**Background:** Enteroendocrine cells (EECs) are rare intestinal epithelial cells that sense luminal nutrients and secrete hormones (e.g., GLP-1, PYY, CCK, secretin) that regulate energy homeostasis, insulin secretion, and food intake. Constitutive EEC deficiency is lethal in neonates due to severe malabsorption, but the physiological role of EECs in healthy adults had not been directly studied. This study aimed to determine the long-term consequences of total EEC loss in adult mice on energy metabolism, intestinal transcriptome, and gut microbiota.
**Methods:** The authors generated an inducible EEC depletion model by treating adult male Neurog3^fl/fl^; Villin-CreER^T2^ mice with tamoxifen (10 mg/30 g body weight, oral gavage, once every second day for 5 days). EEC deletion was confirmed by RT-qPCR, immunofluorescence, and RNA-seq. Mice were fed either a standard chow diet (CD, Safe D04, 3.34 kcal/g) or a high-fat diet (HFD, D12492, 60% kcal from fat). Metabolic phenotyping included weekly body weight, body composition by MRI (4 and 10 weeks post-tamoxifen), food intake and feces production in metabolic cages (5 weeks post-tamoxifen), bomb calorimetry of feces, and blood chemistry (total cholesterol, HDL, LDL, triglycerides, free fatty acids, glycerol, bile acids, lipase). Lipid absorption was assessed by Oil Red O staining of intestinal cryosections. RNA-seq was performed on duodenum and ileum of EEC-deficient and control mice under CD (18 weeks post-tamoxifen, n=4/genotype) and HFD (5 weeks post-tamoxifen, n=4/genotype). EEC-specific transcriptomic responses to HFD were analyzed by sorting eYFP+ EECs from Neurog3^eYFP/+^ mice fed CD or HFD for 4 weeks (n=3/condition). Gut microbiota was profiled by 16S rRNA V3-V4 amplicon sequencing of feces collected before (D0) and 15 and 30 days after tamoxifen (n=8/genotype). Fecal short-chain fatty acids and sterols were quantified by GC-MS. Predictive functional metagenomics was performed using PICRUSt2.
**Key Results:** Tamoxifen treatment efficiently deleted Neurog3 and abolished EEC differentiation, with no effect on goblet cells, Paneth cells, or stem cell marker Lgr5, though villi were shorter in the proximal small intestine. Under CD, EEC-deficient mice lost ~10% of body weight (progressive over 18 weeks) with decreased fat mass (−30%) and increased lean mass (+6%). Food intake was unchanged, but feces production increased and bomb calorimetry showed higher fecal energy excretion, indicating reduced food efficiency. Blood concentrations of total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and free fatty acids were significantly reduced; lipase was decreased; bile acids were unchanged. Fecal cholesterol, lathosterol, and desmosterol were increased. Oil Red O staining showed fewer neutral lipids in the duodenum and more in the ileum of EEC-deficient mice, indicating a spatial delay in lipid absorption. Under HFD, 60% of EEC-deficient mice died within 5 weeks, and the remaining 40% required sacrifice; weight loss was more rapid and severe, with fat mass decreased by −57% and lean mass increased by +15% as early as 4 weeks.
RNA-seq revealed 328 differentially expressed genes (DE) in the duodenum and 598 DE in the ileum of EEC-deficient mice under CD (adjusted P ≤ 0.05, |log2FC| ≥ 0.5). Upregulated genes were enriched for lipid metabolism (cholesterol biosynthesis, triglyceride metabolism), carbohydrate metabolism (glucose transporter Slc2a2/GLUT2, fructose transporter Slc2a5/GLUT5, sucrase-isomaltase Sis), and gluconeogenesis (Gls, Fbp1). The top upregulated gene in the ileum was Pla2g4c (Log2FC = 6.4, adjusted P = 1.36E−20). Antimicrobial peptide genes (Reg3b, Reg3g, Ang4, Defa40) were upregulated mainly in the duodenum. In EECs sorted from HFD-fed mice, 853 genes were DE compared with CD (437 upregulated, 416 downregulated), with 345 genes exclusively upregulated in EECs, including hormone transcripts Pyy, Sct, Cck, Nts, Gip, Vgf and lipid metabolism genes Apoa5, Ptgs2/Cox-2, and Mgll/Magl.
Gut microbiota analysis showed a progressive decrease in α-diversity (richness and Shannon index) in EEC-deficient mice at D15 and D30. β-diversity (Bray-Curtis) was significantly different at D30 (PERMANOVA P = 0.001). At the phylum level, Actinobacteriota and Proteobacteria were enriched by D15. At the family level, Muribaculaceae and Lachnospiraceae declined, while Bacteroidaceae, Lactobacillaceae, Bifidobacteriaceae, Tannerellaceae, Atopobiaceae, and Sutterellaceae increased. Genera enriched in mutants included Bacteroides, Lactobacillus, Bifidobacterium, and Olsenella; depleted genera included Lachnospiraceae, Prevotellaceae, Rikenella, Akkermansia, Muribaculum, and Desulfovibrio. Despite the loss of Lachnospiraceae and Ruminococcaceae (major short-chain fatty acid producers), fecal concentrations of acetate, propionate, butyrate, isobutyrate, and valerate were unchanged. PICRUSt2 predicted increased pathways for carbohydrate metabolism, energy metabolism, and xenobiotic degradation in mutants, and decreased pathways for bacterial motility, antimicrobial resistance, and secondary metabolite biosynthesis.
**Clinical Implications:** This study demonstrates that EECs are critical for efficient lipid absorption and energy homeostasis in adult mice, but that compensatory mechanisms (upregulation of lipogenesis, carbohydrate transport, and gluconeogenesis) can sustain survival under a low-fat diet. The rapid and severe gut microbiota remodeling following EEC loss suggests that enteroendocrine hormones play a direct role in maintaining microbial community structure, independent of dietary changes. These findings may inform the management of human enteric anendocrinosis (NEUROG3 mutations), where patients require parenteral nutrition, by identifying potential adaptive pathways (e.g., enhancing carbohydrate metabolism, modulating microbiota) that could be therapeutically targeted to improve nutrient absorption and reduce dependence on parenteral support. The EEC-specific transcriptomic response to HFD also highlights novel hormone and lipid metabolism genes (GIP, VGF, Apoa5, Ptgs2) that may be relevant to obesity and metabolic disease.