LOSS OF INTESTINAL EPITHELIAL DUOX2 SIGNALING IS ACCOMPANIED BY A REDUCTION IN AKKERMANSIACEAE AND PROMOTES THE DEVELOPMENT OF METABOLIC SYNDROME IN A MICROBIOME-DEPENDENT MANNER.

Objective
To identify genes with differential chromatin accessibility that correlate with small intestinal dietary sugar hyper-absorption.
Methods
Proximal small intestinal tissue samples from either lean healthy (BMI ≤ 25 kg/m²) or obese (BMI ≥ 35 kg/m²) patients were obtained during routine endoscopy procedures or from discarded tissue of patients undergoing bariatric surgery. Enteroid cultures were established and dietary glucose/fructose absorption and gluconeogenesis were measured. The expression of carbohydrate transporters and gluconeogenic enzymes was assessed by qRT-PCR, and measurement of apical-to-basolateral glucose transport in an Amplex Red glucose oxidase assay was used to classify each enteroid cell line into low or high sugar absorption phenotype (5-fold increased dietary sugar absorption). Samples of 9 individuals (6 obese and 3 lean) representing three different phenotypes: Group 1, BMI low, absorption low (n=3); group 2, BMI high, absorption low (n=3); and group 3, BMI high, absorption high (n=3) were subjected to further testing. We performed Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq) and RNA-seq experiments on all 9 enteroid cell lines. ATAC-seq was performed by the Epigenomics Development Lab at Mayo Clinic Rochester. Sequencing and subsequent quality control (QC) was performed by the Mayo Clinic Genome Analysis Core, and differential accessibility analysis was produced by the Mayo Clinic Bioinformatics Core.
Results
Approximately 40 million uniquely mapped read pairs were obtained per library with ~140K peaks identified. Data passed all QC metrics including Encode best practice standards. The transposase-accessible-chromatin domains in group 3 differed markedly from those in groups 1 and 2. Pathway analysis of predicted target gene ratios suggested involvement of the JAK-STAT signaling pathway, bile secretion, and endocrine resistance consistent with the involvement of energy metabolism in the observed differences. Differential accessibility analysis identified an increase in chromatin accessibility of the gene GUCA2A (guanylin), which can activate the downstream target protein kinase G (PKG). In a Drosophila model, a highly active PKG homologue has been previously shown to be associated with high intestinal glucose absorption, independent of food intake levels. PKG stimulation has also been associated with promotion of glucose uptake via the sodium-dependent glucose transporter SGLT1. RNA-seq results for the GUCA2A FPKM values in group 3 were higher than group 2 (P=0.055) (Figure 1).
Conclusion
We have identified elevated GUCA2A transcript in a subset of obese patients with high glucose absorption. GUCA2A may upregulate sugar transporters by activating PKG. Identification of differences in open chromatin and gene transcription may inform future directed inquiries into gene regulation treatment of obesity.

Background: Dysbiosis, an imbalance in microbial composition or function, is a key player in the development of conditions associated with chronic inflammation. The gut epithelium regulates host-microbiome dynamics through a variety of mechanisms, including the release of reactive oxygen species by NADPH oxidases. Dual oxidase 2 (DUOX2) is an antimicrobial NADPH oxidase expressed in the thyroid gland and gut epithelium and its activity is associated with dysbiosis. Loss of function mutations in DUOX2 are linked to the development of congenital hypothyroidism and inflammatory bowel disease. Metabolic syndrome (MetS) is a cluster of conditions characterized by dysbiosis, inflammation, obesity, glucose intolerance, and hepatic steatosis. Dysbiosis in MetS is associated with a reduction in Bacteroidaceae and Akkermansiaceae. Despite this association, the mechanism by which dysbiosis leads to chronic inflammation and MetS is poorly defined. Here, we investigate the relationship between impaired epithelial barrier function and the development of MetS by focusing for the first time on intestinal specific DUOX2 deficiency as a mechanistic link.
Methods: Mice carrying an intestinal epithelial-specific deletion of DUOX2 (DA IEC-KO), and wild-type (WT) littermates were fed a standard diet and euthanized at 24 weeks. DUOX2 activity was determined by Amplex Red. Metabolic alterations were determined by glucose tolerance tests and body, adipose tissue, and liver weight measurements and histological and gene expression assessments. Intestinal permeability was determined by FITC-dextran and microbial translocation assessments. The role of the microbiome was assessed by RNA-sequencing of stool and in antibiotic-treated mice.
Results: DA IEC-KO mice produced less epithelial H₂O₂ and exhibited increased body, liver, and adipose tissue weights. This was accompanied by glucose intolerance and increased plasma triglycerides, lipid deposition in the liver, and lipid droplet size in adipocytes. Expression of macrophage and thermogenesis markers, F4/80, CD68, and UCP1 were altered in the liver and adipose tissue of these mice. DA IEC-KO mice showed altered gut permeability and increased bacterial translocation to the liver and adipocytes. Microbial sequencing revealed that loss of intestinal epithelial DUOX2 activity led to significant differences in alpha and beta diversity, and a marked reduction in the family Akkermansiaceae. Antibiotic depletion of the microbiota abrogated all MetS phenotypes observed.
Conclusion: Our findings suggest that loss of intestinal epithelial DUOX2 activity leads to increased intestinal permeability and MetS in a microbiome dependent manner. These findings are significant in that they elucidate a novel role for epithelial DUOX2 at the host-microbial interface with distant systemic effects.