Society: AGA

The autonomic nervous system, with its two branches the parasympathetic and the sympathetic, plays a pivotal role in the regulation of many physiological responses to maintain metabolic homeostasis in variable target tissues. In general, afferent or sensory fibers transmit metabolic information to the central nervous system, while efferent or motor fibers provide the major neural pathway from the brain to peripheral tissues. An imbalance between the two branches or the two arms of the autonomic nervous system branches is observed in many pathologic conditions. However, new developing technologies in selective electro-stimulation, pharmacogenetics, advanced barbaric endoscopy or new surgical techniques may allow careful manipulation of neuronal tracts of interest for possible future therapeutic purpose. The activation or inhibition of tissue-specific neural fibers can have a significant effect on key metabolic processes such as hepatic or skeletal muscle glucose trafficking, adipose tissue lipolysis and thus weight loss. Even though many techniques in neuroscience and molecular genetics are currently available to study the function of the autonomic nervous system, many aspects are still unknown regarding the extent of regulation of the parasympathetic and sympathetic nervous systems to glucose, lipid and energy homeostasis.

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.

<b>Figure 1. </b><b>Differential accessibility analysis on the 3 groups</b><br /> <b>(A)</b> An increase in transposase-accessible chromatin associated with gene transcription start sites (TSS) for a subset of genes was observed in group 3 vs. groups 1 and 2. <b>(B)</b> Pathway analysis of predicted target gene ratios suggested involvement of JAK-STAT signaling, neuroactive ligand-receptor interactions, and bile secretion in group 3. <b>(C)</b> Differentially accessible <i>cis</i>-regulatory elements of the <i>GUCA2A</i> gene across organoids from lean patients (Ctrl), obese patients with low glucose absorption (Lglu) and obese patients with high glucose absorption (Hglu) visualized by Integrative Genomics Viewer (IGV). The promoter region of <i>GUCA2A</i> is highlighted by the yellow box. <b>(D)</b> Normalized RNA-seq transcript for <i>GUCA2A </i>(FPKM values) in group 3 is higher than group 2 (<i>P</i>=0.055).

Figure 1. Differential accessibility analysis on the 3 groups
(A) An increase in transposase-accessible chromatin associated with gene transcription start sites (TSS) for a subset of genes was observed in group 3 vs. groups 1 and 2. (B) Pathway analysis of predicted target gene ratios suggested involvement of JAK-STAT signaling, neuroactive ligand-receptor interactions, and bile secretion in group 3. (C) Differentially accessible cis-regulatory elements of the GUCA2A gene across organoids from lean patients (Ctrl), obese patients with low glucose absorption (Lglu) and obese patients with high glucose absorption (Hglu) visualized by Integrative Genomics Viewer (IGV). The promoter region of GUCA2A is highlighted by the yellow box. (D) Normalized RNA-seq transcript for GUCA2A (FPKM values) in group 3 is higher than group 2 (P=0.055).

Dilhana Badurdeen

Mayo Foundation for Medical Education and Research

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