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wissenschaft & praxis Abbott-Preisträgerin 2014 Effects of thyroid dysfunction and nitric oxide on insulin sensitive cells A study revealing that thyroid hormones alone or in combination with insulin modulate NOX levels, expression of IL-6 and activation of NF-KB in insulin sensitive cells in a cell type specific manner. Thyroid Dysfunction The thyroid gland is one of the most important endocrine glands in the human body. Thyroid diseases display a wide variety of different disorders, including inflammatory and hormonal diseases of the thyroid gland, autoimmune diseases, malign and benign tumors. Classification guidelines define these diseases upon functional aspects into hyper-, eu-, or hypothyroidism reflecting excessive, normal or defective levels of thyroid hormones. The thyroid gland is part of the hypothalamic-pituitary-thyroid (HPT) axis, which is a hormonal negative feedback control system. The release of the thyroid hormones thyroxine (T4) and triiodothyronine (T3) is regulated by the thyroid stimulating hormone (TSH) which is produced in the anterior pituitary gland. TSH itself is regulated by the thyrotropin releasing hormone (TRH). TRH production in the hypothalamus as well as TSH production in the pituitary gland is suppressed by feedback inhibition via increased T3 and T4 levels. The thyroid gland mostly produces T4, while the more active form T3 is produced from T4 by deoidase enzymes in the peripheral target cells. The diagnosis of thyroid dysfunctions is a combination of clinical symptoms followed by measurements of characteristical laboratory parameters. Once the diagnosis is made, further steps depend on functional aspects of the thyroid gland. Hyperthyroidism is defined as increased circulating levels of T3 and T4 and decreased TSH levels. To restore euthyroidism radioactive iodine (131 I) therapy, antithyroid drugs and thyroidectomy are possible therapies. Hypothyroidism is characterized by low circulating levels of the thyroid hormones T3 and T4 as well as by elevated levels of TSH. To restore euthyroidism thyroid hormones are substituted lifelong in most cases. Thyroid Hormone Action The thyroid hormones T4 and T3 are involved in a wide variety of physiological functions including metabolism, growth and other hormone systems. Thyroid hormones play an important role for human growth, especially for the development of the brain and the skeletal system. Another main thyroid hormone action is to increase metabolism. Under increased metabolic circumstances the cellular oxygen is used to produce adenosine triphosphate (ATP), which transports chemical energy within cells for metabolism. The explosive consumption of ATP leads to metabolic changes affecting the protein, the carbohydrate and the fat metabolism. As a consequence the pancreas, amongst other influenced endocrine glands, is stimulated biomed austria sommer 2014 to secret insulin by increased glucose metabolism. The enhanced fat metabolism leads to increased lipolysis and therefore to increased blood concentration of free fatty acids. The cholesterol metabolism, another aspect of the fat metabolism, is also affected. Cholesterol levels have been shown to be reduced by thyroid hormone action. Another important effect of thyroid hormones is the socalled calorigenic effect, which results in a rising body temperature. Thus, the calorigenic effect is believed to be an important key player in the maintenance of normal body temperature. Further, the rising body temperature also induces heat-dissipating mechanisms like enhanced blood flow and cutaneous vasodilation that in turn increase the renal water reabsorption The aim of this in vitro and with that the blood volume. Therefor, study is to investigate and for the reason that thyroid hormones the pathophysiologialso influence the catecholamines and their cal mechanisms of NOX receptors by triggering their function, they and thyroid hormones affect the cardiovascular system. by using insulin sensitiFor all these reasons thyroid dysfunc- ve cell culture models. tions can contribute to coronary heart diseases (CHD), artherosclerosis, obesity, hyperinsulinaemia and therefor to the metabolic syndrome (MS). The MS is defined as causal relationship between insulin resistance, hypertension, obesity and hypercholesterolaemia. Nitric Oxide NOX is a free radical gas with a very short half-life, produced by a variety of cell types and mediating many different functions. L-arginine is converted to NOX and citrulline by a family of enzymes, called the nitric oxide synthases (NOS). Its most important functions are the control of blood pressure, the vascular tone and growth as well as the regulation of inflammation by reducing leukocyte recruitment and interactions of platelet and leukocytes within the vascular wall. Further, it impairs platelet adhesion and aggregation. Therefore NOX is believed to be a protective factor for atherosclerotic events. The production of NOX can be induced by cytokines and inflammatory mediators like IL-6 or TNFα. NOX is also an important neurotransmitter. Obviously, disturbance in the NOX homeostasis can be associated with a variety of pathologic conditions like thyroid dysfunction, endothelial dysfunction, atherosclerosis, hypertension and cardiovascular diseases and the metabolic syndrome. The Aim The aim of this in vitro study is to investigate the pathophysiological mechanisms of NOX and thyroid hormones by using insulin sensitive cell culture models. Firstly, the impact of thyroid hormones on the nitric oxide pathway will be investigated. Secondly, potential additive effects 11 wissenschaft & praxis concentrations of TSH [10 mU/ml], T3 [10 mM] and T4 [10 mM] in combination with the NOX-donors NOC18 [10 µM] and SNAP [25 µM] which reversed the reduction of NOX to a level comparable to untreated 3T3 cells. In detail both NOX-donors, NOC18 and SNAP, showed similar effects on 3T3 cells. (Figure 1) In contrast, in L6 cells all used concentrations resulted in an increase of NOX except for the cells treated with T3. According to these results L6 cells were treated with the highest used concentrations of TSH [10 mU/ml], T3 [10 mM] and T4 [10 mM] in combination with the the NOX-donors NOC18 [10 µM] and SNAP [25 µM] and the NO-inhibitors L-NAME [10 µM] and L-NMMA [500 µM] which revealed controversial results. In detail, co-treatment with the thyroid hormone T3 and the NOX-donors NOC18 and SNAP resulted in significantly (p<0.001) elevated NOX levels. Controversially, co-treatment with the NOX-inhibitors and the thyroid hormones T3 alone or TSH, T3 and T4 in combination with insulin leaded also to significantly (p<0.05) increased NOX levels. L6 cells treated with TSH and the NOX-inhibitors L-NAME and L-NMMA indicated decreased NOX levels, however not statistically significant. (Figure 2) Conclusion of insulin on these mechanisms will be investigated. Furthermore, the impact on inflammatory markers like IL-6 and the underlying molecular mechanisms, including transcription factor NF-κB will be analyzed. Methods and Results Rat L6 skeletal muscle myoblasts (ATCC: CRL-1485) and mouse 3T3-L1 adipocytes (ATCC: CL-173) were grown as monolayer and differentiated to insulin sensitive cells which were treated in the first experiment for 24 hours with thyroid hormones and insulin and in the second experiment additionally with NOX-donors and NOX-inhibitors. After 24 hours, supernatants were collected to determine NOX (Nitrate/Nitrite Fluorometric Assay, Cayman, USA), IL-6 (Legend Max, BioLegend, USA) and NF-κB (TransAM, Active Motif, Belgium). Representative for all results, the following charts show the NOX measurements in 3T3-L1 adipocytes and L6 skeletal muscle cells. Thyroid hormone treatment showed a highly significant (p<0,001) reduction of NOX in all used concentrations in 3T3 cells. Co-treatment with insulin indicated no additive effects. According to these results, 3T3-L1 adipocytes were treated in a further experiment with the highest used 12 We show here that thyroid hormones alone or in combination with insulin modulate NOX levels, expression of IL-6 and activation of NF-κB in insulin sensitive cells, in a cell type specific manner. NOX can affect the transcription factor NF-κB because of its redox activity. Effects are, at least in part reversible by co-treatment with NOX-donors or NOX-inhibitors, indicating a direct involvement of NOX in these pathophysiological mechanisms. n Maximiliane Haas, BSc. References: G. J. Tortora and B. Derrickson, Principles of Anatomy & Physiology, 13th ed. John Wiley & Sons, Inc., 2011, pp. 696 – 702. R. H. Costa-e-Sousa and A. N. Hollenberg, “Minireview: The neural regulation of the hypothalamic-pituitary-thyroid axis.,” Endocrinology , vol. 153, no. 9, pp. 4128–35, Oct. 2012. P. M. Yen, “Physiological and molecular basis of thyroid hormone action.,” Physiological reviews , vol. 81, no. 3, pp. 1097–142, Jul. 2001. S.-Y. Cheng, J. L. Leonard, and P. J. Davis, “Molecular aspects of thyroid hormone actions.,”Endocrine reviews , vol. 31, no. 2, pp. 139–70, Apr. 2010. “Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report.,” Circulation, vol. 106, no. 25, pp. 3143–421, Dec. 2002. L. Sibal, S. C. Agarwal, P. D. Home, and R. H. Boger, “The Role of Asymmetric Dimethylarginine (ADMA) in Endothelial Dysfunction and Cardiovascular Disease.,” Current cardiology reviews, vol. 6, no. 2, pp. 82–90, May 2010. Fragen zum Literaturstudium finden Sie auf S. 27 sommer 2014 biomed austria