Investigation On Intestinal Lipid Malabsorption Caused By Loss Of Enteroendocrine Cells

Background and aims:Enteroendocrine cells are nutrient sensors in the intestinal tract.They participate in the regulation of various physiological functions by secreting endocrine hormones.Therefore,there is an axis consisting of enteroendocrine cells,endocrine hormones,and target effectors.Researches have shown that loss of enteroendocrine cell will lead to intestinal lipid malabsorption,but which process in intestinal lipid absorption is affected by the loss of enteroendocrine cells is hazy and the underlying mechanism is waiting to be figured out.The protein encoded by the gene Neurogenin3(Ngn3)is a key pre-endocrine factor for the differentiation of enteroendocrine cells.Establishing a mouse model loss of enteroendocrine cells by Ngn3 gene knockout should be helpful to explore the influence of enteroendocrine cell loss on intestinal lipid absorption.However,due to the high mortality rate after birth,the existing Ngn3 intestinal-specific knockout mice model(Ngn3flox/flox;villin-cre mice)needs further optimization.Many enteroendocrine hormones are considered to be involved in regulating intestinal lipid absorption."Enteroendocrine cell loss-enteroendocrine hormone loss-intestinal lipid malabsorption" may be a causal line.The purpose of this study is to explore the influence of enteroendocrine cells on intestinal lipid absorption and try to find out whether there is a key enteroendocrine hormone that can rescue this fat malabsorption caused by the loss of enteroendocrine cells.Methods:1.Establish an optimized enteroendocrine cell-loss mouse model(Ngn3ΔIEC mice):Adult Ngn3flox/flox;villin-creERT2 mice were intraperitoneally injected with 40 μg/g/day tamoxifen for 5 consecutive days to induce Ngn3 gene conditional knockout in intestinal epithelial cells.On the 20th day after induction,mice intestinal epithelial cells and several intestinal segments were collected to analyze Ngn3 gene knockout efficiency,enteroendocrine hormone gene expression,and the changes in the cell number of enteroendocrine cells,paneth cells,and goblet cells.2.Investigation on intestinal lipid malabsorption in Ngn3ΔIEC mice:When Ngn3ΔIEC mice were fed with a normal fat diet(ND),their body weight,daily food intake,and feces excretion were monitored.We investigated whether Ngn3ΔIEC mice were lipid-malabsorptive by detecting serum lipid profiles,carrying out intestinal lipid absorption assay,and analyzing fecal fat content.Besides,we detected the expression of intestinal lipid absorption-related genes to find out whether the core process of intestinal lipid absorption was affected.Finally,Ngn3ΔIEC mice were fed with a 60%high-fat diet(60%HFD),and the lipid-malabsorptive phenotype in Ngn3ΔIEC mice was re-observed under fat overload conditions.3.Enteroendocrine hormone GLP-2 rescue assay:Ngn3ΔIEC mice were intraperitoneally injected with 0.1 μg/g/day GLP-2 for the rescue assay.After GLP-2 pretreatment for 14 consecutive days when Ngn3ΔIEC mice were fed with ND,mice were then challenged with 60%HFD and GLP-2 treatment lasted for 7 days.The body weight of mice was monitored to evaluate the rescue effects of the synthetic enteroendocrine hormone GLP-2.Results:1.Tamoxifen could effectively induce Ngn3 gene conditional knockout in intestinal epithelial cells in mice genotyped Ngn3flox/flox;villin-creERT2.Ngn3 gene knockout resulted in reduced expression of enteroendocrine hormone genes and loss of enteroendocrine cells but did not affect the cell number of paneth and goblet cells.2.When mice were fed with ND,the weight gain of Ngn3ΔIEC mice was slow,and their daily food intake and feces excretion were significantly higher.There was no difference in serum lipid profile between the control and Ngn3ΔIEC groups but evidence from intestinal lipid absorption assay and fecal fat content analysis indicated that Ngn3ΔIEC mice had intestinal lipid malabsorption.The expression of fatty acid transporter gene Cd36 and cholesterol transporter gene Npc1l1 in intestinal epithelial cells of Ngn3ΔIEC mice was reduced.When mice were fed with 60%HFD,the body weight of the Ngn3ΔIEC mice dropped sharply.Their daily food intake,feces excretion,and fecal fat content was increased.Their serum lipid profile was reduced.The expression of fatty acid transporter gene Cd36,cholesterol transporter gene(Npc1l1,Abcg5,Abcg8),triglyceride synthetic gene Dgat1,and apolipoprotein product gene Mttp was reduced.3.GLP-2 pretreatment for 14 days followed by 0.1μg/g/day GLP-2 treatment for 7 days could not rescue the weight loss of Ngn3ΔIEC mice due to intestinal lipid malabsorption when fed with 60%HFD.Conclusions:In this study,we established an adult mouse model with enteroendocrine cell loss through tamoxifen-induced Ngn3 gene knockout in Ngn3flox/flox;villin-creERT2 mice.In this mouse model,the loss of enteroendocrine cells led to intestinal lipid malabsorption.Enteroendocrine cell loss affected the process of lipid uptake by intestinal epithelial cells.With the increase in fat content in food(60%HFD feeding),the intestinal fat-malabsorptive phenotype in Ngn3ΔIEC mice caused by the loss of enteroendocrine cells was more evident.Intraperitoneally treating Ngn3ΔIEC mice with 0.1 μg/g/day synthetic enteroendocrine hormone GLP-2 had no rescue effects on the intestinal lipid malabsorption caused by the loss of enteroendocrine cells.Significance:This study enriches the functional research of enteroendocrine cells and provides a theoretical basis for the treatment of diseases related to intestinal lipid malabsorption.