0013-7227/91/1281-0146$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 1 Printed in U.S.A.

The Effect of Surgical Stress on the in Vitro Metabolism of Thyroxine by Rat Liver, Kidney, and Brain* G. HINTZEf, L. E. BRAVERMAN, AND S. H. INGBARJ Charles A. Dana Research Institute and the Harvard-Thorndike Laboratories of Beth Israel Hospital, Department of Medicine, Beth Israel Hospital and the Harvard Medical School, Boston, Massachusetts 02215; and The Division of Endocrinology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

ABSTRACT. In man, acute stress, like extensive surgery, leads to a rapid and prolonged decrease in serum T3 concentrations. The present study was carried out to investigate the mechanisms that underly the abrupt decrease in T3-neogenesis that occurs in response to acute surgical stress. Male Sprague-Dawley rats, surgically thyroidectomized, and treated with 1.2 ng T4/100 g BW/day, underwent wide vertical and horizontal incisions extending into the abdominal cavity while receiving light ether anesthesia. Different dietary manipulations were performed to investigate the superimposed influence of reduced carbohydrate and caloric intake on T3-neogenesis. The metabolism of 125I T4 labeled in its outer (phenolic) ring was investigated in liver, kidney, and brain homogenates of animals killed 48 h after surgery. In liver, values for the proportion of T4 degraded and the percent generation of T 3 and iodide were unaffected by laparotomy. The percent T3 generation in experiments with 25 nM T4 concentration was 3.7 ± 1.24% (mean ± SD) in fed control animals given free access to 5% glucose, 3.4 ± 0.67% in unoperated controls given a restricted amount of chow and 5%

glucose, and 3.8 ± 0.67% in operated animals given free access

to chow and 5% glucose. As expected, T3 neogenesis in livers from unoperated animals was significantly reduced in rats fasted for 48 h and this reduction was similar in laparotomized rats fasted for 48 h after surgery. As in the liver, no effect of laparotomy on T4 metabolism in kidney and brain homogenates was observed. Finally, serum total T4 and T3 concentrations were not affected by surgery. It is concluded that acute surgical stress in thyroidectomized T4 replaced rats does not influence T4 metaoblism in liver, kidney, and brain homogenates or affect the serum T4 and T3 concentrations. Since thyroid secretion of T4 (and Ts) was eliminated and careful attention was paid to caloric intake in this rat model, previously reported abnormalities in serum thyroid hormone concentrations and T3-neogenesis in various states of nonthyroidal illness in man and rat, including surgery, are probably contributed to by thyroid secretion of T4 (and T3) and caloric deprivation, especially carbohydrate. (Endocrinology 128: 146-152, 1991)

D

of this phenomenon, T3-neogenesis in rat liver in vitro has been a widely used model since in vitro conversion of T4 to T 3 is reduced in livers from starved and diabetic rats (9,10) and this abnormality is reversed or prevented by glucose and insulin, respectively (9,10). Glucose feeding in thyroidectomized, T4 replaced rats causes a significant increase in serum T 3 concentration and the fractional conversion of T4 to T 3 (11). Much less is known of the mechanism(s) that underly the abrupt decrease in T3-neogenesis that occurs in response to acute nonthyroid illness or surgical stress (1220). Therefore, we studied the metabolism of T4 by liver, kidney, and cerebral cortex homogenates from rats subjected to the severe surgical stress of a wide laparotomy. The metabolism of T4 by these three tissues was not affected by this severe surgical stress.

ESPITE the importance of the peripheral conversion of T4 to T 3 (T3-neogenesis) as a determinant of the overall metabolic impact of the thyroid hormones, the mechanism(s) by which a variety of abnormal states influence T3-neogenesis remain incompletely understood (1-3). Studies in man suggest that glucose metabolism is closely linked to the activity of the 5'-monodeiodinase that converts T4 to T3. Starvation, carbohydrate deprivation, and diabetes mellitus are associated with decreased T3-neogenesis (4-7), an abnormality promptly reversed by the administration of food, carbohydrate, and insulin, respectively (5, 6), whereas overnutrition leads to an increased concentration of T 3 (8). In studies

Received July 6,1990. Address requests for reprints to: Dr. Lewis Braverman, Division of Endocrinology and Metabolism, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655. * This work was supported in part by Grant DK-18416 and DK18919 from the NIH. t Recipient of Grant Hi-359/1-2 by Deutsche Forschungsgemeinschaft. X Deceased.

Materials and Methods Hormones and reagents Crystalline sodium salts of T4 and T3 were purchased from Sigma Chemical Co. (St. Louis, MO), and dithiothreitol and 146

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SURGICAL STRESS AND T4 METABOLISM crystalline rT 3 from Calbiochem-Behring Corp. (San Diego, CA). Experiments with radioactive T4 labeled with 125I in its outer (phenolic) ring (125I-T4) were performed using either relatively low (2-6 nM) or high (25 or 75 nM) substrate concentrations. For the former experiments, 125I-T4 (SA, ~400 /iCi/ ng) was either donated by Travenol-Genentech (Cambridge, MA), or purchased from Dupont-New England Nuclear (Boston, MA; SA, 1080-1320 fiCi/ng). Dupont-New England Nuclear provided 125I-T4 of lower SA (150 ixCi/ng) for studies of the metabolism of higher T4 substrate concentrations. RIA kits for measurements of serum T4 and T 3 concentrations were kindly provided by Travenol-Genentech, using hormone free rat serum standards. Animals Experiments were performed in male Sprague-Dawley rats (CD strain; Charles River Breeding Laboratories, Wilmington, MA) weighing 150-200 g. To exclude any influence of variations in thyroid function as a result of the differing experimental conditions being studied, all animals were surgically thyroidectomized 10 days before arrival in our laboratory. All rats were then injected sc daily with T4 (1.2 /ig/100 g BW) for the duration of the experiments. Animals were maintained in the laboratory for 7 days on an ad lib diet of pelleted laboratory chow (Agway, Inc., Syracuse, NY) and were given free access to tap water. Seven different groups of approximately the same weight were then studied as outlined below. Surgical procedure In each experiment, approximately half the rats underwent laparotomy while receiving light ether anesthesia. Wide vertical and horizontal incisions extending into the abdominal cavity were performed. Approximately 5 min later, muscle layers were closed with sutures and the skin with staples, using a 9 mm Autoclip (Clay-Adams, Division of Becton-Dickinson, Persippany, NY). The length of time between induction of anesthesia and completion of surgery was approximately 15 min. Animals were killed 48 h later and tissues taken for studies of T4 metabolism in vitro. Control animals were not anesthetized and were not operated upon. Dietary manipulations Different dietary regimens were employed on the basis of preliminary results summarized in the first part of Results. Surgically treated and control rats either fasted with free access to tap water or had free access to chow and 5% glucose in the drinking water. Since laparotomy caused a decrease in food intake, the access to food and fluid was modestly restricted in other groups of animals. For this purpose, the animals were kept in single cages. The body weight was determined every day as well as the amount of food and fluid they ingested. Animals with limited access in general ingested the total amount of food available. Experimental groups The experimental groups were as follows: operated animals with free access to food and 5% glucose in the drinking water;

147

control animals with free access to food and 5% glucose in the drinking water; control animals with restricted access to food and 5% glucose in the drinking water; operated animals with free access to food and tap water; operated animals with free access to tap water, but fasted for 48 h after surgery; control rats with free access to tap water but fasted for 48 h after surgery; and control rats with free access to food and tap water. Studies of in vitro T4 metabolism At the end of each treatment regimen, animals were lightly anesthetized with ether and killed by decapitation. Blood was collected from the carotid arteries and the serum was separated and stored at —20 C for later analysis of serum T4 and T 3 concentrations. Samples of liver, kidney, and brain were quickly removed, frozen on dry ice, wrapped in foil, and stored frozen at —70 C until used. Usually, specimens were studied within a few days, but in some experiments an interval of 3 or 4 weeks elapsed before study. Control studies revealed that storage of tissues for this period did not affect T4 metabolism. For the in vitro metabolism of 125I-T4, samples were allowed to thaw at room temperature, and weighed segments of liver, kidney, or cerebral cortex were homogenized in Krebs-Ringer phosphate buffer, pH 7.4 (1:3, wt/vol, in liver and kidney; 1:1.5, wt/vol, in brain). The 200 n\ final volume of the reaction mixture comprised 100 ix\ homogenate, 50 /A 40 mM dithiothreitol (final concentration 10 nM), and 50 til 126I-T4-solution containing 0.1-0.4 /iCi 125I (2.0-6.0 nM T4). In some experiments, liver homogenates were also incubated with 0.6-1.8 ixCi of low SA 125I-T4 to yield substrate concentrations of 25 and 75 nM. Incubations were carried out at 37 C for 2 h in a shaking water bath under room air. Since it has been suggested that FFA in serum may inhibit hepatic T3-neogenesis, especially under anerobic conditions (21), some incubations were carried out at 37 C under a constant stream of humidified N2. Reactions were stopped by adding 200 n\ methanol-2 N ammonia (99:1, vol/vol), producing fine granular precipitates which were then uniformly dispersed. Ten microliters of the suspensions were subjected to descending paper chromatography in a hexanetertiary amyl alcohol-2 N ammonia solvent system (1:10:11, vol/vol), together with carrier T4, T3 and iodide, as described previously (9). Strips were then autoradiographed with the aid of an image intensifier for at least 3 days, sufficiently long to permit visualization of all reaction products that contributed at least 2% of the total radioactivity applied. Zones visualized in autoradiographs were excised and counted in a 7-scintillation counter. Values for the percentage degradation of T4 and percentage generation of 125I-labeled products were calculated and then corrected for the proportion of these products and the percentage of 125I-T4 present in the original substrate, as reported previously (9). The latter values were determined from vessels incubated at 37 C for 2 h in the absence of tissue (tissuefree controls) and in vessels to which 200 n\ methanol-2 N ammonia (99:1) were added before the addition of 125I-T4. No significant degradation of 125I-T4 or generation of 125I-T3 was observed in these control preparations. Protein concentrations of homogenates varied between 4 and 5 mg/tube from experiment to experiment (22). However, within a single experiment, values in the several experimental groups did not differ appre-

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SURGICAL STRESS AND T4 METABOLISM

148

ciably. Therefore, reported values for the several indices of T4 metabolism are not corrected for protein concentration, since we did not compare the deiodinative activity of the different tissues but rather exogenous effects within the same tissue. Statistical analyses Statistical analyses were performed with the unpaired Student's t test when only two experimental groups were employed, and analysis of variance, followed by Newman-Keuls test (23), when results in more than two groups were compared. Results Dietary effects of laparotomy

Animals subjected to laparotomy decrease their intake of food and fluids during the 48 h after surgery. Since

T3-neogenesis in vitro is decreased in rats subjected to food deprivation, an effect that can be overcome by providing 5% glucose in the drinking water, preliminary studies were conducted to determine the effect of laparotomy on the intake of food and fluids so that control rats in some subsequent experiments could be restricted to the same food and glucose water intake as occurred in surgically treated rats. During the 48 h after laparotomy, rats consumed 13.5 ± 5.0 g (mean ± SD) chow and 47.5 ± 28.4 ml 5% glucose solution/day. Values in control rats were 17.8 ± 2.4 and 85.8 ± 61.9, respectively. In some groups of animals in subsequent studies, daily food and fluid intake were restricted to the quantities consumed by operated animals. Metabolism of T4 in liver 125

In all experimental groups, I-iodide was the main product of the metabolism of 125I-T4, with lesser proportions of 125I-labeled T3, 3,3'-diiodothyronine and a chromatographically immobile origin material. Since only small proportions of the latter two products were generated under the conditions of these experiments, only results with respect to the generation of iodide and T 3 will be described. Initial experiments were carried out to assess the metabolism of T4 in livers in three groups of rats: control and laparotomized rats given free access to chow and 5% glucose solution, and control rats whose intake of food and glucose solution were restricted to that previously found in animals during the first 48 h of recovery from laparotomy. Studies of 125I-T4 metabolism in each liver homogenate were conducted at total T 4 concentrations of 3 nM, 25 nM, and 75 nM. Values for the proportion of added T4 degraded and the percentage generation of T 3 and iodide decreased as the concentration of T4 substrate increased (Table 1). This was especially evident at the highest concentration

Endo • 1991 Voll28«Nol

of T 4 employed (75 nM). However, at each substrate concentration, none of the indices of T4 metabolism differed significantly among the three experimental groups. These findings indicated that in animals given 5% glucose in tap water to drink, neither laparotomy nor simple reduction in food and glucose intake in unoperated animals comparable to that observed in operated animals, had any effect on hepatic T4 metabolism. The next experiments were undertaken to determine whether the apparent lack of effect of laparotomy on T3neogenesis in animals fed 5% glucose in place of tap water would also be evident in animals given tap water to drink and either fasted totally or allowed free access to chow. Studies were performed in the presence of low (2 nM) and high (75 nM) substrate concentrations. As would be expected from previous studies, T3-neogenesis in livers from surgically treated and control rats were significantly decreased by starvation (9,10). However, at both substrate concentrations, values for the fractional generation of T 3 from T4 were similar in surgically treated and control rats in animals allowed access to food, and were decreased but similar in animals fasted for the 48 h after surgery (Fig. 1). To determine whether laparotomy might inhibit in vitro hepatic T 3 neogenesis under anaerobic conditions, T4 metabolism (3 nM) was conducted in homogenates under a constant stream of N2, rather than in room air. Again, no effect of surgery was observed (data not shown). Kidney Studies comparable to those conducted in liver were carried out in homogenates of kidney from surgically treated and control rats subjected to various dietary regimens. As in the liver, 125Iodide and 125I-T3 were the principal products of the metabolism of 125I-T4 in all experimental groups. The experiments in which surgically treated and control rats were allowed free access to chow and were given 5% glucose in their drinking water and in another group which was given restricted quantities of chow and 5% glucose, indices of T4 metabolism (3 nM substrate concentration) did not differ significantly among the three experimental groups (Table 2A). A similar lack of effect of laparotomy on T 4 metabolism was evident in experiments in which surgically treated and control rats were allowed free access to chow and tap water (Table 2B). No effect of fasting was observed. Cerebral cortex

Homogenates of cerebral cortex degraded T4 far more than liver and kidney homogenates. The products generated also differed from those in liver and kidney (24).

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SURGICAL STRESS AND T4 METABOLISM

149

TABLE 1. Effect of surgery and food restriction on the metabolism of 126 I-T 4 in homogenates of rat liver 126

I-T 4 metabolism (% added 125I-T4; mean ± SD)

Experimental conditions Surgical status

125 I-T 4 degradation

Diet

Fluids

Control Control

Fed Restricted

Operated

Fed

5% glucose Restricted, 5% glucose 5% glucose

125 Iodide generation

126 I-T 3 generation

3 nM substrate concentration 11 13

14.47 ± 4.47° 16.49 ± 3.82*

8.96 ± 3.32 10.67 ± 3.82*

3.55 ± 1.08s 3.95 ± 0.93'

11

15.53 ± 3.29C

9.46 ± 1.99'

3.44 ± 0.88u

25 nM substrate concentration Control Control

Fed Restricted

Operated

Fed

5% glucose Restricted, 5% glucose 5% glucose

11 13

12.38 ± 3.59d 12.28 ± 2.00e

5.60 ± 1.80m 5.90 ± 1.01"

3.66 ± 1.24" 3.39 ± 0.67"

11

12.34 ± 1.73'

5.85 ± 1.13°

3.76 ± 0.671

75 nM substrate concentration Control Control

Fed Restricted

Operated

Fed

5% glucose Restricted, 5% glucose 5% glucose

11 13

8.85 ± 1.90s 9.05 ± 1.91*

4.96 ± 1.30" 5.02 ± 0.89*

2.02 ± 0.68y 1.89 ± 0.74*

11

9.27 ± 1.48'

5.49 ± 0.85r

2.12 ± 0.54aa

Superior letters denote experimental group and function tested. Values shown are mean ± SD of those obtained in the number of animals indicated by n. Statistical analysis by analysis of variance, followed by Newman-Keuls test for comparisons between multiple groups (23). Relevant significant differences ( P values): a us. s < 0.005; b vs. e < 0.05; b vs. h < 0.001; c vs. ' < 0.001;' vs. m < 0.005; ; us. " < 0.001; h vs. n < 0.001; * us. " < 0.001; 'vs.°< 0.001; lvs.r< 0.001;" vs.y< 0.001;' vs. * < 0.001; " vs. M < 0.001;" vs.y< 0.001; w vs. z < 0.001; x vs. M < 0.001.

EFFECT OF STARVATION AND/OR LAPAROTOMY ON T3 -NEOGENESIS IN RAT LIVER 2nMTif 3 -_ 2

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The effect of surgical stress on the in vitro metabolism of thyroxine by rat liver, kidney, and brain.

In man, acute stress, like extensive surgery, leads to a rapid and prolonged decrease in serum T3 concentrations. The present study was carried out to...
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