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Roux-en-Y gastric bypass (RYGB) has become a prominent therapeutic option for long-term treatment of morbid obesity and type 2 diabetes mellitus (T2D). Cross talk and pathogenetic consequences of RYGB-induced profound effects on metabolism and gut microbiome are poorly understood. The aim of the present study therefore was to characterize intra-individual changes of gut microbial composition before and 3 months after RYGB by metagenomic sequencing in morbidly obese patients (body mass index (BMI)>40 kg m − 2 ) with T2D. Subsequently, metagenomic data were correlated with clinical indices. Based on gene relative abundance profile, 1061 species, 729 genera, 44 phyla and 5127 KO (KEGG Orthology) were identified. Despite high diversity, bacteria could mostly be assigned to seven bacterial divisions. The overall metagenomic RYGB-induced shift was characterized by a reduction of Firmicutes and Bacteroidetes and an increase of Proteobacteria. Twenty-two microbial species and 11 genera were significantly altered by RYGB. Using principal component analysis, highly correlated species were assembled into two common components. Component 1 consisted of species that were mainly associated with BMI and C-reactive protein. This component was characterized by increased numbers of Proteobacterium Enterobacter cancerogenus and decreased Firmicutes Faecalibacterium prausnitzii and Coprococcus comes . Functional analysis of carbohydrate metabolism by KO revealed significant effects in 13 KOs assigned to phosphotransferase system. Spearmen’s Rank correlation indicated an association of 10 species with plasma total- or low-density lipoprotein cholesterol, and 5 species with triglycerides. F. prausnitzii was directly correlated to fasting blood glucose. This is the first clinical demonstration of a profound and specific intra-individual modification of gut microbial composition by full metagenomic sequencing. A clear correlation exists of microbiome composition and gene function with an improvement in metabolic and inflammatory parameters. This will allow to develop new diagnostic and therapeutic strategies based on metagenomic sequencing of the human gut microbiome.

Urinary lipidomics may add new valuable biomarkers to the diagnostic armamentarium for early detection of metabolic and kidney diseases. Sources and composition of urinary lipids in healthy individuals, however, have not been investigated in detail. Shotgun lipidomics was used to quantify lipidomic profiles in native urine samples from 16 individuals (eight men, eight women) collected in five fractions over 24 h. All probands were comprehensively characterized by urinary and clinical indices. The mean total urinary lipid concentration per sample was 0.84 μM in men and 1.03 μM in women. We observed significant intra- and interindividual variations of lipid concentrations over time, but failed to detect a clear circadian pattern. Based on quantity and subclass composition it seems very unlikely that plasma serves as major source for the urinary lipidome. Considering lipid metabolites occurring in at least 20% of all samples 38 lipid species from 7 lipid classes were identified. Four phosphatidylserine and one phosphatidylethanolamine ether species (PE-O 36:5) were detectable in almost all urine samples. Sexual dimorphism has been found mainly for phosphatidylcholines and phosphatidylethanolamines. In men and in women urinary lipid species were highly correlated with urinary creatinine and albumin excretion, reflecting glomerular filtration and tubular transport processes. In women, however, lipid species deriving from urinary cells and cellular constituents of the lower genitourinary tract considerably contributed to the urinary lipidome. In conclusion, our study revealed the potential of urinary lipidomics but also the complexity of methodological challenges which have to be overcome for its implementation as a routine diagnostic tool for renal, urological and metabolic diseases.

Bariatric surgery is a well-established approach to improve metabolic disease in morbidly obese patients with high cardiovascular risk. The post-operative normalization of lipid metabolism has a central role in the prevention of future cardiovascular events. The aim of the present study therefore was to characterize changes of plasma lipidomic patterns, consisting of 229 lipid species of 13 lipid classes, 3 months after Roux-en-Y gastric bypass (RYGB) in morbidly obese patients with and without diabetes. RYGB resulted in a 15–32% decrease of body mass index, which was associated with a significant reduction of total cholesterol (TC, −28.3%; P =0.02), LDL-cholesterol (LDL-C, −26.8%; P =0.03) and triglycerides (TGs, −63.0%; P =0.05) measured by routine clinical chemistry. HDL-cholesterol remained unchanged. The effect of RYGB on the plasma lipidomic profile was characterized by significant decreases of 87 lipid species from triacylglycerides (TAGs), cholesterol esters (CholEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), phosphatidylethanolamine ethers (PEOs), phosphatidylinositols (PIs) and ceramides (Cers). The total of plasma lipid components exhibited a substantial decline of 32.6% and 66 lipid species showed a decrease by over 50%. A direct correlation with HbA 1C values could be demonstrated for 24 individual lipid species (10 TAG, three CholE, two LPC, one lysophosphatidylcholine ethers (LPCO) (LPC ether), one PC, two phosphatidylcholine ethers (PCO) and five Cer). Notably, two lipid species (TAG 58:5 and PEO 40:5) were inversely correlated with HbA 1C . LPCO, as single whole lipid class, was directly related to HbA 1C . These data indicate that RYGB-induced modulation of lipidomic profiles provides important information about post-operative metabolic adaptations and might substantially contribute to improvements of glycemic control. These striking changes in the human plasma lipidome may explain acute, weight independent and long-term effects of RYGB on the cardiovascular system, mental status and immune regulation.

Background Serum aldosterone is a causative factor for various metabolic and cardiovascular disorders. Low-density lipoprotein (LDL) is a major cholesterol source for aldosterone steroidogenesis; however, the effect of oxidative modification of LDL on aldosterone release is not known. We studied the effect of hypochlorite-oxidized LDL (oxLDL) on adrenal aldosterone secretion. Methods LDL (native LDL (natLDL)) was obtained from healthy volunteers and oxidatively modified in vitro. NCI-H295R cells were stimulated with natLDL and oxLDL, and the aldosterone release was quantified by radioimmunoassay. Molecular changes were studied with western blot analysis and quantitative RT-PCR analysis. Results NatLDL and oxLDL caused dose-dependent increase in aldosterone release up to threefold. However, the stimulatory effects of modified LDL on aldosterone secretion decreased with increasing degree of LDL oxidation. 24-h incubations with natLDL, mild- and medium-oxidized LDL sensitized the adrenocortical cells to subsequent angiotensin II (Ang II) stimulations by 2.9-, 2.8-, and 2.5-folds, respectively. Heavily oxidized LDL did not sensitize the cells to Ang II stimulations to a similar extent. At the molecular level, the ERK pathway was activated within a minute by both natLDL and oxLDL; however, oxLDL showed a stronger (2.75-fold at 1 and 15min) and longer (15min) activation of ERK than natLDL (twofold). Conclusions This study demonstrates the following: (i) both natLDL and hypochlorite-oxidized LDL utilize ERK pathway to mediate aldosterone release; (ii) mildly oxidized LDL sensitizes the adrenocortical cells to further stimulations by Ang II similar to natLDL that may have a role in pathological processes; (iii) extensive LDL oxidation counteracts adrenocortical aldosterone release.

Aims/hypothesis Glycoxidised LDL has been implicated in the pathogenesis of atherosclerosis, a major complication of diabetes. Since atherogenesis may occur at an early stage of diabetes, we investigated whether circulating LDL isolated from subjects with IGT (n = 20) showed an increased glycoxidation status and explored the proatherogenic effects of LDL samples on macrophages. Subjects and methods We investigated LDL modifications using GC-MS. Murine macrophages were incubated with LDL samples for 1 h, and then mRNA expression rates of the scavenger receptors CD36 and scavenger receptor class B type 1 (SCARB1, formerly known as SR-BI) and transcription factor peroxisome proliferator-activator receptor γ (PPARγ) were quantified by real-time RT-PCR. Results The GC-MS experiments revealed that oxidative modifications of proline, arginine, lysine and tyrosine residues in apolipoprotein B100 were three- to fivefold higher in LDL samples from IGT subjects compared with those from NGT subjects (n = 20). Moreover, LDL glycoxidation estimated by both Nepsilon -(carboxymethyl)lysine (CML) and Nepsilon -(carboxyethyl)lysine (CEL) residues was increased more than ninefold in LDL from IGT subjects compared with samples from NGT subjects. Compared with NGT LDL, IGT LDL elicited a significantly higher CD36 (p < 0.05) and PPARG (p < 0.05) gene expression, whereas SCARB1 mRNA expression was not affected. Conclusions/interpretation These data suggest that IGT is associated with increased glycoxidation of circulating LDL, which might contribute to the conversion of macrophages into a proatherogenic phenotype.

In Vivo Evidence for Increased Oxidation of Circulating LDL in Impaired Glucose Tolerance Steffi Kopprasch 1 , Jens Pietzsch 2 , Eberhard Kuhlisch 3 , Katja Fuecker 4 , Theodora Temelkova-Kurktschiev 5 , Markolf Hanefeld 5 , Helmut Kühne 1 , Ulrich Julius 4 and Jürgen Graessler 1 1 Department of Internal Medicine 3, Carl Gustav Carus Medical School, University of Technology Dresden, Dresden, Germany 2 Institute of Bioinorganic and Radiopharmaceutical Chemistry, Research Center Rossendorf, Dresden, Germany 3 Institute of Medical Informatics and Biometry, Carl Gustav Carus Medical School, University of Technology Dresden, Dresden, Germany 4 Institute and Policlinic of Clinical Metabolic Research, Carl Gustav Carus Medical School, University of Technology Dresden, Dresden, Germany 5 Centre for Clinical Studies GWT, Dresden, Germany Abstract Oxidized LDL (oxLDL) is a key mediator in atherogenesis and a marker of coronary artery disease (CAD). Type 2 diabetes is associated with excessive cardiovascular morbidity and mortality. Because atherogenesis starts before diabetes is diagnosed, we investigated whether circulating oxLDL levels are increased in impaired glucose tolerance (IGT). OxLDL levels were measured in 376 subjects with normal glucose tolerance (NGT), 113 patients with IGT, and 54 patients with newly diagnosed type 2 diabetes. After correction for age and BMI, serum levels of oxLDL were significantly increased in IGT versus NGT subjects ( P = 0.002). OxLDL levels were not associated with the following parameters of the oxidative/antioxidative balance in the blood: total antioxidant capacity, urate-to-allantoin ratio, and circulating phagocyte oxygenation activity. In stepwise multivariate analysis, LDL cholesterol ( P < 0.0005) and triglycerides ( P < 0.0005) were the strongest predictors of circulating oxLDL levels, followed by HDL cholesterol ( P = 0.003), 2-h postchallenge C-peptide ( P = 0.011), fasting free fatty acids ( P = 0.013), and serum paraoxonase activity ( P = 0.035). The strong correlation of oxLDL with LDL cholesterol and triglycerides indicates that LDL oxidation in IGT is preferentially associated with dyslipidemia. OxLDL increase may explain the high atherogenic potency of dyslipidemia in the prediabetic state. Footnotes Address correspondence and reprint requests to Dr. Steffi Kopprasch, University of Technology Dresden, Carl Gustav Carus Medical School, Department of Internal Medicine 3, Pathological Biochemistry, Fetscherstr. 74, D-01307 Dresden, Germany. E-mail: steffi.kopprasch{at}mailbox.tu-dresden.de . Received for publication 21 January 2002 and accepted in revised form 15 July 2002. S.K. and J.P. contributed equally to this work. CAD, coronary artery disease; CPOA, circulating phagocyte oxygenation activity; IGT, impaired glucose tolerance; IMT, intima-media thickness; NGT, normal glucose tolerance; OGTT, oral glucose tolerance test; oxLDL, oxidized LDL; PON, serum paraoxonase activity with paraoxon as substrate; RIAD, Risk Factors in Impaired Glucose Tolerance for Atherosclerosis and Diabetes; TAC, total antioxidant capacity. DIABETES

The uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors of macrophages with resulting foam cell formation is considered a critical event in atherogenesis. Since hypochlorite-oxidized LDL (HOCl-LDL) has been shown to be recognized by macrophages and evidence was provided that HOCl-LDL is internalized via class B scavenger receptors CD36 and SR-BI, the regulatory relationships between CD36, SR-BI, and the nuclear transcription factor PPAR gamma in murine macrophages (RAW 264.7) on exposure to HOCl-LDL were examined. Using the highly sensitive real-time RT-PCR we could demonstrate that HOCl-LDL upregulated CD36 and PPAR gamma levels dose- and time dependently while modulating SR-BI message levels differently in dependence on HOCl-LDL concentration and incubation time. On exposure of macrophages to HOCl-LDL but not native LDL in varying concentrations, a significant positive correlation between CD36 and PPAR gamma ( rho = 0.603, p = 0.001) was observed indicating the presence of a positive feedback mechanism by which HOCl-LDL could promote its own uptake. The transcriptional expression of SR-BI in macrophages was not significantly related to PPAR gamma mRNA levels after treatment with HOCl-LDL suggesting a differential regulation of the two members of the scavenger receptor class B family in response to HOCl-LDL.

Hypochlorite-oxidized low-density lipoprotein (oxLDL) possesses a substantial proinflammatory potential by modulating respiratory burst activities of polymorphonuclear neutrophils (PMN). As evaluated by luminol-amplified chemiluminescence (CL) incubation of 10(6) PMN/ml with 70 nM oxLDL was followed by substantial induction of neutrophil oxidant (ROS) generation. We evaluated the inhibitory capacity of high-density lipoprotein (HDL) and its lipid and protein constituents against the activating effects of oxLDL. At a HDL or apolipoprotein AI/LDL protein ratio of 1.0, native HDL decreased the respiratory burst activation by 64%, followed by trypsinized HDL (57%) and native apoAI (43%). The inhibitory effects of native HDL did not require prior incubation with PMN or with oxLDL suggesting an instantaneously acting protective mechanism in the minute range. OxLDL modulated ROS production not only of resting PMN but also that of activated PMN, as indicated by a 14-fold increase in FMLP-stimulated CL response and a 50% decrease in zymosan-mediated CL answer. HDL itself did not protect PMN from activation by FMLP and zymosan. However, it clearly reduced effects of oxLDL on FMLP-activation and slightly counteracted the oxLDL-mediated decrease in zymosan-induced ROS generation. Taken together, these findings may offer new insight into atheroprotective mechanisms of HDL.

Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of beta-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 x 10(-8) M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 x 10(-5) -3 x 10(-12) M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.

Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of b-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 ï 10-8 M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 ï 10-5 - 3 ï 10-12 M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.[PUBLICATION ABSTRACT]