Dietary iron supplementation modulates jejunal epithelial lineage differentiation and enteroendocrine cell-related responses in suckling piglets

Technical Summary: Dietary Iron Supplementation Modulates Jejunal Epithelial Lineage Differentiation and Enteroendocrine Cell-Related Responses in Suckling Piglets

Problem Statement
Iron is an essential trace element critical for intestinal epithelial homeostasis, yet its specific role in regulating epithelial lineage differentiation during early life remains incompletely understood. While previous research has established that iron status influences intestinal development, barrier function, and epithelial marker expression, it is unclear whether iron availability specifically regulates distinct intestinal epithelial lineages, particularly enteroendocrine cells (EECs). EECs are specialized nutrient-sensing cells that secrete hormones and bioactive peptides, playing a vital role in intestinal maturation. Furthermore, the potential involvement of Peroxisome Proliferator-Activated Receptor gamma (PPARγ) signaling—a known regulator of epithelial differentiation and metabolic adaptation—in iron-mediated intestinal responses requires investigation.

Methodology
The study employed a multi-faceted approach combining in vivo animal trials, transcriptomic analysis, and in vitro organoid models:

• Animal Model: Twenty-four 7-day-old male suckling piglets were randomly assigned to three dietary groups (n=8 per group): Low (basal milk replacer), Medium (basal + 70 mg Fe/kg), and High (basal + 700 mg Fe/kg). The experiment ran from day 7 to day 21.
• Tissue Analysis: Jejunal tissues were collected for histological examination (H&E staining for villus height and crypt depth), quantitative real-time PCR (qPCR) for epithelial lineage markers (LGR5, LYZ, MUC2, ALPI, CHGA), and CHGA immunofluorescence staining to quantify EECs.
• Transcriptomics: RNA-seq was performed on jejunal samples from the Low and High groups to identify differentially expressed genes (DEGs) and enriched pathways. Gene Set Enrichment Analysis (GSEA) was specifically applied to the PPAR signaling pathway.
• Organoid Intervention: Porcine jejunal organoids were cultured and treated with ferric ammonium citrate (FAC) to mimic iron supplementation. To test the role of PPARγ, organoids were co-treated with FAC and troglitazone (a PPARγ agonist). Organoid budding efficiency and gene expression were analyzed.

Key Results

1. Morphology: Dietary iron supplementation did not significantly alter jejunal histomorphology (villus height, crypt depth, or pathological scores) across the treatment groups.
2. Epithelial Lineage Markers: Iron supplementation upregulated the expression of several epithelial lineage marker genes. Specifically, the High group showed increased mRNA levels of goblet cell marker MUC2, Paneth cell marker LYZ, stem cell marker LGR5, and absorptive enterocyte marker ALPI.
3. Enteroendocrine Cell Response: The most pronounced and dose-responsive effect was observed in the enteroendocrine cell marker CHGA. CHGA expression increased progressively from Low to High iron groups. Immunofluorescence confirmed a significant increase in both CHGA fluorescence intensity and the number of CHGA-positive cells in the High iron group compared to the Low group.
4. Transcriptomic Profiling: Transcriptomic analysis revealed 692 differentially expressed genes between Low and High iron groups. These genes were significantly enriched in the PPAR signaling pathway. Notably, GSEA indicated that the PPAR signaling pathway was globally downregulated in the High iron group.
5. Organoid Mechanism: In porcine jejunal organoids, FAC treatment increased CHGA expression. However, when PPARγ was activated via troglitazone, the iron-induced upregulation of CHGA was markedly attenuated, returning to control levels. This effect was specific to CHGA, as troglitazone did not significantly alter the FAC-induced expression of MUC2, LYZ, or LGR5.

Key Contributions

• Lineage-Specific Regulation: The study demonstrates that dietary iron availability modulates jejunal epithelial lineage differentiation in suckling piglets, with a preferential and dose-dependent effect on enteroendocrine cell-related responses (specifically CHGA).
• PPARγ as a Regulatory Node: The research identifies PPARγ-associated signaling as a potential regulatory node in iron-mediated intestinal epithelial development. The data suggests an inverse relationship where high iron suppresses PPAR signaling, which in turn correlates with increased CHGA expression.
• Mechanistic Insight via Organoids: Using a physiologically relevant organoid model, the study provides evidence that PPARγ activation can selectively counteract iron-induced CHGA upregulation, suggesting a direct regulatory interaction between iron status and PPARγ signaling in EEC differentiation.

Significance
The paper claims that these findings provide new insights into the biological role of iron in early-life intestinal epithelial development. Specifically, it highlights that iron acts not only as a metabolic cofactor but as a nutritional signal that reshapes epithelial transcriptional programs, particularly those governing enteroendocrine cells. The study proposes that the "iron–PPARγ axis" may be a critical mechanism in regulating EEC-related differentiation and functional maturation during the suckling period. These results contribute to a deeper understanding of how trace element nutrition influences intestinal barrier integrity and endocrine function in neonatal mammals.