Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction

Abstract

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1β, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

High glucose exposure drives intestinal barrier dysfunction by altering its morphological, structural and functional properties

“…Navabi et al previously suggested the negative effect of glucose on the integrity of the monolayer when they detected a decrease in the epithelial resistance of the LS513 cell line grown in the DMEM medium compared to RPMI medium which contains half the amount of glucose [57]. While some studies have also shown a decrease in TEER in the presence of HG [23,36,37,48,52], other studies have observed that there was no effect [21,38]. Villarroel et al even found that human retinal pigment epithelia cell lines (ARPE-19) showed higher TEER when they were grown in HG medium [27].…”

“…Accordingly, it has been shown in vitro that HG resulted in increased proliferation [26,28,[32][33][34], migration [28,33,35] and reactive oxygen species production [32,33,36], and decreased transepithelial electrical resistance with increased membrane permeability [21,23,36,37]. In a few cases, expressions of intercellular junctional proteins have been investigated at the mRNA and/or protein levels [28,[35][36][37] and/or their subcellular locations have been examined [23,30,38]. Limited studies have assessed the effects of HG on IE barrier functionality, such as mucus secretion or ALP activity and they have mostly been done in vivo [26,37,39,40].…”