Stefanie Lehmann

Long-term exercise training decreases interleukin-6 (IL-6) serum levels in subjects with impaired glucose tolerance: effect of the −174G/C variant in IL-6 gene

OBJECTIVE Exercise training has been shown to have anti-inflammatory effects in patients with type 2 diabetes. Changes in interleukin-6 (IL-6) serum concentrations in response to training could contribute to these beneficial effects. However, there are heterogeneous data on whether circulating IL-6 is altered by exercise training. We therefore hypothesize that genetic factors modify the individual changes in IL-6 levels after long-term training. RESEARCH DESIGN AND METHODS The -174G/C variant in the IL-6 gene was genotyped in 60 subjects with impaired glucose tolerance. For a 12-month interventional study, patients were randomized into three groups: a control group (n=16) was compared with one group, which underwent a standardized training program (n=24) and another group, which was treated with 4 mg rosiglitazone once daily (n=20). At baseline, after 1, 6, and 12 months, we measured anthropometric parameters and serum concentration of IL-6 and, at baseline and after 12 months, we determined glucose tolerance and fitness level. RESULTS Only in subjects carrying the SNP -174C allele did long-term exercise training result in significantly reduced IL-6 serum concentrations. Multivariate linear regression analysis identified the IL-6 genotype as a significant predictor of changes in IL-6 serum concentrations independent of age, gender and improvement in body mass index, hemoglobin (Hb)A(1c), and fitness level in response to training. CONCLUSIONS Genetic variants in the IL-6 gene significantly modify changes in IL-6 serum concentrations in response to long-term exercise training programs. Our data suggest that genetic factors are important determinants for the individual response to anti-inflammatory effects of exercise training.

Combined Serum Proteomic and Metabonomic Profiling After Laparoscopic Sleeve Gastrectomy in Children and Adolescents

AIM The consequences of bariatric surgery on important metabolic and signaling pathways are still poorly understood. The aim of our study was to unravel the network of metabolic changes and obesity-related protein biomarkers in morbidly obese children and adolescents undergoing laparoscopic sleeve gastrectomy (LSG). METHODS In a prospective study, 6 children with morbid obesity who had failed a well-established conservative weight loss therapy underwent LSG. Pre- and 6 months postoperatively, a metabonomic profiling of 163 metabolites by mass spectrometry and protein profiling by ELISA (clusterin [CLU], pigment epithelium-derived factor [PEDF], retinol binding protein 4 [RBP4], paraoxonase 1 [PON1]) was performed to identify biomarkers of important pathways. RESULTS At referral for surgery, the mean age was 14.5 years (range 8-17), mean body mass index (BMI in kg/m(2)) was 48.13 (range 41.1-56.3). All patients showed various metabolic comorbidities. LSG was uneventful in all of them. At 6 months postsurgery, the mean BMI has dropped to 37.1 (range 28.4-40.6). Targeted serum metabonomics resulted in 7 metabolites, which were significantly affected by LSG. Among those, the amino acid phenylalanine was increased, and methionine decreased. The glycero-phospatidylcholine PCaaC38:5 were upregulated, and PCaaC40:2 and PCaaC42:1 were down regulated after 6 months compared with baseline. Further, sphingomyoline (SM) C26:0 and SM C26:1 were decreased after 6 months. The obesity-related biomarkers CLU, PEDF, and RBP4 were decreased, whereas PON1 levels were increased. CONCLUSION LSG leads to changes in amino acids and in lipid metabolism indicated by glycerol-phosphatidylcholines and SM. The pattern of protein biomarkers and metabolites might provide measures for the induced physiological changes and for therapy monitoring.

Serum vaspin concentrations are decreased after exercise-induced oxidative stress.

Elevated visceral adipose tissue-derived serpin (vaspin) serum concentrations are associated with impaired insulin sensitivity, but increase unexpectedly after long-term physical training. We therefore investigated the effect of an acute exercise bout and the effects of vitamin supplementation on chronic exercise effect and on serum vaspin concentrations. We measured serum vaspin and thiobarbituric acid-reactive substances (TBARS) concentrations in 80 individuals before and after a 1-hour acute exercise bout and independently in 40 healthy young men who were randomly assigned to either antioxidant (vitamin C (1,000 mg/day) and vitamin E (400 IU/day)) or to no supplementation after a standardized 4-week physical training program as a post hoc analysis. Serum vaspin concentrations significantly decreased after acute physical exercise as well as after 4 weeks of training in individuals without antioxidants. Changes in vaspin serum concentration correlate with increased TBARS serum concentrations both in response to a 1-hour exercise bout (r = –0.42, p < 0.01) and to the 4-week training (r = –0.31, p < 0.05). Interestingly, supplementation with antioxidants rather increased circulating vaspin levels in response to 4 weeks of exercise. In conclusion, vaspin serum concentrations are decreased by exercise-induced oxidative stress, but not by exercise-associated improvement in insulin sensitivity.

Molecular fingerprint of high fat diet induced urinary bladder metabolic dysfunction in a rat model.

Aims/hypothesis Diabetic voiding dysfunction has been reported in epidemiological dimension of individuals with diabetes mellitus. Animal models might provide new insights into the molecular mechanisms of this dysfunction to facilitate early diagnosis and to identify new drug targets for therapeutic interventions. Methods Thirty male Sprague-Dawley rats received either chow or high-fat diet for eleven weeks. Proteomic alterations were comparatively monitored in both groups to discover a molecular fingerprinting of the urinary bladder remodelling/dysfunction. Results were validated by ELISA, Western blotting and immunohistology. Results In the proteome analysis 383 proteins were identified and canonical pathway analysis revealed a significant up-regulation of acute phase reaction, hypoxia, glycolysis, β-oxidation, and proteins related to mitochondrial dysfunction in high-fat diet rats. In contrast, calcium signalling, cytoskeletal proteins, calpain, 14-3-3η and eNOS signalling were down-regulated in this group. Interestingly, we found increased ubiquitin proteasome activity in the high-fat diet group that might explain the significant down-regulation of eNOS, 14-3-3η and calpain. Conclusions/interpretation Thus, high-fat diet is sufficient to induce significant remodelling of the urinary bladder and alterations of the molecular fingerprint. Our findings give new insights into obesity related bladder dysfunction and identified proteins that may indicate novel pathophysiological mechanisms and therefore constitute new drug targets.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Age and gender specific estimation of visceral adipose tissue amounts from radiological images in morbidly obese patients.

Image-based quantifications of visceral adipose tissue (VAT) volumes from segmented VAT areas are increasingly considered for risk assessment in obese patients. The goal of this study was to determine the power of partial VAT areas to predict total VAT volume in morbidly obese patients (BMI > 40 kg/m2) as a function of gender, age and anatomical landmarks. 130 morbidly obese patients (mean BMI 46.5 kg/m2; 94 females) underwent IRB-approved MRI. Total VAT volumes were predicted from segmented VAT areas (of single or five adjacent slices) at common axial landmark levels and compared with the measured ones (VVAT-T, about 40 slices between diaphragm and pelvic floor). Standard deviations σ1 and σ5 of the respective VAT volume differences served as measures of agreement. Mean VVAT-T was 4.9 L for females and 8.1 L for males. Best predictions were found at intervertebral spaces L3-L4 for females (σ5 = 688 ml, σ1 = 832 ml) and L1-L2 for males (σ5 = 846 ml, σ1 = 992 ml), irrespective of age. In conclusion, VAT volumes in morbidly obese patients can be reliably predicted by multiplying the segmented VAT area at a gender-specific lumbar reference level with a fixed scaling factor and effective slice thickness.

Metabolic in Vivo Labeling Highlights Differences of Metabolically Active Microbes from the Mucosal Gastrointestinal Microbiome between High-Fat and Normal Chow Diet

The gastrointestinal microbiota in the gut interacts metabolically and immunologically with the host tissue in the contact zone of the mucus layer. For understanding the details of these interactions and especially their dynamics it is crucial to identify the metabolically active subset of the microbiome. This became possible by the development of stable isotope probing techniques, which have only sparsely been applied to microbiome research. We applied the in vivo stable isotope approach using 15N-labeled diet with subsequent identification of metabolically active bacterial species. Four-week old male Sprague-Dawley rats were randomly assigned to chow diet (CD, n =15) and high-fat diet (HFD, n =15). After 11 weeks, three animals from each group were sacrificed for baseline characterization of anthropometric and metabolic obesity. The remaining animals were exposed to either a 15N-labeled (n =9) or a 14N-unlabeled experimental diet (n =3). Three rats from each cohort (HFD and CD) were sacrificed at 12, 24, and 72 h. The remaining three animals from each cohort, which received the 14N-unlabeled diet, were sacrificed after 72 h. The colon was harvested and divided into three equal sections (proximal, medial, and distal), and the mucus layer of each specimen was sampled by scraping. We identified the active subset in an HFD model of obesity in comparison with lean controls rats using metaproteomics. In addition, all samples were investigated by 16S rRNA amplicon gene sequencing. The active microbiome of the HFD group showed an increase in bacterial taxa for Verrucomicrobia and Desulfovibrionaceae. In contrast with no significant changes in alpha diversity, time- and localization-dependent effects in beta-diversity were clearly observed. In terms of enzymatic functions the HFD group showed strong affected metabolic pathways such as energy production and carbohydrate metabolism. In vivo isotope labeling combined with metaproteomics provides a valuable method to distinguish the active from the non-active bacterial phylogenetic groups that are relevant for microbiota-host interaction. For morbid obesity such analysis may provide potentially new strategies for targeted pre- or probiotic treatments.

Proteome profiles of HDL particles of patients with chronic heart failure are associated with immune response and also include bacteria proteins

Besides modulation of reverse cholesterol transport, high density lipoprotein (HDL) is able to modulate vascular function by stimulating endothelial nitric oxide synthase. Recently, it could be documented that this function of HDL was significantly impaired in patients with chronic heart failure (CHF). We investigated alterations in the HDL proteome in CHF patients. Therefore, HDL was isolated from 5 controls (HDLhealthy) and 5 CHF patients of NYHA-class IIIb (HDLCHF). Proteome analysis of HDL particles was performed by two-dimensional liquid chromatography-mass spectrometry (SCX/RP LC-MS/MS). In total, we identified 494 distinct proteins, of which 107 proteins were commonly found in both groups (HDLCHF and HDLhealthy) indicating a high inter-subject variability across HDL particles. Several important proteins (e.g. ITGA2, APBA1 or A2M) varied in level. Functional analysis revealed regulated pathways. A minor proportion of bacteria-derived proteins were also identified in the HDL-particles. The extension of the list of HDL-associated proteins allows besides their mere description new insights into alterations in HDL function in diseases. In addition, the detection of bacterial proteins bound to HDL will broaden our view of HDL not only as a cholesterol carrier but also as a carrier of proteins.

New insights into valve-related intramural and intracellular bacterial diversity in infective endocarditis

Aims In infective endocarditis (IE), a severe inflammatory disease of the endocardium with an unchanged incidence and mortality rate over the past decades, only 1% of the cases have been described as polymicrobial infections based on microbiological approaches. The aim of this study was to identify potential biodiversity of bacterial species from infected native and prosthetic valves. Furthermore, we compared the ultrastructural micro-environments to detect the localization and distribution patterns of pathogens in IE. Material and methods Using next-generation sequencing (NGS) of 16S rDNA, which allows analysis of the entire bacterial community within a single sample, we investigated the biodiversity of infectious bacterial species from resected native and prosthetic valves in a clinical cohort of 8 IE patients. Furthermore, we investigated the ultrastructural infected valve micro-environment by focused ion beam scanning electron microscopy (FIB-SEM). Results Biodiversity was detected in 7 of 8 resected heart valves. This comprised 13 bacterial genera and 16 species. In addition to 11 pathogens already described as being IE related, 5 bacterial species were identified as having a novel association. In contrast, valve and blood culture-based diagnosis revealed only 4 species from 3 bacterial genera and did not show any relevant antibiotic resistance. The antibiotics chosen on this basis for treatment, however, did not cover the bacterial spectra identified by our amplicon sequencing analysis in 4 of 8 cases. In addition to intramural distribution patterns of infective bacteria, intracellular localization with evidence of bacterial immune escape mechanisms was identified. Conclusion The high frequency of polymicrobial infections, pathogen diversity, and intracellular persistence of common IE-causing bacteria may provide clues to help explain the persistent and devastating mortality rate observed for IE. Improved bacterial diagnosis by 16S rDNA NGS that increases the ability to tailor antibiotic therapy may result in improved outcomes.

Gastric mucosal devitalization reduces adiposity and improves lipid and glucose metabolism in obese rats

BACKGROUND AND AIMS The gastric mucosa is an endocrine organ that regulates satiation pathways by expression of orexigenic and anorexigenic hormones. Vertical sleeve gastrectomy (VSG) excludes gastric mucosa and reduces gastric volume. Our study aimed to investigate the independent effects of altering gastric mucosa on obesity and its related comorbidities. METHODS Gastric mucosa devitalization (GMD) of 70% of the stomach was achieved by argon plasma coagulation in a high-fat diet rat model and was compared with VSG and sham surgery. In an 8-week follow-up study, we quantified body weight, visceral adiposity, insulin resistance index, cholesterol profiles, and free fatty acid profiles by enzyme-linked immunosorbent assay (ELISA). Following a 2-hour oral glucose tolerance test, the kinetics of ghrelin, glucagon-like peptide-1, peptide YY, and serum and liver bile acid levels were measured. Liver lipid content was quantified by ELISA. RESULTS GMD resulted in significant reductions in body weight, visceral and subcutaneous adipose tissue, and hepatic steatosis as well as an improvement in lipid metabolism. GMD resulted in significant reductions in food intake and intestinal malabsorption of free fatty acids, both contributing to improved body composition and metabolic profile. Mechanistically, GMD resulted in a significant reduction in serum palmitate levels as well as an increase in serum and liver bile acid levels, known to alter glucose and lipid metabolism. Similar changes were noted when VSG rats were compared with sham surgery rats. CONCLUSIONS Devitalization of gastric mucosa, independent of altering gastric volume, was able to reduce obesity-related comorbidities. The gastric mucosa may be a potential target for treating obesity and its associated comorbidities.