t hyroid h ormone s ynthesis, s ecretion, a ction, r eceptors & a ntibodies emily brennan pgy-4,...

THYROID HORMONE SYNTHESIS, SECRETION, ACTION,

RECEPTORS & ANTIBODIES

Emily BrennanPGY-4, EndocrinologyAugust 13, 2014

ObjectivesAt the end of this lecture, you will be able to understand:

• the synthesis of thyroid hormone• thyroid hormone transport and activation at local tissues• the regulation and feedback mechanisms of the thyroid axis• the downstream action of thyroid hormone on tissues• the role and value of thyroid antibodies

Recall: The Thyroid• The thyroid is the largest single

organ specialized for endocrine hormone production

• The thyroid’s function is to secrete an appropriate amount of the thyroid hormone primarily as T4

• The gland is composed of closely packed, spherical units termed follicles

Thyroid Hormone and Related Structures • Comprised iodinated

thyronines• two tyrosine molecules joined

by an ether linkage

• Produced only by the follicular cells of the thyroid

• Iodine is a key structural component of thyroid hormone

Thyrocyte

Thyroid Hormone Synthesis• Requirements for hormone production:• Sodium-Iodide Transporter (NIS)• Iodine• Thyroglobulin (Tg)• Thyroid peroxidase (TPO)

1. Trapping -- Active transport of iodide across the basement membrane into the thyroid cell

Sodium-iodide symporter (NIS)Location: basal membrane of the thyrocyte

Function: actively transports iodide from the blood; maintains concentration of iodide ~30x greater than plasma

Regulation: stimulated by TSH, suppressed by excess iodide

1. Trapping -- Active transport of iodide across the basement membrane into the thyroid cell

PendrinLocation: apical border of the thyrocyte

Function: transports iodide into the membrane-colloid interface to become substrate for thyroid hormonogenesis

Mutation: Pendred syndrome (goitre and congenital deafness)

Iodine• Essential micronutrient consumed in food and

water

• Recommended intake: • 150 mcg adults• 200 mcg pregnant/lactating women• 50-250 mcg children

• When iodine intake is less than 50 mcg/d, a normal-sized thyroid cannot sustain adequate hormone production

• The concentrating ability of the thyroid creates an intrathyroid pool (8-10 mg)

Thyroglobulin• A large glycoprotein molecule, composed of two subunits

• Structure: Includes 140 tyrosyl residues, but only four tyrosyl sites are sterically oriented for effective hormornogenesis in each molecule

• Role: serves in synthesis and storage of thyroid hormone

• Regulation: TSH regulates the expression of the thryoglobulin gene

Thyroglobulinthyroglobulin mRNA translated

into RER↓

glycosylated and transported to golgi apparatus

↓incorporated into exocytic

vesicles↓

fused with basement membrane↓

at apical-colloid border, Tg is iodinated

2. Organification -- oxidation of iodide and iodination of tyrosyl residues in thyroglobulin

Thyroid Perioxase (TPO)A membrane-bound glycoprotein

Function: catalyses both iodide oxidation and covalent linkage of iodine to the tyrosine residues of thyroglobulin

Location: Cell-Colloid Interface

Regulation: gene expression stimulated by TSH

Inhibition: methimazole, carbimazole, and propylthiouracil

3. Coupling -- linking pairs of iodotyrosine molecules within the thyroglobulin to form T3 and T4

The coupling of iodotyrosyl resides is also catalyzed by TPO

Thought to be an intramolecular process involving the oxidation of two iodotyrosyl residues brought into proximity by the tertiary and quatenary structures of thyroglobulin, and it involves a linkage and splitting to form an iodothyronine

Coupling of iodotyrosine molecules

Coupling of iodotyrosine molecules

4. Pinocytosis and then proteolysis of thyroglobulin with release of free iodothyronines and iodotyrosines into circulation

Proteolysis of Thyroglobulin and Thyroid Hormone Secretion

Colloid is engulfed in vesicles by pinocytosis and absorbed into the cell

↓Lysosomes then fuse with the vesicle

↓Releases T4 and T3 and inactive

peptides↓

The biologically active thyroid hormones T4 and T3 enter the

circulation ↓

DIT and MIT are deiodinated and their iodide conserved

5. Deiodination of iodothyroxines within the thyroid cell with conservation and reuse of liberated iodine

6. Intrathyroidal 5’-deiodination of T4 to T3

Summary

Thyroid Hormone Transport• Both T3 and T4 are poorly soluble in water, and therefore,

circulate bound to plasma proteins• 0.04% T4 unbound• 0.4% T3 unbound

• Three major transport proteins:• Thyroxine-binding globulin (TBG)• Transthyretin• Albumin

Thyroxine-Binding Globulin (TBG)• Liver-derived glycoprotein• Each TBG molecule has a single binding site for T4 or T3• It carries about 70% of circulating thyroid hormones (high

binding affinity for T4 and T3)

Conditions Affecting Binding of TBGCongenital• TBG deficiency, X-linked recessive; low total T4/T3, but free hormone levels are

normal; euthyroid• Congenital TBG excess: elevated total T4/T3, normal fT4/fT3, euthyroid

Physiologic• Pregnancy/estrogen – increase the sialic acid content of TBG, decrease metabolic

clearance and elevated TBG levels

Pathophysiologic• Estrogen secreting tumours, OCP, acute hepatitis (increased siailac acid)• Systemic illness – decrease TBG due to cleavage by leukocyte protease, and decrease

binding affinity; lowers hormone concentrations

Drugs• Salicylates, high dose phenytoin, furosemide – bind to TBG, displace T4/T3

Transthyretin• Formerly known as: thyroxine-binding prealbumin• Binds to 10% of circulating T4• Affinity for T4 is 10-fold greater than for T3

• Expressed in the choroid plexus• the major thyroid hormone-binding produced in the CSF

• The dissociation of T4 and T3 from transthyretin is rapid, so that transthyretin is a source of readily available T4

Conditions Affecting Binding• Congenital increased affinity for transthyretin binding for T4

• elevated total T4, but normal fT4• Ectopic production of transthyretin can occur w/ pancreatic and hepatic tumours

• causes euthyroid hyperthyroxinemia

Albumin• Binds to T4 and T3 with a lesser affinity than TBG or

transthyretin, but it has a high plasma concentration• 15% of circulating T4 and T3

• Rapid thyroid hormone dissociation rates from albumin make it a major source of free hormone to tissues

Conditions Affecting Binding• Hypoalbuminia (nephrotic syndrome, cirrhosis) is associated with

a low total T4 and T3, but normal free hormone levels • Familial dysalbuminemic hyperthroxiemia: AD where 25%

albumin has a higher than normal T4 binding affinity results in elevated total T4 level, but normal fT4 = euthyroid

Transport across Cell Membranes• Originally thought to be primarily passive; however, several

specific thyroid hormone transport proteins have been identified:• MCT8 • MCT10• Organic anion transporting polypeptide 1C1 (OATP1C1) – predominantly

in the brain, transports T4 preferentially

• In most cells, 90% of T3 is in the cytosol except the pituitary, where 50% of the T3 is in the nucleus

Metabolism of Thyroid Hormones• Most of the plasma pool of T3

(80%) is derived from:• 5’-deiodination of T4 in the liver,

kidney, and skeletal muscle

• Deiodination of the inner ring of T4 (5-deiodination) produces reverse T3 (metabolically inert)

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Metabolisms of Thyroid Hormone • Three deiodinases enzymes catalyze these reactions:• D1• D2• D3

• permit local tissue and cellular modulation of thyroid hormone action

DeiodinationD1Location: most abundant form, found predominantly in liver, kidney, and lesser extent in thyroid gland, skeletal, and heart muscle, and other tissues

Function: major converter of T4 to T3

Inhibition: PTU (not methimazole), amiodarone and iodinated radiocontrast dye

DeiodinationD2Location: expressed in the brain and pituitary gland, where is maintains constant levels of intracellular T3 in the CNS

Function: maintenance of the level of intraacellular T3 and its neuronal cellular functions

D2 is very sensitive to circulating T4

DeiodinationD3Location: chorionic membranes of the placenta and glial cells in the CNS

Function: inactivates T4 by converting it to rT3 and it inactivates T3 by converting it to 3,3’-T2

Elevated in hyperthyroidism and decreased in hypothyroidism, may help to insulate the fetus and the brain from T4 excess of deficiency

Regulation of Thyroid Function

Regulation of Thyroid FunctionThyrotropin-Releasing Hormone (TRH)• Synthesized in hypothalamus (supraoptic and

supraventricular nuclei)

• Stored in the median eminence and then transported via the pituitary portal venous system to the anterior pituitary

• Role: controls synthesis/release of TSH in the anterior pituitary (binds to G-protein receptor on thyrotrophs to initiate pathway to stimulate for TSH release). Pulsatile secretion, peak midnight-4am

• Negative Control: • TRH gene expression negatively regulated

by T3 and T4• T3/T4 downregulate the TRH receptors in

the pituitary thyrotrophs

Regulation of Thyroid FunctionThyroid-Stimulating Hormone• Structure: Glycoprotein composed of

an alpha and beta subunit, that are noncovalently linked• The alpha subunit is common to FSH and

LH and hCG• The beta subunit is unique to each

hormone, conferring its specific binding properties and biologic activity

• Role: TSH controls thyroid cell growth and hormone production• binding to a specific TSH G-protein

receptor on the basolateral thyrocyte membrane

• activates a cascade responsible for promoting thyroid cell growth and hormone synthesis/release

Effects of TSH on the Thyrocyte• Changes in thyrocyte morphology• induces pseudopods at the follicular cell-colloid border, accelerating

thyroglobulin resorption, which increases thyroid hormone release

• Cell growth• individual cells increase in size, vascularity, and over time, thyroid enlargement

or goiter develops

• Iodine metabolism• TSH stimulates all phases of iodide metabolism (increased uptake, transport,

secretion); increased NIS expression

• Thyroglobulin and TPO mRNA expression• Results in increased incorporation of iodide into MIT, DIT, T3 and T4

TSH RegulationFeedback• T3 concentration in the hypothalamus within the thyrotrophs

cells regulates mRNA expression, TSH translation and T3/T4 release• TRH - controls postranslational glycosylation and release of TSH

Inhibitors of TSH• somatostatin, dopamine, dopamine agonists, high dose glucocorticoids

TSH Receptor Conditions

• Congenital• Familial hyperthyroidism – activating mutation of TSH

receptor• Familial gestational hyperthyroidism – due to structural

similar hCG hormone activated aberrant TSH receptor• Acquired• TSH receptor blocking antibodies (cause hypothyroid)• Graves’ (most common)• Autoantibodies bind and stimulate the TSH receptor

Iodine Autoregulation• The capacity of the thyroid gland to modify its function to the

availability of iodine, independent of pituitary TSH

• Allows maintenance of adequate/appropriate thyroid hormone secretion is varying intake of iodine

• Major adaptation to low iodide intake is preferential synthesis of T3 over T4

• Iodide excess inhibits many thyroidal functions including:• iodide trapping• thyroglobulin iodination (Wolff-Chaikoff)• thyroid hormone release from the gland

Mechanism of Thyroid Hormone ActionGenomic Actions• T3 interacts with its nuclear receptors to regulate gene activity• T3 binds to a specific nuclear thyroid hormone receptor (TR),

which in turns bind to DNA at specific sequences called thyroid hormone response elements (TREs)

• T3 has a 15-fold higher binding affinity for TRs than T4• There are tissue-specific preferences in expression of the various

TRs (difference expression in hypothalamus vs kidney, liver, brain and heart)

Nongenomic Actions• Nongenomic actions mediated by T3 and T4 occur with certain

enzymes

Thyroid Hormone Genomic Actions

Thyroid Hormone ActionsFetal Development• Iodide is found in thyroid tissue

and pituitary TSH appear in the fetus at about 11 weeks

• The high placental content of D3 inactivates most maternal T3 and T4, and very little free hormone reaches fetal circulation

• After 15-18 weeks, the fetus controls most of its own thyroidal secretion

Thyroid Hormone Action

Increases oxygen consumption and heat production by stimulation of Na-K-ATPase in all tissues

Oxygen, Heat, Free Radicals

Stimulates mitochondriogenesis, augmenting the cell’s oxidative capacity

Increases basal metabolic rate

Thyroid Hormone Action

Lowers peripheral vascular resistance, increase intravascular volume increased cardiac output

Increases the rate of myocardial diastolic relaxation

Increases the alpha receptors

Cardiovascular System

Increases the rate of depolarization and repolarization of the SA node, increasing heart rate

Positive inotropic and chronotropic effects on the heart with heightened adrenergic sensitivity

Thyroid Hormone Action

Maintains the ventilatory responses to hypoxia and hypocapnia in the brain stem respiratory center

Pulmonary System

Thyroid Hormone Action

Increased cellular demand for oxygen in hyperthyroidism leads to increased production of EPO and erythropoiesis

Hematopoeitic Effects

Increases the oxygen dissociation from hemoglobin and increases oxygen availability to the tissues

Thyroid Hormone ActionSkeletal System

Stimulates bone turnover and increases bone resorption and to a lesser degree, bone formation

Thyroid Hormone Action

Thyroid hormone promotes gut motility

Gastrointestinal System

Thyroid Hormone Action

In hyperthyroidism, there is increased protein turnover and loss in skeletal muscle

Changes in speed of muscle contraction and relaxation, noted as hyperreflexia or delayed DTR in hypothyroidisms

Neuromuscular System

Fine distal hand tremor is typical in hyperthyroidism

Hyperactivity and sluggishness can be striking

Thyroid Hormone Action

Thyroid hormone regulates the synthesis of pituitary hormones, stimulates growth hormone production, and inhibits TSH

Low thyroid hormones levels can cause delayed puberty by impairing GnRH secretion

Reproductive System

Thyroid Hormone Action

increases hepatic gluconeogenesis and glycogenlysis, and intestinal glucose absorption

?thyroid hormone mediated decreases in insulin sensitivity worse glycemic control

Lipids and Carbohydrate Metabolism

Increase cholesterol synthesis and degradation (largely due to an increase in hepatic LDL receptors, accelerating LDL clearance)

Thyroid Antibodies• Considered the hallmark of the autoimmune thyroid disorders

• Antibodies to thyroglobulin (Tg)• Antibodies to thyroid peroxidase (TPO)• Antibodies directed to TSH-receptor (TSHR)

TPO-Ab and Tg-Ab• Polyclonal antibodies, IgG class• Develop as a secondary response to thyroid injury• Not thought to cause the disease themselves (i.e. they cross the placenta

and do not cause disease in fetus)• Contribute to the disease mechanisms (complement fixing cytotoxicity

or T cell activation)• correlates well with thyroidal damage and lymphocytic infiltration

TPO-Ab and Tg-Ab• Almost all patients with autoimmune thyroiditis are associated with Tg-

Ab and TPO-Ab• May be present in Graves’ as well

• TPO-Ab has a higher affinity and occurs in higher concentrations

• Tg-Ab and TPO-Ab are more common in patients with sporadic goitre, multinodular goiter, or isolated thyroid nodules and cancer than the general population• Non-specific

• Not necessary for evaluation of thyroid function• may be helpful to predict progression of subclinical hypothyroidisms

TSHR-Ab• The presence of antibodies favours an autoimmune cause for

hyperthyroidism, but not sensitive or specific interpretable only as part of the clinical scenario

• Testing for TSHR Ab are the test of choice; they usually are thyroid receptor stimulating antibodies (compete with TSH for binding to its specific receptor site in the cell membrane)• can behave as stimulating, inhibitory or neutral

• Tg-Ab and TPO-Ab are also detectable in 50-90% of patients’ with Graves

Take Home:Requirements for Thyroid Hormone Synthesis:• Sodium-Iodide Transporter (NIS)• Iodine• Thyroglobulin (Tg)• Thyroid peroxidase (TPO)

Steps for Thyroid Hormone Synthesis:• Iodine trapping via the Sodium-Iodide Symporter• Oxidation and organification of thyroglobulin by TPO• Coupling pairs of MIT or DIT within the thyroglobulin molecule to form T3

and T4• De-iodination of MIT/DIT to conserve iodine• Release of T3/T4 into circulation

Take Home:• T3 and T4 are circulate bound to: thyroxine-binding globulin, transthyretinin,

and albumin

• T4 undergoes 5’-deiodination to form T3 (the more biologically potent hormone)

• T3 acts on the cell nucleus to regulate gene expression and protein synthesis.

• Thyroid hormone has targets of action in most tissues in the body

• TSHR-Ab is the most specific for Graves’ disease, and is the mechanism for the disease

• TPO-Ab and Tg-Ab are evidence of thyroidal injury, but are nonspecific for diagnostic purposes

References• Williams Textbook of Endocrinology 12th edition. • Greenspan’s Basic and Clinical Endocrinology.• Endocrine Physiology. Lange. • UpToDate• Boron WF (2003). Medical Physiology: A Cellular And Molecular

Approaoch. Elsevier/Saunders. p. 1300. ISBN 1-4160-2328-3.

Questions?