Fiber-Mediated Nourishment of Gut Microbiota Protects against Diet-Induced Obesity by Restoring IL-22-Mediated Colonic Health

Abstract

Dietary supplementation with fermentable fiber suppresses adiposity and the associated parameters of metabolic syndrome. Microbiota-generated fiber-derived short-chain fatty acids (SCFAs) and free fatty acid receptors including GPR43 are thought to mediate these effects. We find that while fermentable (inulin), but not insoluble (cellulose), fiber markedly protected mice against high-fat diet (HFD)-induced metabolic syndrome, the effect was not significantly impaired by either inhibiting SCFA production or genetic ablation of GPR43. Rather, HFD decimates gut microbiota, resulting in loss of enterocyte proliferation, leading to microbiota encroachment, low-grade inflammation (LGI), and metabolic syndrome. Enriching HFD with inulin restored microbiota loads, interleukin-22 (IL-22) production, enterocyte proliferation, and antimicrobial gene expression in a microbiota-dependent manner, as assessed by antibiotic and germ-free approaches. Inulin-induced IL-22 expression, which required innate lymphoid cells, prevented microbiota encroachment and protected against LGI and metabolic syndrome. Thus, fermentable fiber protects against metabolic syndrome by nourishing microbiota to restore IL-22-mediated enterocyte function.

Keywords: germ-free mice; intestinal inflammation; metabolic syndrome; microbiota encroachment; short-chain fatty acids.

Figure 1. Inulin prevented metabolic syndrome induced by HFD

C57BL/6 male mice were fed chow, HFD, or HFD supplemented with cellulose or inulin for 4 weeks. A) Body mass over time (n=10). B) Epididymal fat (n=10), mesenteric fat (n=5) and subcutaneous fat (n=5) were measured at the end of experiment. C) The epididymal fat was stained with H&E, adipocyte size distribution was determined. D–E) Mice (n=10) were administered glucose, 2 g/kg intraperitoneally following an overnight fast. Blood glucose levels were measured at the indicated time point (D) and area under curve (AUC) calculated (E). F–H) Mice (n=10) were fasted 5 h, the glucose was measured at 0 (F), 30, 60, 90 min after intraperitoneally injected with insulin (G) and area under curve calculated (H). I–J) Fat in the liver (n=5) was observed by oil red staining (I), and quantified by image analysis (J), Scale bars, 50 μm. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01. See also Figure S1.

Figure 2. Enrichment of HFD with inulin increased epithelial cell proliferation and prevented gut atrophy

C57BL/6 male mice were fed chow, HFD, or HFD supplemented with cellulose or inulin for 4 weeks. A) Colon length (n=10). B) Colon mass (n=10). C) The mRNA was extracted from colon of mice fed with chow (n=4), HFD supplemeted with cellulose (n=8) or inulin (n=8), and expression level of ZO-1, Occludin and Claudin-2 was analyzed by RT-PCR. D) Colon histopathologic appearance by H&E staining. Scale bars, 200 μm. E) Colon crypt length (n=5), as shown by a double- headed arrow, was measured. F–G) BrdU was injected intraperitoneally 24 h before euthanizing (n=5). Visualization of BrdU positive cells in proximal colon by fluorescence microscopy following staining with FITC-anti-BrdU (F). Scale bars, 100 μm. Enumeration of BrdU positive cells (G). H–I) The paneth cells in ileum (n=5) were stained for lysozyme (H), and the number of paneth cells per crypt was counted (I), scale bars, 50 μm. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01. See also Figure S2.

Figure 3. Inulin restored microbiota loads and prevented HFD-induced microbiota encroachment

C57BL/6 male mice were fed chow, HFD, or HFD supplemented with cellulose or inulin for 4 weeks and feces collected at 28 days post diets treatment. A) Levels of fecal bacterial DNA were quantitated by qPCR (n=5). B–E) Fecal microbiota composition was analyzed by 16S RNA sequencing (n=4–5). B–D displays relative abundance of bacteria at phylum level. E Global composition as expressed by UniFrac PCoA analysis. F–G) Analysis of microbiota-mucus-epithelial localization in transverse colon via carnoy fixation, FISH, immunohistochemical staining (F). Scale bars, 20 μm. Bacterial epithelial distance was quantified (G). H) Quantitation of colonic Reg3γ by RT-PCR. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01. See also Figure S3.

Figure 4. Microbiota ablation eliminated inulin’s beneficial effects in HFD-induced metabolic syndrome

C57BL/6 male mice (n=10) were fed HFD supplemented with cellulose (HFD-200 Cell) or inulin (HFD-200 Inul) with or without antibiotic cocktail in drink water. A) Body mass over time, B) Colon length, C) Colon weight, D) Epididymal fat and mesenteric fat were measured at the end of experiment. E–G). Glucose tolerance was measured and area under curve (AUC) calculated. H) 5 h fasting glucose. I) Mice were fasted 5 h and intraperitoneally injected with insulin to measure insulin sensitivity. J) Correlation between 5 h fasting glucose and colon weight using data from Figure 1&4. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01; n.s, not significance. See also Figure S4.

Figure 5. Manipulation of SCFA levels in intestine did not impact colonic health or adiposity induced by HFD enriched with cellulose or inulin

A–F) C57BL/6 male mice (n=5) were fed HFD supplemented with cellulose (HFD-200 Cell) or inulin (HFD-200 Inul) with drink water containing β acid or not. A) Colon weight. B) Epididymal fat pad. C) Mesenteric fat pad. D–E). Glucose tolerance was measured and area under curve (AUC) calculated. F) 5 h fasting glucose. G–N) C57BL/6 male mice (n=5) were fed chow, standard HFD or HFD containing inulin while being administered drinking water containing SCFA (L or H as described in material and method) for 28 days. G) Colon weight. H) Epididymal fat pad. I) fat percentage. J) lean percentage. K–L). Glucose tolerance was measured (K) and area under curve (AUC) calculated (L). M) 5 h fasting glucose. N) Mice were fasted 5 h and intraperitoneally injected with insulin to measure insulin sensitivity. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01; n.s, not significance. See also Figure S5.

Figure 6. Inulin’s rescue of HFD-induced colon atrophy and metabolic syndrome is mediated by IL-22

A) C57BL/6 female conventional (n=5) mice and Swiss Webster GF (n=3–4) mice fed indicated diets were euthanized and distal colon cut into small pieces and cultured overnight. The supernatant was used to measure IL-22 by ELISA. B–I) IL-22KO (n=13) male mice were fed chow, HFD supplemented with cellulose (HFD-200 Cell) or inulin (HFD-200 Inul) for 4 weeks. B) Colon length. C) Colon weight. D) Epididymal fat pad. E) Mesenteric fat. F–G) Mice (n=10) were administered glucose, 2 g/kg intraperitoneally following an overnight fast. Blood glucose levels were measured at the indicated time point (F) and area under curve (AUC) calculated (G). H–I) Mice (n=5) were fasted 5 h, the glucose was measured at 0 (H), 30, 60, 90 min after intraperitoneally injected with insulin (I). Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01; n.s, not significance. See also Figure S6 and S7

Figure 7. Inulin’s restoration of microbiota containment and prevention of low-grade inflammation required IL-22

IL-22 KO mice were fed chow, HFD supplemented with cellulose (HFD-200 Cell) or inulin (HFD-200 Inul) for 4 weeks. A) The mRNA was extracted from colon to analyze the expression of Reg3γ. B) Bacterial 16S rRNA in the livers were analyzed by qRT-PCR. C) Analysis of microbiota-mucus-epithelial localization in transverse colon of IL-22 KO mice via carnoy fixation, FISH, immunohistochemical staining. D) Bacterial vs epithelial distance was quantified. E–F) Quantitation of CXCL1, TNF-α and IL-6 in white adipose tissue (WAT) of wild type (E) and IL-22 KO mice (F) by qRT-PCR. Data were expressed as mean ± SEM. Statistical significance was assessed by unpaired Student t test. *p<0.05; **p<0.01; n.s, not significance.