BS2050 Endocrine System Physiology - Thyroid Hormones

Attendance: This lab will be taught on two occasions in the PC laboratory between 1 pm and 4 pm. Students will be divided into two groups A and B and will complete the lab on the allocated day.

LEARN N! "#T$"%ES On the completion o this class students should! understand the ollowing terms! hormone" target cell" hypothalamus" hypothalamic# pituitary portal system" thyrotropin#releasing hormone" thyroid stimulating hormone" thyro$ine and tri#iodothyronine" hypophysectomy" negative eedbac% control. Be able to e$plain how negative eedbac% loops regulate hormone release &nderstand the roles o TS' and thyro$ine in regulating the basal metabolic rate (B)*+. &nderstand how the hypothalamus controls TS' and thyro$ine secretion. ntrod&ction The study o the e ects o hormones in the body is a di icult and time consuming processes because o the comple$ity o the regulatory systems involved and the time#scale o some hormonal e ects. ,urthermore many e ects can only be studied in live animals which need used in large numbers and to be sacri iced or e$perimental purposes in order to build a statistically signi icant picture o the regulatory events. )oreover" technically di icult procedures" such as hypophysectomy" are re-uired to illustrate particular physiological e ects o hormones. There are clearly ethical and legal considerations in carrying out such procedures on e$perimental animals" particularly when the results are already %nown" but it is important that students are aware o the techni-ues which are re-uired in the study o particular endocrine systems. .t has there ore been decided to use computer simulations in order to study this aspect o the course" where you will use virtual animals rather than live ones and where you will be able to carry out surgical operations at the clic% o a /mouse0 and to administer potentially no$ious substances without a 'ome O ice licence1 2ou will carry out the same sort o procedures but in the raction o the time (and cost+ these e$periments will ta%e in the real laboratory situation. Thyroid Hormones There are two ma3or thyroid hormones secreted by the endocrine thyroid gland which is located in the nec%" in ront o the trachea. These are thyro$ine (T4+ and tri#iodothyronine (T4+" which are" uni-uely" hormones which contain iodine atoms which are essential or the endocrine activity o these hormones. .n addition" calcitonin" produced in the para# ollicular cells o the thyroid is involved in the regulation o plasma Ca56 levels. The thyroid hormones are made" in the thyroid epithelial cells" rom two molecules o the amino acid tyrosine which are lin%ed together covalently whilst the tyrosine residues are part o a protein called thyroglobulin. This process re-uires thyroglobulin to be synthesised" iodine concentrated in the cells and modi ied to a orm o iodine which is used to iodinate the tyrosine residue. &nusually" stoc%s o the hormone are stored e$tra# cellularly in colloid ollicles. The modi ied thyroglobulin is then ta%en up by thyroid

epithelial cells and processed by proteolysis and the thyroid hormones released in response to the hormone TS' (thyroid stimulating hormone+.

A molecule o thyroglobulin contains 144 tyrosines" although only a ew o these are actually used to synthesi7e T4 and T4. .odide (.#+" is avidly ta%en up rom blood by thyroid epithelial cells" which have on their outer plasma membrane a sodium#iodide symporter (a transport protein which moves .# into the cell" driven by the 8a6 concentration di erence between the e$tracellular luid and the cytoplasm+. Once inside the cell" iodide is transported into the lumen o the ollicle along with thyroglobulin. Synthesis o thyroid hormones is catalysed by the en7yme thyroid pero$idase" an integral membrane protein present in the apical (colloid# acing+ plasma membrane o thyroid epithelial cells. Thyroid pero$idase catalyses two se-uential reactions! 1. .odination o tyrosines on thyroglobulin (organi ication o iodide+. 5. Synthesis o thyro$ine (or triiodothyronine+ rom two iodotyrosines. By the action o thyroid pero$idase" thyroid hormones accumulate in colloid" on the sur ace o thyroid epithelial cells. Thyroid epithelial cells ingest colloid by endocytosis rom their apical borders. Thyroglobulin is hydrolysed in lysosomes o thyroid epithelial cells thereby liberating ree thyroid hormones.

Actions o' Thyroid Hormones )ost o the thyroid hormone secreted rom the thyroid gland is T4" but T4 is considerably more active. Although some T4 is also secreted" the bul% o the T4 is derived by deiodination o T4 in peripheral tissues" especially liver and %idney. Thyroid hormones are poorly soluble in water and ther ore more than 99: o the T4 and T4 circulating in blood

is bound to carrier proteins" mainly thyro$ine#binding globulin" a glycoprotein synthesi7ed in the liver. T4 and T4 are important or normal growth and development and or energy metabolism. The actions o the hormones are very slow in comparison to most hormones" usually ta%ing days or wee%s to e$ert their e ects. T4 is more biologically active than T4 and its e ects are observed be ore those o T4 and it is believed that T4 is converted to T4 by an en7yme called deiodase whose unction is to remove one o the iodine atoms be ore the thyroid hormone becomes active. Thus T4 is regarded as a pro#hormone" meaning that it is a hormone precursor. Thyroid hormones produce a general increase in the metabolism o carbohydrates" ats and proteins" o ten by the modulation o the e ects o other hormones such as insulin" glucagon" glucocorticoids and the catecholamine hormones. Thyroid hormones are thus o ten described as permissive hormones. The general e ects o thyroid hormones are to cause increased o$ygen consumption and heat production" partly by increasing the turnover o ATP. This is mani ested in a general increase in basal metabolic rate re lecting the action o the thyroid hormones on some organs such as the heart" %idney" liver and muscle but not on others such as the gonads" %idney or spleen. The overall e ect is that thyroid hormones cause an increase in body temperature (calorigenic e ect+ which an important response to a cold environment. Thyroid hormone administration can lead to an increased cardiac output. The hormones also have a critical e ect on bone growth and development in children partly via interactions with growth hormone. Thyroid hormone has ma3or e ect on the development o the brain and C8S in the womb and during the neonatal period. T4 binds to speci ic receptors in the nuclear compartment o target cells. The e ects o thyroid hormone are mediated by the regulation o the transcription o the genes coding or %ey en7ymes and proteins. These transcriptional e ects o T4 can ta%e many days to mani est themselves" or e$ample" the e ects on O5 consumption" heat production ; ree radical ormation. There are also e ects on the cardiovascular system and on lung unction as well as e ects on the sympathetic nervous system where there is an increased sensitivity to adrenaline and noradrenaline. Thyroid hormones also a ect the unction o the haematopoietic" gastrointestinal and s%eletal systems. ,urthermore neuromuscular and neurological development are a ected by thyroid hormones< in other words there is hardly a tissue or organ in the mammalian body which is una ected by thyroid hormones. The important e ects o thyroid hormones are best illustrated in the long#term e ects o thyroid de iciency (or hypothyroidism+ which causes the conditions %nown as my$oedema in adults and cretinism in children. )y$oedema is characterised by a low metabolic rate and low body temperature" slow speech with deep hoarse voice" lethargy" bradycardia (decreased heart rate+" sensitivity to cold and mental impairment. Patients develop a characteristic thic%ening o the s%in which is symptomatic o the condition and is the derivation o the word my$oedema. One possible cause o thyroid de iciency is the lac% o iodide salts in the diet. Thyroid de iciency in children is characterised by gross retardation o growth and o C8S development resulting in mental retardation. 'yperthyroidism (thyroto$icosis+" on the other hand" results in high metabolic rate" high temperature" tachycardia" hyperactivity" and increased appetite associated with weight loss. Thyroto$icosis can be treated can be treated with radioactive iodine =141.> which is ta%en up and concentrated in the thyroid ollicles" the radioisotope emits and radiation and the latter destroys some o the thyroid ollicle endothelial cells" thus reducing thyroid hormone

production. This cytoto$ic e ect is a slow process and may ta%e appro$. 4 months to lower thyroid hormones. Alternatively the thioureylene drugs can be used" such as carbima7ole" methima7ole and propylthiouracil which all have a thiocarbamide group (S#C#8+. These also gradually reduce the output o the thyroid hormones over a 4 wee% period most probably by inhibiting the iodination o tyrosine residues in thyroglobulin by inhibiting thiopero$idase reaction by acting as competitive substrates. Propylthiouracil also may inhibit the deiodination o T4 to T4. Although these drugs cause an inhibition o thyroid organi ication o iodine within 15 hr o administration" the clinical response as measured by a lowering o B)* may ta%e several wee%s. E()erimental Proced&re Loading the PhysioE(5*0 $om)&ter )rogramme Switch on computer and log on to the college networ% Put the C? in the C?#*O) drive Clic% on %y $om)&ter Clic% on PhysioE( 5*0 +,:@o to the bottom o list o iles and then clic% on Start.PhysioE(*e(e 2ou should then see a tic%ing cloc% and clic% on Proceed Clic% on Agree .n the )ain )enu Screen! clic% on 4 Andocrine Systems Physiology 2ou should then be in the )etabolism page which is the e$perimental setup or this particular practical. Clic% on '&ll screen icon or best view.

To e$it the programme clic% on / in the top#right#hand corner Bog o the computer *eturn the PhysioE( $, and man&al to ,r ,a0ies be ore you leave the PC lab Scenario 2ou will be investigating the e ect thyro$ine and TS' on the metabolic rate o an e$perimental animal by investigating the e ect o various treatments on o$ygen consumption. The apparatus used consist o chamber which is a bell#3ar with a closed atmosphere" coupled to a manometer which allow us to measure o$ygen upta%e. The animals are placed in the chamber" weighed and the o$ygen consumption measured over a certain time period with the chamber and manometer connected. The carbon dio$ide evolved is absorbed by soda lime at the bottom o the chamber. At the end o the time period the chamber and manometer are disconnected and the o$ygen consumed measure by using a syringe to in3ect air into the tube" su icient to restore the luid in the manometer columns to their original levels. O$ygen consumption is noted together with the time and the weight o the animal and then the metabolic rate can be calculated in ml O5.hr#1.%g#1. There are three groups o animals to be tested! 1A2 Normal rats3 1B2 thyroidectomi4ed +T(- rats where the thyroid gland has been removed surgically

 1$2 hy)ohysectomi4ed +Hy)o( or H)(- rats where the pituitary gland has been removed. Both these surgical treatments have been carried out some time in advance to allow the animal to recover rom the surgical procedure and the animals have been %ept under constant environmental conditions with plenty o ood and water available. 2ou can record your data on screen or a limited number o e$periments but you will also need to copy your data manually on to the pro ormas provided ma%ing a note o the type o animal used" the body weight" the amount o$ygen evolved in a given time. E()eriment 5: E''ects o' thyroidectomy and hy)o)hysectomy on 6aseline meta6olic rate .n the irst set o e$periments you need to determine the baseline metabolic rates or the three groups o rats 1. &se the mouse to drag the rat into the chamber1 5. )a%e sure that the clam) is open and that the manometer and chamber is connected. . not" clic% on these using the mouse. 4. Clic% on the 7eigh button and record the weight (this will also be recorded temporarily under datasets+. $lose the clam). 4. Clic% on the timer 8 6&tton so that the timer reads any value between 1 and 5 minutes C. Then clic% start and the elapsed time will be shown and you will see an increase in o$ygen upta%e as indicated by the manometer until the timer stops automatically D. Connect the manometer and syringe by clic%ing on the T-connector and then clic% the 8 6&tton under ml O5" and then in3ect until the luid level in the two arms o the manometer are level with one another. E. Record the volume o air that is re-uired to do this and then calculate the o$ygen upta%e relative to the body weight in ml O5.hr#1.%g#1. F. Clic% on record data and ma%e a manual note o your data on the pro ormas provided 9. *epeat this up to 4 times or each group o rats. (There is some /biological variability0 designed into the programme+ Do the metabolic rates differ? Are the differences significant and if they are explain why the treatments have these effects on oxygen consumption. Explain the terms thyroidectomy and hypophysectomy and explain what the effects of these treatments have on the metabolism of these experimental animals. E()eriment 2: E''ect o' thyro(ine on meta6olic rate .nvestigate the e ects o in3ecting thyro$ine into the bloodstream o the e$perimental rats. 8ormally it would involve giving daily in3ections over a period o several wee%s but we are saved all that e ort and trauma. And by clic%ing on clean you can remove all traces o previously in3ected hormone rom circulation1 This would normally ta%e several wee%s or more li%ely you would need to start again with another rat. Select a rat to test Clic% the reset button under apparatus Clic% on the syringe labelled thyro$ine and in3ect it into the rat ,ollow instructions 1#9 as or A$periment 1

 Test the e ects in normal" thyroidectomised and hypophysectomised animals Does thyroxine affect the basal metabolic rate in all groups of animals? Are the effects statistically significant in each case? Explain how thyroxine exerts its effect and why it may take several weeks for these effects to be observed. E()eriment 9: The e''ect o' TSH on %eta6olic Rate Again the e ects o TS' may ta%e a ew wee%s to be observed and will re-uire TS' to be administered two or three times a day (e$pensive and time#consuming+. 2ou can assume that when TS' has been administered to the virtual rat that this procedure has been ollowed. ,ollow the instructions as in e$periment 4 e$cept that this time you in3ect with TS'. Does TS affect the basal metabolic rate in all groups of animals? Explain the differences in the response of each group of animals. Are there any differences between the effects of thyroxine and TS administration and if there are explain why? Explain how TS exerts its effect and why it may take several weeks for these effects to be observed. E()eriment :: E''ect o' )ro)ylthio&racil on meta6olic rate Propylthiouracil is a drug used to treat thyroto$icosis. Again it re-uires several wee%s o treatment be ore any e ect o this drug is seen. A$amine the e ect o administering this drug to all the di erent e$perimental groups. Explain the effects of the drug observed in each case and explain how propylthiouracil exerts its effects on the basal metabolic rate. The computer programme does not allow you to administer treatments simultaneously but you can speculate! with reasoning! what might be the conse"uences of a# in$ecting both TS and propylthiouracil simultaneously and %b# in$ecting thyroxine and propylthiouracil together. ;rite-&) Show your pro# ormas with all the data collected and the results calculated. Grite#up your report under the headings o Res&lts and ,isc&ssion and $oncl&sions. There is no need or an .ntroduction or )ethods section. Please be sure to address the "uestions shown in italics at the end of each experiment. The in ormation you need to answer the -uestions is in the introduction to this e$periment" lecture notes or the te$tboo%. Re'erences and Ac<no7ledgements A 8 )arieb 'uman Anatomy and Physiology Dth ed. Chapter 1D http!HHarbl.cvmbs.colostate.eduHhboo%sHpathphysHendocrineHthyroidHinde$.ht

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