Nutr Res Pract.  2023 Aug;17(4):616-630. 10.4162/nrp.2023.17.4.616.

IPA and its precursors differently modulate the proliferation, differentiation, and integrity of intestinal epithelial cells

• Ismael S
• Rodrigues C
• Santos GM
• Castela I
• Barreiros-Mota I
• Almeida MJ
• Calhau C
• Faria A
• Araújo JR

• KMID: 2545180
• DOI: http://doi.org/10.4162/nrp.2023.17.4.616

Abstract

BACKGROUND/OBJECTIVES
Indole-3-propionic acid (IPA) is a tryptophan-derived microbial metabolite that has been associated with protective effects against inflammatory and metabolic diseases. However, there is a lack of knowledge regarding the effects of IPA under physiological conditions and at the intestinal level.
MATERIALS/METHODS
Human intestinal epithelial Caco-2 cells were treated for 2, 24, and/ or 72 h with IPA or its precursors – indole, tryptophan, and propionate – at 1, 10, 100, 250, or 500 μM to assess cell viability, integrity, differentiation, and proliferation.
RESULTS
IPA induced cell proliferation and this effect was associated with a higher expression of extracellular signal-regulated kinase 2 (ERK2) and a lower expression of c-Jun. Although indole and propionate also induced cell proliferation, this involved ERK2 and c-Jun independent mechanisms. On the other hand, both tryptophan and propionate increased cell integrity and reduced the expression of claudin-1, whereas propionate decreased cell differentiation.
CONCLUSIONS
In conclusion, these findings suggested that IPA and its precursors distinctly contribute to the proliferation, differentiation, and barrier function properties of human intestinal epithelial cells. Moreover, the pro-proliferative effect of IPA in intestinal epithelial cells was not explained by its precursors and is rather related to its whole chemical structure. Maintaining IPA at physiological levels, e.g., through IPA-producing commensal bacteria, may be important to preserve the integrity of the intestinal barrier and play an integral role in maintaining metabolic homeostasis.

Keyword

Caco-2 cells; cell proliferation; indoles; permeability; tryptophan

Figure

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  Fig. 1 Effect of IPA, indole, tryptophan, and propionate at 1, 10, 100, 250, and 500 μM on the viability of Caco-2 cells, following 2 (A), 24 (B), and 72 h treatment periods. Cell viability was determined by the MTT assay. Results are shown as % of control. Data are expressed as mean ± SEM (n = 8).Ctrl, control; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid.

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  Fig. 2 Effect of IPA, indole, tryptophan, and propionate at 500 μM on Caco-2 cells monolayer integrity after a 24 h treatment period. The cell monolayer integrity was determined by assessing the passage of sulfonic acid from the apical to the basolateral compartment. Results are shown as % of control. Data are expressed as mean ± SEM (n = 4).Ctrl, control; etOH, ethanol; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid.*P < 0.05 vs. controls.

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  Fig. 3 Effect of IPA, indole, tryptophan, and propionate at 500 μM on claudin-1 (A), occludin (B), and ZO-1 (C) gene expression in Caco-2 cells after 24 h. Relative expression of these genes was evaluated by quantitative reverse transcription polymerase chain reaction. Results obtained were normalized for the housekeeping gene HPRT and data are expressed as mean ± SEM (n = 3–4).AU, arbitrary units; HPRT, hypoxanthine guanine phosphoribosyltransferase; Ctrl, control; etOH, ethanol; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid; ZO-1, zonula occludens-1.**P < 0.01 and ***P < 0.001 vs. controls.

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  Fig. 4 Effect of IPA, indole, tryptophan, and propionate at 500 μM during the last 72 h of Caco-2 cell differentiation. Cell differentiation was determined by measuring ecto-ALP activity. Results are shown as % of control. Data are expressed as mean ± SEM (n = 8).etOH, ethanol; ALP, alkaline phosphatase; Ctrl, control; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid.*P < 0.05 vs. controls.

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  Fig. 5 Effect of IPA, indole, tryptophan, and propionate at 500 μM on Caco-2 cell proliferation after a 2 h (A) and 24 h (B) treatment period. Cell proliferation was determined by sulforhodamine B assay. Results are shown as % of control. Data are expressed as mean ± SEM (n = 11–24).Ctrl, control; etOH, ethanol; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid.**P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. controls.

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  Fig. 6 Effect of IPA, indole, tryptophan, and propionate at 500 μM on Caco-2 cells expression of ERK2 (A), JNK1 (B), and c-jun (C) after 24 h. Relative expression of these genes was evaluated by qRT-PCR. Results obtained were normalized for the housekeeping gene HPRT and data are expressed as mean ± SEM (n = 4).ERK2, extracellular signal-regulated kinase 2; AU, arbitrary units; HPRT, hypoxanthine guanine phosphoribosyltransferase; Ctrl, control; etOH, ethanol; IPA, indole-3-propionic acid; ACN, acetonitrile; FA, formic acid; JNK1, c-Jun N-terminal kinase.*P < 0.05 and **P < 0.01 vs. controls.

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