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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
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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.
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