Excretion, Metabolism and Enterohepatic Circulation Pathways and Their Role in Overall Thyroid Hormone Regulation in the Rat 1

Transcription

1 AMER. ZOOL., 28: (1988) Excretion, Metabolism and Enterohepatic Circulation Pathways and Their Role in Overall Thyroid Hormone Regulation in the Rat 1 JOSEPH J. DISTEFANO, III Departments of Computer Science & Medicine, University of California, Los Angeles (UCLA), Los Angeles, California SYNOPSIS. Regulation of thyroid, adrenocortical and other hormones secreted by the major endocrine glands in mammals is widely attributed primarily to feedback control relationships with the pituitary, hypothalamus or both, with hepatobiliary and intestinal mechanisms having no more than a passive or excretory role. I present another view of enterohepatic components in thyroid endocrine function, suggesting a functional and more pervasive role for the intestine, in a more complex hierarchical system controlling thyroid hormone levels, effects and economy in the rat, and possibly in other mammals. A central factor is the existence of enterohepatic cycling of these hormones, or their reabsorption from intestinal pools to portal and then systemic blood. This process affects their dynamic behavior throughout the organism, not only hormone economy, because bidirectional transport of hormone between blood and intestine (including large pools in luminal contents) renders all or part of the gut internal to the system regulating thyroid hormones. We review the evidence for and possible significance of this hypothesis, covering specific aspects of hormone level control in the rat, including the deiodination, conjugation and other metabolic pathways, particularly in liver and intestine, and the fecal and urinary excretory (sink) and hormone production (source) pathways. The modulators of enterohepatic subsystem regulation of thyroid hormones are postulated to involve the combined effects of hormone conjugation and degradation processes in liver and their subsequent secretion in bile, coupled with the bacterial deconjugation, the reabsorption and certain hormone storage mechanisms of the intestine. INTRODUCTION In several mammalian species the central role of the hepatobiliary transport mechanism and the enterohepatic circulation in the regulation of cholesterol, bile acids and some other organic substances is well documented. In contrast, regulation of the levels of thyroid, adrenocortical and other hormones secreted by the major endocrine glands is widely attributed primarily to feedback control relationships with the pituitary, hypothalamus or both, with biliary and intestinal components having no more than a passive or excretory role. Several new developments reported in the recent literature on thyroid hormone metabolism and distribution, however, have rekindled interest in the enterohepatic subsystem, suggesting the possibility of a functional role for intestinal components in a more complex hierarchical system controlling thyroid hormone levels and economy. 1 From the Symposium on Comparative Endocrinology of the Thyroid presented at the Annual Meeting of the American Society of Zoologists, December 1986, at Nashville, Tennessee. 373 For thyroid hormones, the intestine and liver are the major organs of the enterohepatic subsystem of the rat (Ratus ratus). Functionally, they are interconnected directly via biliary and by portal venous transport pathways, and indirectly via mesenteric and hepatic circulations exchanging with peripheral plasma (see Fig. 1). The rat has no gallbladder. The central role of the liver as such in thyroid hormone regulation and metabolism has been more extensively studied then that of any other organ, but nearly exclusively in isolation of the intestine. My major emphasis here is on the functionality of the intestine as an integral component of the enterohepatic subsystem and the role it may play in regulating overall thyroid hormone distribution, metabolism and excretion in the rat, and possibly also in other species. For zoologists, this subject matter may be of particular interest, because the thyroid gland and the gastrointestinal tract have many similarities, in phylogeny, embryology and function. The protochordate endostyle, a tubular structure that secretes into the pharynx, is probably the evolutionary precursor of the thyroid, based on

2 374 JOSEPH J. DISTEFANO, III WHOLE-BODY THYROID DISTRIBUTION FOCUS ENTEROHEPATIC SUBSYSTEM DEOHASATIOM DEIODINATIONS. ETC. {DEGRADATION) FIG. 1. A schematic, physio-anatomic view of wholebody thyroid hormone (TH) distribution in the rat, with emphasis on enterohepatic subsystem components and functional interconnections. its iodinating ability possibly even producing thyroid hormones; and the embryological origin of the thyroid in vertebrates is the primitive foregut, the main anlage of the gland developing as a median endodermal downgrowth from the tongue. Furthermore, like the thyroid, the gastric mucosa (and the salivary glands) of the rat and other mammals continuously concentrate iodide, on into adult life, but they do not synthesize iodothyronines, as reported for some gut-like organs in some primitive animals (DeGroot et al., 1984). The presence of iodine-containing compounds in bile has been known since the end of World War I (Kendal, 1919), but it wasn't until the decade following World War II that the role of the gastrointestinal tract in thyroid hormone regulation was actively explored, spurred on by the availability of radioiodinated compounds (Albert and Keating, 1952; Taurog et al., 1952; Briggs et al, 1953; Taurog, 1954; Roche et al., 1956; Van Middlesworth, 1957). Interest and activity in this subject by the mammalian thyroid community during the two following decades were less specific, with the exception of several groups, most notably those of Cottle and Veress (1965, 1971), Chung and Van Middlesworth (1964, 1967), and Galton (Galton and Nisula, 1972; Galton, 1975; Ingbar and Galton, 1975; Nisula et al., 1977). Van Middlesworth (1974) adeptly and comprehensively reviewed the subject of thyroid hormone metabolism and excretion through 1973, with emphasis on the enterohepatic subsystem, and Miller et al. (1978) later published an inciteful review of gut-thyroid interrelationships through Whereas many issues remain unresolved, the literature has been sparse on this subject since then, as interests in the thyroid field have turned to other problems and organ systems. Two of the most intriguing questions about the enterohepatic subsystem vis-d-vis thyroid hormone regulation have never been fully resolved. First, why is so much thyroid hormone found in excreta of normal animals, at least in the rat, the human, and several domestic animals studied? This is important, because it appears to be a relatively uneconomical, or wasteful, process. Second, it is not yet clear whether significant amounts of endogenous thyroid hormones are effectively absorbed from the intestinal lumen to the peripheral circulation under normal conditions. There is convincing evidence that such enterohepatic recycling can occur under various laboratory-induced conditions, and pharmacological doses of thyroid hormones are certainly absorbed when given orally. Although this question also touches on hormone economy, it takes on added importance in the context of overall thyroid system regulatory dynamics. If hormone transport between the intestine (including its luminal contents) and the remainder of the organism is bidirectional, then all or part of the intestine is internal to the system regulating thyroid hormones, and the dynamic characteristics of hormone in the intestine affect corresponding characteristics throughout the organism. This would mean that such intestinal components should be included in any assessment of overall dynamics. The relative importance of intestinal compo-

3 ENTEROHEPATIC REGULATION OF THYROID HORMONES 375 nents in this regulation, however, would depend on specific quantitative characteristics, which in principle may be elucidated from kinetic studies. I shall describe some preliminary efforts toward this goal in subsequent sections, based on work in the Sprague-Dawley rat. In so doing, I will also offer several hypotheses about the role of the intestine in the thyroidal regulatory schema, a synthesis of the results and suggestions of others as well as those from my own laboratory. I am sorry to say, however, that the results of our efforts in this regard have raised more questions than they answer, at least in my own mind. OVERALL HORMONE ECONOMY Thyroid hormones in the rat are thyroxine (T 4 ) and 3,5,3'-triiodothyronine (T 3 ), although there is still controversy about whether T 4 as such has intrinsic hormonal activity. This issue is not particularly pertinent here. Both are synthesized and secreted by the thyroid gland in the rat, but T 4 is the predominant secretory product. Most of the T 3 is generated from T 4 in peripheral tissues, by monodeiodination of T 4 (see below). A precise estimate of the proportion of total T 3 production emanating from T 4 is not yet available, but about a quarter of T 4 production is converted to T 3 (Schwartz et al., 1971; Zimmerman, 1978; DiStefano et al., 1982a, b, 1985). The remaining iodothyronines found in the rat are produced by monodeiodination of T 4 or T 3, sequentially generating reverse-t 3 = rt 3 (3,3',5'-T 3 ), 3, 5-T 2, 3-3'-T 2, 3',5',T 2, 3-T, and 3'-T' (see Fig. 2). The major iodothyronine disposal pathway is deiodination, but several other irreversible metabolic alterations are present in vivo, and excretion pathways are also quite significant for thyroid hormones, as discussed in the next section. It is of interest to note that the fractional rate of deiodinative metabolism of T 4 in the normal rat is fairly constant, and therefore well regulated, over a wide range of exogenous T 4 loads (Ingbar and Galton, 1975). There is also scattered but fairly convincing evidence that the hepatobiliary pathway modulates the excess in the manner of an IODOTHYRONINE 3, 5, 3 1 3, 5, 3\ 5' DEIODINATION FIG. 2. Pathways of iodothyronine deiodination, beginning with T 4 and ending with the thyronine nucleus T o. All outer (phenolic) ring deiodinations, designated ORD, are directed downward and to the left; and inner (tyrosyl) ring deiodinations, designated IRD, are downward and to the right. For T 4, ORD to T 3 is an activating and IRD to rt 3 is a deactivating pathway. "overflow valve," thereby controlling fecal excretory fluxes (Roche et al., 1956; Hillier, 1972; Galton, 1975). This may be part of the mechanism maintaining levels of T 3 derived from T 4 metabolism, and therefore the constancy of thyroid hormonal effects. The important relationships governing overall hormone economy are based on straightforward conservation laws, beginning with: the whole body production rate PR equals the whole body disposal rate DR, when the endogenous system is in steady state. We focus on production first (sources). For T 4, PR 4 = SR 4, the thyroidal secretion rate. For T 3, the production rate is the sum of the thyroidal secretion rate, SR 3, and the whole body rate of production of T 3 converted from T 4, CR 3^. The same is true for the remaining daughter iodothyronines. In summary: PR 4 = SR 4, PR 3 = SR 3 + CR^ and PR; = SRj + CR H, where i denotes rt 3, a specific T 2 or T,, and j its parent iodothyronine (see Fig. 2). Irreversible disposal pathways (sinks) are similarly designated. With MR denoting the

4 376 JOSEPH J. DISTEFANO, III METABOLISM OF THYROXINE (T 4 ) CONJUGATION (Giucurontde. ^. ^S Sulfate) \ y, x \ / DEIODINATION I CH CH ANALOG FORMATION 2 \ COOH DECAR8OXTLATION (to T 3 or rt 3 ) FIG. 3. The thyroxine (T 4 ) molecule and its 4 principle metabolic pathways, in order of their quantitative importance: deiodination, conjugation, acetic acid analog formation and other oxidative processes (ether link cleavage illustrated). (catabolic) metabolic rate and ER the excretion rate, we then have DRj = MRj + ER;, where the subscript i has the meaning given above. Conventional kinetic studies in the whole animal typically permit computation of only total (overall) disposal rate parameters, such as the plasma clearance rate PCR (DiStefano, 1986, 1988). This may be done, for example, by infusing a tracer at a known rate IR* to steady state and measuring the steady state tracer C* and tracee C concentrations in plasma, in which case IR* = DR* = (PCR*)C*. Then, if the tracer perturbation of the endogenous steady state is sufficiently small, PCR for the tracer and tracee are equal and DR = (PCR*)C = (IR*)C/C*. Similarly, if a single trace pulse dose D* is given intravenously, and its disappearing concentration c*(t) is measured along with the steady state endogenous tracee concentration C in plasma, then: DR = (D*)C/ I c*(t) dt. 0 This approach provides SR 4 for T 4, because SR 4 = DR 4 in steady state, but not the production rate PR 3 for T 3, because the multiple source problem demands a more complex solution (DiStefano etal, 1982a, 1985; DiStefano, 1986). On the other hand, if metabolism rates are of interest a common goal in endocrine and metabolic studies, excretion rates ER also must be measured and subtracted from total disposal rates, MR = DR - ER. As noted in the Introduction, thyroid hormone levels in blood and tissues are regulated in part by negative feedback interplay between the thyroid and anterior pituitary, in effect controlling synthesis and secretion of the hormones. This control system is rendered even more complex by the existence of distributed production sites (for T 3 ) in peripheral tissues as well as the thyroid gland (distributed sources). But it must be borne in mind that the various mechanisms controlling their metabolism and excretion, the disposal (sink) processes, are an integral part of the overall system regulating hormone levels and economy. IODOTHYRONINE METABOLISM Iodothyronine metabolic pathways found in vivo include: (a) deiodination, (b) conjugation, (c) transformation to acetic acid analogs, and (d) other oxidative degradation. Deiodination, which is irreversible extrathyroidally, is catalyzed by either a nonspecific single deiodinase (Type I) residing in the endoplasmic reticulum, in

5 ENTEROHEPATIC REGULATION OF THYROID HORMONES 377 which T 4 and its daughters, T 3 's, T 2 's and T,'s (Fig. 2) are sequential monodeiodinated in the outer or the inner ring (Fig. 3), or by one of two other deiodinases specific to the outer ring (ORD Type II) or the inner ring (IRD Type III) (Leonard and Visser, 1986). Deiodination has been demonstrated in numerous tissues of the rat and other species, Type I in particular is found in rat liver (Mol and Visser, 1985a, b). Comprehensive reviews of the deiodination processes occurring in laboratory animal and human tissues can be found in several chapters of a recent book edited by Hennemann (1986). Conjugation of iodothyronines with either glucuronic acid or sulfate, via microsomal glucuronyl or cytoplasmic phenol transferases, occurs with the phenolic hydroxyl group (outer ring, Fig. 3) (Visser et al., 1983; Mol and Visser, 19856). Liver and kidney are important conjugation sites (Taurog et al, 1952; Roche et al., 1956), but conjugates are probably also formed in other tissues, possibly including intestine (Herz et al., 1961), skeletal muscle and skin (author's unpublished observations). Conjugation is a reversible metabolic process and therefore may be viewed as having both hormone deactivating and activating functions. Intestinal bacteria in gut contents have been shown to exhibit exo-enzymic sulfatase and glucuronidase activity; active desulfation in vitro of all iodothyronine sulfates by specific anaerobic bacterial strains collected from the cecum of rat gut and from human feces have recently been reported (Otten et al., 1983; Herder et al., 1984). Conjugated iodothyronines are also deiodinated irreversibly, and a 2-step process, sulfoconjugation followed by deiodination, has been suggested as the primary mechanism for deiodinative metabolism (deactivation) of T 3 to 3,3'-T 2 and T 4 to rt 3 in the liver (Mol and Visser, 1985a, b). Quantitatively, rat bile normally contains both conjugated and unconjugated iodothyronines in a ratio of about 3 to 1; and the ratio of glucuronidated to sulfated hormone is about 10 to 1 (author's unpublished observations). T 4, T 3 and other iodothyronines can be irreversibly transformed in the alanine side chain to acetic acid analogs Tetrac (T 4 A), Triac (T 3 A), etc., via processes which include oxidative deamination and decarboxylation. T 4 A and T S A are present in relatively small quantities in liver, kidney and other tissues in rat and other species, and these analogs also can be subsequently conjugated or deiodinated (Van Middlesworth, 1974; Millers al., 1978). Intestinal flora may decarboxylate as well as deconjugate iodothyronines (White et al., 1968), providing them with a catabolic as well as a locally activating function (via deconjugation to free form) for thyroid hormones. T 4 A and T S A are, however, somewhat of a puzzle when viewed from the perspective of hormone economy. Their formation from T 4 or T s respectively is irreversible, and in this sense analog formation is degradative. They are not deactivating, however, because T 4 A and T 3 A behave in many respects like T 4 and T 3, exhibiting biological potency (Burger, 1986). On the other hand, their kinetics are different than those of the hormones and they may therefore have a more subtle functional role in the hierarchical control of the dynamics of thyroid hormone economy and effects. Other irreversible oxidative degradation pathways of iodothyronine metabolism include the formation of "nonextractable" iodoproteins (NEI) via covalent bonding (Oppenheimer et al., 1972), leukocyte phagocytosis, and ether-link cleavage (ELC) (Burger et al., 1983), which results in a 1-step degradation of the iodothyronine nucleus (Fig. 3). These pathways probably play a quantitatively very minor role in normal animal thyroid hormone metabolism. IODOTHYRONINE EXCRETION The primary excretory pathway for intact iodothyronines is the fecal route, but some hormone also may be excreted in urine when the kidney is not functioning normally. It is fairly common practice in the literature to designate the biliary pathway as excretory, but this is somewhat misleading. Thyroid hormones conjugated in the liver, and possibly also conjugates transported to the liver from other tissues, are secreted into bile, along with some uncon-

6 378 JOSEPH J. DISTEFANO, III jugated hormone, and therein transported into the intestinal lumen, where the conjugates are subjected to hydrolysis by intestinal flora, as noted above. As discussed more fully below, some free hormone is then reabsorbed into blood, rendering the intestinal lumen and gut wall internal pools of iodothyronine distribution. I therefore refer to the transfer of conjugated or free hormone from liver to gut in bile as a transport process via secretion of these substances by hepatocytes into bile. Bile is produced continuously by liver cells, from where it passes along the bile canaliculi and on into the common bile duct, carrying unconjugated and conjugated iodothyronines as well as bile salts, cholesterol and other compounds to the intestine. Bile flow in the conscious adult laboratory rat is less than 1 ml/hr in the day, and about 40% greater at night, during normal feeding (Kuipers et al., 1985). This compares with blood flow to the intestine of about 500 ml/hr (Lee et al, 1986), so the potential is great for a significant mass flux of circulating thyroid hormone to the intestine, depending on the fraction of hormone extracted from blood by the intestine. Measurement of this key parameter has not been reported. Fecal excretion Numerous studies over the past 35 years, the majority based on the use of radioiodinated compounds, have demonstrated that roughly half the total organic iodine contained in the T 4 secreted by the rat thyroid ends up in feces, much in the form of intact hormone, and the other half is the product of complete T 4 degradation, with this iodide recycled back to the thyroid or excreted in urine (Van Middlesworth, 1974; Miller et al., 1978). The fate of T 3 production is about the same. Complete characterization or quantification of the labeled components in radioactively labeled feces, however, was not a part of many early studies. Others were limited by measurement technology or impure tracers, or the doses given were pharmacological, rendering them quantitatively more difficult to interpret. Recent estimates based on detailed chromatographic analyses of radiolabeled feces in stready state; following intravenous injection (DiStefano and Sapin, 1987) or infusion (Boonamsiri et al.,1979) of labeled T 4 or T 3, or in rats isotopically equilibrated with I25 I (Van Middlesworth et al., 1971), have together provided a fairly precise breakdown. About a quarter of endogenous T 4 production in the laboratory rat is excreted as T 4 and the remainder is degraded. Similarly, about a third of the T 9 produced is excreted in feces as T 3, the remainder being degraded. It is also noteworthy that the ratio of T 3 to T 4 found in feces (~0.2) following T 4 injection (DiStefano and Sapin, 1987) is about the same as the whole-body fraction of T 4 converted to T 3 in peripheral tissues (DiStefano et al., 1982a, b; Schwartz et al., 1971). The remaining radioactivity in feces following exogenous T 4 or T 3 injection is mostly iodide (<7%). Some investigators have found small quantities of T 4 A and T 3 A in feces (e.g., Boonamsiri et al., 1979), but using significantly larger (up to 90%) perturbations of endogenous T 4 and T 3 production rates. I would suggest that this may have resulted from the side chain catabolic pathway being invoked in response to hormone "overload" conditions. A possible mechanism for such an occurrence might involve more general amino acid deaminating pathways in the liver, acting only when T s or T 4 levels are high enough to render them susceptible to such enzymatic activity. Conjugates are normally absent from feces, presumably because they appear to be completely hydrolyzed in the intestinal lumen of the rat (Taurog et al., 1952). In contrast to the hormones T 3 and T 4, the nonhormonal iodothyronines rt 3, 3,3'- T 2, 3',5'-T 2 and 3'-T, are nearly completely metabolized, with virtually none appearing in feces (DiStefano and Sapin, 1987.) Urinary excretion Under normal conditions in the rat, no iodothyronines are excreted in urine intact or as conjugates; only iodide is excreted (DiStefano and Sapin, 1987), However,

7 ENTEROHEPATIC REGULATION OF THYROID HORMONES 379 intact hormone can be excreted in urine in some pathological conditions, e.g., proteinuria (Ferguson and Jennings, 1983). Other mammalian species Excretory components are qualitatively similar, but hormone economy does appear to differ somewhat in other species. Irvine (1969) studied T 4 metabolism and excretion in sheep and horses. In sheep, he found: (a) 35-40% of an injected dose of [ 125 I]T 4 appeared in feces, nearly all as unconjugated [ 125 I]T 4, conjugates apparently being hydrolyzed completely in the colon; (b) 38% of the T 4 dose was deiodinated, the iodide released appearing in urinary excreta and the unblocked thyroid; (c) a small amount of T 4 conjugate was also found in urine; and (d) there were no differences between male and female sheep. In the horse, an even greater fraction of T 4 production is fecally excreted (Irvine, 1969). In the human, about 15% to 25% of T 4 and T 3 production is excreted in feces and urine, mostly in feces in unconjugated form (DeGroot et al., 1984). Thus, excretion of thyroid hormones is a substatial fraction of total production in all species studied, with the human excreting only slightly less than the rat. ENTEROHEPATIC CIRCULATION OF THYROID HORMONES Evidence that T 4 and T 3 may be reabsorbed from the intestine into portal blood of the rat is broadly based, but the question of how much hormone is transferred via this route in the intact animal is not fully resolved. Briggs and co-workers (1953) were among the first to demonstrate that some T 4 in rat gut lumen is reabsorbed, with T 4 -glucuronide being not as readily absorbable as free T 4. They suggested that conjugation might be thus permitting elimination of excess T 4 more efficiently. Another interpretation of their observations might be that conjugation possibly protects a large fraction of the total T 4 in gut from binding nearly irreversibly with intraluminal proteins (or other substances) during its passage through the small intestine, prior to deconjugation in the cecum and colon. This would then have the effect of enhancing the amount available for reabsorption, in effect providing a means for temporary storage of hormones within the bowel. Indirect evidence for reabsorption of T 4 was reported by Van Middlesworth (1957), showing that chronically increased loss of fecal bulk decreased plasma T 4 levels and generated goiter in rats. It is now clear that numerous factors affecting the rate of movement of material through the bowel also alter the amount of hormone absorbed; in the rat, the diet is of major import, with increased absorption associated with dietary factors that decrease material flux (Van Middlesworth, 1974). This has been shown to be the case in other species too, including the human (Pinchera et al., 1965; Hays, 1968; Wenzel and Kirschsieper, 1977). Absorption of T 4 and/or T 3 from isolated intestinal loops was studied by Chung and Van Middlesworth (1964, 1967) and by Cottle and Veress (1965, 1971). They showed that unconjugated T 4 and T 3 are absorbed much more readily than conjugated hormone, from small and large intestine, and unconjugated T 3 is absorbed faster than unconjugated T 4. A reassessment of the quantitative data reported for T 4 by Cottle and Veress (1965), however, suggests that most or all of the conjugated hormone they believed was absorbed might have been unconjugated hormone and iodide instead. Bastomsky (1972) reported labeled T 4 absorption from the bowel of the rat in a dual tracer study in which radioactive components in bile and systemic plasma (but not portal plasma) were measured. He postulated that T 4 in his studies was glucuronidated to T 4 G in gut wall and the resulting T 4 G was then preferentially recycled back to gut via portal blood and bile, rather than escaping to the systemic circulation. These indirect results are difficult to assess, because they were reported only in abstract form. Nevertheless, whereas conjugates may be absorbed from gut, or synthesized there and released into portal blood (as suggested by Bastomsky) under some laboratory induced conditions, it remains

8 380 JOSEPH J. DISTEFANO, III questionable whether either of these processes occurs under normal physiological conditions. Recent data from our laboratory, discussed further below, suggest they do not. The majority of studies reporting quantitative estimates of T 4 absorption from the gut of the rat are based on comparisons of biliary and fecal fluxes, the difference assumed to be the hormonal flux absorbed, with the biliary component typically measured following biliary diversion. Myant (1957) and Oppenheimr et al. (1968) reported about half of biliary T 4 is reabsorbed in rats, with the other half excreted in feces. The problem with this approach is that the reabsorption pathway is at least partly broken when the bile is diverted, and the values thus obtained may be either under- or overestimates. The often quoted estimate of about 16% biliary T 4 reabsorption in the human reported by Myant (1956) may be either too large or too small, because biliary diversion was employed. Another problem with most early studies on enterohepatic cycling of thyroid hormones was that biliary/fecal comparisons to estimate absorption neglected the possibility of a circulatory influx of hormone to the gut via the mesenteric arterial blood. However, Irvine (1969) reported that in bile diverted sheep, 24% of an injected dose of T 4 was still excreted in feces, suggesting a significant circulatory as well as biliary influx of hormone into the intestine of sheep. Indirect evidence for a nonbiliary influx of T 4 into the gut of the rat was reported by Galton and Nisula (1972), who also employed the biliary diversion technique. They found a significantly higher steady state fecal efflux of T 4 in a second group of intact rats than in the biliary flux of bilediverted rats, and interpreted this as evidence of no net reabsorption flux under steady state conditions. They also suggested that T 4 may be transferred from blood across the intestinal mucosa and considered the possibility that some T 4 in the bowel also may return to the circulation, especially if a sufficient concentration gradient from lumen to blood is generated by single dose injections (into the lumen). The latter was indeed shown to be the case by Langer and co-workers (1982), who found large increases in plasma T 4 levels following injections of pharmacological doses of T 4 directly into the jejunum or cecum of the rat. The T 4 source in these injections was bile from donor rats injected intravenously with free T 4, and therefore the injected doses contained both conjugated and free T 4, as well as a small amount of T 3 generated from the T 4. They also confirmed that free T 4, and a small amount of free T 3 (at the largest T 4 doses), are absorbed from small and large intestine more from the former, at least under the input "overload" conditions of their studies. In our laboratory, we have recently demonstrated that, in the intact rat under normal physiological steady state conditions, both T 3 and T 4 undergo significant enterohepatic recirculation, and the mesenteric arterial flux of hormone into the intestine is not negligible, as assumed in many earlier studies, but is at least twice as large as the biliary flux (DiStefano and Feng, 1986). In the same series of studies, all summarized below, we have also found a very large pool of T 3 and, to a lesser extent T 4, in the bowel, which is interior to the system regulating overall thyroid hormone economy. We also have recently observed a slightly higher concentration of T 4 and T 3 in portal blood than in cardiac arterial blood of the intact rat under normal steady state conditions, indicative of net absorption of the hormones from the bowel (unpublished observations). In our enterohepatic recycling studies, reported in abstract form (Sternlicht et al., 1984a, b), our goal was to quantify the partitioning of both T 3 or T 4 entering gut in bile into fecal excretion, net "primary" absorption to liver, and net "secondary" absorption to the systemic circulation from the enterohepatic subsystem. Trace doses of labeled T 3 and T 4 were separately infused at a constant rate from an intraperitoneal osmotic minipump cannulated into the duodenum. Feces were collected from days 4 to 7 after implant, with the system in steady state and the rats eating, drinking and functioning normally. On day 7, the rats were sacrificed, blood was collected,

9 ENTEROHEPATIC REGULATION OF THYROID HORMONES 381 T4. T3 PHYSIOLOGY / ANATOMY: A PLAUSIBLE CARICATURE CONVERSION) SECRETION FIG. 4. A more compartmentalized and holistic view of the schematic of Figure 1, depicting thyroid hormone (T 4 or T 3 ) production, distribution and disposal pathways, again featuring the enterohepatic subsystem. The kidney appears separately and all remaining tissues are lumped together in the pool designated RESTO. Gut luminal contents are distinguished from gut wall; fecal excretion is distinguished from possible irreversible luminal degradation and from intracellular degradation in gut wall. Total hormone in gut and other pools includes conjugated as well as free and bound moities. All vascular exchanges of hormone with tissues are also represented physiologically, via arterial and venous transports; and liver and gut exchange hormone via mesenteric arterial and venous (portal) as well as biliary pathways. and gut was excised and cut into several segments. Pooled feces and gut content samples were each homogenized and extracted, and all extracts were chromatographed along with plasma samples and recovery standards. This permitted computation of the percent activities recovered as hormone and metabolites in feces and gut contents. We found 43 ± 13(SD)% of T 3 (n = 7) and 42 ± 9% of T 4 (n = 7) entering gut were excreted as hormone in feces. Therefore, the remainder ( 57%) of each was either portally transported to liver and/or degraded in gut. Most of the T 3 and T 4 found in small gut sections was conjugated, demonstrating unidirectional reabsorption of unconjugated T 3 and T 4, as only unconjugated hormone was infused directly into the duodenum. However, we also found significant T 3 or T 4 in plasma, thus positively demonstrating net primary and net secondary absorption under normal physiological conditions. The product of the plasma clearance rate and labeled hormone concentration in plasma in steady state provided an upper bound on net secondary absorption of hormone to the systemic circulation from the enterohepatic system: 42 ± 8% for T 3, and 49 ± 5% for T 4. In a more comprehensive set of studies, also reported only in abstract form (DiStefano and Feng, 1986), we infused trace amounts of labeled T 3 or T 4 for 7 days from osmotic minipumps implanted subcutaneously, a good approximation of constant intravenous infusions for steady state studies. The rats were sacrificed on day 7 and the following organs were dissected out and analyzed as described above: liver, kidneys, gut wall and contents (separately and in 3 sections upper, lower, cecum), and residual carcass. Systemic and portal plasma and biliary concentrations of hormone, and the bile flow rate, were also measured. Reference to Figure 4 helps to visualize this study. The first objective here was to determine organ and whole-body pool sizes (excluding the thyroid), by direct mea-

10 382 JOSEPH J. DISTEFANO, III surement of everything, a "holistic" viewpoint if you will (DiStefano and Feng, 1986). We found that liver had about 3 times as much T 3 as plasma, and intestinal wall had the same amount as plasma, but intestinal contents had 20 times more T 3 than plasma (all in steady state). It is interesting that this large aliquot of slowly exchangeable T s has often been discarded in whole-body studies, e.g., in Schwartz et al. (1971). The remaining slowly equilibrating pool, residual carcass, or RESTO in Figure 4, contained far less hormone, 20% of total T 3. For T 4, liver and intestine each had about 60% of the amount found in plasma, kidney had very little, and residual carcass had about 50% more T 4 than plasma. So it appears that liver and intestine share the storage function equally for T 4, but far more T 3 is stored in intestine. Summing all the totals and scaling to the loog rat, the measured total body pool size of T 4 of 551 ng compared well with 550 ng determined from conventional plasma kinetic data analysis only, lending credibility to these surprising results. The same was approximately true for T 3. Our second objective in these studies was to evaluate the circulatory and biliary enterohepatic mass fluxes of hormone (Di- Stefano and Feng, 1986). To achieve this goal, the data from earlier studies, namely our plasma kinetic studies (DiStefano et al., 1982a, b; 1985), excretion studies (DiStefano and Sapin, 1987) and enterohepatic cycling studies (Sternlicht et al., 1984a, b) performed in similar rats under the same conditions were combined with data from this study. For T 4, the lower bound for the mesenteric arterial flux was found to be more than two times greater than the biliary flux. For T 3, we also found the mesenteric arterial flux, and also the portal flux, more than twice as large as mean biliary flux of hormone. No conjugates of T 3 or T 4, only unconjugated hormones, were found in portal plasma. In summary, we have found that in the adult laboratory rat maintained under physiological steady state conditions: (a) intestinal thyroid hormone pools are relatively large, especially T 3 in luminal contents (>'/2 of the total body pool of T 3 ); (b) intestinal pools are ultimately exchangeable with hormone in the general circulation, and thus cannot be considered part of irreversible excreta; (c) gut pools are replenished at least twice as fast from blood transport than from bile; (d) little or no conjugated thyroid hormone is absorbed from gut into portal blood; and (e) conventional kinetic analysis using plasma data only, provides good whole-body pool size estimates. Our results also suggest that significant irreversible degradation of T 4 may occur in gut, possibly generating T 3 from T 4. It remains to be seen how these data fit into the overall schema of wholebody thyroid hormone regulation. ENTEROHEPATIC REGULATION OF THYROID HORMONES It is fair to say that the ideas proposed in this section have been influenced in no small part by recent work of several groups in the Netherlands, most notably that of Visser and co-workers over the last several years, the lifelong work of my friend and colleague Lester Van Middlesworth, and the prolific efforts of Dr. Galton, who also participated in this symposium. In an attempt to answer the first question noted in the Introduction, albeit speculatively, it is certainly possible that the apparent wastage of hormone in excreta might be the result of incomplete evolution of an endocrine system that had its probable origins in trie gut. Alternatively, the criterion for optimal regulation of thyroid hormone levels or effects might preclude hormone "economy" in the usually accepted sense, with hormone wastage being a functional property of a dynamically "robust" regulatory system. In addressing the second question, about enterohepatic cycling of hormone, I would propose that some apparent anomolies about the functionality of the liver in this system might be resolved by viewing the liver and intestine as a unit, acting together. In this regard, the luminal contents of the intestine may have a hormone storage function, commonly attributed to the liver, and the two organs acting together may also serve to conserve iodine (the latter idea suggested by Dr. Eales at this symposium),

11 ENTEROHEPATIC REGULATION OF THYROID HORMONES 383 LIVER ART INT WALL MESENTERIC ART FIG. 5. A simplified model of the biochemical/physiological basis of enterohepatic subsystem regulation of thyroid hormones in the rat, a blowup of the enterohepatic portion of Figure 4. Details in the gut are shown for T 3 only; they are essentially similar for T 4. with both processes controlled by a complex sequence of events involving enterohepatic cycling of unconjugated hormones, conjugation and deiodination in the liver, and storage and deconjugation in the intestinal lumen. As a practical matter, the intestinal lumen may be an "ideal" place to store large quantities of hormone, especially T 3, because it is less likely to be toxic there (a rationale suggested to me by L. Van Middlesworth), assuming luminal cells are not target tissues for T 3, in which case local toxicity might be exascerbated. More generally, and based on a synthesis of data selectively reviewed in previous sections, I would propose that the enterohepatic subsystem has a more pervasive role in overall thyroid hormone regulation (as does the pituitary-thyroid axis), precisely and rapidly controlling blood and tissue levels and metabolic effects of the hormone, in response to an oversupply or undersupply of hormone, whether from endogenous or exogenous sources. Overall, the combined effects of hormone conjugation and degradation processes in liver (and possibly other tissues), and their subsequent secretion in bile, coupled with the deconjugation and absorption mechanisms of the intestine, are good candidates for modulators of this regulation. The physiological and biochemical basis for these hypotheses is summarized in the schematic diagram of Figure 5, a more microscopic view of the enterohepatic subsystem portion of the model depicted in Figure 4. Figure 5 is necessarily oversimplified, but it serves to depict the framework and functionality of the model under discussion. We begin with unconjugated T 4 entering liver cells via the hepatic artery (ART) or portal vein at the top left of the figure. Some T 4 (T 4 ) in liver cells is deiodinated to T 3, some is sulfated (T 4 S), some is glucuronidated (T 4 G), some of it remains unconjugated and some may be converted to Tetrac (T 4 A), the latter possibly only when T 4 is in "excess." As noted earlier, the T 4 A "degradation" pathway is paradoxical, because T 4 A has hormonal (or prohormonal) activity. T 4 S is (relatively)

12 384 JOSEPH J. DISTEFANO, III very rapidly deiodinated and therefore deactivated to rt 3 S (outer-ring deiodination (ORD) of T 4 S does not occur), which probably degrades further, freeing additional iodine for recycling to the thyroid or eventual excretion in urine. T 4 G, T 4, small amounts of T 4 S, and possibly rt 3 S and T 4 A, are secreted in bile and thereby transported to the intestinal lumen. Some unconjugated T 4 in the cell returns to the systemic circulation via the hepatic vein (VEN); the other components probably do not follow this pathway. The fate of T 4 and its conjugates in intestine, not detailed in Figure 5, follows the same pathways as those for T 3, described below. When systemic T 4 is in excess, the biliary hormonal mass flux for eliminating the excess appears to be accelerated (Morreale de Escobar, 1967; Hillier, 1972), probably with a smaller than normal relative fraction of hormone reabsorbed. There is also evidence for reduced biliary T 4 flux when hormone is in short supply (Averill and Newman, 1969). Thus, the hepatobiliary pathway may be viewed as a "control valve," capable of responding very effectively and rapidly (within minutes in the rat) to changes in circulating T 4 levels. This mechanism does not appear to be similarly responsive to T 3, however, at least not when T 3 levels are high (Hillier, 1972). T 3 arrives in liver cells from the hepatic artery (ART), portal vein, or from local free T 4 by ORD. In the cell, some T 3 is sulfated (T 3 S), some is glucuronidated (T 3 G), some remains unconjugated, and some may be converted to Triac (T 3 A). As with T 4, deiodination is the major deactivation pathway, chiefly via T 3 S, thus freeing (conserving) iodine. T 3 G, a smaller amount of T 3 and possibly very small amounts of T 3 S and T 3 A are secreted in bile to the gut lumen. Some T 3 in the liver cell returns to the systemic circulation via the hepatic vein (VEN); and, like T 4, probably little or none of the other forms of this hormone return directly to the circulation. Also, as with T 4 A formed from T 4 degradation, it is difficult to assess the role of T 3 A derived from T 3 degradation, because T 3 A also has hormonal activity. The majority of the T 3 is glucuronidated prior to its arrival in the small intestinal lumen. It remains that way until it reaches the cecum (and possibly the lower ileum), where hydrolysis by intestinal bacteria is initiated, a process which continues on into the upper colon. Absorption of T 3 occurs all along the intestine, except possibly in the lower colon where the physical state of the hard fecal pellets may prevent it. Unconjugated T 3 is transferred from the bowel across the mucosal cells and thus returns to the liver unconjugated (under normal conditions) via the mesenteric and then portal venous circulations; T 3 G may also find its way across when it is in substantial excess. The relatively lengthy transit time for passage of large quantitites of T 3 through the gut, much of it in nonabsorbable conjugated form, provides ample opportunity for protected storage. Hydrolysis, and possibly also passive binding to fecal constituents, then provide part of the mechanism for partial reabsorption and partial excretion (in feces), depending on whole-body hormonal requirements. There is only indirect evidence for either T 4 or T 3 degradation locally in gut lumen (dashed lines in Fig. 5), but the possibility of T 3 formation from T 4 is suggested by several factors, most notably by the very much larger steady state T 3 pool in gut lumen compared with the T 4 pool there. This is an intriguing, unanswered question, in part because it is suggestive of a possible hormonal source existing in an unlikely place. Finally, with few exceptions, in particular evidence for thyroid hormone effects on the intestine in development (Henning, 1978; Koldovsky, 1981), little is known about thyroid hormone action on mucosal cells. Perhaps the intestine in toto can provide a rich medium for new explorations into mechanisms of thyroid hormone action, as well as the regulation of their metabolism and whole-body economy. In closing, I wish to remind the reader that the detailed mechanism postulated above for the possible role played by the enterohepatic subsystem in thyroid hormone regulation must be treated as a working model, and Figures 4 and 5 as plausible caricatures. It is a synthesis of scattered data,

13 ENTEROHEPATIC REGULATION OF THYROID HORMONES 385 collected over a long period, and cemented together here by a scientist whose intuition and approach are often influenced by his early training in control systems engineering. While it is based on far more than gut feelings, I offer it more as food for thought than as well-digested, well-founded actuality. It is an area only partially charted, richly endowed with unknowns and, in my opinion, worthy of pursuing vigorously. ACKNOWLEDGMENTS This work was supported by research grants DCB from the National Science Foundation and AM34839 from the National Institutes of Health. Viveca Sapin, Mark Sternlicht, Dr. Dennis Harris, Sheila Sindt, Allyson Pizzo, Thuvan Nguyen, Wendy Morris, Dagan Feng, Hooman Rahimizadeh and Daryoosh Baghai are gratefully acknowledged for their able laboratory assistance. REFERENCES Albert, A. and F. R. Keating The role of the gastrointestinal tract, including the liver, in the metabolism of radiothyroxine. Endocrinology 51: Averill, R. L. W. and W. C. Newman Biliary secretion of thyroxine in rats with thyroiddepressing hypothalamus lesions. Endocrinology 84:950. Bastomsky, C. H The enterohepatic circulation of thyroxine in the rat: A short circuit. Clinical Science, Medical Research Society 42:28P 29P. Boonamsiri, V., J. C. Kermode, and B. D. Thompson Prolonged intravenous infusion of labeled iodocompounds in the rat: 125 I-T 4 and 125 I-T, metabolism and extrathyroidal conversion of T< to T 3. Journal of Endocrinology 82:235. Briggs, F. N., A. Taurog, and I. L. Chaikoff The enterohepatic circulation of thyroxine in the rat. Endocrinology 52: Burger, A. G Nondeiodinative pathways of thyroid hormone metabolism. In G. Hennemann (ed.), Thyroid hormone metabolism, pp Marcel Dekker, Inc., New York and Basel. Burger, A. G., D. Engler, U. Buergi, M. Weissel, and G. Steiger Ether link cleavage is the major pathway of iodothyronine metabolism in the phagocytosing human leukocyte and also occurs in vivo in the rat. Journal of Clinical Investigation 71: Chung, S. J. and L. Van Middlesworth Absorption of thyroxine from the small intestine of rats. Endocrinology 74: Chung, S. J. and L. Van Middlesworth Absorption of thyroxine from the intestine of rats. American Journal of Physiology 212: Cottle, W. H. and A. T. Veress Absorption of biliary thyroxine from loops of small intestine. Can. Journal of Physiol. Pharmacol. 43: Cottle, W. H. and A. T. Veress Absorption of glucuronide conjugate of T s. Endocrinology 88: DeGroot, L., P. R. Larsen, S. Refetoff, and J. B. Stanbury The thyroid and its diseases. Wiley, New York. DiStefano, J. J Modeling approaches and models of the distribution and disposal of thyroid hormones. In G. Hennemann (ed.), Thyroid hormone metabolism, pp Marcel Dekker, Inc., New York and Basel. DiStefano, J. J Hormone kinetic analysis. In L. J. DeGroot (ed.), Endocrinology, 2nd ed. Grune & Stratton, Inc., Florida. (In press) DiStefano, J.J. and D. G. Feng Distribution, metabolism and biliary and circulatory fluxes of thyroid hormones in liver, intestinal wall, luminal contents and kidney of the rat. Program Abstracts of the 61st Mtg. of American Thyroid Association, p. T43. DiStefano.J.J., M.Jang, and M.Kaplan Optimized kinetics of reverse-triiodothyronine distribution and metabolism in the rat: Dominance of large, slowly exchanging tissue pools for iodothyronines. Endocrinology 116: DiStefano.J.J., M.Jang, T. K. Malone.and M.Broutman. 1982a. Comprehensive kinetics of triiodothyronine production, distribution, and metabolism in blood and tissue pools of the rat using optimized blood-sampling protocols. Endocrinology 110: DiStefano, J.J., T. K. Malone, and M.Jang Comprehensive kinetics of thyroxine distribution and metabolism in blood and tissue pools of the rat from only six blood samples: Dominance of large, slowly exchanging tissue pools. Endocrinology 111: DiStefano, J. J., V. Sapin, and D. G. Feng Comparative aspects of the distribution, metabolism and excretion of all outer ring labeled iodothyronines in the rat. Program Abstracts of the 14th Annual Mtg. European Thyroid Association, p. 59. DiStefano, J.J. and V. Sapin Fecal and urinary excretion of 6 iodothyronines in the rat. Endocrinology 121: DiStefano, J. J., V. Sapin, S. Sindt, A. Pizzo, H. Rahimizadeh, and D. Baghai The luminal contents of rat intestine contain large thyroid hormone pools exchangeable with hormones in blood. Program Abstracts of the 69th Mtg. of The Endocrine Society, p Ferguson, D.C. and A. S.Jennings Regulation of conversion of thyroxine to triiodothyronine in perfused rat kidney. American Journal of Physiology, 245 (Endocrinology Metabolism 8):E220- E229. Gallon, V. A Thyroxine metabolism in the rat: Effect of varying doses of exogenous throxine. Acta Endocrinologica 78: Galton, V. A. and B. C. Nisula The entero-

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