Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice – Nature Communications

Deficiency of intestinal Lgr4 reduces body weight and protects mice from HFD-induced obesity

Lgr4 mRNA was detected in a wide variety of tissues, with high abundance in hypothalamus and digestive organs such as pancreas, stomach, jejunum, ileum, and liver (Supplementary Fig. 1a). To determine whether intestinal LGR4 regulates energy metabolism, we generated Lgr4^iKO mice within which Lgr4 was specifically knocked out in intestinal epithelium (Supplementary Fig. 1b–d). Six-week-old male Lgr4^iKO mice and wild type (WT) littermates were fed normal chow diet (NCD) or 60% high fat diet (HFD) for 12 weeks. Compared with WT littermates, significant weight loss and smaller body size were observed in Lgr4^iKO mice fed either NCD or HFD (Fig. 1a, b). However, there was no significant difference in the body condition score (BCS) of Lgr4^iKO mice compared with WT littermates (Fig. 1c). Food intake was substantially reduced only in mice fed NCD but not in animals fed HFD, indicating that the weight loss is not entirely dependent on food intake (Supplementary Fig. 2a). Consistently, Lgr4^iKO mice displayed significantly less fat mass and lean mass (Fig. 1d, e). Fat weight and adipocyte size of subcutaneous white adipose tissue (sWAT) were significantly decreased. mRNA levels of beigeing marker gene Ucp1 was strikingly increased in sWAT of Lgr4^iKO mice fed HFD (Fig. 1f, g). Similar findings were observed in the epidydimal white adipose tissue (eWAT) (Fig. 1h, i). These results suggest that deficiency of intestinal Lgr4 reduces body weight and protects mice from HFD-induced obesity.

Six-week-old male Lgr4^iKO 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 Lgr4^iKO NCD, 7 for WT HFD, and 5 for Lgr4^iKO HFD. WT NCD vs. Lgr4^iKO NCD: P  =  0.0003. WT HFD vs. Lgr4^iKO 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 Lgr4^iKO NCD, 7 for WT HFD, and 5 for Lgr4^iKO 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).

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To further elicit the reason of weight loss in Lgr4^iKO mice, cold exposure and metabolic cage experiments were performed. As shown in Supplementary Fig. 2b, c, rectal body temperature during the 4 °C cold exposure, physical activity, and respiratory quotient (RQ = VCO₂/VO₂) were not significantly altered by deficiency of intestinal Lgr4. These results indicate that weight loss of Lgr4^iKO mice was unlikely due to the alteration in thermogenesis and energy expenditure.

Deficiency of intestinal Lgr4 protects mice from HFD-induced liver steatosis

Next, we examined hepatic lipid metabolism in Lgr4^iKO mice. Liver weight and plasma triglyceride level were significantly decreased in Lgr4^iKO mice fed NCD (Fig. 2a–c). In Lgr4^iKO mice fed HFD, liver weight, plasma and hepatic triglyceride contents, and steatosis evidenced by H&E and oil red O staining were all decreased (Fig. 2d–f). These results suggest that deficiency of intestinal Lgr4 protects mice from HFD-induced hepatic steatosis. Interestingly, hepatic genes relevant to lipogenesis, lipid transport, and β-oxidation remained largely unchanged (Supplementary Fig. 3). The only exception was the upregulation of Pparα (Supplementary Fig. 3). These results indicate that deficiency of intestinal Lgr4 decreases hepatic lipid deposition likely via an extrahepatic mechanism.

Six-week-old male Lgr4^iKO 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. a Liver weight and liver size of NCD-fed mice. n  =  6 for WT NCD and 5 for Lgr4^iKO NCD. *P = 0.0385. b Plasma triglyceride levels of NCD-fed mice. n = 5. *P = 0.0157. c Triglyceride contents and steatosis in liver of NCD-fed mice. n  =  5 for WT NCD and 4 for Lgr4^iKO NCD. d Liver weight and liver size of HFD-fed mice. n  =  6 for WT NCD and 4 for Lgr4^iKO NCD. ^#P = 0.024. e Plasma triglyceride levels of HFD-fed mice. n = 4. ^#P = 0.0236. f Triglyceride contents and steatosis in liver of HFD-fed mice. n = 4. ^#P = 0.0388 for hepatic triglyceride, ^#P < 0.0001 for NAS score.

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Deficiency of intestinal Lgr4 decreases lipid absorption

The primary function of intestine is nutrient absorption. To assess the alteration of lipid absorption, we collected feces from mice, extracted total lipid, then measured fecal triglyceride levels. As shown in Fig. 3a, fecal triglyceride levels were significantly increased in Lgr4^iKO mice fed either NCD or HFD. Since HFD-feeding may alter lipid absorption, mice fed NCD were used in the following experiments. Plasma triglyceride levels and AUC of oral lipid tolerance test (OLTT) were substantially reduced in Lgr4^iKO mice after olive oil gavage (Fig. 3b). Oil red O staining showed that lipid droplets were remarkably decreased in the jejunum of Lgr4^iKO mice 2 h after gavage with 200 μL of olive oil (Fig. 3c). Examination of key intestinal lipid transporters revealed that both mRNA and protein levels of FATP4 and CD36 were substantially reduced (Fig. 3d–f). These results indicate that deficiency of intestinal Lgr4 decreases the expression of lipid transporters, leading to subsequent reduction in lipid absorption.

a–f Six-week-old male Lgr4^iKO 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 or one-way ANOVA. *P < 0.05 vs WT NCD. ^#P < 0.05 vs WT HFD. n = 3–6. a Fecal triglyceride levels. n  =  4 for WT NCD, 4 for Lgr4^iKO NCD, 5 for WT HFD, and 4 for Lgr4^iKO HFD. WT NCD vs. Lgr4^iKO NCD: *P  =  0.0296. WT NCD vs. WT HFD: *P  =  0.0185. WT HFD vs. Lgr4^iKO HFD: ^#P  =  0.011. b Levels of circulating triglyceride and the area under curve in response to oral administration of olive oil in NCD-fed mice. n = 4. *P  = 0.0004 for AUC. c Oil red O staining of intestine 2 h after olive oil gavage and quantitative analysis. n  =  4 for WT NCD and 3 for Lgr4^iKO NCD. *P  = 0.0107. d mRNA levels of lipid absorption markers (Fatp4, Cd36 and Fabp2) in intestine of NCD-fed mice. n  =  5 for WT NCD and 5–6 for Lgr4^iKO NCD. *P  = 0.0483 for Cd36, *P = 0.0104 for Cav1. e Immunohistochemical staining of FATP4 in intestine and quantification of positive area. n = 9. *P  = 0.0004(left) and 0.0075(right). f Western blot and quantification of CD36 protein levels. β-actin was used as internal control. n = 3. *P = 0.0093. g–j The mouse small intestinal epithelial cell line MODE-K cells were transfected with Lgr4 siRNA for 48 h. g Western blot and quantification of LGR4 and FATP4 protein levels. β-actin was used as loading control. n = 3. *P = 0.008 for LGR4, *P = 0.0124 for FATP4. h mRNA levels of lipid absorption markers (Fatp4, Cav1, Fabp1 and Fabp2) in MODE-K cells. β-actin was used as a reference gene. n = 3. *P = 0.0077 for Fatp4, *P = 0.0085 for Cav1. i The triglyceride level in MODE-K cells treated with mixture of oleic acid (0.6 mmol/l) and palmitic acid (0.2 mmol/l). n = 5. j Cells were treated with BODIPY-C₁₂ long-chain fatty acid for 2 h and observed under microscope and the uptake of BODIPY-C₁₂ long-chain fatty acid in MODE-K cells by flow cytometry.

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We next used MODE-K cells as an in vitro model to examine the effects of LGR4 on lipid absorption. Seventy-two hours after Lgr4 siRNA treatment, mRNA and protein levels of LGR4 and FATP4 in MODE-K cells were substantially reduced (Fig. 3g, h). These alterations were associated with a reduction in the concentration of intracellular triglyceride in MODE-K cells treated with a mixture of oleic acid and palmitic acid (Fig. 3i, P = 0.0548). Consistently, intensity of BODIPY fluorescence was also reduced (Fig. 3j). Next, we verified the effect of Lgr4 deletion on lipid uptake in intestinal organoids. The growth process of intestinal organoids was showed in Supplementary Fig. 4a. We found a significant decrease of Fabp1 and Fatp4 mRNA level (Supplementary Fig. 4b) and uptake of BODIPY (Supplementary Fig. 4c) in Lgr4-deficiency intestinal organoids. These results suggest that Lgr4 knockdown decreases lipid uptake.

Intestine-specific knockdown of Lgr4 improves glucose tolerance

In addition to absorption of lipid, we examined whether deficiency of Lgr4 would affect intestinal absorption of carbohydrate. After intraperitoneal injection of glucose, Lgr4^iKO mice exhibited improved glucose tolerance, particularly in the HFD group (Fig. 4a, b). Further, insulin resistance index (HOMA-IR) was decreased and insulin sensitivity index (HOMA-IS) increased in Lgr4^iKO mice fed HFD (Fig. 4c), suggesting that the increment in glucose tolerance may be attributed to improved peripheral insulin sensitivity. Interestingly, levels of blood glucose after oral glucose administration were not significantly altered compared with littermate control mice (Fig. 4d, e). The differential results of OGTT and IPGTT indicates that intestinal glucose absorption was increased in Lgr4^iKO mice. As shown in Fig. 4f, g, both mRNA and protein levels of GLUT2 were substantially elevated. These results suggest that deficiency of intestinal Lgr4 increases glucose absorption via up-regulation of glucose transporter, GLUT2.

Six-week-old male Lgr4^iKO 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 Lgr4^iKO NCD, 4 for WT HFD, and 4 for Lgr4^iKO 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 Lgr4^iKO 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 Lgr4^iKO NCD, 6 for WT HFD, and 4 for Lgr4^iKO 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 Lgr4^iKO NCD, 6 for WT HFD, and 4 for Lgr4^iKO 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.

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Deficiency of intestinal Lgr4 decreases enterocytes selective for long-chain fatty acid absorption while increasing carbohydrate-absorptive enterocytes

Intestinal epithelial populations are crucial for dietary lipid and carbohydrate absorption. To determine the mechanism underlying the decrease of lipid absorption and concurrent increase of carbohydrate absorption, we analyzed the cellular heterogeneity of intestinal epithelia using the single cell RNAseq. After quality control of data filtering, intestinal epithelial populations were re-clustered using Seurat. According to the reported marker genes⁷, stem cells, TA cells, absorptive enterocytes, goblet cells, Paneth cells, enteroendocrine cells and tuft cells were defined (Fig. 5a, b). To further elucidate the order of differentiation between the epithelial populations, pseudotime analysis was analyzed and the cell markers of each population were projected on pseudotime axis. As shown in Fig. 5c, the differentiation of each population is well in line with the known order. These results confirm the accuracy for our definition of intestinal epithelial populations.

Six-week-old male Lgr4^iKO 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 Lgr4^iKO 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.

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To investigate the cellular heterogeneity, absorptive enterocytes were re-clustered. Long-chain fatty acid-absorptive, and carbohydrate-absorptive enterocytes were defined by the expression of Cd36 and Fatp4、Glut2 and Sglt1 respectively (Fig. 5d). The statistical analysis for the proportions of absorptive cells revealed a decrease in enterocytes selective for long-chain fatty acid absorption and an increase in enterocytes elective for carbohydrate absorption in intestine of Lgr4^iKO mice (Fig. 5e).

LGR4 regulates differentiation of intestinal stem cells via Wnt and Notch signaling pathways

Absorptive enterocytes derive from the differentiation of intestinal stem cells. LGR4 has been found to be highly expressed in intestinal stem cells and TA cells⁸. To investigate the effect of intestinal LGR4 on stem cell differentiation, the proportions of each intestinal epithelial population was analyzed via single cell RNAseq. According to analysis of the number, the marker genes expression and specific staining, we found that deficiency of intestinal Lgr4 increased the proportion of absorptive enterocytes and TA cells, while decreasing the proportion of stem cells, goblet cells, Paneth cells, enteroendocrine cells and tuft cells (Supplementary Figs. 5, 6 and Fig. 6a). This observation was further confirmed by experiments using organoids derived from WT and Lgr4 deficient mice (Supplementary Fig. 6l). Consistent with the decrement in the number of Paneth cells and Tuft cells, intestinal barrier disruption (Supplementary Fig. 7a–d) and alteration of the microbiota (Supplementary Fig. 8) were observed in Lgr4^iKO mice. In addition, Lgr4 deficiency supressing the apoptosis indicates that the survival time of enterocytes is increased (Supplementary Fig. 7e).

a–i Six-week-old male Lgr4^iKO mice and littermates were fed normal chow diet for 12 weeks. Single cell RNA sequencing was used to obtain intestinal epithelium single cell transcriptome data from 18-week-old Lgr4^iKO mice and littermates. n = 3. *P < 0.05 vs WT. a The proportions of stem cells, TA cells, absorptive cells, goblet cells, paneth cells, enteroendocrine cells and tuft cells. b t-SNE plot showing absorptive progenitor cell marker genes expression (left) and heatmap showing UMI value (right). The transcription expression levels were calculated as UMI value. c t-SNE plot showing secretory progenitor cell marker genes expression (left) and heatmap showing UMI value of Dll1 (right). d mRNA levels of absorptive progenitor cell and secretory progenitor cell markers in intestine of NCD-fed mice. n = 4–5 for WT, and 5–6 for Lgr4^iKO. Results were expressed as mean ± SD. e Pseudotime showing Wnt target genes expression (left) and heatmap showing UMI value (right). f Western blotting detecting nuclear and cytosol levels of β-catenin protein. The relative expression level was quantified using Image J software. Results were expressed as mean ± SD. g mRNA levels of downstream genes of the Notch signaling pathway (Math1 and Hes1) detected by RT-qPCR. n = 5 for WT, and 6 for Lgr4^iKO. Results were expressed as mean ± SD. h Pseudotime showing Notch related genes expression (left) and heatmap showing UMI value (right). i Western blot and quantification of HES1 protein levels. β-actin was used as loading control. Results were expressed as mean ± SD. j Wnt and Notch related genes of the mouse small intestinal epithelial cell line MODE-K cells treated with the Notch activator VPA (P4543-25G, Sigma). *P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák’s multiple comparisons test. ^*P = 0.0001 for Axin2, ^*P = 0.0035 for Ccnd1, ^*P = 0.0003 for Hes1, ^*P < 0.0001 for Hey1. k mRNA levels of lipid absorption markers of MODE-K cells treated with the Wnt inhibitor IWR-1 (HY-12238, MedChemExpress). *P < 0.05 vs NC. n = 3. Statistical analysis by two-way ANOVA with Šídák’s multiple comparisons test. ^*P = 0.0178.

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Since the differentiation of intestinal stem cells into absorptive and secretory progenitors is regulated by Notch and Wnt signaling respectively^9,10, we next examined the alteration of these two signaling pathways in Lgr4^iKO mice. As shown in Fig. 6b and c, cells highly expressing the absorptive progenitor genes (Ccnb1, Cdc20, Cenpa, Cdkn3, Ube2c, Aurka and Ccna2) substantially increased, whereas cells highly expressing the secretory progenitor gene (Dll1) decreased. Consistently, mRNA levels of absorptive progenitor marker and secretory progenitor marker genes were strikingly increased and decreased respectively (Fig. 6d) in intestine of Lgr4^iKO mice. These results indicate that deficiency of intestinal Lgr4 increases proportion of absorptive progenitors while concurrently decreases proportion of secretory progenitors.

The differentiation of secretory progenitors is activated by Wnt-β-catenin signaling, whereas the differentiation of absorptive progenitors depends on Notch signaling^{9,11,12,13,14}. Consistently, cells highly expressing the Wnt target genes such as Sox9, Sox4, Ascl2, Myc, Ube2c, and Lgr5, and the UMI value of these genes were decreased in Lgr4^iKO mice (Fig. 6e). Further, nuclear translocalization of β-catenin was decreased in intestine epithelia of Lgr4^iKO mice (Fig. 6f). mRNA levels of Math1, the downstream target of β-catenin was decreased (Fig. 6g). On the other hand, cells highly expressing Notch related genes such as Notch1, Notch4, Dtx1, Dtx3, Dll4, and Mfng and the UMI value of these genes were significantly increased (Fig. 6h). mRNA and protein levels of HES1, the downstream target of Notch was increased (Fig. 6i). As shown in Fig. 6j, activation of Notch signaling by VPA significantly suppresses the Wnt signaling evidenced by the decrement of its relevant target genes. This observation suggests a link between Wnt and Notch signaling. In addition, inhibition of Wnt signaling substantially decreases expression of lipid uptake genes Fatp4 in MODE-K cells (Fig. 6k). This observation suggests that inhibition of Wnt signaling may decrease lipid absorption. These results indicate that LGR4 regulates the differential differentiation of intestinal stem cells into absorptive and secretory cells via Notch and Wnt signaling pathways.

Next, we further explored the relationship between LGR4 and Notch signaling in our research. In MODE-K cells, we quantified the mRNA levels of genes in Notch signaling, and found that levels of Psen1, Aph1a and Nedd4 were significantly changed (Supplementary Fig. 9a). We then further confirmed the increment of PSEN1 mRNA (Supplementary Fig. 9b) and protein levels (Supplementary Fig. 9c) in IEC6 cells. PSEN1 is an active component of γ-secretase, which is responsible for the cleavage of Notch and next-step function of NICD. These observations suggest that deficiency of LGR4 enhances the expression of Psen1 and thus stimulating the function of Notch signaling. The canonical pathway of LGR4 function is mediated by the nuclear translocation of β-catenin and then the transcriptional regulation of TCF/LEF transcription factor family. Using UCSC Genome Browser Home and JASPAR database, we predicted that TCF7L2 may bind to the promoter region of Psen1. Therefore, we constructed pcDNA3.4-Tcf7l2 and pGL3-Psen1 promoter plasmids and cotransfected them with pRL-TK into 293T cells. We found that Psen1 relative luciferase activity was significantly reduced in condition of Tcf7l2 overexpression (Supplementary Fig. 9d). In organoids, Lgr4 deficiency increased Psen1 mRNA level (Supplementary Fig. 9e). Suppression of Wnt signaling by IWR-1 and activation of Notch using VPA mimicked, to some extent, the effects of Lgr4 deficiency on differentiation of intestinal stem cells to lipid absorptive cells (Supplementary Fig. 9f and Supplementary Fig. 9g). Further, suppression of Notch signaling using DAPT attenuated the differentiation of intestinal stem cells into lipid absorptive cells (Supplementary Fig. 9h–j). Together, these results suggest that deficiency of LGR4 may activate Notch signaling via β-catenin and TCF7L2 mediated transcriptional regulation of Psen1.

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• Source: https://www.nature.com/articles/s41467-024-48622-5