Hepatic and ileal levels of targeted bile acids are altered in the murine myosin Vb knockout model of microvillus inclusion disease (MVID)

Abstract

Background: Myosin Vb (Myo5b) is a molecular motor that traffics key cargo to the apical membrane of epithelial cells. Mutations in Myo5b that affect its function result in microvillus inclusion disease (MVID) in humans, which causes life-threatening diarrhea. Patients with MVID frequently present with liver cholestasis – a disorder resulting from reduced or blocked bile flow. Bile - primarily composed of cholesterol-derived bile acids (BAs) - is an important liver secretion that aids in the digestion of dietary fats and fat-soluble vitamins in the intestine. Primary BAs (such as cholic (CA) and chenodeoxycholic acid (CDA)) are synthesized in the liver and secreted into the intestine. In the intestine, primary bile acids are often converted by bacteria to secondary BAs (such as deoxycholic acid (DCA) and lithocholic acid (LCA)). Currently, the etiology of liver cholestasis and alterations in BA composition in the setting of loss of functional Myo5b is unknown. We hypothesized that Myo5b knockout (KO) mice would be unable to properly transport BAs in the liver and have lower levels of primary and secondary BAs in the intestine. Methods & Results: Liver and ileal contents was collected from Myo5b KO and littermate controls (n=3/genotype) and the tissue was weighed for normalization purposes. Each tissue sample was placed into individual homogenization tubes containing 100 mg of 1.4 mm ceramic beads and a volume equivalent to a tissue density of 200 mg/mL of ice-cold methanol. Tissue samples were then homogenized, tissue debris was pelleted by centrifugation, and the tissue sample supernatants were diluted in an Internal Standard solution prior to LC-MS/MS analysis. The BA (e.g., CA, β-MCA, CDCA, DCA, UDCA, LCA, GCA, GCDCA, GDCA, GUDCA, GLCA, TLCA, TCA, TCDCA, TDCA, TUDCA) content of the tissue sample extracts were quantified using a reverse-phase chromatography-tandem mass spectrometry (LC-MS/MS) method. Significant decreases in the concentrations of primary BAs (CA, β-MCA, GCA, TCA) and secondary BAs (DCA, TUDCA, TCDCA) were measured in the homogenized liver sample extracts collected from the Myo5b KO mice when compared to littermate control mice. Interestingly, we measured increased concentrations of primary BAs (CA and β-MCA) and one secondary BA (DCA) in the ileal tissue collected from the Myo5b KO mice when compared to littermate controls. We speculated that intestinal BAs could be accumulating due to loss of the apical BA transporter ASBT in the intestine. Immunostaining revealed loss of ASBT in the intestinal epithelium of adult mice with an intestinal specific deletion of Myo5b; indicating that Myo5b may regulate BAs levels via delivery of BAs transporters. Conclusion: Collectively, these data demonstrate the utility of our method for measuring the BA content of hepatic and GI tissue sample extracts in murine models of human disease, and the results suggest that the loss of functional Myo5b impairs liver BA production Financial support for this project was provided by an NIH-K01DK121869 and startup funds from MUSC (A Engevik). The Texas Children's Hospital Department of Pathology provides salary support to Texas Children's Microbiome Center-Metabolomics laboratory staff, and purchased the reagents, the consumables and durable supplies, and the LC-MS/MS equipment described. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.