Effects of T3 and 3-iodothyronamine (T1AM) on cellular metabolism, and influence of serum proteins on T1AM assay.

Thyroxine (T4) is the predominant form of thyroid hormone (TH). In target tissues, T4 is enzymatically deiodinated to 3,5,3′-triiodothyronine (T3), a high-affinity ligand for the nuclear TH receptors TRα and Trβ. T3 modulates genes transcription via activation of TRα and TRβ. Non-genomic effects have also been described. In 2004 the research groups of professors Scanlan Grandy and Zucchi discovered an endogenous thyroid hormone derivative called 3-iodothyronamine (T1AM). They proved that at nanomolar concentrations it can activate trace amine associated receptors 1 (TAARs)[1] and it may also interact with other targets, such as plasma membrane transporters, mitochondrial proteins and vesicular biogenic amine transporters [2]–[4]. Endogenous T1AM has been detected in human and rodent blood and tissues samples by liquid chromatography coupled mass spectrometry (LC/MS/MS) [5]. Its endogenous levels are a matter of argument due to the challenges that its accurate quantification poses. For this reason, so far a worldwide adopted extraction method has not been established. Circulating T1AM has so far been considered to be largely bound to apolipoprotein apoB100 [6]. The first T1AM functional effect to be discovered was severe hypothermia [7]. This effect is associated to a decrease in oxygen consumption and a reduction of the respiratory quotient (CO2/O2), which reflects the relationship between glucose and fatty acid oxidation, resulting in a shift from carbohydrate to lipid as energy source [8]. The molecular mechanisms underlying T1AM effects are still unknown, however Mariotti and colleagues [9] analyzed gene expression profiles in adipose tissue and liver of T1AM chronically treated rats and found significant transcriptional effects involving sirtuin genes, which regulate important metabolic pathways. Therefore, the first aim of this work was to compare the effect of T1AM and T3 chronic treatment on mammalian sirtuin expression in hepatoma cells (HepG2) and isolated hepatocytes. Isolated rat hepatocytes were obtained by liver in-situ collagenase perfusion. Sirtuin expression was determined by Western Blot analysis in cells treated for 24 h with 1-20 µM T1AM or T3. In addition, cell viability was evaluated by MTT test upon 24 h treatment with 100 nM to 20 µM T1AM or T3. In HepG2, T1AM significantly reduced SIRT1 and SIRT4 protein expression at 20 µM while T3 strongly decreased the expression of SIRT1 (20 µM) and SIRT2 (any tested concentration). In primary rat hepatocytes T1AM, did not affect protein expression whereas T3 decreased SIRT2 at 10 µM. The extent of MTT-staining was moderately but significantly reduced by T1AM, particularly in HepG2 cells, in which the effect occurred at concentration starting from 100 nM. T3 reduced MTT staining in HepG2 but not in isolated hepatocytes. T1AM and T3 differently affected sirtuin expression in hepatocytes. Since SIRT4 is an important regulator of lipid and glucose metabolism, whereas SIRT1 and SIRT2 have a key role in regulating cell cycle and tumorigenesis, our observations are consistent with the shift from carbohydrates to lipids induced by T1AM and indicate a potential new role of T1AM in modulating tumor proliferation. The second part of this project was aimed at clarifying the issue that, so far, every research group working on this molecule has encountered when trying to accurately quantifying T1AM endogenous levels. These difficulties were usually attributed to problems in extraction or other pre‐analytical steps. Most researchers have developed various workaround for this issue. For example, on cell culture experiments, to avoid the presence of serum proteins in the culturing media, experiments have often be performed with unphysiological protein‐free media. The second goal of this project was therefore to evaluate the effect of serum protein on the recovery of exogenous T1AM. Cell culture media (Krebs buffer, DMEM, FBS, DMEM+FBS, used either in the absence or in the presence of NG108‐15 cells) and other biological matrices (rat brain and liver homogenates, human plasma and blood) were spiked with T1AM and/or deuterated T1AM (d4‐T1AM) and incubated for times ranging from 0 to 240 min. Samples were extracted using a liquid/liquid method and analysed using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to assay T1AM and some of its metabolites. For the first time in the history of this molecule, in FBS‐containing buffers, an exponential decrease in T1AM levels was observed over time. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4‐T1AM decreased over time at a much lower rate, reaching 50‐70% of the baseline at 60 min. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide, however, the process could not be reverted. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 min, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human whole blood. Conclusions. On the whole, these results suggest binding and sequestration of T1AM by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenousT1AM.