Isobutyrylcarnitine as a Biomarker of OCT1 Activity and Interspecies Differences in its Membrane Transport

Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC. In this study, we wanted to confirm the suggested use of IBC as an endogenous biomarker of OCT1 activity and contribute to a better understanding of the mechanisms behind the association between blood concentrations of carnitine derivatives and OCT1 genotype. Blood and urine IBC concentrations were quantified in healthy volunteers regarding intra- and interindividual variation and correlation with OCT1 genotype and with pharmacokinetics of known OCT1 substrates. Furthermore, IBC formation and transport were studied in cell lines overexpressing OCT1 and its naturally occurring variants. Carriers of high-activity OCT1 genotypes had about 3-fold higher IBC blood concentrations and 2-fold higher amounts of IBC excreted in urine compared to deficient OCT1. This was likely due to OCT1 function, as indicated by the fact that IBC correlated with the pharmacokinetics of known OCT1 substrates, like fenoterol, and blood IBC concentrations declined with a 1 h time delay following peak concentrations of the OCT1 substrate sumatriptan. Thus, IBC is a suitable endogenous biomarker reflecting both, human OCT1 (hOCT1) genotype and activity. While murine OCT1 (mOCT1) was an efflux transporter of IBC, hOCT1 exhibited no IBC efflux activity. Inhibition experiments confirmed this data showing that IBC and other acylcarnitines, like butyrylcarnitine, 2-methylbutyrylcarnitine, and hexanoylcarnitine, showed reduced efflux upon inhibition of mOCT1 but not of hOCT1. IBC and other carnitine derivatives are endogenous biomarkers of hOCT1 genotype and phenotype. However, in contrast to mice, the mechanisms underlying the IBC-OCT1 correlation in humans is apparently not directly the OCT1-mediated efflux of IBC. A plausible explanation could be that hOCT1 mediates cellular concentrations of specific regulators or co-substrates in lipid and energy metabolism, which is supported by our in vitro finding that at baseline intracellular IBC concentration is about 6-fold lower alone by OCT1 overexpression.

Systems Biology Analysis Reveals Eight SLC22 Transporter Subgroups, Including OATs, OCTs, and OCTNs

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as “drug” transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.

OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics

OCTN is a small subfamily of membrane transport proteins that belongs to the larger SLC22 family. Two of the three members of the subfamily, namely, OCTN2 and OCTN1, are present in humans. OCTN2 plays a crucial role in the absorption of carnitine from diet and in its distribution to tissues, as demonstrated by the occurrence of severe pathologies caused by malfunctioning or altered expression of this transporter. These findings suggest avoiding a strict vegetarian diet during pregnancy and in childhood. Other roles of OCTN2 are related to the traffic of carnitine derivatives in many tissues. The role of OCTN1 is still unclear, despite the identification of some substrates such as ergothioneine, acetylcholine, and choline. Plausibly, the transporter acts on the control of inflammation and oxidative stress, even though knockout mice do not display phenotypes. A clear role of both transporters has been revealed in drug interaction and delivery. The polyspecificity of the OCTNs is at the base of the interactions with drugs. Interestingly, OCTN2 has been recently exploited in the prodrug approach and in diagnostics. A promising application derives from the localization of OCTN2 in exosomes that represent a noninvasive diagnostic tool.

Effect of L-carnitine against acute mitoxantrone toxicity in mice

Various experiments were performed in our laboratory to define a possible role for carnitine derivatives in mitoxantrone (MX) therapy. We report here the results of the effect of L-carnitine (LCAR) on the lethal toxicity of MX in mice. MX was administered intravenously at doses of 7, 9, 11, 13, 15 or 17 mg kg−1 either alone or in combination with LCAR at a single intravenous dose of 200 mg kg−1. The dependence of the probability of death on various doses was evaluated for the MX-LCAR combination compared to MX alone. From these experiments, the following lethal dose fifty (LD50) values were calculated: LD50 for MX alone was 15.2 mg kg−1, whereas in combination with LCAR it increases to 21.8 mg kg−1. The relative toxicity given as the ratio of the LD50 of both MX alone and the combination of MX-LCAR was 69.7%. The results of our experiments unequivocally show the effect of LCAR on acute toxic doses of MX.