Fig 2: Enteric neurons highly express IP6K2 in the mammalian GIT.A, expression analysis of IP6K1-3 in intestinal cell subsets using publicly available scRNA-seq datasets. Relative expression (log scale) of IP6K1-3 among human embryonic enteric cells (upper panel) and their neural cell subsets (lower panel), obtained by analysis of human embryonic intestinal cells scRNA-seq datasets, are shown (39). The size and color of the dots represent the percentage of cells that express IP6K1-3 mRNA and their average abundances within a cluster, respectively. B, UMAP-based unsupervised clustering of recently reported mouse embryonic (E18.5) ENS data (40) (lower left panel). Assignment of cell identities was based on the expression of signature genes as described in the literature: Sox10 (Progenitor), Ascl1 (Neuroblast), Elavl4 (Neuron), Plp1 (Enteric glia), and Dhh (SCP). Relative expression (log scale) of IP6K1-3 among the ENS clusters (upper panel) are shown. C, immunohistochemical analysis of IP6K2 expression in the duodenal muscularis externa of C57BL/6J mice. The neuronal marker HuC/D was also detected to identify enteric neurons in the myenteric plexuses. Open arrowheads indicate double-positive cells. Two different areas of confocal microscopy images are shown for clarity since enteric neurons exist sparsely on the GIT tissue sections. In addition, differential interference contrast images were overlaid onto the respective merged fluorescent images to identify cell contours and clarify the location where the double-positive cells exist in the GIT tissue. The scale bar represents 10 μm. E, embryonic day; ENS, enteric nervous system; GIT, gastrointestinal tract; SCP, Schwann cell precursor; UMAP, uniform manifold approximation and projection.

Fig 3: IP6K2-dependent enhanced IP7metabolism exists in the gut and duodenal muscularis externa.A, schematic illustration of the experimental workflow. C57BL/6J mice were fed a standard diet or fasted for 48 h to deplete dietary IPs in the GIT. These mice were sacrificed to collect stomach and three consecutive 5-cm segments of the proximal GIT (duodenum, jejunum, ileum). The muscularis externa containing myenteric plexus as well as total tissues in the proximal GITs were subjected to HILIC-MS/MS analysis. B, the IP7/IP6 ratios in the muscularis externa and total tissue of four proximal GIT segments of C57BL/6J mice under the two different conditions. The values shown represent the mean ± SD of four independent experiments. C, schematic illustration of the experimental workflow. IP6K2−/− and WT mice fasted for 48 h were sacrificed to collect four proximal GIT segments (stomach, duodenum, jejunum, ileum), which were then subjected to isolate muscularis externa. D, the IP7/IP6 ratios in the muscularis externa of the four GIT segments of IP6K2−/− and WT mice. The values shown represent the mean ± SD of four independent experiments and are expressed relative to those for WT mice. Asterisks indicate statistical significance (p < 0.05, Student’s t test) compared with WT mice. GIT, gastrointestinal tract; HILIC, hydrophilic interaction liquid chromatography; MS/MS, tandem mass spectrometry.

Fig 4: IP6K2−/−mice show significant impairment of IP7metabolism in the proximal gastrointestinal tract.A, schematic depiction of the IP6K2 genomic locus in IP6K2−/− and WT mice (left panel) and the experimental workflow (right panel). IP6K2 exons and introns are represented as boxes and lines, respectively. B–D. the concentrations of IP6 (B) and IP7 (C), and IP7/IP6 ratios (D) in the central nervous system, gastrointestinal tract, and other organs of male IP6K2−/− and WT mice. The values shown represent the mean ± SD of five independent experiments and are expressed as pmol per mg of organ weight. Asterisks indicate statistical significance (p < 0.05, Student’s t test) compared with WT mice. HILIC, hydrophilic interaction liquid chromatography; MS/MS, tandem mass spectrometry.

Fig 5: IP6K2-IP7axis is crucial for certain neurotranscriptome profile associating with enteric nervous system development and functioning.A, transcript levels of two different neuronal genes (Drd5 and Cckbr) in the CNS and GIT of IP6K2−/− and WT mice. Data were normalized to 18S rRNA level. The values shown represent the mean ± SD of three (CNS of IP6K2−/−, and CNS and GIT of WT mice) and five (GIT of IP6K2−/− mice) independent experiments and are expressed relative to those of WT mice. Asterisks indicate statistical significance (p < 0.05, Student’s t test) compared with WT mice. B, schematic illustration of the experimental workflow. IP6K2−/− and WT mice were sacrificed to collect the duodenal muscularis externa. High-quality total RNAs isolated from these tissues (each n =3) were subjected to whole transcriptome analysis by high-throughput RNA sequencing. C, Gene Set Enrichment Analysis of the enriched gene signature in IP6K2−/− duodenal muscularis externa. Cell type signature gene sets (C8 in The Molecular Signatures Database ver7.5.1; http://www.gsea-msigdb.org/gsea/msigdb/index.jsp) were used for this analysis. Horizontal dashed line indicates nominal p-value 0.05, and vertical lines indicate normalized enriched score (NES) ± 1.2 cutoff. Gene sets assigned to neural progenitor cells and oligodendrocyte progenitor cells (source data are derived from Zhong et al. (79)), neural stem cells and glial cells (Fan et al. (80)), and mature neurons (inhibitory neurons, Cao et al. (81); GABAergic and dopaminergic neurons, La Manno et al. (82)) with nominal p < 0.05 and NES > 1.2 or < −1.2 are labeled in colored dots. D, qPCR validation of RNA-Seq results for seven neuronal genes significantly accumulated or depleted in IP6K2−/− duodenal muscularis externa. qPCR data were normalized to β-actin level. The values shown represent the mean ± SD of six independent experiments and are expressed relative to those for WT mice. Asterisks indicate statistical significance (p < 0.05, Student’s t test) compared with WT mice. E, transcript levels of three different neuronal genes (Drd5, Cckbr and Npy4r) in the duodenal muscularis externa of IP6K2−/− and WT mice. Data were normalized to β-actin level. The values shown represent the mean ± SD of six independent experiments and are expressed relative to those for WT mice. Asterisks indicate statistical significance (p < 0.05, Student’s t test) compared with WT mice. CNS, central nervous system; GIT, gastrointestinal tract.