Proteomic Analysis Reveals Changes in Tight Junctions in the Small Intestinal Epithelium of Mice Fed a High-Fat Diet

Abstract

The impact of a high-fat diet (HFD) on intestinal permeability has been well established. When bacteria and their metabolites from the intestinal tract flow into the portal vein, inflammation in the liver is triggered. However, the exact mechanism behind the development of a leaky gut caused by an HFD is unclear. In this study, we investigated the mechanism underlying the leaky gut related to an HFD. C57BL/6J mice were fed an HFD or control diet for 24 weeks, and their small intestine epithelial cells (IECs) were analyzed using deep quantitative proteomics. A significant increase in fat accumulation in the liver and a trend toward increased intestinal permeability were observed in the HFD group compared to the control group. Proteomics analysis of the upper small intestine epithelial cells identified 3684 proteins, of which 1032 were differentially expressed proteins (DEPs). Functional analysis of DEPs showed significant enrichment of proteins related to endocytosis, protein transport, and tight junctions (TJ). Expression of Cldn7 was inversely correlated with intestinal barrier function and strongly correlated with that of Epcam. This study will make important foundational contributions by providing a comprehensive depiction of protein expression in IECs affected by HFD, including an indication that the Epcam/Cldn7 complex plays a role in leaky gut.

Keywords: Cldn 7; Epcam; intestinal epithelial cells; non-alcoholic fatty liver disease; proteomic analysis; tight junctions; upper small intestine.

Figure 1

Mouse model of high-fat diet (HFD) induced nonalcoholic fatty liver. (A) Body weight gain of HFD- and control diet (CD)-fed mice. (B) The concentration of blood fluorescein isothiocyanate (FITC)-dextran following oral administration to mice. (C–F) Liver weight, Liver/body weight ratio, serum total cholesterol (TC), and serum aspartate aminotransferase (ALT) of the HFD and CD groups. (G) Hematoxylin and eosin (H&E) staining of livers from HFD- and CD-fed mice. Scale bar: 100 μm. Red Arrowheads show ballooning cells. (H) The non-alcoholic fatty liver disease activity score (NAS) is the sum of the lobular inflammation, ballooning, and steatosis scores according to the H&E score. (I) Area of liver steatosis of HFD- and CD-fed mice. All data represent the mean ± standard error of the mean. Student’s t-test, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 4, CD group; n = 5 HFD group.

Figure 2

H&E staining of the upper (A) and lower (B) small intestines from HFD- and CD-fed mice. Scale bar: 100 μm.

Figure 3

Representative immunohistological ZO-1 stained images of the upper small intestine of HFD- and CD-fed mice. Scale bar: 50 μm.

Figure 4

High-throughput deep proteomics of intestinal epithelial cells (IECs) in the upper and lower small intestines. (A) Principal component analysis (PCA) for proteomes of IECs from the upper small intestine. (B) PCA for proteomes of IECs from the lower small intestine. Green and red areas show 95% confidence regions.

Figure 5

High-throughput deep proteomics of IECs from the upper small intestine of HFD- and CD-fed mice. (A) Volcano plot of differentially expressed proteins (DEPs). Red and blue dots represent upregulated and downregulated proteins, respectively. (B) Functional annotation clustering of DEPs by DAVID. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEPs.

Figure 6

Heatmaps of DEPs include “GTP binding” (GO: 0005525, (A)) and “Tight Junction” (mmu 04530, (B)).

Figure 7

Correlations of claudin (Cldn)7 with intestinal permeability (A) and Epcam (B). Red and black dots indicate samples from HFD- and CD-fed mice, respectively (A; n = 4, CD group; n = 5 HFD group, B; n = 8, CD group; n = 10).

Figure 8

Representative immunohistological Epcam stained images of the upper small intestines from HFD- and CD-fed mice. Scale bar: 50 μm. Arrowheads indicate decreased Epcam signal at the plasma membrane of IECs of HFD-fed mice.