Metabolic Reprogramming by 3-Iodothyronamine (T1AM): A New Perspective to Reverse Obesity through Co-Regulation of Sirtuin 4 and 6 Expression.

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes.

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

Background 3-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 methods Sirtuin (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. Results In 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. Conclusions T1AM 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.

Assay of Endogenous 3,5-diiodo-L-thyronine (3,5-T2) and 3,3'-diiodo-L-thyronine (3,3'-T2) in Human Serum: A Feasibility Study.

3,5-diiodo-L-thyronine (3,5-T2) is an endogenous derivative of thyroid hormone with potential metabolic effects. It has been detected in human blood by immunological methods, but a reliable assay based on mass spectrometry (MS), which is now regarded as the gold standard in clinical chemistry, is not available yet. Therefore, we aimed at developing a novel ad-hoc optimized method to quantitate 3,5-T2 and its isomers by MS in human serum. Serum samples were obtained from 28 healthy subjects. Two ml of serum were deproteinized with acetonitrile and then subjected to an optimized solid phase extraction-based procedure. To lower background noise, the samples were furtherly cleaned by hexane washing and acetonitrile precipitation of residual proteins. 3,5-T2 and its isomers 3,3'-T2 and 3',5'-T2 were then analyzed by HPLC coupled to tandem MS. Accuracy and precision for T2 assay were 88-104% and 95-97%, respectively. Recovery and matrix effect averaged 78% and +8%, 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, while 3',5'-T2 was not detected. 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 a subset of patients in which these hormones were assayed. In conclusion, our method is able to quantify 3,5-T2 and 3,3'-T2 in human serum. Their concentrations lie in the subnanomolar range, and a significant correlation was detected between these two metabolites in healthy individuals.

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.

Recovery of 3-Iodothyronamine and Derivatives in Biological Matrixes: Problems and Pitfalls.

BACKGROUND: 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 Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes. METHODS: Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (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 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters. RESULTS: In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. 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 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, 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 blood. CONCLUSIONS: These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

Effect of Hypothyroidism and Hyperthyroidism on Tissue Thyroid Hormone Concentrations in Rat.

BACKGROUND AND OBJECTIVE: The present study was aimed at determining the effects of experimental hypothyroidism and hyperthyroidism on tissue thyroid hormones by a mass spectrometry-based technique. METHODS: Rats were subjected to propylthiouracil treatment or administration of exogenous triiodothyronine (T3) or thyroxine (T4). Tissue T3 and T4 were measured by liquid chromatography tandem mass spectrometry in the heart, liver, kidney, visceral and subcutaneous adipose tissue, and brain. RESULTS: Baseline tissue T3 and T4 concentrations ranged from 0.2 to 20 pmol ∙ g(-1) and from 3 to 125 pmol ∙ g(-1), respectively, with the highest values in the liver and kidney, and the lowest values in the adipose tissue. The T3/T4 ratio (expressed as a percentage) was in the 7-20% range in all tissues except the brain, where it averaged 75%. In hypothyroidism, tissue T3 was more severely reduced than serum free T3, averaging 1-6% of the baseline versus 30% of the baseline. The extent of tissue T3 reduction, expressed as percentage of the baseline, was not homogeneous (p < 0.001), with liver = kidney > brain > heart > adipose tissue. The tissue T3/T4 ratio significantly increased in all organs except the kidney, averaging 330% in the brain and 50-90% in the other tissues. By contrast, exogenous T3 and T4 administration produced similar increases in serum free T3 and in tissue T3, and the relative changes were not significantly different between different tissues. CONCLUSIONS: While the response to increased thyroid hormones availability was similar in all tissues, decreased thyroid hormone availability induced compensatory responses, leading to a significant mismatch between changes in serum and in specific tissues.