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

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

Biochemical basis of ischemic heart injury and of cardioprotective interventions

Cardioprotective interventions are defined as interventions able to increase myocardial resistance to ischemia. The authors approach the issue of cardioprotection on the basis of the present knowledge about the biochemical mechanisms responsible for the injury produced by myocardial ischemia or ischemia-reperfusion. Reversible and irreversible injury are distinguished. The former is largely accounted for by the direct consequences of reduced ATP synthesis, which causes decreased ATP phosphorylation potential, acidosis and phosphate accumulation. The biochemical mechanisms leading to irreversible injury include osmotic overload, production of toxic lipid metabolites, cytosolic calcium overload, and generation of reactive oxygen species, which lead to membrane disruption, mitochondrial dysfunction and possibly to the activation of apoptotic pathways. The major effect of the classical cardioprotective agents (nitrates, beta adrenergic antagonists, calcium channel blockers) consists in affecting ATP demand/supply ratio in such a way as to delay the decrease in ATP phosphorylation potential. Other drugs have been introduced, which allegedly interfere directly with the mechanisms responsible for irreversible ischemic injury. These include 3-ketoacyl-CoA tiolase inhibitors, modulators of intracellular calcium channels, ionic exchanger inhibitors, free radical scavengers, caspase inhibitors, purinergic agonists, K(+)(ATP) channel openers, and modulators of mitochondrial permeability transition. The results obtained with these substances in experimental models and in the clinical setting are discussed. Special attention is devoted to angiotensin converting enzyme inhibitors, whose direct cardioprotective properties has recently been demonstrated

Vascular Progenitor Cells: From Cancer to Tissue Repair

Vascular progenitor cells are activated to repair and form a neointima following vascular damage such as hypertension, atherosclerosis, diabetes, trauma, hypoxia, primary cancerous lesions and metastases as well as catheter interventions. They play a key role not only in the resolution of the vascular lesion but also in the adult neovascularization and angiogenesis sprouting (i.e., the growth of new capillaries from pre-existing ones), often associated with carcinogenesis, favoring the formation of metastases, survival and progression of tumors. In this review, we discuss the biology, cellular plasticity and pathophysiology of different vascular progenitor cells, including their origins (sources), stimuli and activated pathways that induce differentiation, isolation and characterization. We focus on their role in tumor-induced vascular injury and discuss their implications in promoting tumor angiogenesis during cancer proliferation and migration

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

Chronic carbamazepine selectively downregulates cytosolic phospholipase A2 expression and cyclooxygenase activity in rat brain

BACKGROUND: Carbamazepine is a mood stabilizer used as monotherapy or as an adjunct to lithium in the treatment of acute mania or the prophylaxis of bipolar disorder. Based on evidence that lithium and valproate, other mood stabilizers, reduce brain arachidonic acid turnover and its conversion via cyclooxygenase to prostaglandin E(2) in rat brain, one possibility is that carbamazepine also targets the arachidonic acid cascade. METHODS: To test this hypothesis, carbamazepine was administered to rats by intraperitoneal injection at a daily dose of 25 mg/kg for 30 days. RESULTS: Carbamazepine decreased brain phospholipase A(2) activity and cytosolic phospholipase A(2) protein and messenger RNA levels without changing significantly protein and activity levels of calcium-independent phospholipase A(2) or secretory phospholipase A(2). Cyclooxygenase activity was decreased in carbamazepine-treated rats without any change in cyclooxygenase-1 or cyclooxygenase-2 protein levels. Brain prostaglandin E(2) concentration also was reduced. The protein levels of other arachidonic acid metabolizing enzymes, 5-lipoxygenase and cytochrome P450 epoxygenase, were not significantly changed nor was the brain concentration of the 5-lipoxygenase product leukotriene B(4). CONCLUSIONS: Carbamazepine downregulates cytosolic phospholipase A(2)-mediated release of arachidonic acid and its subsequent conversion to prostaglandin E(2) by cyclooxygenase. These effects may contribute to its therapeutic actions in bipolar disorder

Characterization of 3-Iodothyronamine In Vitro Dynamics by Mathematical Modeling

3-Iodothyronamine (T1AM) is regarded as a hormone-like substance thanks to its endogenous nature, itsinteraction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T1AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T1AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T1AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide(semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T1AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T1AM dynamics. We also estimated that T1AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T1AM degradation, while no significant changes were observed in experiments with SSAO inhibitors

Uptake and metabolic effects of 3-iodothyronamine (T1AM) in hepatocytes

3-Iodothyronamine (T1AM) is an endogenous relative of thyroid hormone with profound metabolic effects. In different experimental models, T1AM increased blood glucose, and it is not clear whether this effect is entirely accounted by changes in insulin and/or glucagone secretion. Thus, in the present work, we investigated the uptake of T1AM by hepatocytes, which was compared with the uptake of thyroid hormones, and the effects of T1AM on hepatic glucose and ketone body production. Two different experimental models were used: HepG2 cells and perfused rat liver. Thyronines and thyronamines (T0AMs) were significantly taken up by hepatocytes. In HepG2 cells exposed to 1 μM T1AM, at the steady state, the cellular concentration of T1AM exceeded the medium concentration by six- to eightfold. Similar accumulation occurred with 3,5,3′-triiodothyronine and thyroxine. Liver experiments confirmed significant T1AM uptake. T1AM was partly catabolized and the major catabolites were 3-iodothyroacetic acid (TA1) (in HepG2 cells) and T0AM (in liver). In both preparations, infusion with 1 μM T1AM produced a significant increase in glucose production, if adequate gluconeogenetic substrates were provided. This effect was dampened at higher concentration (10 μM) or in the presence of the amine oxidase inhibitor iproniazid, while TA1 was ineffective, suggesting that T1AM may have a direct gluconeogenetic effect. Ketone body release was significantly increased in liver, while variable results were obtained in HepG2 cells incubated with gluconeogenetic substrates. These findings are consistent with the stimulation of fatty acid catabolism, and a shift of pyruvate toward gluconeogenesis. Notably, these effects are independent from hormonal changes and might have physiological and pathophysiological importance