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

The role of tandem mass spectrometry in clinical chemistry: quantification of steroid hormones and vitamin D

Nowadays tandem mass spectrometry coupled to liquid chromatography is considered as the "gold standard" technique for steroid hormones quantification in biological fluids. Its emerging role in the clinical laboratories is mainly due to its capability to overcome the main limitations of the widespread immunoassays (IAs), providing, at the same time, the simultaneous quantification of several steroids of interest. This is a very important feature, as it allows, just in a single analysis, the monitoring of some key steroids in a metabolic pathway. In this chapter, we will describe the instrumental layout to be used in the measurement of clinically relevant steroids or steroid panels, and we will discuss the advantages and disadvantages of tandem mass spectrometry with respect to the main IA-based assays. The quantification of the main metabolites of vitamin D, 25-hydroxyvitamin D3, which is considered a steroid hormone, is also discussed in the chapter. Applications concerning 11β-HSD enzyme activity, 21-hydroxylase deficiency in newborns, hypovitaminosis D in patients with heart failure and vitamin D intoxication are discussed

LTI Models for 3-Iodothyronamine Time Dynamics: A Multiscale View.

3-Iodothyronamine (T1AM) is a novel relative of thyroid hormone that plays a role in critical body regulatory processes, such as glucose metabolism, thermal regulation and heart beating. This work was aimed at characterizing time dynamics of T1AM and its catabolite 3-iodothyroacetic acid (TA1) in different biological scales with LTI (Linear Time Invariant) models. Culture medium samples coming from culture of H9c2 murine cells and perfusion liquid samples from perfused rat heart were collected after the injection of a T1AM bolus. T1AM and TA1 concentrations in the samples were assayed with high performance liquid chromatography coupled to tandem mass spectrometry. Kinetic constants relative to T1AM transport and conversion were estimated with Weighted Least Squares method. We found that these constants can be related with an allometric power law depending on mass, with a negative exponent of -0.27± 0.19, implying that the velocity of conversion and internalization of T1AM decrease with increasing of system mass

Cardioprotection by ouabain and digoxin in perfused rat hearts

This work was aimed at determining the cardioprotective effect of digitalis glycosides in rat heart, and to relate it with Na+, K+-ATPase inhibition and ERK1/2 activation. Isolated working rat hearts were perfused in the presence of ouabain or digoxin, which were used at concentrations ranging from 10(-8) to 10(-5) M. The hearts were then subjected to 30 minutes of global normothermic ischemia followed by 120 minutes of retrograde reperfusion; irreversible tissue injury was determined on the basis of triphenyltetrazolium chloride staining. Significant cardioprotection was observed with 10(-7) M and 10(-5) M ouabain (ischemic injury averaged 7.0 +/- 3.5% and 8.3 +/- 0.6% versus 37.3 +/- 2.0% in controls. P < 0.01 in each case). Hearts treated with digoxin showed decreased ischemic injury at 10(-6) M and 10(-5) M (18.0 +/- 1.5% and 14.2 +/- 1.0%, P < 0.01 versus control in both cases). In parallel experiments, ERK2 phosphorylation was increased by 10(-7) to 10(-5) M ouabain, while ERK1 and ERK2 phosphorylation was increased by 10(-6) to 10(-5) M digoxin. The cardioprotective effect was not related to Na+, K+-ATPase inhibition, since Rb+ uptake was not significantly different between control and treated hearts

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

3,5-diiodo-L-thyronine increases glucose consumption in cardiomyoblasts without affecting the contractile performance in rat heart

3,5-diiodo-L-thyronine (T2) is an endogenous derivative of thyroid hormone that has been suggested to regulate energy expenditure, resting metabolic rate and oxygen consumption with a mechanism that involves the activation of mitochondrial function. In this study, we focused on the cardiac effects of T2, which have been poorly investigated so far, by using both in vitro and ex vivo models. As a comparison, the response to T3 and T4 was also determined. Rat cardiomyoblasts (H9c2 cells) were used to determine T2, T3, and T4 uptake by high-performance liquid chromatography-tandem mass spectrometry. In the same experimental model, MTT test, crystal violet staining, and glucose consumption were investigated, using T2 concentrations ranging from 0.1 to 10 μM. To assess cardiac functional effects, isolated working rat hearts were perfused with T2, T3, or T4 in Krebs-Ringer buffer, and the hemodynamic variables were recorded. T2 was taken up by cardiomyoblasts, and in cell lysate T2 levels increased slowly over time, reaching higher concentrations than in the incubation medium. T2 significantly decreased MTT staining at 0.5-10 μM concentration (P < 0.05). Crystal violet staining confirmed a reduction of cell viability only upon treatment with 10 μM T2, while equimolar T3 and T4 did not share this effect. Glucose consumption was also significantly affected as indicated by glucose uptake being increased by 24 or 35% in cells exposed to 0.1 or 1.0 μM T2 (P < 0.05 in both cases). On the contrary, T3 did not affect glucose consumption which, in turn, was significantly reduced by 1 and 10 μM T4 (-24 and -41% vs control, respectively, P < 0.05 and P < 0.01). In the isolated perfused rat heart, 10 μM T2 produced a slight and transient reduction in cardiac output, while T3 and T4 did not produce any hemodynamic effect. Our findings indicate that T2 is taken up by cardiomyoblasts, and at 0.1-1.0 μM concentration it can modulate cardiac energy metabolism by increasing glucose consumption. Some evidence of toxicity and a transient impairment of contractile performance are observed only at 10 μM concentration. These effects appear to be specific for T2, since they are not reproduced by T3 or T4