ObjectiveBacterial translocation to various organs including human adipose tissue (AT) due to increased intestinal permeability remains poorly understood. We hypothesised that: (1) bacterial presence is highly tissue specific and (2) related in composition and quantity to immune inflammatory and metabolic burden.DesignWe quantified and sequenced the bacterial 16S rRNA gene in blood and AT samples (omental, mesenteric and subcutaneous) of 75 subjects with obesity with or without type 2 diabetes (T2D) and used catalysed reporter deposition (CARD) – fluorescence in situ hybridisation (FISH) to detect bacteria in AT.ResultsUnder stringent experimental and bioinformatic control for contaminants, bacterial DNA was detected in blood and omental, subcutaneous and mesenteric AT samples in the range of 0.1 to 5 pg/µg DNA isolate. Moreover, CARD-FISH allowed the detection of living, AT-borne bacteria. Proteobacteria and Firmicutes were the predominant phyla, and bacterial quantity was associated with immune cell infiltration, inflammatory and metabolic parameters in a tissue-specific manner. Bacterial composition differed between subjects with and without T2D and was associated with related clinical measures, including systemic and tissues-specific inflammatory markers. Finally, treatment of adipocytes with bacterial DNA in vitro stimulated the expression of TNFA and IL6.ConclusionsOur study provides contaminant aware evidence for the presence of bacteria and bacterial DNA in several ATs in obesity and T2D and suggests an important role of bacteria in initiating and sustaining local AT subclinical inflammation and therefore impacting metabolic sequelae of obesity.

BackgroundThere is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity.MethodsSerum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects.ResultsAs zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family.ConclusionOur study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Background The microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery. Methods Subjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH). Results The contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiometabolic risk parameters. CARD-FISH allowed the detection of living bacteria in blood samples in obesity. Conclusions We show that the circulating bacterial signature reflects metabolic disease and its improvement after bariatric surgery. Our work provides contaminant-aware evidence for the presence of living bacteria in the blood and suggests a putative crosstalk between components of the blood and metabolism in metabolic health regulation.

Keywords: diabetes -lipid metabolism -pre-Haptoglobin 2 -ELISA -intestinal permeability 25 -properdin -obesity -metabolic diseases 26 27 Nonstandard abbreviations: HP, haptoglobin; T2D, type 2 diabetes; WHR, waist-to-hip 28 ratio.Abstract 30 BACKGROUND. There is increasing evidence for the role of impaired intestinal 31 permeability in obesity and associated metabolic diseases. Zonulin is an established serum 32 marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to 33 investigate the relationship between circulating zonulin and metabolic traits related to obesity. 34METHODS. Serum zonulin was measured by using a widely used commercial ELISA kit in 35 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In 36 addition, haptoglobin genotype was determined in DNA samples from all study subjects. 37 RESULTS.As zonulin concentrations did not correlate to the haptoglobin genotypes, we 38 investigated the specificity of the zonulin ELISA assay using antibody capture experiments, 39 mass spectrometry and Western blot analysis. Using serum samples that gave the highest or 40 lowest ELISA signals, we detected several proteins that are likely to be captured by the 41 antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. 42 We used increasing concentrations of recombinant pre-haptoglobin 2 and complement C3 as 43 one of the representative captured proteins and the ELISA kit did not detect either. Western 44 blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies 45 rose against zonulin showed a similar protein recognition pattern but with different intensity 46 of detection. The protein(s) measured using the ELISA kit was (were) significantly increased 47 in patients with diabetes and obesity and correlated strongly with markers of the lipid and 48 glucose metabolism. Combining mass spectrometry and Western blot analysis using the 49 polyclonal antibodies used in the ELISA kit, we identified properdin as another member of 50 the zonulin family. 51 CONCLUSIONS. Our study suggests that the zonulin ELISA does not recognize pre-52 haptoglobin 2, rather structural (and possibly functional) analogue proteins belonging to the 53 mannose-associated serine protease family, with properdin being the most likely possible 54 candidate. 55 56 The "intestinal barrier" is an established term, defined as a functional entity separating the gut 57 lumen from the inner host, and consisting of mechanical, humoral, immunological, muscular 58 and neurological elements. Intestinal barrier dysfunction is a characteristic feature of 59 pathological states such as inflammatory bowel disease, celiac disease, nonalcoholic 60 steatohepatitis and ulcerative colitis (1, 2). There is also emerging evidence for the role of 61 impaired intestinal permeability in metabolic diseases including obesity and type 2 diabetes 62 (T2D) (3-5). It has been hypothesized that gut bacteria and bacterial endotoxins may disrup...