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As  faculty  of  Weill  Cornell  Medical  College,  we  are  commi8ed  to  providing  transparency  for  any  and  all  external  rela<onships  prior  to  giving  an  academic  presenta<on.    I  do  not  have  a  financial  interest  in  commercial  products  or  services  related  to  the  subject  of  this  lecture.        

James  R.  Hurley,  MD  

Outline

• Evolution of the thyroid gland • Anatomy of the human thyroid • Synthesis and secretion of thyroid hormones • Control of thyroid hormone secretion • Nuclear actions of thyroid hormones • Effect of deiodinases on thyroid hormone action • Metabolic actions of thyroid hormones • Thyroid hormones and fetal development

Overview

• Thyroid hormones are essential to life. - Required for successful pregnancy - Control fetal growth and maturation - Control growth during childhood and adolescence - Principal regulator of metabolism

• Abnormal thyroid function is common - 6% of the US population have abnormal levels of

thyroid hormone

• There are two thyroid hormones -  T4 (levo-thyroxine) contains 4 iodine atoms -  T3 (tri-iodothyronine) contains 3 iodine atoms

• Synthesized in the thyroid gland from iodine and protein -  Secreted into the circulation

• Synthesis and secretion of T4 & T3 is controlled by the pituitary hormone TSH

-  Thyroid Stimulating Hormone or thyrotropin

Thyroid Hormones

Thyroid Hormone Action

• T4 and T3 are actively transported into cells -  T4 is converted into T3 by removal of the 5’ iodine

- Catalyzed by a 5’deiodinase

• T3 enters the nucleus where it binds to nuclear receptors -  Initiates transcription of T3 responsive genes

Control of TSH

• Thyrotropin Releasing Hormone (TRH) stimulates TSH release from the anterior pituitary thyrotrophs

-  Synthesized by the paraventricular nuclei in the hypothalamus -  Reaches the anterior pituitary directly via the hypothalamic-

pituitary portal system • Thyroid hormones control TSH release in a classic negative

feedback system - High levels cause suppression of TSH release -  Low levels permit an increase in TSH release

•  TSH is the best test for evaluation of thyroid function

Iodine

• Iodine is required for thyroid hormone synthesis - A normal thyroid takes up about 40 mcg daily -  The recommended daily intake is 150 mcg - 220 mcg for pregnant women

• Iodine in the diet is 100% absorbed -  Iodine not used for thyroid hormone synthesis is

rapidly excreted in the urine

Iodine

• Iodine is a trace element -  Most of the earth’s iodine is in the oceans

• Iodine is only present in the the top layers of the soil -  Uplift and erosion or glaciers remove the iodine-rich upper

layers and create areas of iodine deficiency

• 2.2 billion people (40% of the world’s population) are at risk for iodine deficiency

Iodine Deficiency

• Mild iodine deficiency can result in thyroid enlargement (goiter) and thyroid nodules

-  Present in 30% of individuals in iodine deficient regions • Severe deficiency can cause hypothyroidism in the fetus

-  Resulting in failure of normal brain development -  The most avoidable cause of mental retardation

• Very severe deficiency can result in cretinism -  A specific form of physical and mental retardation

• Can be prevented by iodine supplementation of salt or bread

Evolution of the thyroid gland

• Protochordates produce T4 & T3 in an endostyle – a pouch below the pharynx

-  T4 & T3 are synthesized on the surface of the endostyle cells -  Mucous carries T4 & T3 into the gut where they are absorbed

• Bony fish have groups of thyroid hormone secreting cells scattered along the ventral aorta

• Higher vertebrates have an encapsulated thyroid gland which is under neuroendocrine control

Historical Background

• 1656 – Thomas Wharton described the thyroid gland -  Named it the “glandulae thyroideae” -  Suggested that its purpose was “to beautify the neck”

• 1896 – Baumann found a high iodine content in the thyroid -  Extracted a compound he named “thyrojodin” -  Effective in the treatment of hypothyroidism

• 1914 – Thyroxine (T4) was crystallized by Kendall • 1926 –T4 was synthesized by Harrington • 1958 – Tri-iodothyronine (T3) was found by Pitt-Rivers

Human Thyroid Gland

• The thyroid is located in the lower anterior neck -  It has two lobes which lie on either side of the larynx -  A thin connecting segment anterior to the trachea is called

the isthmus

• An adult thyroid weighs 6-20 grams -  Dependent on body size and iodine supply

• The thyroid receives 2% of the cardiac output -  May increase greatly in severe hyperthyroidism

Human Thyroid Embryogenesis

• 3rd week - the thyroid appears as a downward growth from the region of the primitive pharynx

• 4th – 12th week- the thyroid migrates caudally -  Failure of migration can result in an ectopic location

• 7th week – neural crest cells from the ultimobranchial bodies enter the thyroid as calcitonin secreting C cells

• 10-12th week – follicle formation begins -  Thyroid concentrates iodine & synthesis of T4 & T3 begins

• 18th week – neuroendocrine control is established -  significant secretion of T4 and T3

Microscopic Anatomy

• The thyroid gland is composed of small spherical structures called follicles

- The follicle wall is formed by epithelium one cell thick - T4 and T3 are synthesized by the follicular cells - T3 and T4 are stored in the lumen of the follicle in the

form of colloid

Microscopic Anatomy

• The follicles are surrounded by a rich capillary network

- Delivers iodine to the follicular cells -  Transports T4 and T3 secreted by the follicular cells to

the general circulation • Between the follicles are nests of C-cells

- Migrate into the thyroid during embryogenesis -  Secrete calcitonin, a calcium lowering hormone

TSH Effects on the Thyroid

• TSH (Thyroid Stimulating Hormone) stimulates all steps in the synthesis and secretion of thyroid hormones

• TSH action is initiated by binding to specific receptors in the basal membrane of thyroid follicular cells

Copyright ©2005 American Society for Clinical Investigation

Davies, T. F. et al. J. Clin. Invest. 2005;115:1972-1983

TSHR structure

Iodide Transport

• Iodide is actively transported into follicular cells by the sodium-iodide transporter (NIS)

-  Located in the basal membrane • Couples the inward “downhill” translocation of two Na+

with the inward “uphill” translocation of one I- Driven by the inwardly directed sodium gradient

generated by Na+/K+ ATPase • Iodide crosses the apical membrane into the follicular

lumen by a mechanism that is not well characterized

Thyroglobulin

• A large protein containing many tyrosines -  Synthesized by ribosomes in follicular cells - Glycosylated in the Golgi apparatus -  Transported to the apical membrane in small vesicles - Released into the follicular lumen

• Binding of iodine to tyrosine on thyroglobulin is the first step in thyroid hormone synthesis

Thyroid Hormone Synthesis

• Thyroid hormone synthesis takes place on the luminal side of the apical membrane

- Outside of the follicular cells in the colloid • All steps are catalyzed by thyroid peroxidase (TPO)

-  Located in the apical membrane

Thyroid Peroxidase

• Antibodies to thyroid peroxidase (TPOAb) - Marker for presence of autoimmune thyroid diseases - Hashimoto’s thyroiditis – major cause of hypothyroidism - Graves’ disease – major cause of hyperthyroidism

• A target for drugs used in the treatment of hyperthyroidism -  Anti-thyroid drugs (ATD)

Step 1 - Organification

• Iodide is first raised to a higher oxidative state by thyroid peroxidase

-  The reaction requires hydrogen peroxide (H2O2) generated by a flavoprotein oxidizing system

• The reactive iodide binds to tyrosines which are part of the thyroglobulin molecule to form iodotyrosines:

- Monoiodotyrosine or MIT - Diiodotyrosine or DIT

• This induces a conformational change in thyroglobulin

Step 2: Coupling

• Two iodotyrosines are joined via an ether link to form iodothyronines:

- Coupling two DIT molecules yields T4 - Coupling one MIT and one DIT yields T3 - This reaction is also catalyzed by thyroid peroxidase

• Iodinated thyroglobulin containing MIT,DIT, T4 and T3 is stored in the follicular lumen as colloid

Secretion of Thyroid Hormone

• Vesicles containing thyroglobulin are taken into follicular cells by pinocytosis

• Lysosomes fuse with the vesicles, digest thyroglobulin and release T4, T3, MIT and DIT

-  T4 and T3 diffuse into the surrounding capillaries -  MIT and DIT are deiodinated and the released iodide is

recycled for thyroid hormone synthesis • 80% of secreted thyroid hormone is T4 and 20% is T3

-  In iodine deficiency and hyperthyroidism the fraction of T3 is higher

Circulating T4 and T3

• Over 99 % of circulating T4 and T3 are bound to carrier proteins

-  Only 0.03% of T4 & 0.3% of T3 are free and available for transport into cells

• Protein bound and free hormones are in equilibrium -  As free hormones enter cells they are immediately replaced

• Result: a stable supply of T4 and T3 despite changes in the rate of secretion or metabolism

Transport Across Cell Membranes

• It was previously assumed that T4 and T3 crossed cell membranes by diffusion

• Recently several membrane-based transporters have been described

• MCT8 is the best characterized -  Found in many tissues -  Cells transfected with MCT8 cDNA have a marked

increase in both uptake and efflux of T4 and T3

MCT8 Mutations

•  MCT8 gene mutations are associated with a rare X-linked recessive syndrome first described in 1944

- Allan-Herndon-Dudley Syndrome is manifested by: - Severe mental retardation - Absent speech - Low muscle tone – inability to walk - Low levels of T4 - High levels of T3 - Slightly high TSH

T3 – The Active Thyroid Hormone

• 80% of thyroid hormone secretion is T4 • Circulating levels of T4 are 60 times greater than T3

BUT • T3 is responsible for most thyroid hormone actions

Discovering the Genomic Action of T3

•  1966 – T3 was found to stimulate DNA dependent RNA polymerase activity and to increase synthesis of new RNA

•  1974 – High-affinity low-capacity binding sites for T3 were discovered in the nuclei of rat tissues and cultured GH cells

Conclusions: 1. T3 is the active thyroid hormone at the cellular level 2. Nuclear receptors for T3 (TRs) mediate transcriptional

activation of T3 responsive genes

T3 Nuclear Receptors (TRs)

• The binding domain for T3 is on the carboxy terminal end -  T3 binding induces dissociation of repressors, binding of

activators and activates transcription • The DNA binding domain is located centrally

-  Interacts with the thyroid hormone response element (TRE) of T3 regulated genes

• Heterodimerization of TRs with retinoid X receptors( RXRs) enhances transcriptional activation

T3 Responsive Genes

• Positively regulated genes - When T3 is bound transcription is activated - Transcription is suppressed in absence of T3 binding

• Negatively regulated genes - When T3 is bound transcription is suppressed - Transcription is activated in absence of T3 binding

• Binding of co-activators enhances gene expression • Binding of co-repressors suppresses gene expression

T3 Nuclear Receptors (TRs)

• TRβ gene encodes three T3 binding isoforms - TRβ1, TRβ2 and TRβ3

• TRα gene encodes one T3 binding isoform - TRα1

• Cellular expression of TR isoforms is both tissue dependent and developmentally regulated

- Thyroid hormone action is modified in different tissues and at different stages of development

TRβ gene mutations

• Cause Resistance to Thyroid Hormone (RTH) • The clinical syndrome is variable and may include:

-  High T4 and T3 with normal or high TSH -  Goiter -  Short stature -  Decreased IQ -  Hearing loss -  Mild hyperthyroid symptoms -  AD/ADHD -  Dyslexia

Effect of Deiodinases

• The strength of thyroid hormone signaling depends on the level of TR occupancy.

-  In most cells 50% of T3 bound to TR comes from serum T3

-  The remaining 50% is produced locally from T4 by deiodinases

• Deiodinases control TR occupancy and thus thyroid hormone signaling in cells

- Allow cells to customize their own T3 footprint

Type 1 Deiodinase (D1)

• Located in the plasma membrane -  Deiodinates both inner and outer ring (both 5 and 5’ activity) -  Has both activating (T4 to T3) and inactivating (T4 to rT3

and T3 to T2) actions • T3 generated by D1

-  Equilibrates rapidly with serum T3 -  Contributes little T3 directly to TR occupancy

• Up-regulated by T4 -  Source of over 50% of circulating T3 in hyperthyroidism -  Cause of high T3/T4 ratio in hyperthyroidism

Type 2 Deiodinase (D2)

• The major activating deiodinase -  Converts T4 to T3

• Located in the endoplasmic reticulum –close to nucleus -  T3 generated by D2 contributes about 50% to TR occupancy

and equilibrates slowly with serum T3 • D2 levels can be varied rapidly due to its short half life

-  The half life is reduced by high T4 (suppressing T3 generation) and prolonged by low T4 (enhancing T3 generation)

-  Creates a rapid feedback loop which stabilizes intracellular T3

Type 3 Deiodinase (D3)

• Major inactivating deiodinase for both T4 and T3 - Converts T4 to reverse T3 and T3 to T2 -  Located in the plasma membrane

• Decreases thyroid hormone action at the cellular level -  Cells expressing D3 have lower TR occupancy and a gene

expression profile typical of hypothyroid cells

• Up-regulated by T3 -  Protects tissues from excess thyroid hormone -  Especially important in fetal development

Changes in D3 Expression

• Increased in hyper and decreased in hypothyroidism -  Contributes to T3 homeostasis

• Increased in myocardium and brain during hypoxia and ischemia

-  slows cell metabolism when oxygen supply is limited • Induced in liver and skeletal muscle by severe illness

-  Results in low circulating T3 and high reverse T3 -  Protects against the catabolic effect of T3 -  Part of the abnormalities seen in the “euthyroid sick” syndrome

Changes in D3 Expression

• Massive expression occurs in the early phases of regeneration after tissue injury

-  Lower TR occupancy by T3 favors cellular proliferation vs differentiation

• Highly expressed in hemangiomas in children -  can exceed the ability of the thyroid to synthesize thyroid

hormone resulting in “consumptive hypothyroidism” • Expressed in a number of human tumors

-  May play a role in initiating tumors by suppressing differentiation

Control of T3 Availability

• Availability of T3 to nuclear receptors (TRs) is controlled by the relative activities of D2 & D3

- Largely independent of circulating T3 & T4 • Expression of both enzymes is regulated in a

tempero-spatial and tissue-specific manner -  resulting in varying levels of T3 action in individual

tissues and at distinct times during development

Thyroid Hormones in the Brain

• Neurons express D3 but not D2 - Dependent on T3 generated by glial cells which is

translocated to neurons by a paracrine mechanism -  Sufficient to overwhelm D3 in the neurons

• Generation and translocation of T3 are potential control points for thyroid hormone signaling in brain

Thyroid Hormones in Brain

• Glial derived T3 in neurons is modulated by coordinated changes in D2 & D3 activity

- Hypoxia increases D3 in neurons - Which decreases T3 and slows metabolism

-  Inflammation increases D2 in glial cells - Which increases T3 and stimulates metabolism - This plays a role in the ”euthyroid sick” syndrome

Cardiac Effects of Thyroid Hormones

• T3 increases the rate and force of cardiac muscle contraction • Cardiac genes controlled in part by T3 include those for:

-  β-adrenergic receptors -  Ca++-ATPase -  Adenyl cyclase -  Na+/K+ -ATPase -  Sarcoplasmic reticulum -  α-myosin heavy chains

• T3 also has non-genomic actions in the heart including -  Increasing Ca++ transport in sarcoplasmic reticulum -  Effects on Na+ and K+ ion channels

Role of Thyroid Hormones in Metabolism

• Heat is produced from energy lost during the synthesis and metabolism of ATP

-  All species produce some heat – obligatory thermogenesis • Birds and mammals (homeotherms) need to produce more heat to

maintain a constant body temperature -  Basal thermogenesis

• Additional heat is required during cold exposure -  Facultative thermogenesis

•  In homeotherms 30% of heat production is dependent on T3 -  Via increased ATP consumption and decreased efficiency of ATP

synthesis

Facultative Thermogenesis

• Extra heat needed during cold exposure is generated by brown adipose tissue (BAT) and muscle

-  BAT is the major source of extra heat in rodents and human infants

-  Mitochondria in BAT contain uncoupling protein (UCP) which reduces the coupling of ATP synthesis to oxidation

-  Gene for UCP is stimulated by T3 • BAT contains D2

-  During cold exposure increased norepinephrine stimulation results in an increase in D2 and T3 generation

-  Higher T3 results in an increase in UCP and heat production

Non-Genomic Effects of Thyroid Hormones

•  Initiated by binding of T4 or T3 to cellular proteins other than TRs

-  Effects are rapid - do not require protein synthesis • Stimulation of membrane transporters has been best studied -

these include: -  Calcium ATPase -  Adenlate cyclase -  Glucose transporters

• T4 and T3 also have non-genomic effects on thermogenesis

Fetal Thyroid Development

• 12 weeks -  Thyroid follicles have formed -  Tg, NIS, TPO and TSH receptors are present -  iodine uptake begins

• Secretion of T4 & T3 is minimal before 18 weeks -  When the hypothalmic-pituitary axis becomes functional

• Before 18 weeks the fetus is entirely dependent on maternal thyroid hormone

-  Maternal hypothyroidism in early pregnancy causes abnormal brain development in the fetus

Deiodinases and Differentiation

• In the fetus nuclear receptors (TRs) act as developmental switches

-  Unoccupied TRs maintain cell proliferation and suppress differentiation

• High levels of D3 in the placenta and most fetal tissues keep T3 and TR occupancy low

-  Prevent differentiation

• Tissues expressing D2 can generate T3 locally -  initiate differentiation by increasing TR occupancy -  Fetal D2 expression varies both spatially and temporally

Changes after Full-term Delivery

• TSH rises abruptly to 60-70 µU/ml by 6 hours - Declines to normal by one week

• Total and free T4 levels peak above normal around day 7

-  decline to normal by day 28 • T3 rises rapidly to normal during the first week

- Continues to rise slowly to day 28 • Reverse T3 declines rapidly to low levels

Changes After Premature Delivery

• The changes in TSH and thyroid hormone levels are blunted in premature infants

• Low T4 levels are common -  Infants < 31 weeks may have no increase in T4 at birth -  T4 may be undetectable in very premature infants

• TSH is usually normal -  May reflect immaturity of the hypothalamic-pituitary-

thyroid axis

Congenital Hypothyroidism

•  Incidence is between l:2,500 and 1:4,000 births -  Caused by: thyroid agenesis or ectopy

absence of a thyroidal enzyme

• Can be detected by testing TSH and T4 at birth -  Screening programs are in place in most countries

• Early treatment with thyroid results in normal mental function -  In utero the brain is protected by maternal T4 and up-

regulated D2 • Failure to treat results in severe mental retardation

-  The brain is not fully mature until 2 years of age

Nuclear Accidents

•  I-131 is used to treat hyperthyroidism and thyroid cancer -  As it decays it emits β particles which damage thyroid cells -  The T1/2 of I-131 is 8 days

• Nuclear reactors contain huge amounts of I-131 -  I-131 represented 15% of the radioactivity released in the

Chernobyl accident in 1986 •  Iodine is highly volatile

-  I-131 released by overheated nuclear fuel rises into the atmosphere and returns to earth as fallout

• Milk is the major source of I-131 ingested by humans -  I-131 on vegetation is ingested by cows and concentrated by

their mammary glands

Thyroid Cancer After Chernobyl

• 4 years after Chernobyl an increase in thyroid cancer was detected in children in the contaminated areas

-  By 20 years there was an excess of 4,000 thyroid cancers in children exposed to I-131

-  Age dependent with the youngest at greatest risk

• Radiation dose to the thyroid is increased in children -  Radioiodine uptake is higher and the thyroid is smaller

• Children have increased susceptibility -  Dividing cells are more sensitive to radiation carcinogenesis

Iodine Prophylaxis after Reactor Accidents

• A normal thyroid gland concentrates 20-30% of ingested iodine (and radioiodine)

-  Often over 50% in areas of iodine deficiency • Uptake of I-131 by the thyroid can be almost completely

blocked by flooding the body with stable iodine -  Usually in the form of potassium iodide

• After the Chernobyl accident there was no restriction on drinking milk and no distribution of potassium iodide for several weeks