Cell-mediated reduction of protein and peptide hydroperoxides to reactive free radicals.

Radical attack on proteins in the presence of O(2) gives protein hydroperoxides in high yields. These peroxides are decomposed by transition metal ions, reducing agents, UV light and heat, with the formation of a range of reactive radicals that are capable of initiating further damage. Evidence has been presented for the formation of alcohols as stable products of peroxide decomposition, and these have been employed as markers of oxidative damage in vivo. The mechanism of formation of these alcohols is unclear, with both radical and nonradical pathways capable of generating these products. In this study we have investigated the reduction of peptide and protein hydroperoxides by THP-1 (human monocyte-like) cells and it is shown that this process is accompanied by radical formation as detected by EPR spin trapping. The radicals detected, which are similar to those detected from metal-ion catalyzed reduction, are generated externally to the cell. In the absence of cells, or with cell-conditioned media or cell lysates, lower concentrations of radicals were detected, indicating that intact cells are required for rapid hydroperoxide decomposition. The rate of radical generation was enhanced by preloading the cells with ascorbate, and this was accompanied by intracellular formation of the ascorbate radical. It is proposed that decomposition of some amino acid and peptide hydroperoxides occurs extracellularly via the involvement of a cell-surface reducing system, such as a trans-plasma membrane electron transport system (TPMET) either directly, or indirectly via redox cycling of trace transition metal ions.

Markers of protein oxidation: different oxidants give rise to variable yields of bound and released carbonyl products.

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed on the C-3 carbons of Ala, Val, Leu, and Asp residues undergo beta-scission to give backbone alpha-carbon radicals, with the release of the side- chain as a carbonyl compound. We now show that this is a general mechanism that occurs with a wide range of oxidants. The quantitative significance of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride, and a peroxynitrite generator, 3-morpholinosydnonimine hydrochloride, gave lower overall carbonyl yields, with released carbonyls predominating over protein-bound species similar to that observed with metal ion/H2O2 systems.

Assessment of tocopherol metabolism and oxidative stress in familial hypobetalipoproteinemia.

BACKGROUND: Vitamin E supplementation has been recommended for persons with familial hypobetalipoproteinemia (FHBL), a rare disorder of lipoprotein metabolism that leads to low serum alpha-tocopherol and decreased LDL-cholesterol and apolipoprotein (apo) B. We examined the effect of truncated apoB variants on vitamin E metabolism and oxidative stress in persons with FHBL. METHODS: We studied 9 individuals with heterozygous FHBL [mean (SE) age, 40 (5) years; body mass index (BMI), 27 (10) kg/m2] and 7 normolipidemic controls [age, 41 (5) years; BMI, 25 (2) kg/m2]. We also studied 3 children-2 with homozygous FHBL (apoB-30.9) and 1 with abetalipoproteinemia-who were receiving alpha-tocopherol supplementation. We used HPLC with electrochemical detection to measure alpha- and gamma-tocopherol in serum, erythrocytes, and platelets, and gas chromatography-mass spectrometry to measure F2-isoprostanes and tocopherol metabolites in urine as markers of oxidative stress and tocopherol intake, respectively. RESULTS: Compared with controls, persons with FHBL had significantly lower fasting plasma concentrations of total cholesterol [2.4 (0.2) vs 4.7 (0.2) mmol/L], triglycerides [0.5 (0.1) vs 0.9 (0.1) mmol/L], LDL-cholesterol [0.7 (0.1) vs 2.8 (0.3) mmol/L], apoB [0.23 (0.02) vs 0.84 (0.08) g/L], alpha-tocopherol [13.6 (1.0) vs 28.7 (1.4) micromol/L], and gamma-tocopherol [1.0 (0.1) vs 1.8 (0.3) micromol/L] (all P < 0.03). Erythrocyte alpha-tocopherol was decreased [5.0 (0.2) vs 6.0 (0.3) micromol/L; P < 0.005], but we observed no differences in lipid-adjusted serum tocopherols, erythrocyte gamma-tocopherol, platelet alpha- or gamma-tocopherol, urinary F2-isoprostanes, or tocopherol metabolites. CONCLUSION: Taken together, our findings do not support the recommendation that persons with heterozygous FHBL receive vitamin E supplementation.