Purpose: Identification of endogenous substrates of disease-associated transporters may help clarifying both their pathophysiological function in vivo and diagnostic biomarkers for the diseases since their substrate compounds may contribute to homeostatic regulation. Carnitine/organic cation transporter (OCTN1/SLC22A4) is genetically associated with various chronic diseases including Crohn’s disease (CD), and the C1672T (L503F) single nucleotide polymorphism of OCTN1 gene is associated with an increased susceptibility in CD. Although OCTN1 is ubiquitously expressed in the body including gastrointestinal tract, relatively higher expression of OCTN1 was detected in CD14 positive immune cells which may be associated with disease states in CD. Possible association of OCTN1 with CD has also been suggested by the up-regulation of its protein expression in inflamed intestinal tissues of both CD patients and dextran sodium sulfate (DSS)-induced colitis mice. These phenomena imply that certain endogenous substrates of OCTN1 may play disease-associated roles. The purpose of the present study was to identify endogenous substrates of OCTN1 that are associated with gastrointestinal inflammation. Untargeted metabolome analysis is the most common tool to identify endogenous substrates, although this approach can also lead to nonspecific identification of unrelated compounds. Therefore, we attempted to improve conventional metabolome approach by combination of transporter- and structure-selective metabolomics strategies.
Methods: Inflammatory intestinal tissue extracts were isolated from DSS-induced colitis mice and used as a source for OCTN1 substrates. Human OCTN1 overexpressing HEK293 cells (HEK293/OCTN1) and mock cells were exposed to the inflammatory intestinal tissue extracts and used to concentrate OCTN1 substrates into the cells by the uptake activity of the transporter in HEK293/OCTN1 cells (transporter-selective metabolomics). Amino group-selective derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) of the extract of HEK293/OCTN1 and mock cells was performed to label SLC22A4 substrates, since most of the OCTN1 substrates contain amino groups in their structures. Precursor ion scanning against the common product ion of APDS-labelled compound, m/z 121, was performed by LC-TQ-MS/MS to comprehensively detect APDS-labeled compounds (structure-selective metabolomics). Peaks with higher intensities in HEK293/OCTN1 cells than those in mock cells were selected as candidates of OCTN1 substrates. Parent and product mass scanning were then performed by LC-TOF-MS to obtain chromatograms and MS spectrums with accurate mass, which was then compared with MS spectrums of database and standard compounds. Cellular uptake studies of [3H]spermine using HEK293/OCTN1, HEK293/L503F-OCTN1, and mock cells were performed to examine whether the selected compound is transported by OCTN1. Spermine concentrations in plasma and organs were measured by-LC-TQ-MS/MS after derivatization using APDS and compared between wild type and octn1 gene knockout mice (octn1-/-) to investigate the possible involvement of OCTN1 in spermine homeostasis in vivo. Inflammatory responses in primary cultured peritoneal macrophages isolated from wild type and octn1-/- mice after exposure to lipopolysaccharide (LPS) were examined by measuring mRNA expression of inflammatory cytokine genes to identify the effect of octn1 gene deletion on inflammation.
Results: After precursor ion scanning, 7,538 ion peaks were detected in HEK293/OCTN1 or mock cell samples. Among these peaks, 10 peaks had significantly higher signal intensities in HEK293/OCTN1 cells compared to the mock cells. Further following analyses were next performed to identify the m/z 342.4, which showed relatively highest signal intensities. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. The uptake of [3H]spermine in HEK293/L503F-OCTN1 cells was time-dependently increased and was higher than that in mock cells at most of the evaluated spermine concentrations. The uptake of [3H]spermine became saturated as the spermine concentration increased. OCTN1, especially the L503F variant, may interact with spermine, which was consistent with previous findings that spermine could inhibit OCTN1-mediated acetylcholine uptake in OCTN1-reconstituted liposomes. Spermine concentration in peripheral blood mononuclear cells (PBMCs) containing CD14 positive population from octn1-/- was significantly lower than in wild-type mice, suggesting possible involvement of OCTN1 in spermine homeostasis in PBMCs. On the other hand, LPS-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1-/- mice was lower than in wild-type mice, implying that octn1 gene deletion may partially accelerate recovery from inflammation in macrophages. The higher spermine uptake by L503F-OCTN1 might provoke acrolein, the toxic metabolite of spermine, accumulation and its toxicity in the cells, and this may partially explain the higher risk of CD in L503F-OCTN1 variant possessing patients. Further studies will be needed to clarify the possible involvement of OCTN1-mediated spermine uptake in the development of CD.
Conclusion: The combination of transporter- and structure-selective metabolomics strategies may provide a novel approach to identify endogenous substrates of cation transporters.