(P30-007-25) Effects of Inositol Phosphates on Zinc Uptake in a Human Enterocyte Cell Line

Objectives: Phytic acid (IP6), a well-known inhibitor of Zn absorption, is hydrolyzed in the gut by microbial and host enzymes into lower-phosphorylated inositol phosphates (IPs) and myo-inositol (MI). While IPs differ in phosphorylation levels, their specific effects on dietary Zn absorption and cytosolic Zn signaling in intestinal epithelial cells remain poorly understood. This study investigated the phosphorylation-dependent effects of IPs on Zn uptake using a zinc-reporter enterocyte model developed with genetically encoded cytosolic Zn sensors.

Methods: Human C2bbe1 cells were electroporated with PB-NES-ZapCV2 (encoding a cytosolic Zn sensor) using the piggyBac transposon system. Stable transfectants were selected with blasticidin to establish a reporter cell line. Cells were pretreated with IPs or MI and subsequently exposed to Zn in culture media at an inhibitory molar ratio of 20:1 (IPs:Zn). Cytosolic Zn kinetics were monitored in real-time by measuring Förster resonance energy transfer (FRET) intensities using live-cell fluorescence microscopy.

Results: The FRET ratio remained stable following IP addition but increased after Zn supplementation in all groups, peaking within 30 minutes before gradually declining. The control group without IP pretreatment showed the steepest initial slope. Pretreatment with IP6 significantly inhibited Zn uptake, as indicated by minimal FRET ratio increases, while IP5 caused a similarly strong inhibition. IP4 and IP3 led to moderate inhibition, whereas IP1 and IP2 had negligible effects, with FRET ratios comparable to those of MI and control groups.

Conclusions: These findings reveal a phosphorylation-dependent gradient in Zn uptake inhibition, with higher phosphorylated IPs exerting stronger effects. It highlights the relationship between zinc bioavailability and the extent of IP6 hydrolysis, which is regulated by microbial and host enzyme activities. This suggests the potential role of genes encoding these enzymes in modulating dietary zinc bioavailability, aligning with nutrigenetics perspectives. Future studies will investigate long-term cytosolic zinc fluctuations and the broader implications of IP-mediated zinc nutrition.

Funding Sources: This research is supported by SEED Grant from the Food and Human Health, and the Hatch Grant, both from USDA.