Palmitic acid induces enhanced stemness in canine colonoids.
In this study, PA, a well-established main component of the HFD [7, 26], was used to model the effect of HFD in vitro. First, 3D organoids were used to examine the effect of PA on the stemness of intestinal organoids following PA exposure. Canine colonic organoids, also known as colonoids, were cultured in organoid medium. This culturing process was maintained from the point of passaging until day 4, allowing the organoids to reach a suitable level of maturity. Subsequently, the medium was substituted with PA-containing medium, and the organoids were incubated for a duration of 24 hours.
Figure 1A and B demonstrate the alteration in organoid dimensions following exposure to PA. At concentrations of 30µM and 60µM, PA elicited a notable impact on organoid size. Specifically, the organoid dimensions increased by 2.66-fold at 30 µM (p < 0.01) and 2.22-fold at 60 µM (p = 0.02) in comparison to the control group.
In order to explore the underlying cause of the altered organoid size, we conducted an investigation into whether increased stemness played a role. The gene expression of LGR5, a prominent stem cell marker, using quantitative reverse transcription polymerase chain reaction (qPCR) (Fig. 1C) was performed. Notably, the expression level of LGR5 was found to be significantly increased by exposure to 30 µM PA (1.61 times: p < 0.01), although such an increase was not observed at 60 µM (p = 0.26). Building upon these observations, we established 30 µM as the dosage that manifests a representative effect of PA. This aligns with earlier in vivo and in vitro investigations involving mice and dogs, as evidenced by previous studies[7, 34]. Consequently, we opted to employ 30 µM PA for subsequent experiments.
Influence of palmitic acid on the proliferative activity of canine colonoids.
To demonstrate increased stemness, we employed 5-ethynyl-2-deoxyuridine (EdU) assay to assess the proliferative activity of colonoids. Following a 24-hour exposure to 30 µM PA, an EdU assay was conducted. The rate of EdU-positive cells exhibited a significant increase upon PA exposure (2.22 times: p < 0.01), providing conclusive evidence of PA-induced heightened stemness (Fig. 2A and 2B).
Palmitic acid exposure and its impact on intestinal barrier integrity.
Subsequently, we examined the influence of PA on intestinal barrier function. To achieve this, a colonoid-derived monolayer was established by seeding single cells enzymatically isolated from Matrigel-grown colonoids onto a cell culture insert, a method previously documented [36]. These organoid-derived monolayers were cultured in organoid medium until day 5, a point at which the transepithelial electrical resistance (TEER) value reached a stable state (Supplemental Fig. 1) [36]. On day 5, the apical medium was substituted with a PA-containing medium, and the assessment of epithelial barrier integrity was conducted 24 hours subsequent to this medium alteration.
While no overt disparity in cellular morphology was discernible between the Control and PA groups, as observed through phase contrast microscopy (Fig. 3A), a significant reduction in TEER value was evident after a 24-hour PA exposure (0.79 times: p = 0.02) (Fig. 3B).
Palmitic acid exposure leads to downregulation of tight junction protein expression.
To elucidate the underlying cause behind the observed decline in epithelial barrier integrity, substantiated by TEER measurements, the expression levels of E-cadherin and ZO-1 were evaluated by immunocytochemistry. These are proteins that play a crucial role in maintaining the integrity and function of cell-cell junctions, particularly in epithelial tissues [37, 38].
In the control group, both ZO-1 and E-cadherin displayed distinct staining patterns at cell boundaries. However, within the PA-exposed group, E-cadherin exhibited staining comparable to the control, whereas ZO-1 demonstrated a conspicuous reduction in fluorescence intensity (Fig. 4A).
The quantification of these fluorescence intensities using Image J, revealed no significant variance in fluorescence intensity concerning E-cadherin However, a marked reduction in fluorescence intensity was detected for ZO-1 within the PA group (0.78 times: p < 0.01) (Fig. 4B).