Impact of a high‑fat diet on intestinal stem cells and epithelial barrier function in middle‑aged female mice

Abstract

A high‑fat diet (HFD) or obesity‑promoting diet is closely associated with metabolic diseases and intestinal tumors, particularly in middle‑aged individuals (typically 45‑64 years old). The intestinal epithelium constitutes a barrier that separates the host from the food and microbiota in the gut, and thus, a dysfunctional epithelium is associated with a number of diseases. However, the changes caused to the function of intestinal epithelium in response to an HFD have not been well‑studied to date. In the present study, middle‑aged female mice (12 months old) fed an HFD for a period of 14 weeks were used to determine the effects of HFD on the intestine. Characteristics including the body weight, fat deposition, glucose metabolism, inflammatory state and intestinal morphology were assessed, while the intestinal stem cell (ISC) counts and the ability of isolated intestinal crypts to form organoid bodies in 3D culture were examined. Intestinal epithelial barrier function, including secretory defense, tight junctions and cell apoptosis, were also studied. Morphologically, the HFD resulted in a mild reduction in the length of villi of the small intestine, the colon length and the depth of colon crypts. In addition, the ISC counts were increased in the small intestine and colon in HFD‑fed mice. The ability of crypts to grow into organoids (mini‑guts) was also increased in crypts obtained from mice fed an HFD, while HFD compromised the epithelial barrier function of the colon. These results demonstrated how an HFD affects the intestinal epithelium and highlighted the need to carefully consider dietary patterns.

Keywords: high-fat diet; intestinal stem cells; organoids; barrier function; middle-aged female mice.

Figure 1.

Characteristics of middle-aged (12-month-old) female C57BL/6 mice fed an HFD for 14-weeks. (A) Representative appearance of Con and HFD mice. (B) Body weights were determined weekly. (C) oGTT and (D) ITT results. (E) Representative images of iBAT, eWAT and iWAT. (F) Tissue weight of iBAT, eWAT and iWAT from the two mouse groups. mRNA expression levels of (G) F4/80 and (H) MCP-1 inflammation-associated genes in eWAT. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001, vs. Con group. HFD, high-fat diet; Con, control; oGTT, oral glucose tolerance test; ITT, insulin tolerance test; iBAT, interscapular brown adipose tissue; eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue.

Figure 2.

HFD alters the intestinal morphology. Mice were fed an HFD for 14-weeks, and then the small intestines and colons were removed. (A) Representative images (scale bar, 2 cm), and (B) quantified length of the small intestine. (C) Representative H&E staining images of the crypt and villi in the jejunum (scale bar, 50 µm; magnification, ×40). (D) Villus length and (E) crypt depth in the small intestine. (F) Representative images of the colon of mice in the two groups (scale bar, 2 cm), and (G) quantified colon length. (H) H&E staining images of colonic crypt (scale bar, 50 µm; magnification, ×40), and (I) quantified colonic crypt depth. Data are presented as the mean ± standard deviation. *P<0.05 vs. Con group. HFD, high-fat diet; Con, control; H&E, hematoxylin and eosin.

Figure 3.

An HFD increases the ISC count and crypt function in the small intestine of mice. Small intestinal crypts and villi were isolated and epithelial cell suspensions were dissociated into single cells. (A) Representative flow cytometry analysis showing Lgr5 expression in the CD326 cell population obtained from the small intestine. (B) ISCs defined as Lgr5^high, and (C) progenitor cells defined as Lgr5^low in the entire small intestine. (D) mRNA expression of Lgr5 in the small intestine. (E) Small intestinal crypts were isolated and cultured in Matrigel to develop organoid colonies. Representative images of small intestinal organoids at days 0, 4 and 6 are displayed (scale bar, 100 µm; magnification, ×20). (F) Organoids formed per crypt and (G) crypt-domains per organoid on day 4. (H) Organoids formed per crypt and (I) crypt-domains per organoid on day 6. Data are presented as the mean ± standard deviation. *P<0.05 and ****P<0.0001, vs. Con group. HFD, high-fat diet; ISC, intestinal stem cell; Con, control.

Figure 4.

An HFD increases ISC count in the colon. Colon crypts were isolated, and epithelial cell suspensions were dissociated into single cells. (A) Representative flow cytometry analysis showing Lgr5 expression in the CD326 cell population obtained from the colon. (B) ISCs defined as Lgr5^high, and (C) progenitor cells defined as Lgr5^low in the entire colon. (D) Representative images of immunofluorescence staining for Lgr5, seen as red fluorescence, with DAPI used as a counterstain (scale bar, 50 µm; magnification, ×40). (E) Percentage of Lgr5⁺ cells among the crypt cells, and (F) the number of Lgr5⁺ cells per a crypt. (G) mRNA expression of Lgr5. (H) Representative images of immunohistochemical staining for Ki67 (scale bar, 100 µm; magnification, ×100), and (I) quantified Ki67⁺ cells as a percentage of the crypt cells. (J) mRNA expression of Ki67 in the colon. (K) Colon crypts were isolated from Con and HFD mice and cultured in Matrigel to develop organoid colonies. Representative images of colonic organoids at days 0, 4 and 6 are displayed (scale bar, 100 µm; magnification, ×20). Organoids per crypt at (L) day 4 and (M) day 6 are shown. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01, vs. Con group. ISC, intestinal stem cell; HFD, high-fat diet; Con, control.

Figure 5.

Barrier function of the small intestine was not affected by HFD. Goblet and Paneth cell numbers, and cell apoptosis were analyzed to determine the barrier function of the small intestine. (A) Representative flow cytometry analysis of Muc2 expression in CD326 cell populations. (B) Percentage of goblet cells in the small intestine. (C) Ileal sections were stained for goblet cells using PAS (scale bar, 500 µm; magnification, ×20). (D) Percentage of area stained as PAS-positive among the total villi-crypts sections. (E) Quantification of the number of goblet cells per villus-crypt unit. (F) Representative flow cytometry analysis showing CD24 expression in CD326 cell populations. (G) Percentage of Paneth cells in small intestinal crypts. (H) Crypt-derived organoids of Paneth cells, including lysozyme-positive cells, assessed using immunofluorescence (blue, DAPI; red, lysozyme; scale bar, 200 µm; magnification, ×40). (I) Quantification of Paneth cells in small intestinal crypt-derived organoids. (J) mRNA expression levels of Muc2 in the small intestine of the Con and HFD mice. HFD, high-fat diet; Con, control; PAS, Periodic Acid-Schiff; Muc2, mucin 2.

Figure 6.

Cell apoptosis in the small intestine was not affected by HFD. (A) Ileal sections stained for apoptotic cells (scale bar, 100 µm; magnification, ×20). Quantification of apoptotic cells among (B) villi and (C) crypt sections. (D) Expression of P21 in the small intestine, examined using immunohistochemistry (scale bars, 100 µm; magnification, ×40). Quantification of P21 positive cells as a percentage of the (E) crypts and (F) villi. (G) Immunohistochemical assay for P53 in the small intestine (scale bars, 100 µm; magnification, ×40). Quantification of P53 positive cells as a percentage of the (H) crypts and (I) villi. (J) P21 and (K) P53 mRNA expression levels in the small intestine. Data are presented as the mean ± standard deviation. HFD, high-fat diet; Con, control.

Figure 7.

An HFD compromises the barrier function of the colon. Goblet cell number, tight junctions and cell apoptosis were analyzed to determine the barrier function of the colon. (A) Representative flow cytometry analysis, showing Muc2 expression among the CD326 cell populations. (B) Proportion of goblet cells. (C) Histochemical PAS staining of goblet cells (scale bar, 100 µm; magnification, ×20), (D) percentage of area stained as PAS-positive among the colonic crypts, and (E) quantification of the average number of goblet cells per crypt. (F) mRNA expression of Muc2 in the colon. (G) Representative images of immunofluorescence staining for ZO-1 seen as red fluorescence, with DAPI used as the counterstain (scale bar, 50 µm; magnification, ×40), and (H) quantification of red fluorescence. (I) Representative images of immunofluorescence staining for Claudin-2 seen as red fluorescence, with DAPI used as the counterstain (scale bar, 100 µm; magnification, ×40), and (J) quantification of red fluorescence. (K) mRNA expression of Claudin-2 in the colon. *P<0.05, **P<0.01 and ***P<0.001, vs. Con group. HFD, high-fat diet; Con, control; PAS, Periodic Acid-Schiff; Muc2, mucin 2.

Figure 8.

Increased apoptosis in the colon of HFD-fed mice. (A) Colon sections were stained for apoptotic cells using TUNEL assay (scale bar, 50 µm; magnification, ×40), and (B) the quantified number of apoptotic cells is shown. (C) Immunohistochemical staining for P21 expression in the colon (scale bars, 100 µm; magnification, ×40), and (D) quantified percentage of P21-positive crypt cells. (E) Representative images of immunohistochemical staining for P53 (scale bars, 100 µm; magnification, ×40), and (F) quantified percentage of P53-positive crypt cells. (G) P21 and (H) P53 mRNA expression levels in the colon. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01, vs. Con group. HFD, high-fat diet; Con, control.