Thyroid hormone synthesis and secretion

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Thyroid Hormone Synthesis and Secretion
Françoise Miot, PhD, IRIBHM, Université Libre de Bruxelles (ULB), Campus Erasme, BatC,
808,route de Lennik, B-1070 Brussels, Belgium ; fmiot@ulb.ac.be;
Corinne Dupuy, PhD, Oncogenese thyroidienne, Institut Gustave Roussy, FRE2939 CNRS, 39
rue Camille Desmoulins, 94805 Villejuif, France dupuy@igr.fr
Jacques E Dumont, MD, PhD, IRIBHM, Université Libre de Bruxelles (ULB), Campus Erasme,
BatC, 808,route de Lennik, B-1070 Brussels, Belgium ; jedumont@ulb.ac.be
Bernard A. Rousset, PhD. Faculté de Médecine Lyon-Est, Site Laennec - UMR 664 Inserm,
69372 LYON Cedex 08 Fédération de Biochimie et Biologie Spécialisée, Hôpital Edouard-Herriot,
REVISED 1 July 2010
INTRODUCTION
The thyroid contains two hormones, L-thyroxine (tetraiodothyronine, T4) and L-triiodothyronine (T3) (Figure
2-1, below). Iodine is an indispensable component of the thyroid hormones, comprising 65% of T 4's
weight, and 58% of T3's. The thyroid hormones are the only iodine-containing compounds with
established physiologic significance in vertebrates.
Fig. 2-1: Structural formula of thyroid hormones and precursor compounds
The term "iodine" occasionally causes confusion because it may refer to the iodine atom itself but also to
molecular iodine (I2). In this chapter "iodine" refers to the element in general, and "molecular iodine" refers
to I2. "Iodide" refers specifically to the ion I-.
Ingested iodine is absorbed through the small intestine and transported in the plasma to the thyroid,
where it is concentrated, oxidized, and then incorporated into thyroglobulin (Tg) to form MIT and DIT and
later T4 and T3 (Figure 2-2). After a variable period of storage in thyroid follicles, Tg is subjected to
proteolysis and the released hormones are secreted into the circulation, where specific binding proteins
carry them to target tissues. This chapter discusses these broad steps as: (a) iodine availability and
absorption; (b) uptake of iodide by the thyroid; (c) oxidation of iodide, which involves the thyroperoxidase
1
(TPO), H2O2, and H2O2 generation; (d) Tg, whose iodination leads to hormone formation; (e) storage of
thyroid hormones in a Tg-bound form; (f) Tg breakdown and hormone release; (g) control of synthesis
and secretion by iodine supply and TSH; and (h) effects of drugs and other external agents on the
process.
Fig. 2-2: The iodide cycle. Ingested iodide is trapped in the thyroid, oxidized, and bound to
tyrosine to form iodotyrosines in thyroglobulin (TG); coupling of iodotyrosyl residues forms T4
and T3. Hormone secreted by the gland is transported in serum. Some T 4 is deiodinated to T3.
The hormone exerts its metabolic effect on the cell and is ultimately deiodinated; the iodide is
reused or excreted in the kidney. A second cycle goes on inside the thyroid gland, with
deiodination of iodotyrosines generating iodide, some of which is reused without leaving the
thyroid.
The production of thyroid hormones is based on the organization of thyroid epithelial cells in functional
units, the thyroid follicles. A single layer of polarized cells (Fig. 2-4A.) forms the enveloppe of a spherical
structure with an internal compartment, the follicle lumen. Thyroid hormone synthesis is dependent on the
cell polarity that conditions the targeting of specific membrane protein, either on the external side of the
follicle (facing the blood capillaries) or on the internal side (at the cell-lumen boundary) and on the
tightness of the follicle lumen that allows the gathering of substrates and the storage of products of the
reactions.Thyroid hormone secretion relies on the existence of stores of pre-synthetized hormones in the
follicle lumen and cell polarity-dependent transport and handling processes leading to the delivery of
hormones into the blood stream.
2
IODINE AVAILABILITY AND TRANSPORT
The daily iodine intake of adult humans varies from less than 10 µg in areas of extreme deficiency to
several hundred milligrams for some persons receiving medicinal iodine. Milk, meat, vitamin preparations,
medicines, radiocontrast material, and skin antiseptics are important sources (Table 2-1) (1, 2). In the
United States, the average intake in 1960 was about 100-150 µg/day, then rose to 200-500 µg/day in the
following decade. It is currently about 150 µg/day (3). The use of iodate as a bread conditioner in the
baking industry greatly increased average iodine consumption; this additive has been replaced more
recently by other conditioners that do not contain iodine. Iodophors as sterilizing agents in the milk
industry also added much iodine to the food chain, but this source may also be diminishing. In the USA
and elsewhere, most consumers are unaware of the amount of iodine they ingest. Commerce and
manufacturing technology rather than health dictate the presence of iodine in most products. The
amounts of iodine are usually unrevealed, and changes in them unannounced.
In the USA, where iodized salt use is optional, about 70% of the population consumes table salt
containing approximately 76 ppm iodine (76 mg I/kg salt). Most prepared food in the USA and Europe
uses uniodized salt (Switzerland and Macedonia are exceptions) and only about 15% of the daily salt
intake is added at the table, so iodized salt in these areas makes only a modest contribution to daily
iodine intake (4). The National Health and Nutrition Examination Surveys (NHANES) showed that the
median national urinary iodine excretion in the USA in samples collected between 1988 and 1994 was
145 µg/L, a marked decrease from the 321 µg/L in a similar survey two decades before (5, 6). Preliminary
estimates from the most recent NHANES (2001) are about 160 µg/L. The Total Diet Study of the U.S.
Food and Drug Administration reported a parallel decrease in iodine consumption between 1970 and
1990 (7) . These fluctuations in iodine intake result from changes in societal and commercial practices
that are largely unrecognized and unregulated. Canada mandates that all salt for human consumption
contain KI at 100 ppm (76 ppm as iodine). Calculations of the representative Canadian diet in 1986
estimated slightly over 1 mg iodine/person/day, of which iodized salt contributed over half (8). Urinary
iodine excretion in a group of men in Ottawa in 1990 was less than 50% of that in the Canadian national
survey of 1975 (9), suggesting a decrease in dietary intake there as well as in the USA. Some countries
have areas with very high iodine intake (10), from dietary custom (e.g., seaweeds in Japan) or from
iodine-rich soil and water (e.g., a few places in China). But until recently, many countries have had some
degree of iodine deficiency (11) in at least part of their territory. This is now being corrected by the
widespread programs of iodine prophylaxis promoted by ICCIDD (12).
Too much iodine increases the incidence of iodine-induced hyperthyroidism, autoimmune thyroid disease
and perhaps thyroid cancer. Too little causes goiter, hypothyroidism and their consequences i.e.features
of the so-called iodine deficiency disorders (5). The global push to eliminate iodine deficiency in the
current decades has put both excess and deficiency of iodine in the spotlight. Some countries have
already moved rapidly from severe iodine deficiency to iodine excess, while others are only now
recognizing iodine deficiency as a problem (5, 12). Their experience, as well as that in the USA and
Canada, emphasizes the need for continued monitoring to assess trends in iodine intake.
Medicinal sources can provide iodine in amounts much larger than those consumed in an average diet
(Table 2-1). For example, 200 mg of amiodarone contains 75 mg of iodine. Radiographic contrast
materials typically contain grams of iodine in covalent linkage, and significant amounts (milligrams) may
be liberated in the body. Skin disinfectants (e.g., povidone iodine) and iodine-based water purification
systems can greatly augment iodine intake. At the other end, some individuals with little consumption of
dairy products and of iodized salt have low iodine intakes.
3
Table 2-1. Some common sources of iodine in adults USA (1,2)
Dietary iodine Daily intake (µg)
Dairy products 52
Grains 78
Meat 31
Mixed dishes 26
Vegetables 20
Desserts 20
Eggs 10
Iodized salt 380
Other iodine sources (µg)
Vitamin/mineral prep (per tablet) 150
Amiodarone (per tablet) 75,000
Povidone iodine (per mL) 10,000
Ipodate (per capsule) 308,000
Most dietary iodine is reduced to iodide before absorption throughout the gut, principally in the small
intestine. Absorption is virtually complete. Iodinated amino acids, including T4 and T3, are transported
intact across the intestinal wall. Short-chain iodopeptides may also be absorbed without cleavage of
peptide bonds (13). Iodinated dyes used in radiography are absorbed intact, but some deiodination
occurs later. Except in the postabsorptive state, the concentration of iodide in the plasma is usually less
than 10 µg/L. Absorbed iodide has a volume of distribution numerically equal to about 38% of body weight
(in kilograms) (14), mostly extracellular, but small amounts are found in red cells and bones.
The thyroid and kidneys remove most iodide from the plasma. The renal clearance of iodide is 30-50 mL
plasma/min (14-16) and appears largely independent of the load of iodide or other anions. In certain
species, such as the rat, large chloride loads can depress iodide clearance. In humans, renal iodide
clearance depends principally on glomerular filtration, without evidence of tubular secretion or of active
transport with a transfer maximum (17). Reabsorption is partial, passive, and depressed by an extreme
osmotic diuresis. Hypothyroidism may decrease and hyperthyroidism may increase renal iodide
clearance, but the changes are not marked (14, 18).
On iodine diets of about 150 µg/day, the thyroid clears iodide from 10-25 mL of serum (average, 17 mL)
per minute (14). The total effective clearance rate in humans is thus 45-60 mL/min, corresponding to a
decrease in plasma iodide of about 12%/hr. Thyroidal iodide clearance may reach over 100 mL/min in
iodine deficiency, or as low as 3 or 4 mL/min after chronic iodine ingestion of 500-600 µg/day.
The salivary glands and the stomach also clear iodide and small but detectable amounts appear in sweat
and in expired air. Breast milk contains large amounts of iodide, mainly during the first 24 hours after
ingestion (19). Its content is directly proportional to dietary iodine. For example, in one part of the USA
with community adult iodine intake of about 300 µg daily per person, breast milk contained about 18 µg
iodine/dL, while in an area of Germany consuming 15 µg iodine per capita daily, the breast milk iodine
concentration was only 1.2 µg/dL (20). Milk is the source of virtually all the newborn's iodine, so milk
substitutes need to provide adequate amounts.
UPTAKE OF IODINE BY THE THYROID
Thyroid cells extract and concentrate iodide from plasma (21, 22). As shown in Fig. 2-3, shortly after
administration, radioiodide is taken up from the blood and accumulates within thyroid follicular cells. About
4
20% of the iodide perfusing the thyroid is removed at each passage through the gland (23). The normal
thyroid maintains a concentration of free iodide 20 to 50 times higher than that of plasma, depending on
the amount of available iodine and the activity of the gland (24). This concentration gradient may be more
than 100:1 in the hyperactive thyroid of patients with Graves' disease. The thyroid can also concentrate
other ions, including bromide, astatide, pertechnetate, rhenate, and chlorate, but not fluoride (25, 26).
Fig. 2-3: Radioautographs of rat thyroid sections. Animals
received iodide shortly before sacrifice, and radioautographs
of thyroid sections were coated with emulsion after being
stained by the usual methods. The radioautographs indicated
the presence of iodide primarily over the cells at these early
time intervals. (From Pitt-Rivers, R.J., S.F. Niven, and M.R.
Young, in Biochemistry, 90:205, 1964, with permission of the
author and publisher.)
The protein responsible for iodide transport, the so-called sodium/iodide symporter or NIS, is located at
the basolateral plasma membrane of thyrocytes (Fig. 2-4.). NIS-mediated I - accumulation is a Na+-
dependent active transport process that couples the energy released by the inward translocation of Na +
down to its electrochemical gradient to the simultaneous inward translocation of I- against its
electrochemical gradient. The maintenance of the Na+ gradient acting as the driving force is insured by
5
Na+-K+-ATPase. NIS belongs to the sodium/glucose cotransport family as the SLC5A5 member. Iodide
transport is energy-dependent and requires O2. Ouabain, digitoxin, and other cardiac glycosides block
transport in vitro (27, 28). Iodide uptake by thyroid cells is dependent on membrane ATPase. During
gland hyperplasia, iodide transport usually varies concordantly with plasma membrane Na+-K+-activated,
ouabain-sensitive ATPase activity (29).
NIS cDNA was first cloned in rat FRTL-5 cells by Dai et al. (30). The rat NIS gene gives rise to a 3kb
transcript with an open reading frame of 1,854 nucleotides encoding a polypeptide chain of 618 amino
acids. The mature protein is a glycoprotein with an apparent molecular mass of 85kDa (31, 32).It has 13
membrane spanning domains, with the carboxy terminus in the cytoplasm and the amino terminus located
outside the cells (33). In the model of Levy et al. (34), a Na + ion first binds to the transporter which, in the
presence of iodide, forms a complex that then transfers iodide and two Na+ ions to the cell interior.
The human NIS gene located on chromosome 19 (35) codes for a protein of 643 amino acids that is 84%
homologous with rat NIS (36). The mouse NIS polypeptide chain (37) has the same size (618 amino
acids) as the rat NIS. At variance with other species, three different transcripts are generated from the
porcine NIS gene by alternative splicing (38); the main form encodes a polypeptide of 643 amino acids as
human NIS.
Functional studies clearly show that NIS is responsible for most of the events previously described for
iodide concentration by the thyroid. TSH stimulates NIS expression (39, 40) and iodide transport (31, 32).
TSH exerts its regulatory action at the level of transcription through a thyroid-specific far-upstream
enhancer denominated NUE (NIS Upstream Enhancer) that contains binding sites for the transcription
factor Pax8 and a cAMP response element-like sequence. This original demonstration made on the rat
NIS gene (41) has now been extended to human (42) and mouse (43) NIS genes. It has been suggested
that TSH could also regulate NIS expression at post-transcriptional level (44). This remains to be
confirmed. Data from TSH receptor-null mice (44-46) clearly show that TSH is required for expression of
NIS. Moderate doses of iodide in the TSH-stimulated dog thyroid inhibit expression of the mRNAs for NIS
and TPO, while not affecting that for Tg and TSH receptor (40) . The decrease in thyroid iodide transport
resulting from excess iodide administration (escape from the Wolff-Chaikoff effect, see further) is related
to a decrease in NIS expression (40, 47). Both NIS mRNA and NIS protein are suppressed by TGFb,
which also inhibits iodide uptake (48, 49). Reviews focus on NIS and its functional importance {Dohan,
2003 50 /id;Riedel, 2001 56 /id}.
Several mutations in the NIS gene causing defective iodide transport have been reported in humans (52-
59). The most commonly found mutation corresponds to a single base alteration T354P in the ninth
putative transmembrane domain of NIS (55). Site directed mutagenesis of rat NIS cDNA to substitute for
threonine at residue 354 and transfection into COS cells lead to loss of iodide transport activity (60).
Other mutations lead to truncated NIS (58) or to alterations of membrane targeting of the NIS protein
(59) . NIS expression is increased in Grave’s disease and hyperactive nodules (61-63) and decreased in
adenomas and carcinomas (64, 65) appearing as cold nodules at scintigraphy.In hypofuctioning benign or
malignant tumors, the impairment of iodide transport would result from both transcriptional and post-
transcriptional alterations of NIS expression (66).Other tissues that concentrate iodide also show NIS
expression, including salivary glands (67) and mammary glands (68, 69).
Iodide supply of follicular lumen involves a two-step transport process: the active transport across the
basolateral plasma membrane of thyrocytes by NIS and a passive transport across the apical plasma
membrane. The protein(s) insuring the second step is (are) not yet identified. A potential iodide
transporters has been proposed: pendrin (70, 71). Pendrin, encoded by the PDS gene (72) and
composed of 780 amino acids, is expressed in different organs including kidney, inner ear and thyroid. In
the thyroid, pendrin is a 110kDA membrane glycoprotein (73), selectively located at the apical plasma
membrane (74).Its activity as transporter of anions including iodide has been demonstrated in different
experimental systems (71, 75-77). Pendrin belongs to the SLC family under the reference SLC26A4.
However, the implication of pendrin in thyroid iodide transport remains uncertain for several reasons.
First, there is still no direct demonstration of a pendrin-mediated efflux of iodide from thyrocytes to the
6
follicular lumen. Second, the genetic alterations of the PDS gene found in patients with the Pendred
syndrome, which lead to a loss of the anion transport activity of pendrin and to a constant and severe
hearing loss, only have a moderate impact on the thyroid functioning, generally a euthyroid goiter (78).
Third, PDS knock-out mice (79) do not show any thyroid dysfunction. In summary, contrary to NIS for
which the anion selectivity (25) corresponds to what was expected, the ion selectivity of thyroid pendrin
remains to be elucidated. In the thyroid as in the kidney, pendrin could act primarily as a
chloride/bicarbonate anion exchanger.
Fig. 2-4: NIS-mediated transport of iodide. A, immunolocalization of the human NIS protein at the
basolateral plasma membrane of thyrocytes in their typical follicle organization. B, schematic
representation of the membrane topology of the NIS polypeptide chain deduced from secondary
structure prediction analyses (33). C, transport of iodide from the extracellular fluid (or plasma) to
the thyroid follicle lumen. The uptake of iodide at the basolateral plasma membrane of thyrocytes
must be active; it operates against an electrical gradient (0 - 50 mV) and a concentration gradient,
[ I- ]c being higher than extracellular [ I- ]. The transport of iodide from the cytoplasm to the follicle
lumen should be a passive process, the electrical and concentration gradients being favorable.
7
Iodide that enters the thyroid remains in the free state only briefly before it is further metabolized and
bound to tyrosyl residues in Tg. A significant proportion of intrathyroidal iodide is free for about 10-20
minutes after administration of a radioactive tracer (80), but in the steady state, iodide contributes less
than 1% of the thyroid total iodine. A major fraction of the intrathyroidal free iodide pool comes from
deiodination of MIT and DIT; this iodide is either recycled within the thyroid or leaked into the circulation.
Some data suggest that iodide entering the gland by active transport segregates from that generated by
deiodination of Tg within the gland (81, 82). Once in the thyroid, iodide is organically bound at a rate of 50
to 100% of the pool each minute (24, 83). The proportion of an iodide load that is bound varies little,
despite wide shifts in daily intake. In contrast, NIS activity is sensitive to both iodine availability and TSH
stimulation, and transport rather than intrathyroidal binding is the controlling factor in making iodide
available for hormonogenesis.
IODINE IN OTHER TISSUES
The thyroid is not the only organ to concentrate iodine; the others endowed with this capacity are salivary
glands, gastric mucosa, mammary glands, and choroid plexus. Ductal cells of the salivary glands express
NIS (67) . The plasma membrane of the mammary gland epithelium contains a NIS protein with a
molecular mass different from that of thyroid NIS (~75 kDa vs ~90 kDa). In the mammary gland, NIS is
processed differently after translation and subjected to regulation by lactogenic stimuli (68).It has been
reported that over 80% of human breast cancer samples expressed this symporter.As it is absent in
normal non-lactating tissue, NIS may represent a marker for breast malignancy and even a possible
target for radioiodine therapy (69). The thyroid, salivary glands, and gastric mucosa share a common
embryologic derivation from the primitive alimentary tract and, in each of these tissues; iodide transport is
inhibited by thiocyanate, perchlorate, and cardiac glycosides. TSH stimulates transport only in the thyroid.
An active transport for iodide in the gastric mucosa has an obvious value because it provides iodine to the
circulation for use in the thyroid. Active concentration by the breast helps transfer iodide to milk. Iodide
concentration by the choroid plexus and salivary glands does not have any obvious physiologic benefit,
but needs to be remembered for possible insights into pathways as yet undiscovered.
Iodine, particularly in the form of I2, may enter additional metabolic pathways outside the thyroid. Rats
administered I2 orally showed much less circulating free iodide and much more iodine bound to proteins
and lipids than did animals given iodide (84). In another comparison of I 2 versus iodide, administration of
iodide to iodine-deficient rats eliminated thyroid hyperplasia much more efficiently than did I2. Additionally,
I2 decreased lobular hyperplasia and periductal fibrosis in the mammary glands, while iodide increased
the former and had no effect on the latter (85).
THYROPEROXIDASE (TPO)
After concentrating iodide, the thyroid rapidly oxidizes it and binds it to tyrosyl residues in Tg, followed by
coupling of iodotyrosines to form T4 and T3. The process requires the presence of iodide, a peroxidase
(TPO), a supply of H2O2, and an iodine acceptor protein (Tg).
Thyroperoxidase oxidizes iodide in the presence of H2O2. In crude thyroid homogenates, enzyme activity
is associated to cell membranes. It can be solubilized using detergents such as deoxycholate or digitonin.
The enzyme activity is dependent on the association with a heme, the ferriprotoporphyrin IX or a closely
related porphyrin (86, 87). Chemical removal of the prosthetic group inactivates the enzyme, and
recombination with the heme protein restores activity (88). The apoprotein from human thyroid is not
always fully saturated with its prosthetic group (89). Some congenitally goitrous children have poor
peroxidase function because the apoprotein has weak binding for the heme group (89).
Antibodies directed against the thyroid "microsomal antigen," which are present in the serum of patients
with autoimmune thyroid disease (AITD), led to identification of TPO. These antibodies were found to
react with proteins of 101-107 kDa and to immunoprecipitate thyroid peroxidase (TPO), thus identifying
microsomal antigen as TPO (90-94). A monoclonal antibody to purified microsomal antigen or antibodies
8
directed againt thyroperoxidase were then used to clone human TPO (95-97). Different laboratories then
cloned TPO from various species: pig (98), rat (99), and mouse (100). Kimura et al. (95) cloned two
different cDNAs of humanTPO.TPO1 coded for a protein of 933 residues and TPO2 was identical to
TPO1 except that it lacked exon 10 and was composed of 876 residues. Both forms occur in normal and
abnormal human thyroid tissue. The C-terminal portion of the proteins exhibits a hydrophobic segment
(residues 847-871), likely corresponding to a transmembrane domain; thus, TPO has a short intracellular
domain and most of the polypeptide chain is extracellular (Fig. 2-5A.). TPO1 is active, but TPO2 appears
enzymatically inactive because it does not bind heme, degrades rapidly, and fails to reach the cell surface
in transfected cell lines (101). Different degradative pathways exist for the two forms (102). Several other
TPO variants resulting from exon skipping have been identified; they appear either active or inactive
(103). Pig TPO contains 926 amino acids (98) ; mannose-rich oligosaccharide units occupy four of its five
glycosylation sites (104).
Human TPO, which has 46% nucleotide and 44% amino acid sequence homology with human
myeloperoxidase, clearly belongs to the same protein family. The TPO gene resides on chromosome
2p13, spans over 150 kbp, and has 17 exons (105). Several types of mutations in the TPO gene cause
diminished iodide organification (see Chapter 16). As NIS, Tg, and the TSH receptor (TSHr), TPO
expression is controlled by the TSH cAMP pathway (106) through thyroid-specific transcription factors.
These include TTF-1, TTF-2, and Pax-8 (107, 108). Tg and TPO genes have the same binding sites for
TTF-1, TTF-2, and Pax-8 in their promoters, and the genes for both have TTF-1 sites in enhancer
regions.
TPO synthesized on polysomes is inserted in the membrane of the endoplasmic reticulum and undergoes
core glycosylation. TPO is then transported to the Golgi where it is subjected to terminal glycosylation and
packaged into transport vesicles along with Tg (109) (Fig. 2-6). These vesicles fuse with the apical
plasma membrane in a process stimulated by TSH. TPO delivered at the apical pole of thyrocytes
exposes its catalytic site with the attached heme in the thyroid follicular lumen (110). TPO activity is
restricted to the apical membrane, but most of the thyroid TPO is intracellular, being located in the
perinuclear part of the endoplasmic reticulum (111, 112). Most of this intracellular protein is incompletely
or improperly folded; it contains only high mannose-type carbohydrate units, while the membrane TPO
has complex carbohydrate units. Glycosylation is essential for enzymatic activity (112). Chronic TSH
stimulation increases the amount of TPO and its targetting at the apical membrane (113).
9
Fig. 2-5: Schematic representation of the membrane topology of Thyroperoxidase, TPO (A) and
NADPH thyroid oxidase, ThOX (Duox) (B) at the apical plasma membrane of thyrocytes. C,
hypothetical reaction scheme for TPO. H2O2 is presumed to oxidize the free enzyme with a loss
of two electrons leading to the formation of complex I. Iodide binds to complex I, is oxidized and
form complex II, which then reacts with a tyrosyl residue of Tg, Tyr-Tg.The newly-formed I 0 and
Tyr0-Tg free radicals interact to form MIT-Tg and the enzyme returns to its free state. I2 may be
generated from two cxidized iodine atoms
H2O2 GENERATING SYSTEM
By definition, a peroxidase requires H2O2 for its oxidative function. A large body of older work (reviewed in
(114) investigated possible sources using various in vitro models (114-116). It was already suggested in
1971 that H2O2 would be produced at the apical plasma membrane of the thyrocyte by an enzyme that
requires calcium and NADPH originating from the stimulation of the pentose phosphate pathway (117).
Further biochemical studies showed that the enzymatic complex producing H2O2 for TPO is a membrane-
bound NADPH dependent flavoprotein (118-122). H2O2 produced by this NADPH-dependent protein is the
limiting step of protein iodination and therefore of thyroid hormone synthesis when iodide supply is
sufficient (123-125). In human thyroid, the H2O2 production and iodination process are stimulated by the
calcium-phosphatidylinositol pathway (125). The quantity of H2O2 produced is important especially in
10
stimulated thyrocytes; it is comparable to the ROS production of activated leukocytes. While the activated
leukocyte lives a few hours, the life of an adult thyrocyte is 7 yr (126, 127). Thus thyroid cells may be
exposed to high doses of H2O2 and have to adapt to it by developing highly regulated generator and
efficient protective systems.
More than twenty years passed between the initial biochemical studies and the cloning of Duox as the
catalytic enzymatic core of the H2O2 thyroid generating system. By two independent molecular strategies
Duox enzymes were uncovered from the thyroid. Starting from a purified fraction of pig thyroid membrane
bound NADPH flavoprotein, the team of C. Dupuy isolated p138 Tox which turned out to be Duox2 lacking
the first 338 residues(128). Simultaneously, De Deken et al cloned two cDNAs encoding NADPH
oxidases using the strategy based on the functional similarities between H2O2 generation in the
leukocytes and the thyroid according to the hypothesis that one of the components of the thyroid system
would belong to the known gp91phox gene family and display sequence similarities with gp91phox, now
called NOX2. Screenings of two cDNA libraries at low stringency with a NOX2 probe enabled the
isolation of two sequences coding for two NADPH oxidases of 1248 and 1548 amino acids respectively
initially named Thox1 and Thox2 (129). The encoded polypeptides display 83% sequence similarity and
are clearly related to gp91phox (53 and 47% similarity over 569 amino acids of the C-terminal end) . The
whole protein is composed of : a N-terminus ecto-sequence of 500 amino acids showing a similarity of
43% with thyroperoxidase (hence named Dual oxidase-Duox in the present terminology); a first
transmembrane segment preceding a large cytosolic domain which contains two calcium binding EF-hand
motifs; the C-terminal portion componed of six transmembrane segments, harbouring the four His and two
Arg characteristic of the Nox family protein heme binding site and the conserved FAD- and NADPH-
binding sites at the extreme C-terminal cytolic portion (Fig. 2-5B.). Duox proteins are localized at the
apical plasma membrane of the thyrocyte as fully glycosylated forms (~190kDa) and in the endoplasmic
reticulum as high mannose glycosylated forms (~180kDa) (Fig. 2-6C).
Duox1 and Duox2 genes are co-localized on chromosome 15q15.3, have opposite transcriptional
orientations and are separated by a ~16kb region (Fig. 2-6A). Duox1 gene is more telomeric, spans 36 kb
and is composed of 35 exons; two first of them are non-coding. Duox2 spans 21.5 kb and is composed of
34 exons; the first being non-coding (130).
In addition to thyroid, Duox expression is reported in several tissues: Duox1 is expressed in lung epithelia,
in oocytes (131-133) and Duox2 in gastrointestinal mucosa and salivary glands (134, 135). Multiple
functions are attributed to Duox enzymes: airway fuid acidification (136), mucin secretion (137), wound
healing {Boots, 2009 3226 /id;Luxen, 2009 3225 /id} and innate hoste defense (140-142).
Most of the time Duox activity is associated to a peculiar peroxidase activity like in oocyte with the
ovoperoxidase involved in the fertilization process or with the lactoperoxidase in lung epithelia or in the
gut (141-144) . Beside these killing mechanisms, Duox and H2O2 are certainly also involved in the
interaction between host mucosa and bacteria to maintain mucosal homeostasis e.g. in bronchi and
intestine (140, 145). In the thyroid, the specificity of the thyroid hormone machinery using Duox lays on
TPO. Thus colocalization of Duox and TPO and their probable association at the apex of the thyrocyte
would increase the efficiency of H2O2 producer-consumer system (146, 147).
Onset of Duox expression study in thyroid embryonic development pointed Duox as a thyroid
differentiation marker. The proteins involved in the synthesis of thyroid hormones are expressed just after
the thyroid precursor cells have completed their migration from the primitive pharynx and reached their
final location around the trachea (148, 149). This final morphological maturation begins in mouse with the
expression of Tg at embryonic day 14 followed one day later (E15) by the expression of TPO, NIS, TSH
receptor and Duox concomitant with the apparition of iodinared Tg (46, 150).
Until 2006, the major obstacle for molecular studies of Duox was the lack of a suitable heterologous cell
system for Duox correctly expressed at the plasma membrane in its active state. Several cell lines
transfected with Duox1 and/or Duox2 showed Duox expression completely retained in the endoplasmic
reticulum in their immature form without displaying any production of H2O2 (151). HEK293 cells
transfected with Duox2 generate rather small quantities of superoxide anions in a calcium-depnedent
manner (134). The reconstitution of a Duox-based functional H2O2 generating system requires a
maturation factor called DuoxA. The two human DuoxA paralogs were initially identified as thyroid specific
expressed genes by in silico screenings of multiple parallel signature sequencing data bases (152). The
11
two genes are located on chromosome 15 in the Duox1/Duox2 intergenic region in a tail to tail orientation,
DuoxA1 facing Duox1 and DuoxA2 facing Duox2 (Fig. 2-6A.). DuoxA2 ORF spans 6 exons and encodes
a 320 amino acid protein predicted to compose five transmembrane segments, a large external loop
presenting N-glycosylation sites between second and third transmembrane helices and a C-terminal
cytoplamic region (Fig. 2-6B). DuoxA1 gene was initially annotated “homolog of Drosophila Numb-
interacting protein: NIP”. Four alternatively spliced DuoxA1 variants have been identified (153). One of the
most expressed transcript, DuoxA1α, is the closest homolog of DuoxA2 and encodes a 343 amino acid
protein (58% identity of sequence with DuoxA2) adopting the same predicted structure.
In heterologous system DuoxA proteins in the absence of Duox are mainly retained in the endoplasmic
reticulum. When co-transfected with Duox they cotransported with Duox to the plasma membrane where
they probably form complexes. Only the Duox1/DuoxA1 and Duox2/DuoxA2 pairs produce the highest
levels of H2O2 as they undergo the glycosylation steps through the Golgi. Duox2/DuoxA1 pair does not
produce H2O2 but rather superoxide anions and Duox1/DuoxA2 is unable to produce any ROS. This
means that the Duox activators promote Duox maturation but also are parts of the H 2O2 generating
complex (154, 155).
The reconstitution of this functional H2O2 producing system has been useful to measure and compare the
intrinsic enzymatic activities of Duox1 and Duox2 in relationship with their expression at the plasma
membrane under stimulation of the major signalling pathways active in the thyroid. It has been shown that
the basal activity of both isoenzymes is totally depending on calcium and functional EF-hands calcium
binding motifs. However, the two oxidase enzymatic activities are differently regulated after activation of
the two main signalling cascades in the thyroid. Duox1 but not Duox2 activity is stimulated by the cAMP
dependent cascade triggered by forskolin (EC50=0.1µM) via protein kinase A-mediated phosphorylation
on serine 955 of Duox1. In contrast, phorbol esters, at low concentrations, induce Duox2 phosphorylation
via protein kinase C activation associated with high H2O2 generation (EC50= 0.8nM) (156). These results
suggest that both Duox proteins could be involved in thyroid hormone synthesis by feeding H 2O2 to TPO
to oxidize iodide and couple iodotyrosines.
Defects in Duox and/or DuoxA were rapidly recognized possible causes of congenital hypothyroidism
(CH) due to thyroid dyshormonogenesis in patients born with a hyperplastic thyroid or developing a goiter
postnatally when T4 treatment is delayed after birth.
The first screening of mutations in Duox genes in 2002 was performed on 9 patients who had idiopathic
congenital hypothyroidism with positive ClO4- discharge (>10%), one with permanent and 8 with transient
hypothyroidism (157). They were identified in the Netherlands by neonatal screening and followed up to
determine the evolution of CH with the time. One of the patients with total organification defect (TIOD)
presented a permanent hypothyroidism and the 8 others presented a transient hypothyroidism with a
partial organification defect (PIOD). Of these last 8 patients 3 harboured heterozygous nonsense or
frameshift mutations (Q686X, R701X, S965fsX994) meaning that a single defective Duox2 allele can
cause haploinsufficency resulting in mild transient CH. It is noteworthy that this hypothyroid status was
limited to the neonatal period, when thyroid hormone requirement is the highest, and was not detectable
in adulthood since adult heterozygotes in theses families presented normal TSH serum levels. The only
case with severe permanent CH was homozygous for a nonsense mutation (R434X= protein devoid of
the catalytic core) leading to the conclusion at this time of a complete inability to synthesize thyroid
hormone in absence of Duox2. No mutation was detected in Duox1.
With the increasing number of reported Duox2 mutations in CH, it becomes more and more difficult to
make the correlation between genotype and phenotype as initially described.
Indeed, subsequent studies have shown a link between biallelic Duox2 defects and PIOD. Patients with
compound heterozygous missense (R376W) and a nonsense mutation (R842X), leading to a presumed
non functional protein showed PIOD with mild and persistent hyperthyrotropinemia. This suggests that
Duox1 can compensate at least partially for the defect in Duox2 (158). Varela et al. described also two
cases of permanent CH with compound heterozygous missense and nonsense or splicing mutations
(Q36H and S965fsX994; G418fsX482 and g.IVS19-2A>C conducting to inactive proteins) responsible for
congenital goiter with a PIOD (159).
12
The phenotype-genotype correlation suggested by the work of Moreno et al is further not respected
anymore in two different studies. Maruo et al described a series of transient CH characterized by biallelic
defects in Duox2: in one family, four siblings were compound heterozygous for early frameshift mutations
(L479SfsX2 and K628RfsX10) resulting in a presumed complete loss of Duox2 activity (not tested at this
time). Three of them had low free T4 at birth, mild thyroid enlargement. The thyroid hormone replacement
therapy ceased to be necessary by 9yr of age (160). A French-Canadian patient with a transient CH
initially detected by neonatal screening presented a compound heterozygozity for a hemizygous missense
mutation (G1518S) inherited from the father and a deletion removing the part of the gene coding for the
catalytic core of Duox2 inherited from the mother. In vitro test proved that the missense mutant protein
was totally inactive (161). This case provides further evidence that permanent or transient nature of CH is
not directly related to the number of inactivated Duox2 alleles.
The first homozygous nonsense mutation in DuoxA2 (Y246X) that resulted in a non-functional protein
tested in vitro has been found to be responsible of a permanent mild CH in a Chinese patient with a
dyshormonogenic goiter (155). The mild phenotype can be explained by a partial maintenance of H2O2
production by Duox2/DuoxA1 as demonstrated in vitro.
The variety of observerd phenotypes associated with Duox2 and now DuoxA2 mutations suggest that the
manifestation of Duox2 defects could likely be influenced by the environmental factors like iodine intake or
by the activation of Duox1 or DuoxA1 in peculiar circumstances.
A
B
C
Duox2 DuoxA2 DuoxA1 Duox1
21.5kb 4kb 12kb 36kb
Chromosome: 15q15.3
Fig 2-6: A, Localization of Duox and DuoxA genes on chromosomes 15q15.3. B, Schematic
representation of the predicted structure of DuoxA (from (152). C, Immmunolocalization of human
Duox and TPO at the apical membrane of the thyrocyte (upper: Duox immunostaining,
middle:preimmune serum, lower:TPO immunostaining) (129).
13
THYROGLOBULIN (Tg)
Thyroglobulin is the most abundant protein in the thyroid gland; its concentration within the follicular
lumen can reach 200-300 g/L. Its main function is to provide the polypeptide backbone for synthesis and
storage of thyroid hormones (162). It also offers a convenient depot for iodine storage and retrieval when
external iodine availability is scarce or uneven. Neosynthesised Tg polypeptide chains entering the lumen
of the rough endoplasmic reticulum (RER) are subjected to core glycosylation, dimerise and are
transferred to the Golgi where they undergo terminal glycosylation (Fig. 2-7.). Iodination and hormone
formation of Tg occur at the apical plasma membrane-lumen boundary and the mature hormone-
containing molecules are stored in the follicular lumen, where they make up the bulk of the thyroid follicle
colloid content.
Fig. 2-7: A polarized thyroid epithelial cell synthesizing soluble proteins, Tg (▲) and lysosomal
enzymes (X) and membrane proteins, NIS (┴) and TPO (°). The polypeptide chain(s) generated by RER
membrane-bound polysomes, enter the lumen of RER for the former and remain inserted into the RER
membrane for the latter. Inside the lumen of RER, newly-synthesized proteins undergo core
14
glycosylation and by interacting with chaperones acquire their conformation. Proteins are then
transported to the Golgi apparatus (G), where terminal glycosylation and other post-translational
reactions take place. In the Trans-Golgi network (TGN), mature proteins undergo sorting processes
and are packed into transport vesicles. The vesicles carrying soluble proteins (inside the vesicle) and
membrane proteins (as integral vesicle membrane protein) deliver them at the appropriate plasma
membrane domain: the apical domain (1) and (2) or the basolateral domain (4). Vesicles carrying
lysosomal enzymes (3) conveyed their content to prelysosomes or late endosomes (LE) and
lysosomes (L). Apical plasma membrane proteins may reach their final destination by an alternative
route involving a transient transfer to and then a retrieval and transport (*) from the basolateral
membrane domain to the apical domain.
The Tg peptide chain derives from a gene of more than 200 kbp located on chromosome 8 in humans.
The human Tg gene consisting of 48 exons (163) gives rise to a 8.5kb transcript that translates a 2,749
residue peptide (in addition to a 19-residue signal peptide) (164, 165). The primary structure deduced
from cDNA is also known for bovine, rat, and mouse (166-168). The biochemical traits of human Tg have
been reviewed in (169). The N-terminal part of Tg has regions of highly conserved internal homology (10
motives of about 60 amino acids) which appears in several other proteins and are referred to as
‘thyroglobulin type-1 domains’. Such domains have been found to be potent inhibitors of cysteine
proteases (170). This finding might be of importance, because these proteases are active in Tg
proteolysis (see below). It has been suggested that this region of the Tg molecule may modulate its own
degradation and hormone release (171). In the Tg-type 1 repeats, cysteine and proline residues are found
in constant position; they may have an important role in the tridimensional structure of the protein. The
proximal region of the C-terminal half portion of Tg contains five repeats of another type of cysteine-rich
motives. The presence of a high number of cysteine residues in Tg, involved for most of them in disulfide
bonds, probably gives rise to peculiar structural constraints. The C-terminal portion of Tg is homologous
with acetylcholinesterases (172). Because binding to cell membranes is one feature of
acetylcholinesterases, perhaps Tg C-terminus has a similar role. It was very recently reported that the
acetylcholinesterase-homology region of Tg could function as a dimerization domain (173).Furthermore,
three highly conserved thioredoxin boxes have been identified in mammalian Tg between residues 1,440
and 1,474; these boxes might be involved in disulfide bond formation leading to intermolecular cross-
linking of Tg molecules inside the follicle lumen (174) . Tg gene expression is controlled by the same
thyroid-specific transcription factors that regulate synthesis of TPO (107). The three most important are
TTF-1, TTF-2, and Pax-8, and they bind at the same sites in Tg as they do in TPO. Hydrogen peroxide
might be a regulatory factor of Tg expression, based on experimental work showing increased Tg
promoter activity with reduced Pax-8 and TTF-1 (175-178). If substantiated, this proposal offers another
point of integration between H2O2 generation and transcription of NIS, Tg and TPO genes, all of which
being regulated by TSH.
Maturation of the Tg polypeptide chain begins while still on the RER. It undergoes core glycosylation and
then monomers fold into stable dimers. Arvan and co-workers (179-184) have mapped this process and
emphasize the role of molecular chaperones. The latter are essential for folding the new Tg molecules,
and those that are folded improperly are not allowed to proceed further. The principal molecular
chaperones are BiP, GRP 94, ERP 72, and calnexin. Only Tg molecules that pass this quality control
system unscathed can proceed towards the secretory pathway. Glycosylation is a key event in Tg
maturation. Carbohydrates comprise about 10% of Tg weight (185). Human Tg may contain four different
types of carbohydrate units. The "polymannose" units consist only of mannose and N-acetylglucosamine.
The "complex unit" has a core of three mannose residues with several chains of N-acetylglucosamine,
galactose, and fucose or sialic acid extending from them. Both these types of unit are common in
glycoproteins and are linked to peptide through an asparagine-N-acetylglucosamine bond. About three
quarters of the potential N-glycosylation sites in human Tg are occupied, mostly with the complex unit
(186). Two additional units have been found in human Tg; one contains galactosamine and is linked to
the hydroxyl group of serine, the other is a chondroitin sulfate unit containing galactosamine and
glucuronic acid (187) .
15
Failure in Tg folding can lead to disease as in the cog/cog mouse; these animals have a large thyroid with
a distended ER and sparse Tg storage in follicles (188). Their Tg shows abnormal folding and decreased
export from the ER in association with increased levels of several molecular chaperones. In the Tg cDNA
of cog/cog mouse, Kim et al.(167) identified a single base substitution that changes leucine to proline at
position 2,263. Correction of this defect by site-directed mutagenesis returned Tg export to normal in
transfected cells. The cog/cog mouse is an example of endoplasmic reticulum storage disease (189).
Other examples are cystic fibrosis, osteogenesis imperfecta, familial neurohypophyseal diabetes
insipidus, insulin receptor defect, growth hormone receptor defect, and a variety of lipid disorders (190). In
each situation, the underlying defect appears to be a mutation in the coding sequence of exportable
proteins. The ER retains the abnormal proteins, which cannot then proceed for further maturation. Several
reports describe a similar pathogenesis for cases of congenital goiter and hypothyroidism in humans,
although these are not as well characterized. Ohyama et al. (191) investigated a five-year-old euthyroid
goitrous boy with high thyroidal radioiodine uptake, a positive perchlorate discharge test, apparently
normal H2O2 generation and peroxidase activity in gland tissue, and low amounts of Tg in thyroid tissue
overall, but large amounts in the RER. In another report, two hypothyroid goitrous sibs had a 138 bp
segment missing between positions 5,590-5,727 in hTg mRNA, translating into a Tg polypeptide chain
that lacked 46 residues (192). A third example described four subjects with congenital hypothyroid goiter
from two unrelated families (193). Their thyroid tissue showed accumulation of Tg intracellularly with
distension of the ER and large increases in activity of specific molecular chaperones, but with failure of Tg
to reach the Golgi or the follicular lumen; this case was put forward as an ER storage disease similar to
the cog/cog mouse (193).
Tg also contains sulphur and phosphorus. The former is present in the chondroitin sulfate and the
complex carbohydrate units, although its form and role are not known (194). Several studies have
reported phosphate in Tg, up to 12 mol. per mol Tg. Of this, about half is in the complex
carbohydrate units, the remainder is present as phosphoserine and phosphotyrosine (195-197).
This may relate to protein kinase A activity (198).
THYROGLOBULIN IODINATION AND HORMONE SYNTHESIS
The step preliminary to thyroid hormone formation is the attachment of iodine to tyrosyl residues in Tg to
produce MIT and DIT. This process occurs at the apical plasma membrane-follicle lumen boundary and
involves H2O2, iodide, TPO, and glycosylated Tg. All rendezvous at the apical membrane to achieve Tg
iodination (Fig. 2-8.).
16
Fig. 2-8: Iodination of Tg at the apical plasma membrane-follicle lumen boundary.The scheme
does not account for the relative size of the intervening molecules
First, iodide must be oxidized to an iodinating form. An extensive literature has sought to identify the
iodinating species, but the issue is still not resolved (see (199) for a detailed review). One scheme
proposes that oxidation produces free radicals of iodine and tyrosine, while both are bound to TPO to
form MIT which then separates from the enzyme (Fig.2-5C). Further reaction between free radicals of
iodine and MIT gives DIT. Experimental studies by Taurog (199) and others suggest that the TPO
reduction occurs directly in a two electron reaction. A second proposal, based on studies of rapid spectral
absorption changes (87, 200, 201), is that TPO-I+ is the iodination intermediate and that the preferred
route is oxidation of TPO by H2O2 followed by two electron oxidation of I- to I+, which then reacts within a
tyrosine. As a third possibility, Taurog (199) proposed a reaction between oxidized TPO and I - to produce
hypoiodite (OI-), which also involves a two electron reaction. Whatever the precise nature of the iodinating
species, it is clear that iodide is oxidized by H2O2 and TPO, and transferred to the tyrosyl groups of Tg. All
tyrosine residues of Tg are not equally accessible to iodination. The molecule has about 132 tyrosyl
residues among its two identical chains; at most, only about 1/3 of the tyrosyls are iodinated. As isolated
from the thyroid, Tg rarely contains more than 1% iodine or about 52 iodine atoms.
The final step in hormone synthesis is the coupling of two neighbouring iodotyrosyl residues to form
iodothyronine (Fig. 2-9). Two DIT form T4; one DIT and one MIT form T3. Coupling takes place while both
acceptor and donor iodotyrosyl are in peptide linkage within the Tg molecule.The reaction is catalyzed by
TPO, requires H2O2 (202-205) and is stringently dependent on Tg structure (206).The generation of the
iodothyronine residue involves the formation of an ether bond between the iodophenol part of a donor
tyrosyl and the hydroxyl group of the acceptor tyrosyl (Fig 2-10). After the cleavage reaction that gives the
iodophenol, the alanine side chain of the donor tyrosyl remains in the Tg polypeptide chain as
dehydroalanine (207-209). Observations both in vivo and in vitro show an appreciable delay in coupling
after initial formation of iodotyrosines. A typical distribution for a Tg containing 0.5% iodine (a normal
amount for iodine-sufficient individuals) is 5 residues MIT, 5 of DIT, 2.5 of T4 and 0.7 of T3 (162). More
iodine increases the ratios of DIT/MIT and T4/T3, while iodine deficiency decreases them.
17
Fig. 2-9: Synthesis of hormone residues (coupling of iodotyrosines) in Tg at the apical plasma
membrane-follicle lumen boundary. The scheme does not account for the relative size of the
intervening molecules
Fig. 2-10: Possible coupling reaction sequence. Oxidation of iodotyrosines may produce
iodotyrosyl radicals. The free radicals could combine to generate the iodothyronine residue (at the
tyrosine acceptor site) and a dehydroalanine residue (at the tyrosine donor site), which in the
presence of H2O converts into a serine
The distribution of hormone among several sites in the Tg molecule has been studied in a number of
species (162, 210-213). The most important is at tyrosyl 5, quite close to Tg N-terminus. It usually
contains about 40% of Tg total T4. The second most important site is at tyrosyl 2554, which may contain
for 20-25% of total T4. A third important site is at tyrosyl 2747, which appears favored for T3 synthesis in
some species. Tyrosyl 1291 is prominent in T4 formation in guinea pigs and rabbits and very responsive
18
to TSH stimulation. Incremental iodination of low iodine hTg in vitro, with lactoperoxidase as surrogate for
TPO, led to the identification of the favored sites for iodination (214). Small increments of iodine go first to
tyrosyl residues 2554, 130, 685, 847, 1448, and 5, in that order. Further addition increases the degree of
iodination at these sites, iodinates some new tyrosyls, and results in thyroid hormone formation at
residues 5, 2554, 2747, and 685, with a trace found at 1291, in that quantitative order. These data
identified the most important hormonogenic sites in hTg, and also the favored sites for early iodination.
The same work recognized three consensus sequences associated with iodination and hormone
formation: i) Asp/Glu-Tyr at three of the four most important sites for hormone synthesis, ii) Ser/Thr-Tyr-
Ser associated with hormone formation, including the C-terminal hormonogenic site that favors T3 in
some species and iii)Glu-X-Tyr favoring early iodination, although usually not hormone formation.(Fig. 2-
11)
Fig. 2-11: Diagram of the human Tg polypeptide chain; residue numbers refer to the human cDNA
sequence; (a) sites forming T4 (sites A,B,D) (solid circles) and/or T3 (site C) (solid square); (b)
early iodinated sites (solid triangles); (c) other iodinated sites (open triangles).
Identifying the donor tyrosyls has attracted considerable investigational interest over the past several
decades. The fact that some tyrosyls are iodinated early but do not go on to provide the acceptor ring of
T4 makes them potential donor candidates (214). On the basis of in vitro iodination of an N-terminal
cyanogen bromide Tg peptide, Marriq et al. (215) concluded that residue 130 was a donor tyrosine for the
major hormonogenic site at Tyr5. This conclusion was challenged by Xiao et al. (216) in a similar in vitro
system. A baculovirus system expressing the 1-198 fragment of Tg, either normal or mutated on tyrosyl
residues, showed that iodination of a fragment containing tyrosyls only at residue 5, 107 and 130 formed
T4 as did the intact normal peptide, but this fragment could also form T4 with substitutions at residue 5 or
130 (217). Dunn et al.(218) who incorporated 14C-Tyr into beef thyroid slices followed by in vitro
iodination and trypsin digestion of the N-terminal portion of Tg localized pyruvate (as a derivative of
dehydroalanine) to residue 130 by mass spectrometry. They proposed that Tyr130 was the donor tyrosine
for the most important hormonogenic site at Tyr5. Gentile et al. (219) used mass spectrometry to identify
a peptide containing dehydroalanine at tyrosine 1375 of bTg and proposed this tyrosine as the donor for
the hormonogenic site at residue 1291. Donors for the other major hormonogenic sites have not yet been
identified.
19
In addition to its role as component of the iodoamino acids, iodine is associated with cleavage of peptide
bonds of Tg, at least in vitro (162). This has been attributed to generation of free radicals during oxidation
(220). Exposure of Tg to reducing agents yields an N-terminal peptide of about 20-26kDa, depending on
the animal species, that contains the major hormonogenic site of Tg (221). This peptide appears in
parallel with iodination or may slightly precede it (222). Further addition of iodine cleaves the 26kDa
further, to produce an 18kDa (on human Tg), an event that also occurs with TSH stimulation (222). Thus,
iodination-associated cleavage appears to be part of the maturation of the Tg molecule. These discrete N-
terminal peptides have been found in all vertebrate Tg examined so far (211).
The amount of iodine has important effects on thyroid hormone production (223). The initial reaction
between TPO and H2O2 produces the so-called "compound I," which oxidizes iodide and iodinates Tg.
Next, the two reactants form compound II, which is necessary for the coupling reaction to make thyroid
hormones. However, if excessive iodine is present, conversion to compound II does not take place, and
hormone synthesis is impaired. (Fig. 2-12) Other iodinated compounds occasionally inhabit the thyroid.
Thyroalbumin excited considerable interest several decades ago. This is an iodinated albumin, shown to
be serum albumin that is iodinated in the thyroid (224). Occasionally, large amounts are found in certain
thyroid diseases, including Hashimoto's thyroiditis (225), congenital metabolic defects (226),
thyrotoxicosis (227) and thyroid carcinoma (228). In all these cases, there are abnormalities in thyroid
structure which might explain the access of serum albumin to intrathyroidal iodination sites
Fig. 2-12: Demonstration of the Wolff-Chaikoff block induced by iodide in the rat. Animals were
given increasing doses of stable iodide. There was at first an increase in total organification, but
then, as the dose was increased further, a depression of organification of iodide and an increase
in the free iodide present in the thyroid gland occurred.
However, in physiological conditions, serum albumin can reach thyroid follicle lumina by transcytosis i.e.
basolateral endocytosis and vesicular transport to the apical plasma membrane (229). The thyroid also
iodinates lipids and many different iodolipids have been described after high doses of iodide in vitro (230,
231). Of particular interest is 2-iodohexadecanal (232, 233). It occurs in the thyroid of several species
following administration of KI, and its amount increases linearly with additional iodine, in contrast to
iodination of Tg which eventually is inhibited by excess iodide. This compound inhibits the action of
NADPH oxidase, which is responsible for H2O2 production (234, 235). These findings suggested that
iodination of lipids impairs H2O2 production and, therefore, decreases further Tg iodination. This is the
most probable mechanism for the Wolff-Chaikoff effect (124).
20
HORMONE STORAGE
Tg molecules vectorially delivered to the follicule lumen by exocytosis accumulates to reach uncommun
concentrations i.e. 0.3-0.5 mM.The mechanism operating such a “packaging” is unknown. Water and ion
extraction from the follicle lumen might represent an active process leading toTg concentration. As the
follicle lumen is a site of Ca++ accumulation (236, 237), the high degree of compaction of lumenal Tg might
depend on electrostatic interactions between Ca++ and anionic residues of Tg, which is an acidic protein.
Stored Tg molecules undergo iodination and hormone formation reactions at the apical plasma
membrane-lumen boundary (237-239), where TPO and H2O2 generating system reside. The mature Tg
molecules, now containing MIT, DIT, T4 and T3, remains extracellular in the lumen of thyroid follicles.
Turnover of intrafollicular material or so-called colloid varies greatly with gland activity. For normal
humans, the organic iodine pool (largely in intrafollicular material), turns over at a rate of about 1% per
day (14). When the turnover increases, less Tg is stored, and with extreme hyperplasia, none is evident
and the entire organic iodine content may be renewed daily (14). In this situation, secretion of Tg and
resorption of Tg (see below) probably occur at similar rates and only tiny amounts of intrafollicular
material are present at any time.
Thyroglobulin as usually isolated from the thyroid is chiefly the 19S 660kDa dimer that has been
glycosylated and iodinated. Iodination and hormone formation of Tg is more complex than generally
thought because of the slow diffusion of molecules that are in a colloidal state in the follicle lumen. It has
been reported that TSH alters the hydrodynamic properties of intrafollicular Tg molecules (240, 241). The
diffusion coefficient of Tg which is about 26mm2 / sec in water would only be in the order of 10-100mm2 /
hour in the thyroid follicle lumen. There is evidence for the presence of insoluble Tg in the form of
globules of 20-120 microns, at a protein concentration of almost 600 mg/mL, in the lumen of thyroid
follicles of different animal species (242). In human, about 34% of the gland Tg would be in this form
(243). In pig, insoluble Tg contains more iodine than did the 660kDa Tg, and had virtually no thyroid
hormone (244). Insoluble Tg has many internal crosslinks through disulfide bonds, dityrosine, and
glutamyl-lysine bonds, the latter generated by transglutaminase (245). The formation of Tg multimers that
probably results from oxidative processes might be limited by the presence of molecular chaperones such
as the protein disulfide isomerase (PDI) and BiP in the follicle lumen (246).
THYROGLOBULIN ENDOCYTOSIS
To be useful, thyroid hormones must be released from Tg and delivered to the circulation for action at
their distant target tissues. Depending on numerous factors including - the supply of iodide as substrate,
the activity of enzymes catalyzing hormone formation, the concentration and physico-chemical state of Tg
- the hormone content of lumenal Tg molecules varies to a rather large extent. Tg molecules newly
arrived in the follicle lumen with no or a low hormone content would co-exist with “older” Tg exhibiting up
to 6-8 hormone residues. The downstream processes responsible for the production of free thyroid
hormones from these prohormonal molecules must therefore adequately manage the use of these
lumenal heterogeneous Tg stores to provide appropriate amounts of hormones for peripheral utilization.
One would expect to find i) control systems preventing excess hormone production that would result from
the processing of excessive amounts of prohormonal Tg molecules and ii) checking systems avoiding the
use of Tg molecules with no or a low hormone content.
21
Fig. 2- 13: Visualization of Tg endocytosis by in vitro reconstituted thyroid follicles obtained from
porcine thyrocytes in primary culture. Purified porcine Tg molecules labeled by covalent
coupling of fluorescein were microinjected into the lumen of a follicle. A and B, phase contrast
and fluorescence images taken at the time of microinjection. C and D, fluorescence images of
the top (C) and the bottom (D) of the follicle after 2hr of incubation. Fluorescently-labeled Tg is
present inside thyrocytes.
The way the thyroid follicle proceeds to generate free hormones from stored hormone containing Tg
molecules has been known for a long time. Tg molecules are first taken up by polarized thyrocytes (Fig.
2-13) and then conveyed to lysosomal compartments for proteolytic cleavage that release T4 and T3 from
their peptide linkages. The first step represents the limiting point in the thyroid hormone secretory
pathway. Over the last decade, there has been substantial improvement in the knowledge of the cellular
and molecular mechanisms governing the internalization or endocytosis and intracellular transport of the
prohormone, Tg. The evolution has first been to consider that it could proceed via a mechanism different
from phagocytosis, also named macropinocytosis, evidenced in rats under acute TSH stimulation
(reviewed in (247)). Results obtained in rats and dogs have been for a long time extrapolated to the
different animal species including human. There is now a number of experimental data indicating that in
the thyroid of different species under physiological circumstances, basal internalization of Tg, mainly if not
exclusively, occurs via vesicle-mediated endocytosis or micropinocytosis (reviewed in (248)), while
macropinocytosis results from acute stimulation (Fig. 2-14) (249, 250) .
22
Fig. 2-14: Schematic representation of the two modes of internalization of Tg; Micropinocytosis
(on the right) and Macropinocytosis or phagocytosis (on the left). Intralumenal Tg stores
potentially subjected to endocytosis are composed of (recently secreted) non-iodinated Tg,
iodinated Tg (Tg-I) and iodinated Tg containing iodothyronine residues (Tg-Ith).Abbreviations
are: CV, Coated Vesicle; EE, Early Endosome; LE, Late Endosome; L, Lysosome; Pp,
Pseudopod; CD, Colloid Droplet; PL, Phagolysosome. The scheme on the right indicates the
three possible routes of transport of internalized Tg molecules reaching the EE: transport to LE,
recycling towards the follicle lumen and transcytosis i.e.transport towards the basolateral
plasma membrane.
The internalization process starts with the organization of microdomains at the apical plasma membrane
of thyrocytes; these microdomains or pits, resulting from the recruitment and assembly of proteins
(clathrin, adaptins…) on the cytoplasmic side of the membrane, invaginate to finally generate coated
vesicles after membrane fission. Lumenal Tg molecules, either free or associated to membrane proteins
acting as Tg receptors, enter the pits and are then sequestrated into the newly-formed vesicles (251-253).
Tg internalization via vesicle-mediated endocytosis is regulated by TSH (252). The vesicles lose their coat
and, through a complex fusion process, deliver their content into a first type of endocytic compartments,
the early apical endosomes (254) (Fig 2-15). In these compartments, Tg molecules probably undergo
sorting on the basis of recognition of different physico-chemical parameters either linked or independent
such as the hormone content, exposed carbohydrates, conformation of peptide domains… A step of
sorting appears as a prerequisite for subsequent differential cellular handling of Tg molecules. It has been
shown that internalized Tg molecules can follow different intracellular pathways. Part of Tg molecules are
conveyed via a vesicle transport system to the second type of endocytic compartments, late endosomes
or prelysosomes. This route ending to lysosomes corresponds to the Tg degradation pathway for the
generation of free thyroid hormones. It is reasonable to think that Tg molecules following this route are the
more mature molecules (with a high hormone content) but, this has not been firmly demonstrated. The
other Tg molecules with no or a low hormone content, present in early apical endosomes, enter either of
23
the two following routes; they are recycled back into the follicle lumen through a direct vesicular transport
towards the apical plasma membrane (255) or via a two-step vesicular transport to the Golgi apparatus
and then to the apical plasma membrane (256). Alternately, Tg molecules are transported and released at
the basolateral membrane domain of thyrocytes via transcytotic vesicles (242, 257); a process accounting
for the presence of Tg in plasma. The orientation of Tg molecules towards one or the other of these three
routes requires the presence of receptors. However, one route could simply convey Tg molecules that are
not selected for entering the other pathways.
Receptors involved in Tg endocytosis may operate at the apical plasma membrane for Tg internalization
and downstream in apical early endosomes for Tg sorting. The requirement and/or the involvement of
apical cell surface receptors has long been debated. Most investigators now recognize that receptors are
not needed for internalization since Tg is present at a high concentration at the site of vesicle formation.
So, Tg molecules are most likely internalized by fluid-phase endocytosis and not by receptor-mediated
endocytosis. On the contrary, if apical membrane Tg receptors exist, their function would be to prevent
the internalization of sub-classes of Tg molecules (258, 259). As it is not conceivable that internalized Tg
molecules could enter the different intracellular routes, described above, at random, Tg receptors must
exist in early apical endosomes. A detailed review on potential Tg receptors has been made by Marino
and Mc Cluskey (248).
The first candidate receptor, initially described by Consiglio et al.(260, 261) was later identified as the
asialoglycoprotein receptor composed of three subunits (RLH1,2 and 3). This receptor binds Tg at acidic
pH and recognizes both sugar moities and peptide determinants on Tg (262). As low-iodinated Tg
molecules are known to have a low sialic acid content, this receptor could be involved in sorting immature
Tg molecules for recycling to the follicle lumen. A second receptor, still not identified, named N-
acetylglucosamine receptor (263, 264), presumably located in sub-apical compartments, interacts with Tg
at acidic pH; it could also act as a receptor for recycling immature Tg molecules back to the follicle lumen.
A third receptor; megalin, has more recently been discovered in the thyroid and has been the subject of
extensive studies yielding convincing data (248, 265-268). Megalin is an ubiquitous membrane protein
belonging to the LDL receptor family. It is located in the apical region of thyrocytes and its expression is
regulated by TSH. Megalin, that binds multiple unrelated ligands, interacts with Tg with a high affinity.In
vitro and in vivo data indicate that Megalin is involved in the transcellular transport or transcytosis of Tg
molecules, possibly with a low hormone content (269).
From the properties and subcellular location of these receptors, one can propose an integrated view of
the sorting processes that would operate in early apical endosomes. The asialoglycoprotein receptor
and/or the less defined N-acetylglucosamine receptor would recognize immature Tg for recycling and
megalin would interact with Tg subjected to apical to basolateral transcytosis. The remaining Tg
molecules would enter, without sorting, the functionally important pathway i.e. the prelysosome-lysosome
route.
Under TSH stimulation, macropinocytosis would be triggered and would become operative in Tg
internalization. Pseudopods representing extensions of the apical plasma membrane project into the
follicle lumen and pinch off to form a resorption vacuole known as colloid droplet (270) .The colloid
droplets then deliver their content to lysosomes. Pseudopod formation is one of the earliest effects of TSH
on the gland, evident within several minutes after administration (271, 272). In most species but perhaps
not in rat, TSH stimulates macropinocytosis through the activation of the cyclic AMP cascade (273, 274).
24
Fig. 2-15: Transmission electron microscope observations of apical endocytic structures in
thyrocytes. Top: coated pits at the apical plasma membrane. Bottom: an early endosome located
in the apical region. Bars, 200 nm.
25
PROTEOLYTIC CLEAVAGE OF THYROGLOBULIN
Internalized Tg molecules that are conveyed to lysosome compartments are subjected to diverse
hydrolytic reactions leading to the generation of free thyroid hormones and to complete degradation of the
protein. Given its composition, Tg is likely the substrate for the different lysosomal enzymes: proteases,
glycohydrolases, phosphatases, sulfatases.... Efforts have been made to identify proteases involved in
the release of hormonal residues from their peptide linkage in Tg. Endopeptidases such as cathepsin D,
H and L (275-282) are capable of cleaving Tg.
Initial cleavage would bring into play endopeptidases and resulting products would be further processed
by exopeptidases. Dunn et al. (278) showed that cathepsin B has exopeptidase activity as well as an
endopeptidase action (278, 280). These investigators tested the activities of human enzyme preparations
against the 20kDa N-terminal peptide from rabbit Tg, which contains the dominant T4 site at residue 5.
Extended cathepsin B incubation produced the dipeptide T4-Gln, corresponding to residues 5 and 6 of
Tg. The combination of cathepsin B with the exopeptidase dipeptidase I released T4 from this dipeptide,
although lysosomal dipeptidase I alone was not effective. Thus, the combination of cathepsin B and
lysosomal dipeptidase I was sufficient to release free thyroid hormone from its major site at residue 5. The
exopeptidase lysosomal dipeptidase II may also be involved in release of free T4, but from a site in Tg
other than residue 5 (280). Thus, Tg probably undergoes selective cleavage reactions at its N- and C-
terminal ends to release iodothyronines that are located nearby (280, 283). Starting from highly purified
preparations of thyroid lysosomes, Rousset et al.(284-286) have identified intralysosomal Tg molecules
with very limited structural alterations but devoid of hormone residue. One may think that proteolysis of Tg
occurs in two sequential steps; i) early and selective cleavages to release T3 and T4 residues and ii)
delayed and complete proteolysis. The reduction of the very high number of disulfide bonds might be the
limiting reaction between the two steps. The nature and the origin of the reducing compounds acting on
Tg are not known. Noteworthy, the possibility of proteolytic cleavage of Tg inside the follicle lumen, before
internalization, has been proposed (287-290) but not yet confirmed by other groups.
After Tg digestion, T4 and T3 must go from the lysosomal compartments to the cytoplasm and from the
cytoplasm out of the cell to enter the circulation. It has been postulated for decades that thyroid hormones
are released from thyrocytes by simple diffusion. There are many objections to this view (291). One of
these comes from the chemical nature of iodothyronines; T4 and T3, which are generally considered as
lipophylic compounds possess charges on both their proximal (amino acid side chain) and distal
(phenolate) parts. As now known for the entry of thyroid hormones in peripheral target cells, the exit of
thyroid hormones from thyrocytes probably involves membrane transporter(s). Details of hormone
transport across the lysosomal membrane and then across the basolateral plasma membrane are
unknown, including whether it is an active or passive process. At present, only a lysosomal membrane
transporter for iodotyrosines has been reported (292, 293). Nevertheless the role of newly cloned
peripheral tissue thyroid transporters (294, 295) in this process remains to be defined.
The type I and type II iodothyronine 5'-deiodinase is present in the thyroid (63, 296, 297) and deiodinate
about 10% of T4 to T3. The extent of this intrathyroidal deiodination is increased when the thyroid is
stimulated by TSH (298, 299). Estimates of average normal secretion for euthyroid humans are 94-110
µg T4 and 10-22 µg T3 daily (300). The thyroid may also convert some T4 to 3,3'5'-T3 (reverse T3) within
the thyroid. About 70% of the Tg iodine content is in the form of DIT and MIT, so this represents an
important part of the intrathyroid iodine pool. Rather than lose it to the circulation, the thyroid deiodinates
MIT and DIT and returns most of iodide to the intrathyroidal iodide pool. The responsible enzyme i.e. the
iodotyrosine deiodinase is an NADPH-dependent flavoprotein with a estimated molecular weight of about
42kDa (301) and recently identified as DEHAL1(302-305). About 3-5 times more iodide is formed inside
the gland each day by this deiodinase than enters the cell from the serum (14). The importance of the
internal recycling of iodide is demontrated by congenitally goitrous subjects who harbour mutations in
DEHAL gene (305) and cannot deiodinate iodotyrosines. These patients are successfully treated with
large amounts of iodide (306). Some iodine is lost from the gland through inefficiency of its recycling by
26
the iodotyrosine deiodinase (14, 86, 300, 307). This leak may increase as the thyroid adapts to a high
daily iodine intake (308), possibly as an autoregulatory process to prevent excessive Tg iodination. Much
more iodide can be lost from diseased glands. Ohtaki et al. (86) found that some iodide leaks from all
glands, including normal ones, but that the amount increases markedly with gland iodine content,
presumably reflecting a dependence on dietary iodine intake. Fisher et al. (300) reported that about 38
µg iodide was released when the mean T4 secretion was 53 µg/day.
Among other products which are released or leak out from the thyroid, there is Tg (309, 310). The
secretion of Tg is clinically important. Its presence in serum can be detected by a routine assay and
provides a sensitive (although not always specific) marker for increased thyroid activity. Attempts have
been made to determine the biochemical characteristics of circulating Tg molecules in terms of iodine
content (311), structural integrity (312) and hormone content (313). Serum levels are elevated in patients
with hyperplastic thyroid or thyroid nodules including differentiated thyroid cancer. Tg measurement can
identify congenital hyperplastic goiter, endemic goiter, and many benign multinodular goiters, but its
greatest application is in the follow-up of differentiated thyroid cancer (314). Most papillary and follicular
cancers retain some of the metabolic functions of the normal thyrocyte, including the ability to synthesize
and secrete Tg. Subjects who have differentiated thyroid cancer treated by surgery and radioiodine
should not have normal thyroid tissue left, and therefore, should not secrete Tg. If Tg is found in their
serum, it reflects the continuing presence either of normal tissue, unlikely after its previous ablation, or of
thyroid cancer. The depolarized cancer cells presumably secrete Tg directly in intercellular space.
Tracking serum Tg levels is probably the most sensitive and practical means for the follow-up of such
patients. It is more sensitive when the subject is stimulated by TSH. Until recently, this could only be done
by withdrawal of thyroid hormone and consequent symptomatic hypothyroidism, but now recombinant
human TSH can be administered to enhance the sensitivity of the serum Tg and thyroid scan (315-320).
CONTROL OF HORMONE SYNTHESIS
The most important controlling factors are iodine availability and TSH. Inadequate amounts of iodine lead
to inadequate thyroid hormone production, increased TSH secretion and thyroid stimulation, and goiter in
an attempt to compensate. Excess iodide acutely inhibits thyroid hormone synthesis, the Wolff-Chaikoff
effect (223), apparently by inhibiting H2O2 generation, and therefore, blocking Tg iodination (123). A
proposed mechanism is that the excess iodide leads to the formation of 2-iodohexadecanal (235), which
is endowed with an inhibitory action on H2O2 generation.
TSH influences virtually every step in thyroid hormone synthesis and release. In humans the effects on
secretion appear to be mediated through the cAMP cascade (see chapter 1) while the effects on
synthesis are mediated by the Gq/phospholipase C cascade (321). Elsewhere in this chapter, we have
mentioned instances of TSH regulation. To summarize, TSH stimulates the expression of NIS, TPO, Tg
and the generation of H2O2 , increases formation of T3 relative to T4, alters the priority of iodination and
hormonogenesis among tyrosyls and promotes the rapid internalization of Tg by thyrocytes. These
several steps are interrelated and have the net effects of increasing the amount of iodine available to the
cells and of making and releasing a larger amount and a more effective type of thyroid hormone (T3).
Anti-thyroid drugs are external compounds influencing thyroid hormone synthesis. The major inhibitory
drugs are the thionamides: propylthiouracil and methimazole. In the thyroid, they appear to act by
competing with tyrosyl residues of Tg for oxidized iodine, at least in the rat (199). Iodotyrosyl coupling is
also inhibited by these drugs and appears more sensitive to their effects than does tyrosyl iodination.
SUMMARY
The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the
regulation of metabolic processes throughout the body. As at any factory, effective production depends on
three key components - adequate raw material, efficient machinery, and appropriate controls. Iodine is the
critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the
27
circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma
membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in
the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key
proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls
serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where
it reduces H2O2, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to
tyrosyls in Tg. H2O2 is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial
iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in
peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid
hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and
lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and
exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about
70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available
for recycling within the gland. TSH is the important stimulator that affects virtually every stage of thyroid
hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic
machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply,
either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide
oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can
produce goiter and hypothyroidism.
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  • ... Большая часть поступившего йода требуется для производства тироидных гормонов. Так, массовая доля йода в молекуле Т4 составляет 65% (Rousset et al., 2015). Для эффективной продукции гормонов необходимо: достаточное поступление самого йода, эффективная транспортная машина и надлежащий контроль. ...
  • ... The process requires iodine and tyrosine, which are both obtained from the diet; tyrosine is also produce by the body. Iodine is the rate limiting agent in the process (Rousset et al., 2015). Iodide is absorbed from bloodstream and oxidised by thyroid peroxidase (TPO) and hydrogen peroxide to produce iodine. ...
    Thesis
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    Abstract Diabetes mellitus (DM) is a group of aetiologically different metabolic defects characterized by hyperglycemia resulting from defect in insulin secretion as well as insulin action or both. Occasionally other endocrine disorders like abnormal thyroid hormone level are found in diabetes. Diabetes and thyroid disorders have been shown to mutually influence each other and associations between both conditions have long been reported. This study has been done at The National Diabetes Center of AL-Mustansiria University from October 2017 to April 2018. Ninety men with age ranged from (35-65) years were involved in this study. Divided into four groups ; G1(15 healthy control), G2 (15 patients with type 2 DM), G3 (30 patients with type 2 DM and hypothyroidism), and G4 (30 patients with type 2 DM and hyperthyroidism). Blood samples were collected from each individual via vein puncture to assess fasting blood glucose (FBG), glycated hemoglobin (HbA1c), Insulin, Glucagon, and levels of thyroid hormones: thyroid stimulating hormone (TSH), Thyroxine (T4), and Triiodothyronine (T3). The result showed highly significant (p < 0.01) increase in the level of TSH in diabetic with hypothyroidism group, and highly significant (p < 0.01) decrease in level of TSH in diabetic with hyperthyroidism group when compared to diabetic and control groups. And there was no significant difference between diabetic group and control group. Level of T4 and T3 showed highly significant (p < 0.01) increase in diabetic with hyperthyroidism group and highly significant (p < 0.01) decrease in diabetic with hypothyroidism group when compared to the diabetic group and healthy control group. Furthermore, there was no significant difference between diabetic group and healthy control group. The result showed highly significant (p < 0.01) increase in the level of insulin, insulin resistance, and glucagon in diabetic with hyperthyroidism group when compared to diabetic II with hypothyroidism group, diabetic group, and healthy control group. Also there was highly significant (p < 0.01) increase in the level of insulin, insulin resistance, and glucagon in diabetic with hypothyroidism group and diabetic group when compared to the healthy control group. while the results showed no significant difference increase in the level of insulin, insulin resistance, and glucagon between diabetic with hypothyroidism group and diabetic group. The result showed highly significant (p < 0.01) increase in the levels of fasting blood glucose (FBG), and HbA1c in diabetic with hyperthyroidism group, diabetic with hypothyroidism group, and diabetic group when compared to the healthy control group.
  • ... Thyroid hormones synthesis takes place in the thyroid follicles. Iodine in the form of iodide is absorbed from the blood stream to the follicles lumen of thyroid cells the TG is produced thyroid's endoplasmic reticulum then the tyrosine residues molecule of the TG protein fuse with iodine and form monoiodotyrosine (MIT) and diiodotyrosine (DIT) they still only chain of peptide (Rousset et al., 2015). The iodide penetrate and enter to the thyrocyte by sodium\ iodide symporter the iodide is oxidized into iodine for the process of formation DIT and MIT is going by thyroid peroxidase (TPO) this conversion requires hydrogen peroxidase (H 2 O 2 ) then the iodination of TG is take place. ...
    Thesis
    Full-text available
    Abstract Chronic kidney disease (CKD) is a serious public health concern, due to its prevalence among the populations world and its effect on increased mortality and patients recurrent hospitalizations .CKD is a permanent loss of kidney function diagnosed when the glomerular filtration rate (GFR) is under 60 ml\min\1.73m2 for more than three months and this disease is divided into 5 stages based on GFR level. Endocrine disorders such as abnormal thyroid hormone level and abnormal parathyroid hormone level were detected the patients included in this study were at the stage 4 which they did not need dialysis yet. The study was done at Kidney Transplant and Dialysis Center in medical city from October 2018 to March 2019. Eighty five subjects with age ranged from (40 to 65) years were involved in this study. And they were divided into two groups; G1 (25 healthy control) and G2 (60 patients with CKD) Blood samples were collected from each individual to assess urea, creatinine, uric acid, calcium, phosphorus, potassium and levels of thyroid hormones (thyroxin T4, and Triiodothyronine T3), thyroid stimulating hormone TSH and parathyroid hormone PTH while GFR and creatinine clearance were calculated. Urea, creatinine and uric acid showed highly significant (p < 0.01) increase in CKD patient in compares with control group and showed highly significant (p < 0.01) decrease in eGFR and creatinine clearance in CKD patients. The result showed highly significant (p < 0.01) increase in the level of PTH, phosphorus and potassium while significant (p < 0.5) decrease in calcium level was detected in CKD .The level of T3 and T4 showed highly significant (p < 0.01) decrease and highly significant (p < 0.01) increase in level of TSH in patients with CKD in compare with control group.
  • ... Nonetheless, as part of the "underlying mechanism", they point to a different level and may also point at intermediates, excited states, transition states, Figure 6. The biosynthesis of thyroid hormones and their impact on healthy muscle growth [5,23,26,27]. This scheme is adapted from two standard pharmacology textbooks, namely, Brenner and Stevens' Pharmacology 5th edition p. 368 and Basic & Clinical Pharmacology 14th edition p. 688, and purely for illustration purposes, has been expanded to include some additional formulae and conversions. ...
    Article
    Full-text available
    Talk of mechanisms is ubiquitous in the natural sciences. Interdisciplinary fields such as biochemistry and pharmacy frequently discuss mechanisms with the assistance of diagrams. Such diagrams usually depict entities as structures or boxes and activities or interactions as arrows. While some of these arrows may indicate causal or componential relations, others may represent temporal or operational orders. Importantly, what kind of relation an arrow represents may not only vary with context but also be underdetermined by empirical data. In this manuscript, we investigate how an analysis of pharmacological mechanisms in terms of producing and underlying mechanisms—as discussed in the contemporary philosophy of science—may shed light on these issues. Specifically, we shall argue that while pharmacokinetic mechanisms usually describe causal chains of production, pharmacodynamics tends to focus on mechanisms of action underlying the in vivo effects of a drug. Considering the action of thyroid gland hormones in the human body as a case study, we further demonstrate that pharmacodynamic schemes tend to incorporate entities and interactions on multiple levels. Yet, traditional pharmacodynamic schemes are sketched “flat”, i.e., non-hierarchically. We suggest that transforming flat pharmacodynamic schemes into mechanistic multi-level representations may assist in disentangling the different kinds of mechanisms and relations depicted by arrows in flat schemes. The resulting Baumkuchen model provides a powerful and practical alternative to traditional flat schemes, as it explicates the relevant mechanisms and relations more clearly. On a more general note, our discussion demonstrates how pharmacology and related disciplines may benefit from applying concepts from the new mechanist philosophy to guide the interpretation of scientific diagrams.
  • ... The iodide uptake is a sodium-dependent active transport process that couples the energy released by the inward movement of sodium down its electrochemical gradient to the simultaneous inward movement of iodide against its electrochemical gradient. [10] The sodium gradient is maintained by sodium-potassium ATPase, which requires oxygen to provide energy; therefore, iodide transport is energy-dependent, hence requiring oxygen. [10] Iodide is a necessary component in the synthesis of thyroid hormone, which occurs inside the thyroid gland. ...
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    Full-text available
    Despite hormone replacement therapy with levothyroxine sodium, some patients continue to complain of overt signs and symptoms of hypothyroidism, despite having normal labs. In some cases, there is no identifiable correlation with comorbidity and/or medication interfering with an optimal response; tissue hypothyroidism may be the reason for the observed signs and symptoms in these patients. We report a case of a 54-year-old Afro-Caribbean female who has been diagnosed with hypothyroidism for the past 26 years but was started on hormone replacement therapy with levothyroxine after bilateral partial lobectomy 19 years ago. The patient is currently on the standard levothyroxine therapy in tablet form, yet for the past 16 years have been complaining of persistent signs and symptoms of hypothyroidism; fatigue, dry skin, brittle hair and nails, cold intolerance, constipation, and unintentional weight gain. Normal laboratory findings and thyroid function tests confirm that the patient is given optimal doses of treatment. This case may suggest the presence of tissue resistance to levothyroxine, resulting in the persistence of signs and symptoms of hypothyroidism.
  • ... Increases in T3 or T4 inhibit production of TSH and TRH by the pituitary and hypothalamus, respectively. Inversely, as T3 and T4 decrease, TSH and TRH genes are activated (31). In the bloodstream, THs are almost entirely bound to serum distributing proteins, such as transthyretin (TTR), albumin or thyroxin-binding globulin (TBG). ...
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    Full-text available
    Plant Protection Products, more commonly referred to as pesticides and biocides, are used to control a wide range of yield-reducing pests including insects, fungi, nematodes, and weeds. Concern has been raised that some pesticides may act as endocrine disrupting chemicals (EDCs) with the potential to interfere with the hormone systems of non-target invertebrates and vertebrates, including humans. EDCs act at low doses and particularly vulnerable periods of exposure include pre- and perinatal development. Of critical concern is the number of pesticides with the potential to interfere with the developing nervous system and brain, notably with thyroid hormone signaling. Across vertebrates, thyroid hormone orchestrates metamorphosis, brain development, and metabolism. Pesticide action on thyroid homeostasis can involve interference with TH production and its control, displacement from distributor proteins and liver metabolism. Here we focused on thyroid endpoints for each of the different classes of pesticides reviewing epidemiological and experimental studies carried out both in in vivo and in vitro. We conclude first, that many pesticides were placed on the market with insufficient testing, other than acute or chronic toxicity, and second, that thyroid-specific endpoints for neurodevelopmental effects and mixture assessment are largely absent from regulatory directives.
  • ... Thyroid gland extracts iodine from blood and combines amino acid tyrosine to make T3 and T4 which is under the control of both pituitary gland and hypothalamus via thyroid stimulating hormone (TSH) and TSH releasing hormone (TRH), respectively. 2,3 Thyroid hormone synthesis and secretion is mainly regulated by TSH secreted by pituitary gland and thereby protects the body from thyroid gland related diseases like hypo-and hyperthyroidism. ...
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    Full-text available
    p class="abstract"> Background: Thyroid gland is a key part of endocrine system and it performs its functions via two most important thyroid hormones thyroxine (T4) and triiodothyronine (T3). Thyroid gland is mainly regulated by thyroid-stimulating hormone (TSH). Povidone-iodine (polyvinylpyrrolidone-iodine, PVP-I) mouthwash is commonly used to treat infections of the oral cavity and oropharynx and iodine released from PVP-I can interfere with thyroid function. In this study the effect of brief treatment with povidone-iodine mouth wash on thyroid function was assessed. The aim of the present study was to assess whether iodine is absorbed through oral transmucosal route and interfere with TSH in serum. Methods: Fifty one patients with acute and chronic pharyngitis and tonsillitis were recruited and out of which forty-seven patients were treated with 20 ml of PVP-I mouthwash twice daily for 3 weeks and blood was collected from the respective patients before and after treatment with PVP-I. Serum thyroid stimulating hormone concentration was measured from the collected blood samples of the patients. Results: In the present study there was a small increase in serum TSH concentration during the therapy with PVP-I but the concentration determined was within the normal range. Conclusions: Based on the results of this study we conclude that the use of PVP-I for a brief period transiently increase TSH value and prolonged use should be avoided in people with an increased risk of thyroid dysfunction and other autoimmune disorders.</p
  • ... The thyroid gland releases two hormones i.e. thyroxine or tetraiodothyronine (T4) and triiodothyronine (T3) (Miot, Dupuy, Dumont, & Rousset, 2010). They regulate energy metabolism of tissues of heart, liver, kidneys and skeletal muscles. ...
  • ... The thyroid gland releases two hormones i.e. thyroxine or tetraiodothyronine (T4) and triiodothyronine (T3) (Miot, Dupuy, Dumont, & Rousset, 2010). They regulate energy metabolism of tissues of heart, liver, kidneys and skeletal muscles. ...
    Article
    The current research was designed to determine single as well as combined effects of zinc and probiotic on biochemical parameters of broilers. A total of 192- day old broiler chicks were randomly divided into six groups: Control, Zn30 (30 mg Zn/kg diet), Zn60 (60 mg Zn/kg diet), Pro (0.1 g/kg Protexin®), Com30 (30 mg Zn/kg diet + 0.1 g/kg Protexin®) and Com60 (60 mg Zn/kg diet + 0.1 g/kg Protexin®). Com60 showed highest catalase activity and lowest malondialdehyde level in comparison to other supplemented groups. A significant increase in serum proteins was observed with all the supplementations in comparison to the control group. Protexin significantly increased serum T3 levels as compared to other supplemented groups. On the other hand, serum T4 concentration was significantly increased with both Zn60 and Com60. Com60 significantly increased urea while both the combinations showed significantly increased uric acid and decreased creatinine concentrations in serum. There was no significant change in serum glucose, ALT and AST concentrations with any supplementation. It can be concluded that protexin and zinc supplementations are beneficial for improving health status of broilers as depicted from the thyroid hormones level and no hepatotoxicity, yet their synergistic effects need to be studied further
  • Chapter
    The human endometrium is the main target organ for the ovarian steroidal hormones. The pivotal role of sex hormones in the development, growth and maintenance of the normal physiological endometrium is well established. Aberrations in the endometrial hormonal milieu due to endogenous or exogenous factors influence endometrial carcinogenesis and cancer progression. Emerging evidence suggests that other non-steroid hormones are involved in endometrial carcinogenesis via altering the tumour microenvironment and facilitating tumour progression. Understanding the intricate relationship between these hormones in endometrial carcinogenesis could improve the current therapeutic options and lead to the designing of new strategies for the prevention and treatment of endometrial cancer in the era of evolving hormone therapy. This chapter focuses on the influences of both ovarian steroid hormones and the other non-steroidal hormones in endometrial cancer.
  • Article
    The Na+/I−symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I− into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458–460). Because mature NIS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (∼90–110 kDa) than nonglycosylated NIS (∼50 kDa). Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putativeN-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50–90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn225 is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH2 terminus faces extracellularly as well. A new secondary structure model consistent with these findings is proposed.
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