Effetti delle tironamine sul tessuto epatico

La 3-iodotironamina (T1AM), è un metabolita endogeno che s’ipotizza derivare dalla decarbossilazione e deiodinazione dell’ormone tiroideo tiroxina (T4). La somministrazione di T1AM esogeno porta ipotermia, riduzione della gittata e della frequenza cardiaca; è stata inoltre osservata una riduzione del quoziente respiratorio, dato dal rapporto tra CO2 prodotta e O2 consumato, indice dell’aumentata ossidazione di acidi grassi e quindi di uno spostamento dai carboidrati agli acidi grassi come sorgente metabolica preferenziale. La somministrazione di T1AM modula inoltre la secrezione di insulina, è stato infatti osservato che l’iniezione intraperitoneale di T1AM nei topi, genera un incremento del livello di glucosio nel sangue e diminuisce i livelli di insulina. L’osservazione in letteratura, che topi spontaneamente obesi trattati cronicamente con T1AM mostrassero, a livello del fegato, una variazione nell’espressione delle sirtuine e dell’enzima glucochinasi, ha portato a indagare più in dettaglio sulla variazione nell’espressione di tali proteine in un modello di fegato. Lo scopo di questa tesi è stato dunque quello di valutare tramite Western Blot, la variazione nell’espressione proteica delle sirtuine e della glucochinasi in cellule trattate con T1AM. Per effettuare questo studio sono state utilizzate due linee cellulari come modello di fegato, le HepG2 (carcinoma epatocellulare umano) ed epatociti primari di ratto ottenuti tramite perfusione con collagenasi. Tutti gli esperimenti sono stati poi ripetuti sulle stesse linee cellulari trattate però con T3, molecola strutturalmente simile alla 3-iodotironamina, derivante dalla tiroxina. Inizialmente sono stati svolti saggi di vitalità cellulare, mediante il test MTT, su cellule di entrambe le linee cellulari trattate con T3 o T1AM, così da avere una preliminare idea degli effetti di queste due molecole sulla cellula in toto. Nelle cellule trattate con T1AM è stata notata una diminuzione della vitalità cellulare, che risulta preferenziale nelle cellule tumorali piuttosto che negli epatociti primari, questo effetto richiederà però ulteriori indagini. Il trattamento delle HepG2 e degli epatociti primari con T3 non sembra invece avere lo stesso effetto indotto dalla T1AM e la vitalità cellulare rimane pressoché la invariata. In entrambe le linee trattate con T1AM è stata osservata una sottoespressione della sirtuina 4, confermando risultati già precedentemente osservati nel fegato di topi spontaneamente obesi trattati cronicamente con T1AM. L’espressione proteica della glucochinasi non è invece risultata alterata in nessuna delle linee analizzate trattate con T3 o T1AM. Inoltre sono state osservate una diminuzione nell’espressione della sirtuina 1, comune a cellule HepG2 trattate con T1AM o T3, e una riduzione dell’espressione della sirtuina 2 solo in presenza di T3 in entrambe le linee cellulari. Considerato che variazioni di espressione delle sirtuine 1 e 2 sono correlate alla tumorigenesi a livello epatico e in altri tessuti, una maggior comprensione del meccanismo regolatorio mediato dalla T1AM e dalla T3 potrebbe essere utile nella terapia antitumorale

3-Iodothyronamine and 3,5,3′-triiodo-L-thyronine reduce SIRT1 protein expression in the HepG2 cell line

AbstractBackground3-Iodothyronamine (T1AM) is an endogenous messenger chemically related to thyroid hormone. Recent results indicate significant transcriptional effects of chronic T1AM administration involving the protein family of sirtuins, which regulate important metabolic pathways and tumor progression. Therefore, the aim of this work was to compare the effect of exogenous T1AM and 3,5,3′-triiodo-L-thyronine (T3) chronic treatment on mammalian sirtuin expression in hepatocellular carcinoma cells (HepG2) and in primary rat hepatocytes at micromolar concentrations.Materials and methodsSirtuin (SIRT) activity and expression were determined using a colorimetric assay and Western blot analysis, respectively, in cells treated for 24 h with 1–20 μM T1AM or T3. In addition, cell viability was evaluated by the MTTtest upon 24 h of treatment with 0.1–20 μM T1AM or T3.ResultsIn HepG2, T1AM significantly reduced SIRT 1 (20 μM) and SIRT4 (10–20 μM) protein expression, while T3 strongly decreased the expression of SIRT1 (20 μM) and SIRT2 (any tested concentration). In primary rat hepatocytes, T3 decreased SIRT2 expression and cellular nicotinamide adenine dinucleotide (NAD) concentration, while on sirtuin activity it showed opposite effects, depending on the evaluated cell fraction. The extent of MTT staining was moderately but significantly reduced by T1AM, particularly in HepG2 cells, whereas T3 reduced cell viability only in the tumor cell line.ConclusionsT1AM and T3 downregulated the expression of sirtuins, mainly SIRT1, in hepatocytes, albeit in different ways. Differences in mechanisms are only observational, and further investigations are required to highlight the potential role of T1AM and T3 in modulating sirtuin expression and, therefore, in regulating cell cycle or tumorigenesis

Thyroid hormone derivates T1AM and 3,5-T2: their effects on rat heart, NG108-15 and U-87 MG cell lines.

The term “thyroid hormones”, classically referred to both 3,5,3′-triiodothyronine (T3) and thyroxine (T4), seems nowadays to be simplistic; some T3 and T4 metabolites, particularly 3,5diiodothyronine (3,5-T2) and 3-iodothyronamine (T1AM), are independent chemical messengers, with specific metabolic effects. In this PhD thesis we focused on the effects of these derivates on different tissues. In the first project we evaluated the effects of T1AM on the glutamatergic pathway, the main excitatory system in brain. A cancer hybrid cell line of mouse neuroblastoma and rat glioma (NG108-15) and a human glioblastoma cell line (U-87 MG) were used as in vitro model and treated with different T1AM concentration for 24h, alone or in combination with resveratrol 10 µM and/or amyloid β peptide (25-35) 10 µM. Firstly, we characterized cell lines for the expression of receptors implicated in glutamatergic pathway with real time PCR (qRT-PCR) and Western blot. Both cell lines expressed AMPA, NMDAR1 and EphB2, but only U-87 MG expressed TAAR1, the putative T1AM receptor. Using LC-MS-MS we discovered that both lines were able to take up T1AM and, rapidly catabolized it to TA1. In both cell lines, T1AM showed a slightly but significant cytotoxic action starting from 0.1 μM, that increase is presence of β-amyloid (10 µM), but not resveratrol (10 µM) evaluated by MTT test. We then evaluate glucose consumption using a glucose assay and observed in the NG108-15 a metabolic effect mediated by T1AM (p<0.05). For protein expression and post-translation modifications Western blot was used and an increase in the phosphorylation of Ca-calmodulin-dependent protein kinase (CaMK) II (pCaMKII/total CaKMII, p<0.05) in NG108-15 cell line was observed. In association with resveratrol T1AM could increase the expression of PKC (p<0.001 vs RSV) in the same cell line, a synergic effect showed exclusively in presence of both T1AM and resveratrol at all tested thyronamine concentration. In U-87 MG T1AM induce the phosphorylation of the transcriptional factor cAMP response element-binding protein (CREB) (PCREB/total CREB p<0.05). Our results indicated that these two nervous cell lines express receptors implicated in glutamatergic system and might be used as biochemical model to study its post synaptic signaling cascade. T1AM had a minimal cytotoxic effect and it was able to induce different post-translational modification in neuronal cell lines. T1AM might activate mechanisms of action which included increasing CaMKII phosphorylation and PKC expression in NG108-15 while in U-87 MG induced the activation of the transcriptional factor CREB. We then focused on another endogenous thyroid hormone derivate, the 3,5-diiodo-l-thyronine (3,5-T2) and its effect on heart which have been poorly investigated so far. It’s well understood that 3,5-T2 is able to regulate energy expenditure, resting metabolic rate and oxygen consumption with a mechanism that might involve mitochondria. We decided to evaluate the functional metabolic, and toxic effect of 3,5-T2 using both in vitro and ex vivo models of cardiac preparations. As comparison for our results we also evaluated the response to T3 and T4. As cell culture we selected the H9c2 cells (rat cardiomyoblasts) to determine 3,5-T2, T3, and T4 uptake using LC-MS-MS. We treated cells with 3,5-T2 (0.1 to 10 µM) and evaluated cell viability using MTT test and crystal violet staining. We also investigate a possible 3,5-T2 metabolic effect performing a glucose and hexokinase assay. In the end we measured the cardiac functional effects, perfusing isolated working rat hearts with 3,5-T2, T3, or T4 in Krebs-Ringer buffer and recording hemodynamic variables. H9c2 cells took up 3,5-T2, in cell lysate and the analyte levels increased slowly over time. 3,5-T2 significantly decreased MTT staining at 0.5–10 µM concentration, an effect confirmed by the crystal violet staining only at 10 µM T2, while equimolar T3 and T4 did not share this effect. In cells exposed to 0.1 or 1.0 µM of 3,5-T2 glucose uptake increased by 23% or 30% (p < 0.05). On the opposite side, T3 did not affect glucose consumption which was significantly reduced by 1 and 10 µM T4 (−24 and −41%, respectively, p < 0.01 and p < 0.0001). In the isolated perfused rat heart, 10 µM T2 produced a transient and slight reduction in the cardiac output and aortic flow (p<0.05), while thyroid hormone did not induce any hemodynamic change. Our findings demonstrate that 3,5-T2 was taken up by cardiomyoblasts, and in a concentration range between 0.1 µM and 1.0 µM modulated cardiac energy metabolism increasing glucose accumulation. Furthermore, we observed some evidence of cytotoxicity and a transient impairment of contractile performance only at the highest 3,5-T2 concentration tested (10 µM). These effects seem to be specific for 3,5-T2, since they are not reproduced by thyroid hormone. In the end we develop a novel ad-hoc optimized method to quantify T2 isomers using LC-MSMS in human serum. T2 isomers (3,5-T2 and 3,3’-T2) has been detected in human blood using immunological methods, but until now a reliable assay based on mass spectrometry was not available. We obtained 2 mL of serum samples from 28 healthy subjects. The serum was firstly deproteinized with acetonitrile and then exposed to a solid phase extraction-based procedure. Then samples were furtherly cleaned by hexane washing and subjected to another step of deproteinization with acetonitrile to precipitate residual proteins. Both isomers were then analyzed by high performance liquid chromatography coupled to tandem mass spectrometry. We developed a method with 88–104% accuracy, 95–97% precision, 78% recovery and a matrix effect average of +8%. In the serum sample 3,5-T2 was detected with a concentration averaged (mean ± SEM) 41 ± 5 pg/mL and 133±15 pg/mL for 3,3′-T2. Furthermore, we observed a significant correlation between 3,5-T2 and 3,3′-T2 concentrations (r = 0.540. p < 0.01), while no significant relation was observed with thyroid hormone. In conclusion, this method can quantify both T2 isomers in human serum using a reliable assay based on LC-MS-MS. The concentrations of these isomers lie in the subnanomolar range and show a significant correlation in healthy subject

Effects of thyroid hormones and 3-iodothyronamine on sirtuin expression in hepatocytes

Background: 3-iodothyronamine (T1AM) is an endogenous messenger chemically related to thyroid hormone. Among its functional effects a shift from carbohydrates to lipids as principal energy resource has been observed. Recent results indicate significant transcriptional effects of chronic T1AM administration involving genes of the sirtuin family. Sirtuins regulate important metabolic pathways involved in apoptosis, stress resistance, energy metabolism. Therefore the 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. Methods:. Isolated hepatocytes were obtained by liver in-situ collagenase perfusion. Sirtuin expression was evaluated by Western Blot analysis in cells treated for 24h with 1-20µM T1AM or T3. In addition, cell viability was evaluated by MTT test upon 24h treatment with 0.5nM to 20µM T1AM or T3. Results: Protein expression: In HepG2, T1AM significantly reduced SIRT1 and SIRT4 expression at 20µM while T3 strongly decreased the expression of SIRT1 (20µM), and SIRT2 (any concentration tested). In primary rat hepatocytes T1AM decreased SIRT4 expression (10-20µM) whether T3 decreased SIRT2 at 10µM. Cell viability: T1AM caused a moderate but significant reduction in the number of viable cells particularly in HepG2 cells in which the effect occurred at concentration starting from 5nM that did not caused any change in sirtuin expression. T3 did not affect cell viability in both HepG2 and isolated hepatocytes. Conclusions: T1AM and T3 differently affect sirtuin expression in hepatocytes. Since SIRT1 and SIRT4 are important regulator of lipid and glucose metabolism, whereas SIRT2 has a key role in regulating cell cycle and genomic integrity, our observations are consistent with the shift from carbohydrates to lipids induced by T1AM. T1AM has also a moderate effect on cell viability in HepG2 cells which seems however independent from sirtuin modulation

Recovery of 3-iodothyronamine and derivatives in biological matrices: problems and pitfalls

Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely DMEM supplemented with fetal bovine serum (FBS), and to other biological matrices

3-iodothyronamine (T1AM) induces changes on glutamatergic postsynaptic signaling pathway

Objective: Exogenous 3-iodothyronamine (T1AM), a derivative of thyroid hormone, produces antiamnestic and prolearning effects in mice. Glutamatergic neurotransmission, the major excitatory system in brain, plays a key role in regulating neuroplasticity, learning and memory, and it is often compromised in neurological disorders. In the present work, we characterized the gene expression profile of two different neuronal cell lines and we evaluated the effects of T1AM on the expression of proteins involved in glutamatergic signaling pathway. Methods: A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG108-15) and a human glioblastoma cell line (U-87 MG) were used. We first characterized the in vitro model by analyzing gene expression of several proteins involved in the glutamatergic postsynaptic cascade by real time PCR. Cell lines were then treated with T1AM for 24h, ranging from 0.1 to 10 μM, alone or in combination with 10 µM resveratrol (RSV) and/or 10 µM amyloid β peptide (25-35). Cell viability, glucose consumption, protein expression, (cell calcium content) and cAMP production were assessed. Results: NG108-15 and U-87 MG cell lines expressed receptors and other proteins belonging to the glutamatergic post synaptic signaling pathway, while only U87 cells expressed TAAR1, one of the putative T1AM receptors. In NG 108-15 cells, T1AM in combination with RSV upregulated PKC protein expression vs the baseline (P<0.05). Furthermore, in the same cell line, T1AM decreased glucose consumption (p<0.01 vs control), without affecting cell viability. In U-87 MG cells, T1AM improved pCREB/CREB ratio (p<0.01) without altering glucose consumption. Cell viability and cAMP concentration were significantly increased at 0.1 M T1AM. Conclusions: Our results show that in in vitro models of neuronal cells T1AM may produce changes in the post synaptic signaling cascade of the glutamatergic system

Effects of 3-iodothyronamine (T1AM) on signaling pathways involved in synaptic plasticity and neuroprotection.

In mouse, the administration of 3-iodothyronamine (T1AM) had an antiamnestic and prolearning effect. In addition, T1AM rescued long term potentiation (LTP) in enthorinal cortex exposed to the toxic effect of β-amyloid. In the present paper we used a neuronal cell line to evaluate the effect of T1AM on the expression calcium calmodulin dependent kinase II (CamKII) and protein kinase C (PKC), two key players involved in LTP. The effects on sirtuin 1 (SIRT1), allegedly responsible for the neuroprotective effect of resveratrol, were also investigated. Methods: A hybrid line of cancer cells of mouse neuroblastoma and rat glioma (NG108-15) was used and treated with 100 nM to 10 M T1AM for 24 h, alone or in combination with 10 µM resveratrol and/or 10 µM amyloid β peptide (25-35). Protein expression and post-translational modifications were investigated by Western blot technique. Glucose consumption was also measured upon 4 h treatment. Results: NG108-15 expressed TAAR1, the putative T1AM receptor, as well as AMPA and NMDA receptors, that are involved in the signaling cascade responsible of LTP. T1AM (10 M) induced CaMKII phosphorylation (p<0.001), but had no direct effect on PKC and SIRT1 expression. However, in the presence of β-amyloid, T1AM increased PKC and SIRT 1 expression at 1M and 10 M concentration, respectively, although β-amyloid alone had no significant effect. The response to resveratrol was also modified by T1AM, since resveratrol inhibited PKC expression while the association of resveratrol and T1AM (100 nM) produced a remarkable increase vs the baseline (P<0.05). Furthermore an increase in glucose consumption was observed at the highest T1AM concentration (p<0.01). Conclusions: In NG108-15 cells T1AM activated CaMKII and had complex effects on the response to β-amyloid and resveratrol

Assay of endogenous 3,5-diiodo-L-thyronine (3,5-T2) and 3,3’-diiodo-L-thyronine (3,3’-T2) in human serum.

Background: 3,5-diiodo-L-thyronine (3.5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been occasionally detect in human blood using High Performance Liquid Chromatography- tandem Mass Spectrometry (HPLC-MS/MS), but the results have been quite variable and quality control data were often missing. In this study we used an ad-hoc optimized Solid Phase Extraction (SPE)-based extraction method which allowed us to quantitate 3,5-T2 and its isomer 3,3’-T2 in human serum. Methods: Serum samples were obtained from 28 patients (8 healthy volunteers and 20 women undergoing endocrinological screening and found to be euthyroid). Two ml of serum were deproteinized with acetonitrile and then extracted using an SPE based process. To lower background noise, after extraction the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and 3,3’-T2 were analyzed by a Sciex API4000 mass spectrometer coupled to an Agilent Infinity 1290 LC system using isotope dilution method. In 20 patients total T3 and T4 were also assayed by HPLC-MS/MS. Results: Accuracy and precision for 3,5-T2 assay were 88-104% and 95-97%, respectively. Recovery, matrix effect and process efficiency averaged 78%, 108%, and 84% respectively. 3,5-T2 was detected in all samples and its concentration averaged (mean±SEM) 41±5 pg/ml, i.e. 78±9 pmol/l. In the same samples the concentration of 3,3’-T2 averaged 133±15 pg/ml, i.e. 253±29 pmol/l. 3,5-T2 concentration was significantly related to 3,3’-T2 concentration (r=0.540, P<0.01), while no significant correlation was observed with either T3 or T4 in the subset of patients in which these hormones were assayed. Conclusion: Our optimized extraction method is able to quantify 3,5-T2 and 3,3’-T2 in human serum. Their concentrations are in the subnanomolar range, and a significant correlation was detected between these two metabolites in healthy individuals

Effects of 3,5-diiodo-L-thyronine on the cardiac tissue

Background: 3,5-diiodo-L-thyronine (T2) is an endogenous derivative of thyroid hormone. Its physiological role is unclear, but it has been suggested to regulate energy expenditure, resting metabolic rate and oxygen consumption. Thyromimetic effects on the myocardial tissue have also been reported. In this study we evaluate T2 cardiac effects using both in vitro and ex-vivo models . Methods: To investigate T2 effect on cellular metabolism, MTT test and glucose consumption assay were performed on cultured rat cardiomyoblast (H9c2) cells. T2 cellular uptake was also evaluated using High Performance Liquid Chromatography- tandem Mass Spectrometry (HPLC-MS/MS). To assess cardiac functional effects, isolated working rat hearts were perfused with T2 (0.1-10μM) using glucose as energy source and hemodynamic parameters were evaluated for 40min. Results: MTT test results showed that T2 (5nM-10µM) induced a significant increase in cell metabolism (p<0.0001). Glucose consumption was also significantly affected (p<0.01) since we observed an increase in the range of 15% (100nM) to 18% (1μM) compared to control group. HPLC-MS/MS results showed that in the incubation medium T2 (100nM or 1μM) T2 concentration remained nearly constant over time while in cell lysate T2 increased, reaching a steady state after about 60min (0.5 nM, with T2 100nM) or 240min (15nM with T2 1μM) with a recovery of about 90%. Notably, T2 did not produce any significant change in cardiac output nor in heart rate. Conclusions: Our findings indicate that T2 is taken up by cardiomyoblasts and it may modulate cardiac energy metabolism, increasing glucose consumption without affecting the contractile performance