The thyroid hormones A thesis concerning their action

J. theor. Biol. (1978) 73, 81-100

The Thyroid Hormones : A Thesis Concerning their Action A. J. HULBERT Department of Biology, University of Wollongong, Wollongong, N.S. W., Australia 2500 (Received 9 August 1977, and in revisedform

8 December 1977)

It is proposed that the thyroid hormones act on cellular membranes altering the level of unsaturation of the membrane fatty acids. These changes in membrane composition affect membrane fluidity and hence membrane function. Many of the effects of the thyroid hormones can then be explained as secondary results of a change in membrane structure and function. Such an action also explains the differences in membrane composition and membrane function between homeotherms and poikilotherms. These hormones are also involved in the tolerance of low temperature during hibernation.

1. Introduction The ground squirrel Spermophilus tridecemlineatus completely ceases release of hormones from its thyroid gland just before the hibernating season (Hulbert & Hudson, 1976). For a variety of reasons this “shut-down” of thyroid hormone secretion was suggested to be necessary for changes in membrane lipid composition to occur so that, at the low body temperatures of hibernation, this animal’s membranes would remain in a fluid state. Since that study and further work which showed that thyroid hormones do in fact influence the composition, structure and function of mitochondrial membranes in a non-hibernator (Hulbert, Augee & Raison, 1976), I have become convinced that the primary locus of thyroid hormone action is cellular membranes and more specifically on the membranes’ fatty acids. Initially hesitant about proposing such an action, but recently imbued with the spirit of a prefatory chapter in the Annual Review of Physiology (Burton, 1975) favouring the “publication of integrative concepts and generalizations, even if advanced by non-experts”, I shall present such a thesis concerning the action of the thyroid hormones. The literature concerning the thyroid is vast and this article in no way purports to be comprehensive, but is rather a selective review of critical 81

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aspects of thyroid hormone action. The thyroid gland secretes two hormones, triiodothyronine and thyroxine (Fig. 1) the effects of which are both complex and diverse. There are reversible and irreversible effects and the hormones act on most of the cells and tissues of the mammalian organism. Although the thyroid hormones are found in all vertebrates, it is only in the homeothermic (warm-blooded) animals that most effects are observed. This complexity has led to many hypotheses, but at present there is no generally accepted theory that explains the actions of these hormones at the cellular level. The main emphasis has been placed on the regulation of protein synthesis. One theory is that the thyroid hormones regulate protein synthesis by an action at the level of transcription (Tata, 1969) whilst others suggest they act at the level of translation, either directly (Carter, Faas & Wynn, 1975) or indirectly, via an initial action on mitochondria (Sokoloff, 1970). The nuclear site of action has been supported by the findings of nuclear “receptors” for thyroid hormones in recent years (Oppenheimer & Surks, 1975) but the specificity of such “receptors” and their physiological relevance has recently been called into doubt (Tata, 1975). It is suggested here that there are multiple sites of action in the cell and that these are the various membranes of the cell and its organelles. It is proposed that thyroid hormones associate with membrane lipids, that in the process of being deiodinated they change the relative proportions of saturated and unsaturated fatty acids and that this change in membrane composition affects membrane structure and membrane fluidity which in turn affect the different functions of these various cellular membranes. I shall examine the subcellular localization and the metabolism of the thyroid hormones, then their main physiological and biochemical effects and lastly their role in “warm-blooded” and “cold-blooded” vertebrates as well as hibernators. Firstly, however, something should be said regarding

L-thyroxine

3,5,3’-triiodothyronine Ho~o~cH2-I”cooH

FIG. 1. The chemical structure of the thyroid hormones.

ACTION

current concepts of biological in membrane function.

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membranes and the role of membrane lipids

2. The Role of Lipids in Membrane Structure and Function Modern thinking on biological membranes, whether they be plasma membranes or the membranes of intracellular organelles, centres around the “fluid-mosaic” model (Singer & Nicolson, 1972). In this model (Fig. 2) the membrane consists of a bilayer of lipid molecules containing a number of protein molecules. Some of the proteins are deeply embedded in the bilayer matrix, some may in fact penetrate right through the bilayer (both of these are called integral proteins), whereas others are only loosely attached to the membrane (called peripheral proteins). The lipid bilayer provides the basic permeability barrier whereas the various enzymatic and transport properties of the membrane are associated with the proteins. An important feature of this model is that individual lipid molecules are laterally mobile within the bilayer and that membrane lipids normally exist in a fluid (liquidcrystalline) phase. Many proteins are also capable of lateral diffusion within the membrane. The fluidity of a biological membrane is related to both the degree of unsaturation and the length of the various fatty acid chains that constitute the inner part of the membrane. The fluidity of lipid bilayers and biological membranes that are composed of a variety of fatty acid chains have been examined using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) techniques (Lee, 1975). It is possible in some experimental systems to alter the fatty acid composition of cellular membranes (Hazel, 1973; Liepkalns & Spector, 1975) and various workers have studied the effects of such changes in membrane

FIG. 2. A diagrammatic representation of the “fluid-mosaic” model for biological membranes. The large molecules represent proteins, whilst the small molecules represent lipids. “Boundary lipids” are shaded (adapted from Singer & Nicolson, 1972).

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composition on membrane function. In young rats it is possible to vary the composition of erythrocyte membrane fatty acids by manipulation of dietary fat. The apparent permeability of such altered erythrocytes is correlated with the fatty acid make-up of their membrane lipids (Walker & Kummerow, 1964). Just as the permeability properties of a membrane are influenced by its fatty acid composition so are the transport and enzymatic functions of the membrane proteins. Many membrane proteins require lipids to be present for full enzymic activity (Coleman, 1973). Some membrane proteins require the presence of a layer of tightly bound lipid (boundary lipids) to stabilize them in their active form (Jost, Griffith, Capaldi & Vanderkooi, 1973). The activity of membrane proteins can be influenced by the fluidity of the lipid bilayer in which they are embedded. For example, changes in membrane fatty acid composition and thus membrane fluidity can alter the activity of the plasma membrane (Na+ + K+)-ATPase in some cells (Solomonson, Liepkalns & Spector, 1976). At normal body temperatures the membrane lipids in homeothermic (warmblooded) animals exist in the liquid-crystalline (fluid) phase. However, as temperature is lowered the membrane lipids undergo a phase transition, passing from the liquid-crystalline state through a transition of mixed liquid-crystalline and gel phases and finally they pass into a predominantly gel (solid) phase. This phase transition for rat liver mitochondrial membranes starts at about 23°C and finishes at approximately 8°C (Raison & McMurchie, 1974). The interaction between membrane lipids and membrane function is also clearly demonstrated by the increase in Arrhenius activation energy (Ea) of membrane-associated enzymes below the temperature limits of the lipid phase transition (Raison, 1973). It should be emphasized that only relatively small changes in membrane fatty acids are required for significant changes in both the phase transition temperatures and in membrane function to occur (Raison, 1973). 3. Cellular Localization

of Thyroid Hormones and their Metabolism

Hillier (1970) has demonstrated that the thyroid hormones bind to phospholipid membranes and he has also shown that there is a strong correlation between in vivo tissue thyroxine concentration and the phospholipid content of various tissues from the laboratory rat. Phospholipid content of a tissue can be regarded as an index of its membrane density. This binding of the thyroid hormone is very rapid and appears to be related to the hydrophobic nature of the aromatic portion of the two molecules (see Fig. 1). Autoradiographic evidence, using cultures of developing nervous tissue, suggests that, within the cell, thyroxine is bound at multiple sites. It was

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found that the cell membrane, mitochondria, ribosome-“mixed” endoplasmic reticulum, nucleus and synapse were labelled in order of decreasing intensity. These sites were all labelled as early as 15 min after hormone exposure and there was increasing accumulation of the hormone at all sites after longer exposure (Manuelidis, 1972). Similarly, an analysis of the various subcellular fractions of rat liver cells also suggests that thyroxine is distributed at multiple cellular sites. Over half of the intracellular thyroxine is bound to microsomes (= endoplasmic reticulum+plasma membrane). Both the nucleus and the mitochondria also show significant binding of hormone and the remaining thyroxine is found in the supernatant fraction. Phenobarbital administration, which dramatically increases the amount of smooth endoplasmic reticulum in the liver, also dramatically increases the amount of thyroxine bound by this subcellular fraction (Schwartz, Bernstein & Oppenheimer, 1969). The finding of thyroxine at these multiple cell sites supports the thesis that the thyroid hormones act at multiple cell sites, specifically cellular membranes. Much recent attention has been devoted to the in vitro binding of thyroid hormones to nuclear “receptors” molecules which have been thought to be analogous to steroid hormone receptors. The information regarding nuclear receptors for thyroid hormones has been reviewed elsewhere (Oppenheimer & Surks, 1975). As mentioned previously, Tata (1975) has recently questioned both the specificity and physiological relevance of such “receptors”. Although binding of thyroid hormones to nuclear proteins does occur no link between this binding and gene expression (which represents hormone action in the steroid hormone analogy) has been shown. The thyroid hormones can be metabolized by a number of processes, one of which is deiodination. A correlation between hormone deiodination and hormone action has been reported by Galton 8z Ingbar (1962) who noted that thyroxine had no calorigenic effect in poikilothermic animals which do not deiodinate the hormone. The current thesis suggests that in the process of exerting their action the thyroid hormones are deiodinated at cellular membranes. Oppenheimer, Shapiro, Schwartz & Surks (1971) reported a supposed dissociation between the deiodination of thyroxine and its action. They found that increased hepatic deiodination of thyroxine in phenobarbital-treated rats was not associated with an increased hormonal effect. The increased deiodination is a microsomal event whereas the effects they measured were mitochondrial events. If, as previously suggested, the thyroid hormones act at multiple cell sites then one would expect that such an increase in microsomal binding and deiodination of thyroxine would result in less thyroxine for other cellular membranes and therefore a reduction in its effects on mitochondria. Their results in fact show significant decreases

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in mitochondrial enzyme activity in all tissues measured and thus support the present thesis. Deiodination takes place in many tissues but predominantly in liver and kidney (De Groot & Stanbury, 1975). Both these tissues are membrane dense as measured by their high phospholipid content. At the subcellular level deiodination in the liver takes place predominantly in microsomes (Hesch, Brunner & Soeling, 1975). Mitochondria also have deiodination activity (Rall, Robbins and Lewallen, 1964) and it is known that thyroid hormones will bind to erythrocyte plasma membrane lipids (Segal, Carter, Singh & Kydd, 1975) and undergo a membrane-linked deiodination (Reinwein & Durrer, 1972). Deiodination of thyroid hormones varies in detail in different tissues and it may be that not all deiodinating mechanisms are linked with hormonal action. Jorgensen (1970) reports some alkyl derivatives of thyronine which show thyromimetic activity. It is interesting that although these molecules do not contain iodine at the 3’ or 5’ position (but do have it at the 3 and 5 position) the alkyl derivatives which show the greatest thyromimetic activity have a hydrophobic character similar to that of the natural thyroid hormone. Triiodothyronine is more hydrophobic than thyroxine (Hillier, 1970) and this may be connected with its greater physiological potency. Presumably both D-thyroxine and L-thyroxine have the same hydrophobicity ; however, only L-thyroxine is hormonally active (Jorgensen, 1970). This is probably due to only the L-isomer being able to undergo enzymatic deiodination. The above suggests that it is the enzymatic removal of the inner two iodine atoms from both triiodothyronine and thyroxine that is important in the exertion of hormonal action and that the other features of these molecules (and their derivatives) are probably important in the determination of the right degree of “hydrophobicity”. Although very little is known about the exact molecular details of the process recent studies on the deiodination of thyroxine by rat liver microsomes have shown that in vitro deiodination is associated with the formation of lipid peroxides (Nakano, Tsutsumi & Ushijima, 1971). Although lipid peroxides may not be formed in vivo it is a very interesting finding, because lipid peroxide formation requires unsaturated fatty acids. Thus it links the possible removal of unsaturated fatty acids from membrane lipids with hormonal metabolism. In this thesis I propose no stoichiometry for the deiodination-change in fatty acid process; however, I do not feel it should be assumed it is necessarily 1 :I. Although the precise molecular details of the process are unknown it has now been shown by several independent workers that a number of membranes can have their relative unsaturation changed by physiological manipulation of thyroid status (Patton & Platner, 1970;

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HORMONES

Ricquier, Mory & Hemon, 1975; Chen & Hoch, 1976; Shaw & Hoch, 1976, 1977; Steffen & Platner, 1976). 4. Effects of the Thyroid Hormones

The next three sections will attempt to explain the various effects of the thyroid hormones as effects that can be associated with a membrane site of action and they will also show, where possible, that many of these membrane-associated events are indeed influenced by the nature of the surrounding membrane lipids. (A)

EFFECTS

ON RESPIRATION-THE

“CALORIGENIC

EFFECT”

The elevation of metabolism is the most conspicuous effect of the thyroid hormones and is called its “calorigenic effect”. Complete thyroidectomy can eventually lead to as much as a 50% decrease in basal metabolic rate. All tissues but brain, testes and spleen appear to take part in this elevated metabolism and all classes of foodstuffs, protein and lipid are the substrates (Sokoloff, 1970). Much attention has therefore been focused on the effect of the thyroid hormones on mitochondria. The activity of several components of the mitochondrial respiratory chain is increased by the thyroid hormones (Wolff and Wolff, 1964), and they are also known to stimulate mitochondrial protein synthesis (Primack, Buchanan & Tapley, 1970). Although some of the increased activity may be due to more mitochondrial enzymes there are also direct effects, since it has been shown that thyroxine will stimulate mitochondrial oxygen consumption in vitro even when protein synthesis is inhibited (Buchanan, Primack & Tapley, 1971). The stimulation of protein synthesis by the thyroid hormones will be discussed in the next section. The fact that the mitochondrial respiratory chain is influenced by the lipids of the inner mitochondrial membrane is illustrated by the temperatureinduced phase transition that has been observed in mitochondria from various homeotherms (Raison & McMurchie, 1974). It has been recently shown that the thyroid hormones can change the fatty acid composition of mitochondrial membranes and thus membrane fluidity and membrane function (Hulbert, Augee & Raison, 1976). The data from this paper are presented in Fig. 3 whereby it can be seen that both the Arrhenius activation energy, Eu, of succinate oxidase (a multi-enzyme complex that includes most of the respiratory chain) and the upper limit of the phase transition are related to the fatty acid composition of the mitochondrial membrane. It is understandable that the Ea of a series of enzymic interactions which take

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thyroidectomired I

7

60 -

I I

1

t t

iA

42 : n,40-0.e .- I q

c

1

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:4 20rol .E 4 ::

ii

thyroxinetreated I

control

a

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0 75 Percent

I

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of

50

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acids

FIG. 3. Relationship between the relative unsaturation of rat liver mitochondrial membrane fatty acids and (i) the upper limit of the phase transition (top), and (ii) the Arrhenius activation energy of succinate oxidase in the same mitochondria (bottom). The changes in all parameters were brought about by manipulation of thyroid status (centre). All data are taken from Hulbert, Augee & Raison (1976).

place within the hydrophobic interior of a membrane should be influenced by the nature of this micro-environment. The fact that significant changes in membrane composition occurred within 12 hours after a single in uivo injection of thyroxine support the current proposal especially when compared with the rather lengthy periods required before other thyroid hormone effects become evident (Tata, 1969). In the early 195Os, the mechanism of thyroid thermogenesis was proposed to be via the uncoupling of oxidative phosphorylation which would result in an elevated mitochondrial oxygen consumption without a corresponding increase in ATP synthesis. This theory has been generally discarded because the measurement of uncoupling required high doses of the thyroid hormones, Whether the thyroid hormones cause some loosening of coupling in the normal

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situation is still controversial (Hoch, 1971). Any proposal for thyroid hormone action should be able to explain the “uncoupling” of oxidative phosphorylation that can be caused by high doses of thyroid hormones. An action on the mitochondrial membrane would seem most appropriate especially since it now appears that the coupling of electron transport and phosphorylation occurs via a hydrogen ion gradient that is created across the inner mitochondrial membrane (Lehninger, 1975). Furthermore, it has been shown by dietary manipulation of membrane fatty acids in the yeast Succharomyces cerevisiae that a large decrease in unsaturation of mitochondrial membrane fatty acids (the sort of change expected by large doses of the thyroid hormones) uncouples oxidative phosphorylation. This uncoupling is completely reversible and is due solely to changes in membrane lipid composition (Linnane, Haslam & Forrester, 1972). Changes in the composition of the mitochondrial membrane would simultaneously affect many different mitochondrial properties. One of the most dramatic effects of the thyroid hormones is on the mitochondrial enzyme, glycerol phosphate dehydrogenase. This enzyme is a flavoprotein tightly bound to the inner mitochondrial membrane, which together with a soluble glycerol phosphate dehydrogenase found in the cytoplasm, forms a shuttle for transferring reducing equivalents across the outer mitochondrial membrane (Lehninger, 1975). It is of interest that, although there are dramatic increases in the activity of the mitochondrial membrane-bound enzyme following administration of thyroid hormones, the soluble enzyme is unaffected (Lee, Takemori and Lardy, 1959). Another mitochondrial activity stimulated by thyroid hormones is the ADP-ATP carrier (Babior, Creagan, Ingbar & Kipnes, 1973). This is located in the inner mitochondrial membrane and is responsible for the passive exchange of ADP and ATP across the inner mitochondrial membrane. Its behaviour is known to be affected by changes in fluidity of membrane lipids (Pfaff, Heldt & Klingenberg, 1969). Similarly, a membrane site of action would also explain the swelling of mitochondria that can be induced in vitro by very low concentrations of the thyroid hormones (Lehninger, 1959). Recently, emphasis on the “calorigenic effect” has shifted from ATP production to ATP utilization; from the mitochondrial membrane to the plasma membrane. It has been shown that a large part (but not all) of the calorigenic effect of the thyroid hormones can be accounted for by stimulation of the Na+ pump (Ismail-Beigi & Edelman, 1970, 1971). Thyroid hormone stimulation of the sodium pump in renal cortical tissue (Lo &z Edelman, 1976) and in heart tissue (Philipson & Edelman, 1977) seems to be due, not to changes in the surrounding membrane lipid, but due to increased synthesis of the Na+/K+-ATPase enzyme. The thyroid hormones

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can also stimulate the transfer of other ions, for instance they stimulate both calcium and magnesium transport in liver tissue (Wallach, Bellavia, Gamponia & Bristrim, 1972). The activities of numerous enzymes are affected by these hormones (Wolff & Wolff, 1964) and since they stimulate protein synthesis, it would be necessary to determine for each individual enzyme whether hormonallystimulated changes in its activity are due to an increase in protein synthesis or a more direct activation via the surrounding membrane lipid (if the particular enzyme is membrane bound). Of course in some instances both may be applicable. (B) EFFECTS

ON PROTEIN

SYNTHESIS,

GROWTH

AND

DEVELOPMENT

In young mammals, thyroid hormones profoundly influence growth and development. Fetal or post-natal hypothyroidism can have irreversible effects on the central nervous system leading to cretinism. A dramatic effect of the thyroid hormones is their role in amphibian metamorphosis. Most of these effects on growth and development are thought to be mediated by the action of the thyroid hormones on protein synthesis. Within two hours after administration of triiodothyronine, microsomal protein synthesis is enhanced in liver tissue (Sokoloff, Roberts, Januska & Kline, 1968). The stimulus occurs at the level of translation and has been studied extensively in o&o. Initially it was thought to be dependent on a specific “mitochondrial factor”, but recent work has shown it is a direct action at the polysomal level requiring an ATP-generating system in the incubation medium (Carter et al., 1975). Protein synthesis, as measured by amino acid incorporation has been shown to be influenced by the fluidity of the membrane with which the polysome is associated during both microsomal (Towers, Kellerman, Raison & Linnane, 1973) and mitochondrial protein synthesis (Towers, Raison, Kellerman & Linnane, 1972). It has also been shown that thyroid hormones affect microsomal membrane fatty acid composition in the same manner as they do mitochondrial membrane fatty acids (Steffen & Platner, 1976). It is thus conceivable that the stimulatory effects of the thyroid hormones on translation may be via an action of membrane lipids. The polysomal fraction used by Carter et al. (1975) in their recent in vitro studies contained phospholipid (approximately 5% of the phospholipid found in unfractionated microsomes) and in this respect it would be interesting to know if the composition of these lipids is changed by the thyroid hormones. The thyroid hormones are also known to stimulate in vivo incorporation of labelled precursor into nuclear RNA in rat liver (Tata & Widnell, 1966). Incorporation data from in viva labelling can, however, be complicated by

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several factors. For instance, it assumes that the permeability of the cell membrane to the labelled precursor is not rate limiting. Observed changes in labelled RNA with time may in fact be due to changes in the permeability characteristics of the cell membrane to the labelled precursors rather than a direct stimulation at the transcriptional level. This is illustrated in recent work by Gee1 (1975) who found an increased incorporation of labelled precursors into RNA in the brain of young hypothyroid rats relative to controls. He also found an elevated precursor pool size in these rats and suggested that the thyroid hormones influence labelled RNA synthesis by regulating the intracellular uptake of labelled precursors at the cell membrane. In adult mammals changes in brain function induced by hypothyroidism are reversible. On the other hand, changes caused by hypothyroidism in fetal life or during infancy are sometimes irreversible and appear to be related to depressed synthesis of particular proteins. These normally appear at relatively fixed times during cerebral development and are associated with myelination of nervous tissue (Wysocki & Segal, 1972). This area has been reviewed by others (Dunn, 1972). Recent work has suggested that some of the enzymes involved in the synthesis of myelin sheath lipids undergo a degree of in uioo regulation via changes in the composition of membrane phospholipids with which the enzymes are associated (ConstantinoCeccarini & Suzuki, 1975). An extensive treatment of the role of the thyroid in amphibian metamorphosis is beyond the scope of this paper and is covered elsewhere (Cohen, 1970; Frieden, 1967). Griswold 8z Cohen (1972) have more recently shown an increase in the enzyme RNA polymerase following thyroxine treatment in tadpoles. The hormone stimulated increase in activity of this enzyme has a relatively long lag time and these authors suggest that thyroxine may have “an effect at some other level of cellular function before RNA polymerase activity is stimulated”. The increased activity of this enzyme could be due to a general increase in protein synthesis, possibly caused by thyroxine acting at the level of translation. Much attention has been focused on the role of thyroid hormones in the initiation of urea synthesis in the liver in metamorphosis during which the amphibian changes from the ammonotelic metabolism of the tadpole to the ureotelic metabolism of the adult (Cohen, 1970). In this respect it is interesting that recent work suggests that mitochondrial membranes could be involved in the regulation of urea synthesis in rat liver cells (Meijer et al., 1975). Although there would appear to be a direct effect of the thyroid hormones on translation (Carter et al., 1975) stimulation of protein synthesis is probably not solely due to an effect on translation. For example the increase in

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RNA polymerase activity mentioned above would probably have an effect at the transcriptional level but this would be indirect. Recent work (Ku&, Sippel & Feigelson, 1976) has demonstrated a thyroid stimulated increase in messenger RNA specific for the hepatic protein, azu globulin. This might suggest the thyroid hormones act pretranslationally, however, in order to determine whether this effect is close to the primary action of the hormones it would be necessary to measure the effect sooner than the 2-10 day period after hormone treatment used in the study. This would also help to lessen the possibility that the increase in messenger RNA is mediated via a thyroid hormone effect on other hormones which are known to exert pretranslational control of gzU globulin synthesis (Sippel, Feigelson & Roy, 1975). The thyroid hormones do not equally stimulate the synthesis of all cellular proteins and unfortunately the present thesis has nothing to propose for this situation except to suggest that, at least initially, only those proteins synthesized on membrane-bound ribosomes may be among those affected. (C)

OTHER

EFFECTS

The thyroid hormones have many effects besides those already mentioned, and many of these are also explicable in terms of a membrane site of action. For instance, some clinically important reflexes appear to be slowed in hypothyroidism (Costin, Kaplan & Ling, 1970) and the excitability of the central nervous system is reduced (Woodbury, 1954). Labelled thyroxine accumulates at the synapse as well as other cell sites (Manuelidis, 1972) and there is electrophysiological evidence that thyroxine alters synaptic transmission (Ignatov, 1970). The thyroid status of rats affects their spontaneous behavioural activity and such effects have been correlated with an influence of these hormones on the catecholamine synapse in the central nervous system (Emlem, Segal & Mandell, 1972). Thyroid hormones act at the neuromuscular junction lowering both the amplitude of miniature end-plate potentials and the resting membrane potential (Hofmann & Denys, 1972). Recent measurements of events at individual acetylcholine receptors in muscle cells have shown aspects of their behaviour to be very much influenced by the fluidity of the surrounding membrane lipids (Lass & Fischbach, 1976). Muscle becomes noticeably inefficient in the hyperthyroid state. The thyroid hormones stimulate the Ca2+ transport system in rabbit muscle (Suko, 1973) and by this effect influence muscle contraction. Work on the calcium transport system has shown that its activity is very much affected by the composition of the surrounding membrane lipids (Warren et al., 1974) and thus it is possible that the thyroid hormones exert their influence on this membrane-bound system by changing membrane lipid composition. Thyroid hormones also influence various membrane-associated events in

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the heart. It is a familiar clinical observation that the heart rate is increased in hyperthyroidism. Pacemaker cells from the isolated hearts of hyperthyroid rabbits show an increased rate of depolarization, a decreased duration of the action potential and hence a faster beat rate when compared to normal rabbits (Johnson, Freedberg & Marshall, 1973). The beat rate of isolated heart from normal rabbits appears to be influenced by the fluidity of heart membrane lipids (McMurchie, Raison & Cairncross, 1973). The thyroid hormones are also known to increase the conduction velocity of the cardiac impulse through the atrioventricular node (Shahawy, Stefadouros, Carr & Conti, 1975). Wildenthal (1972) has shown that triiodothyronine acts directly on heart tissue to increase its sensitivity to catecholamines. Catecholamines act on heart tissue via the adenyl cyclase system which is located in the plasma membrane and has been shown to have a definite requirement for the presence of phospholipids in order that its sensitivity to the catecholamines be operative (Levey, 1971). It is conceivable that the reported direct effect of the thyroid hormones on this membrane-bound cardiac adenyl cyclase system (Levey & Epstein, 1968) is mediated via an action on the surrounding membrane lipids. Adipose tissue responds to catecholamine by increasing the breakdown of stored fats and this response is mediated via another membrane-bound adenyl cyclase system. Hypothyroid subjects show a markedly diminished lipolytic response. It has been reported that the activity of the membranebound adenyl cyclase is affected by the thyroid hormones (Grill & Rosenquist, 1973) although other authors contest this finding (Correze, Laudat, Laudat & Nunez, 1974). Recently it has been suggested that the thyroid hormones exert their influence on adipose tissue by regulating the activity of a different membrane-associated enzyme, a phosphodiesterase enzyme (Armstrong, Stouffer & van Inwegen, 1974). This membrane-associated enzyme would be responsible for the destruction of cyclic AMP produced by the membrane-associated adenyl cyclase following catecholamine stimulation. Whatever is the final outcome of this disagreement, it would appear that the cell membrane remains a reasonable site of action for the thyroid hormones in adipose tissue. 5. The Role of the Thyroid in Cold-blooded and Warm-blooded Vertebrates

So far, I have concentrated discussion on the effects of the thyroid hormones in “warm-blooded” (homeothermic) vertebrates and, except for a mention of amphibian metamorphosis, have ignored any effects these

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hormones may have in “cold-blooded” (poikilothermic) vertebrates. I intend now to compare the effects of the thyroid hormone in these two groups and then to compare the nature of cellular membranes in these two groups. There is evidence that in several species of fish the thyroid hormones stimulate growth and protein synthesis. The effect, though definite, is limited and the pervasive effects of the thyroid hormones on mammalian growth are not seen in fish (Etkin & Gona, 1974). Thyroid hormones increase the incorporation of labelled amino acids into plasma, liver and gill protein (Narayansingh & Eales, 1975) in fish but this effect is not dramatic. It is of interest that this study showed that thyroid hormones change the pool size of labelled amino acid after their in vivo administration. Such changes are consistent with an action of the thyroid hormones on the cell membrane. Apart from their previously mentioned role in amphibian metamorphosis, there appear to be no studies that indicate the hormones from the thyroid gland stimulate the growth of reptiles or non-metamorphosing amphibians. Early work showed that the thyroid hormones had negligible effects on the general metabolism of poikilothermic vertebrates (Etkin & Gona, 1974). Unlike the situation in homeothermic vertebrates there appeared to be no “calorigenic effect” in poikilotherms. More recently it has been shown that in a variety of amphibians and reptiles, the general metabolic response of the tissues of poikilotherms to the thyroid hormones is dependent on their temperature (Maher, 1965, 1967; Packard & Packard, 1975). For instance thyroid hormones have a calorigenic effect in lizards when they are kept at 30°C but have no measurable effect at 20°C (Maher, 1965). Since most work on poikilothermic vertebrates is on animals at room temperature, it seems fair to assume that these studies have, in most cases, been on animals in which the thyroid hormones have no effect on metabolism. It is therefore of some interest that the membrane lipids of poikilotherms are more unsaturated than the corresponding membrane lipids of homeothermic vertebrates (Richardson & Tappel, 1962; Natochin, Leont’ev & Maslova, 1975). Such differences in membrane lipid composition correlate with differences in membrane function. Although homeothermic vertebrates show temperature induced changes in membrane-associated activities such as mitochondrial respiration, (Na+ + K+)-ATPase and heart rate, which correlate with temperature-induced changes in the state of the membrane lipids, poikilothermic vertebrates show no such changes in membrane-associated events, presumably because their membrane lipids undergo no temperatureinduced phase transition (McMurchie et al., 1973; Lyons & Raison, 1970). These differences in membrane structure and function are easily explained if the thyroid hormones exert their effects in homeotherms but not in poikilotherms (at room temperature). When thyroid function is impaired in homeo-

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therms many cellular membranes revert to a condition similar to that found in poikilotherms, that is, they become more unsaturated (Hulbert, Augee & Raison, 1976; Steffen 8z Platner, 1976; Shaw & Hoch, 1976). It is understandable that a poikilotherm would possess a membrane lipid composition that would allow its membranes to remain in a fluid (liquidcrystalline) state over a wide range of body temperatures. With the attainment of a constant body temperature, the homeothermic vertebrates were freed of this constraint and it became physiologically feasible for their membrane lipids to become less unsaturated. But what evolutionary advantage was gained by such a change in membrane lipids? A guide to the answer possibly lies in Fig. 3, where it can be seen that decreasing the unsaturation of mitochondrial membrane lipids results in a lowering of the “Ea” of mitochondria oxidation of succinate (in the region of a “fluid” membrane). The Ea of succinate oxidation by mitochondria from a wide range of vertebrates is related to the phase transition temperature of the mitochondrial membranes which is in turn related to the lipid composition of the membranes (Raison, personal communication). If other membrane-associated events were affected likewise then we may perceive a definite advantage in this evolutionary change in membrane lipid composition.

6. The Role of the Thyroid in Hibernation

and its Implications

Unlike poikilotherms, homeotherms cannot withstand low body temperatures and generally die if kept at body temperatures of 15-20°C for any extended period of time (Swan, 1974). At these temperatures the cellular membranes of homeotherms have undergone their temperature-induced phase transition and their membrane-associated functions are dramatically impaired. It is probable that such temperature-induced changes are an important factor in death at these low temperatures. There are some homeotherms, however, the hibernators, which are capable of tolerating low body temperatures. They revert to a “poikilothermic-like” condition and use such hypothermia as an energy-saving mechanism. Hibernating ground squirrels (Spermophilus) will allow their body temperature to drop to just above freezing and readily tolerate such a level. The obvious questions concern the nature of the membrane lipids in these animals and their effects on membrane-associated events. During the active season the liver mitochondrial membranes of ground squirrels are typical of other homeotherms in that they show a temperature-induced phase transition that commences at about 23°C. During the hibernating season, however, the

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upper temperature limit of this phase transition decreases to below 4°C (Raison & Lyons, 1971). Obviously there has been a change in membrane lipids between the seasons. Liver mitochondrial fatty acids are more unsaturated in hibernating ground squirrels than active ground squirrels (Lerner, Shug, Elson & Shrago, 1972). There is also an increase in unsaturation of fatty acids, as well as changes in the types of phospholipids, in the heart of hibernating ground squirrels (Aloia, Pengelley, Bolen & Rouser, 1974). The cellular membrane lipids of these animals have reverted to a “poikilothermic” condition and thus remain fluid even at very low body temperatures. Prior to the hibernating season, the thyroid gland of ground squirrels ceases secretion of the thyroid hormones and probably allows such changes in membrane lipids to occur (Hulbert & Hudson, 1976). Other membrane functions also change in hibernation. For example, erythrocytes from hibernating ground squirrels are much less permeable to urea than erythrocytes from active ground squirrels (Chilian & Tollefson, 1976). Another hibernator, the echidna (Tachyglossus aculeatus), has been shown to change the unsaturation of its mitochondrial membrane lipids between the active and hibernating state and such changes are correlated with changes in membrane fluidity and membrane function (McMurchie & Raison, 1975). Thyroid function in this animal is being investigated. Extensive studies of the cold resistance of the hamster’s brain during hibernation have shown that it is very much associated with a qualitative change in the nature of (Na” +K+)-ATPase (Goldman & Albers, 1975), and that this change is due to alterations in the composition of membrane lipids in the hamster’s brain prior to hibernation (Goldman, 1975). Such changes in brain membrane lipids may be mediated by changes in thyroid function. Some of the implications of work in this area are intriguing. For example, it may be possible by manipulation of thyroid status to prepare an individual for tolerance of a low body temperature. Such changes in membrane lipids would take some time but could make hypothermic surgery considerably less dangerous. It is of interest that the extensive feeding of unsaturated fatty acids to laboratory rats is known to lower the temperature at which their heart stops beating (Huttunen & Johansson, 1963). As well, it could mean that body organs from hypothyroid subjects are better organs for transplants in that they will keep better during low temperature storage. A study examining the effects of thyroid hormones on the preservation of stored kidneys for later transplantation concluded that these hormones are not beneficial in such circumstances and in fact “seem to further the histological changes observed in stored kidneys” (Villasante et al., 1971). Obviously it would be of advantage to examine the effect of hypothyroidism on tissue storage.

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7. Summary and Conclusions In the present paper I have presented a thesis that the thyroid hormones exert their cellular effects by acting on the various membranes of the cell. It is proposed that in the process of being deiodinated the thyroid hormones change the unsaturation of membrane fatty acid and that such changes affect both the permeability and catalytic activities associated with the membrane. It is also proposed that this action of the thyroid hormones explains a basic difference in the membrane composition and membrane function of homeotherms and poikilotherms and is also involved in the tolerance of low body temperatures by hibernators. Such an action has medical implications and possible applications. The present thesis is based mainly on “circumstantial evidence” (though, I believe, strong evidence) thus some parts of it may, in time, be shown to be either unduly emphasized or just untrue. However, it is hoped that its overall thrust will prove to be correct. My personal pleasure during the conception and development of these ideas has been best described by Prince Petr Kropotkin, that marvellous Russian scientist-revolutionist who, at the turn of the century, said, “There are not many joys in human life equal to the joy of the sudden birth of a generalization, illuminating the mind after a long period of patient research. What has seemed for years so chaotic, so contradictory, and so problematic takes at once its proper position within an harmonious whole. Out of a wild confusion of facts and from behind the fog of guesses-contradicted almost as soon as they are born-a stately picture makes it appearance, like an Alpine chain suddenly emerging in all its grandeur from the mists which concealed it the moment before, glittering under the rays of the sun in all its simplicity and variety, in all its mightiness and beauty.” (Kropotkin, 1899). I thank Professor A. D. Brown and Dr K. J. Raison for reading and commenting on the manuscript and Dianne Kelsey for typing it. This work was supported by a grant from the University of Wollongong Research Fund. REFERENCES ALOIA, R. C., PENGELLEY, E. T., B~LEN, J. L. & ROUSER, G. (1974). Lipids 9, 993. ARMSTRONG, K. J., STOUFFER, J. E. & VAN INWEGEN, R. G. (1974). J. biol. Chem. 249,4226. BABIOR, B. M., CREAGAN, S., INGBAR, S. H. & KIPNES, R. S. (1973). Proc. natn. Acud. Sci.

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