Fig 1: Intestine-specific Lgr4 knockout improves glucose tolerance.Six-week-old male Lgr4iKO mice and littermates were fed normal chow diet (NCD) or 60% high fat diet (HFD) for 12 weeks. Results were expressed as mean ± SD. *P < 0.05 vs WT NCD. #P < 0.05 vs WT HFD. n = 3–6. a, b Intraperitoneal glucose tolerance test and the area under curve of mice fed NCD or HFD. n = 3 for WT NCD, 3 for Lgr4iKO NCD, 4 for WT HFD, and 4 for Lgr4iKO HFD. Statistical analysis by two-way ANOVA with Šídák’s multiple comparisons test for IPGTT. #P = 0.0007. c Homeostatic model assessment of insulin resistance (HOMA-IR) and homeostatic model assessment of insulin sensitivity (HOMA-IS) of HFD-fed mice. HOMA-IR = plasma insulin (μU) × glucose (mmol/l)/22.5. HOMA-IS = 1/HOMA-IR. n = 5 for WT HFD, and 3 for Lgr4iKO HFD. Student’s t test (two-side) was used for un-paired analysis. #P = 0.0371 for HOMA-IR. #P = 0.0234 for HOMA-IS. d, e Oral glucose tolerance test and the area under curve in mice fed NCD or HFD. n = 6 for WT NCD, 5 for Lgr4iKO NCD, 6 for WT HFD, and 4 for Lgr4iKO HFD. f mRNA levels of carbohydrate absorption markers (Glut1, Glut2, Glut5 and Sglt1) in intestine of mice fed NCD or HFD. n = 5 for WT NCD, 5–6 for Lgr4iKO NCD, 6 for WT HFD, and 4 for Lgr4iKO HFD. Statistical analysis by two-way ANOVA with Šídák’s multiple comparisons test. *P < 0.0001. #P = 0.0007 for Glut2 and #P < 0.0001 for Glut5. g Immunohistochemical staining of GLUT2 in intestine and quantification of positive area. n = 9.
Fig 2: Metformin could reduce tumorigenesis through LGR4. (a). The expression of LGR4 was detected upon metformin treatment. (b). The proliferation of HCC by LGR4 overexpression in metformin-treated HCC cell lines. (c). The migration of HCC by LGR4 overexpression in metformin treated HCC cell lines; scar bar = 100 μm. (d). The invasion of HCC by LGR4 overexpression in metformin-treated HCC cell lines; scar bar = 50 μm. (e). The expression of catenin by LGR4 overexpression overexpressed metformin-treated HCC cell lines. ∗∗p < 0.01, ∗∗∗p < 0.001. PcDNA, an empty vector.
Fig 3: The effect of LGR4 on the heterogeneity of intestinal absorptive cells.Six-week-old male Lgr4iKO mice and littermates were fed normal chow diet (NCD) for 12 weeks. Single cell RNA sequencing was used to obtain intestinal epithelium single cell transcriptome data from 18-week-old Lgr4iKO mice and littermates. n = 3. a t-SNE plot showing stem cell, TA cell, absorptive cell, goblet cell, paneth cell, enteroendocrine cell and tuft cell marker genes expression in intestinal epithelium. b Defining cell populations with marker gene expression. c Pseudotime ordering on intestinal epithelium cells. d Re-clustering absorptive cells. Enterocytes selective for absorption of long-chain fatty acid, carbohydrate, or both were defined by the expression of Cd36、Fatp4、Glut2 and Sglt1. e The proportions of long-chain fatty acid-absorptive enterocytes, carbohydrate-absorptive enterocytes, and long-chain fatty acid and carbohydrate-absorptive enterocytes.
Fig 4: Deficiency of intestinal Lgr4 protects mice from HFD-induced obesity.Six-week-old male Lgr4iKO mice and littermates were fed normal chow diet (NCD) or 60% high fat diet (HFD) for 12 weeks. Results were expressed as mean ± SD and analyzed by the t-test (two-side). *P < 0.05 vs WT NCD. #P < 0.05 vs WT HFD. n = 4–11. a, b Body weight and body size of mice fed NCD or HFD. n = 11 for WT NCD, 5 for Lgr4iKO NCD, 7 for WT HFD, and 5 for Lgr4iKO HFD. WT NCD vs. Lgr4iKO NCD: P = 0.0003. WT HFD vs. Lgr4iKO HFD: P = 0.005. c Body condition score (BCS) of mice fed NCD or HFD. n = 5. *P = 0.0476. d, e Fat mass and lean mass of mice fed NCD or HFD. n = 5 for WT NCD, 4 for Lgr4iKO NCD, 7 for WT HFD, and 5 for Lgr4iKO HFD. d Fat mass: *P = 0.0268. Lean mass: *P = 0.0027. e Fat mass: #P = 0.0048. Lean mass: #P = 0.006. f–i Fat mass, H&E staining, adipocyte size and mRNA levels of beigeing marker genes in sWAT (Ucp1, Ucp3 and Pgc1α) of mice fed either NCD (f: n = 5–6, *P = 0.0196) or HFD (g: n = 4–6, #P = 0.0279 for sWAT weight, #P <0.0001 for frequency, #P = 0.0115 for Ucp1), as well as in eWAT of mice fed NCD (h, n = 4–6, *P = 0.0005) or HFD (i, n = 4–6, #P = 0.0222 for eWAT weight, #P = 0.047 for Ucp1).
Fig 5: LGR4 switching genes RNAi reduces the antitumor effect caused by metformin. (a). The proliferation of HCC by LGR4 switching genes RNAi in metformin-treated HCC cell lines. (b). The migration of HCC by LGR4 switching genes RNAi in metformin-treated HCC cell lines; scar bar = 100 μm. (c). The invasion of HCC by LGR4 switching genes RNAi in metformin-treated HCC cell lines; scar bar = 50 μm. (d). The expression of catenin was induced by LGR4 switching genes by RNAi metformin-treated HCC cell lines. ∗∗p < 0.01, ∗∗∗p < 0.001. PcDNA, an empty vector.
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