Short-Term Overfeeding with Dairy Cream Does Not Modify Gut Permeability, the Fecal Microbiota, or Glucose Metabolism in Young Healthy Men.

Background: High-fat diets (HFDs) have been linked to low-grade inflammation and insulin resistance. Objective: The main purpose of the present study was to assess whether acute overfeeding with an HFD affects insulin sensitivity, gut barrier function, and fecal microbiota in humans. Methods: In a prospective intervention study, 24 healthy men [mean ± SD: age 23.0 ± 2.8 y, body mass index (in kg/m2) 23.0 ± 2.1] received an HFD (48% of energy from fat) with an additional 1000 kcal/d (as whipping cream) above their calculated energy expenditure for 7 d. Insulin sensitivity (hyperinsulinemic euglycemic clamp), gut permeability (sugar and polyethylene glycol absorption tests, plasma zonulin), and gut microbiota profiles (high-throughput 16S rRNA gene sequencing) were assessed before and after overfeeding, and 14 d after intervention. Additionally, inflammation markers such as high-sensitivity C-reactive protein, lipopolysaccharide-binding protein, leptin, high-molecular-weight adiponectin, calprotectin, regulated on activation normal, T cell expressed and secreted (RANTES), and monocyte chemoattractant protein-1 were measured in plasma by ELISA. Finally, lipid parameters were analyzed in serum by a laboratory service. Results: Although participants gained 0.9 ± 0.6 kg (P < 0.001) body weight, overnutrition was not associated with a significant change in insulin sensitivity (M value and glucose disposal). Overfeeding for 7 d resulted in elevated serum total (10.2%), LDL (14.6%) and HDL (14.8%) cholesterol concentrations (P < 0.01). In contrast, fasting plasma triglyceride significantly declined (29.3%) during overfeeding (P < 0.001). In addition, there were no significant changes in inflammatory markers. Urine excretion of 4 sugars and polyethylene glycol, used as a proxy for gut permeability, and plasma concentration of zonulin, a marker of paracellular gut permeability, were unchanged. Moreover, overfeeding was not associated with consistent changes in gut microbiota profiles, but marked alterations were observed in a subgroup of 6 individuals. Conclusions: Our findings suggest that short-term overfeeding with an HFD does not significantly impair insulin sensitivity and gut permeability in normal-weight healthy men, and that changes in dominant communities of fecal bacteria occur only in certain individuals. The study was registered in the German Clinical Trial Register as DRKS00006211.

Development of a stable isotope dilution LC-MS assay for the quantitation of multiple polyethylene glycol (PEG) homologues to be used in permeability studies.

A new quantitation method based on a multiple stable isotope dilution assay (SIDA) was developed for polyethylene glycol (PEG) homologues from PEG mixtures with average molecular weights (MW) of 400, 1500, 3000 and 4000Da in urine. Seven [(13)C4(2)H4] and two [(13)C8(2)H8]PEG homologues were synthesized and served as labelled internal standards for SIDA. PEG oligomers were resolved by reversed phase high performance liquid chromatography (RP-HPLC) coupled to mass spectrometry (MS) in multiple ion (MI) scan modus. Very low limits of detection (LODs) in a range of 0.4-12ng/mL were achieved for the single homologues. Higher PEG homologues showed increased LODs and LOQs and less effective recovery (77-87%) than PEG with lower molecular masses (95-121%). Precision (relative standard deviation) varied between 3 and 13% and showed no dependence of the chain length. The method was successfully applied to human and mice urine samples. Beside an accurate quantitation of single PEG homologues it was possible to show an alteration in the MW distribution in urine samples compared to the dosed PEG solutions. The highest MW, with which a PEG can pass the intestinal wall (so called "cut off") for humans appeared to be higher than for mice.

Diet-induced obesity causes metabolic impairment independent of alterations in gut barrier integrity.

SCOPE: The causal relationship between diet-induced obesity and metabolic disorders is not clear yet. One hypothesis is whether the obese state or high-fat diet per se affects intestinal barrier function provoking metabolic comorbidities. METHODS AND RESULTS: In three independent experiments with AKR/J, SWR/J, or BL/6J mice, we addressed the impact of genetic background, excess body fat storage, duration of high-fat feeding, and quality/quantity of dietary fat on glucose tolerance and gut barrier integrity in vivo and ex vivo. Impaired glucose tolerance in diet-induced obese BL/6J and AKR/J mice was not accompanied by an altered intestinal barrier function. Enforced dietary challenge by prolonged feeding and increasing fat quantity in BL/6J mice still failed to aggravate metabolic and intestinal deterioration. Despite a low-grade inflammatory status in adipose tissue, barrier function of BL/6J mice fed lard high-fat diet revealed no evidence for a diet-induced loss in barrier integrity. CONCLUSION: None of our results provided any evidence that gut barrier function is a subject to dietary regulation and obesity per se seems not to cause gut barrier impairment.

High fat diet accelerates pathogenesis of murine Crohn's disease-like ileitis independently of obesity.

BACKGROUND: Obesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn's disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn's disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn's disease-like ileitis. METHODS: TNF(ΔARE/WT) mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors. RESULTS: HFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNF(ΔARE/WT). Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria. CONCLUSIONS: HFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn's disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.

Development of analytical methods for the determination of tenuazonic acid analogues in food commodities.

Analogues of the Alternaria mycotoxin tenuazonic acid (TA, biosynthesized by the fungus from the amino acid isoleucine) derived from valine (ValTA), leucine (LeuTA), alanine (AlaTA) and phenylalanine (PheTA) were synthesized and characterized by mass spectrometry (MS) and (1)H nuclear magnetic resonance (NMR) spectroscopy. Concentrations of stock solutions were determined by quantitative (1)H NMR (qHNMR). Two analytical methods based on high performance liquid chromatography (HPLC) and MS detection were developed, one with derivatization with 2,4-dinitrophenylhydrazine (DNPH) and one without derivatization. Limits of detection (LODs) were between 1-3 µg/kg (with derivatization) and 50-80 µg/kg (without derivatization). Respective limits of quantitation (LOQs) were about three times higher. Beside TA, the analogues LeuTA (about 4% of TA content) and ValTA (about 10% of TA content) were found in highly contaminated sorghum infant cereals and sorghum grains. Other analogues were not detected. Quantification of LeuTA and ValTA was performed using [(13)C6,(15)N]-TA as internal standard and matrix matched calibration. Recovery was between 95±11% and 102±10% for both compounds. Precision (relative standard deviation of triplicate sorghum cereal analyses three times during 3 weeks) was 7% for TA (912±60 µg/kg), 17% for LeuTA (43±8 µg/kg) and 19% for ValTA (118±22 µg/kg). These results indicate that several TA-like compounds, which are not yet characterized in aspects of their toxic properties, were detected in sorghum based infant food highly contaminated with TA, already.

Content of the Alternaria mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay.

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [(13) C6,(15) N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 µg/kg (fruit juices), 1.0 µg/kg (cereals) and 17 µg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 µg/kg), 3.5% (whole wheat flour; 36 µg/kg) and 0.9% (curry powder; 215 µg/kg). The median content of TA in the analyzed samples was 1.8 µg/kg (fruit juices), 16 µg/kg (cereals) and 500 µg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).