Decrease of n6/n3 PUFA Ratio Augmented Growth and Improved Markers of Intestinal Barrier Integrity in Small Intestinal Organoids Derived from Naïve and Alcohol‐Fed Mice

Abstract

IntroductionIntestinal stem cells (ISCs) play an important role in gut homeostasis and function, including the maintenance of gut barrier integrity. Disruption of the intestinal barrier, specifically tight junctions, results in “leakage” of microbe‐derived compounds/toxins such as lipopolysaccharide (LPS) into the blood stream leading to endotoxemia and liver injury. We have previously demonstrated that both alcohol and nutritional factors are key players in alcohol‐induced intestinal and liver pathology. In the present study, we aimed to investigate the effects of alcohol and modulation of the n6/n3 PUFA ratio on ISC homeostasis. To this end, we used transgenic fat‐1 mice, which endogenously decrease the n6/n3 ratio by converting n6 PUFAs to n3.Hypothesisintestinal organoids derived from fat‐1 mice will show improved growth and proliferation, as well as attenuation of alcohol‐associated alterations in markers of tight junctions and antimicrobial defense.Materials and MethodsIntestinal crypts were isolated from small intestines of naïve or chronic ethanol‐fed WT C57BL/6 and fat‐1 mice. Crypts were cultured for seven days in StemCell IntestiCult growth media supplemented with gentamicin. Growth of ex vivo 3‐dimensional organoids was monitored daily by measuring cross‐sectional area, which was calculated digitally in ImageJ software. RNA was isolated with the Qiagen RNeasy kit, converted to cDNA, and used to quantify ISC growth/proliferation, tight junction, and antimicrobial defense markers by RT‐qPCR.ResultsCrypts derived from naïve fat‐1 mice grew organoids that were significantly larger than those derived from naïve WT mice after four days of culture, whereas ethanol had no significant effect. ISC markers Lgr5 and Bmi1 were significantly upregulated in organoids derived from fat‐1 as compared to WT mice in both pair‐fed and ethanol‐fed animals. No difference between WT and fat‐1 mice was seen in the expression of Axin2, a downstream target of the pro‐growth/proliferation Wnt/β‐catenin pathway. Expression of tight junction proteins ZO1 and Occludin were upregulated in ethanol‐fed fat‐1 mice as compared to their ethanol‐fed WT littermates. Bpi1, an antimicrobial peptide, was significantly increased in both ethanol‐fed and pair‐fed fat‐1 mice compared to WT animals. A similar trend was seen with Muc2, a marker of intestinal goblet cells.ConclusionsDecrease in the n6/n3 PUFA ratio and a significant increase in endogenous n3 PUFAs resulted in augmented growth of small intestinal organoids derived from naïve fat‐1 compared to WT mice. Chronic alcohol exposure compromised growth and proliferation in WT but not fat‐1 mice, as shown by expression of Lgr5 and Bmi1. Similarly, expression of tight junction and antimicrobial defense markers was greater in mice with higher endogenous n3 PUFA levels.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.