Effects of thyroidectomy and triiodothyronine administration on rat liver alcohol dehydrogenase

GASTROENTEROLOGY

Effects of Thyroidectomy and Triiodothyronine Administration Liver Alcohol Dehydrogenase ESTEBAN MEZEY and JAMES

Hepatic alcohol dehydrogenase is the principal enzyme which catalyzes the oxidation of ethanol in vivo. A number of studies suggest that the hepatic activity of alcohol dehydrogenase is regulated by various hormones. Striking increases in liver alcohol Received June 20. 1980. Accepted October 15, 1980. Address requests for reprints to: Esteban Mezey, M.D., Balti-

more City Hospitals, 4940 Eastern Avenue, Baltimore, Md. 21224. This work was supported by grant AA00628 from the United States Public Health Service. 0 1981 by the American Gastroenterological Association 00165085/81/030586-09$02.50

on Rat

J. POTTER

Deoartment of Medicine. Baltimore Citv Hospitals School of Medicine, Baltimore, Maryland

The effect of thyroidectomy on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination was determined in the rat. Thyroidectomy resulted in a marked increase in liver alcohol dehydrogenase activity. Three isoenzymes of alcohol dehydrogenase activity were demonstrated in thyroidectomized animals by starch gel electrophoresis, as compared with two in sham-operated control animals. Triiodothyronine administration decreased the enzyme activity in control animals, and suppressed the enhanced activity in thyroidectomized animals. Inhibition of alcohol dehydrogenase by triiodothyronine in vitro was found to be competitive with respect to NAD’ and uncompetitive with respect to ethanol in both control and thyroidectomized animals. Thyroidectomy did not result in any changes in the rate of ethanol elimination. The cytosolic free NAD+/NADH ratio decreased after ethanol administration in both control and thyroidectomized animals, while the mitochondrial-free NAD+/NADH ratio decreased only in the control animals. These results indicate that the thyroid is a repressor of liver alcohol dehydrogenase activity. A defect in the transfer of reducing equivalents from the cytosol to the mitochondria appears to limit the rate of ethanol elimination in thyroidectomized animals with increased liver alcohol dehydrogenase activity.

1981:80:566-74

and the Johns Hopkins

University

dehydrogenase were recently observed in the rat af(2) ter immobilization stress (l),hypophysectomy and castration (33. The increases found after hypophysectomy and castration were suppressed by the administration of growth hormone (2) and testosterone (3,4), respectively. In earlier studies thyroidectomy (5) and the chronic administration of propylthiouracil (6) were also reported to increase rat liver alcohol dehydrogenase. Thyroidectomy decreases pituitary and serum growth hormone (7) and hence, may affect liver alcohol dehydrogenase by this indirect mechanism. Thyroxine and triiodothyronine are known to inhibit horse liver alcohol dehydrogenase in vitro (8,9), and to depress rat liver alcohol dehydrogenase in vivo (6,10,11). Rates of ethanol elimination have been found to be increased in hyperthyroid patients (12) and after the administration of thyroxine or triiodothyronine in some (10,13,14), but not most studies (6,15-17). The purpose of this study was to determine the effect of thyroidectomy on the activity of liver alcohol dehydrogenase and on rates of ethanol elimination.

Materials and Methods Animals and Treatments Thyroidectomized and sham-operated male Sprague-Dawley rats weighing between 100 and 120 g were obtained from Charles River Breeding Laboratories (Wilmington, Mass.). The animals were placed in separate wire mesh cages in a room at a controlled temperature of 20% with light/dark cycles alternating every 12 h, beginning at 790 AM. They were provided water and Purina chow ad libitum. The water given to the thyroidectomized animals contained 1% calcium chloride. Ten days after surgery these animals were started on subcutaneous injections of hormones or isovolumetric amounts of saline. Eight thyroidectomized and eight sham-operated animals received 3,3’,B-triiodo-L-thyronine, sodium salt (Sigma Chemical Co.,St. Louis, MO.), 15 fig/

March 1981

THYROIDECTOMY AND LIVER ALCOHOL DEHYDROGENASE

100 g of body wt twice a day, while an equal number of thyroidectomized and sham-operated animals received isovolumetric amounts of normal saline. The remaining eight thyroidectomized animals received bovine growth hormone (0.8-1.0 USP U/mg, Miles Laboratories Inc., Elkhart, Ind.) 0.5 mg/lOO g of body wt twice a day. The injections were given for 8.5 days, and the animals were killed 2 h after the last injection, between 1O:OOand 11:00 AM.

Enzyme

Determinations

The animals were killed by decapitation. The livers were removed, rinsed in 1.15% KCl, and weighed. Liver tissue was minced and homogenized in a Potter-Elvehjem with 4 vol of 0.25 M sucrose in 0.1 M Tris-HCl buffer pH 7.4. The homogenate was centrifuged at 9000 g for 10 min. The resulting precipitate was discarded, and the supernatant was centrifuged at 106,000 g for 80 min. The supernatant fraction obtained at this point was separated for assay of alcohol dehydrogenase activity. For the determination of the activity of the microsomal ethanol oxidizing system, the microsomal pellet was washed with 8 ml of the above buffer and then centrifuged at 106,000 g for 80 min. The washed pellet was finally resuspended in 8 ml of 0.1 M NaH,PO,-K,HPO, buffer, pH 7.4. Alcohol dehydrogenase activity was determined in the 106,000 g supernatant at 30°C by the method of Bonnichsen and Brink (18). The volume of the reaction mixture was 3 ml and consisted of 0.01 M pyrophosphate buffer pH 10.3, 10 mM ethanol, 0.41 mM NAD+, and 0.1 ml of a l/ 5 dilution of the supernatant. A blank reaction without ethanol was run in each case. The change in optical density at 340 nm was recorded for 10 min in each case. The alcohol dehydrogenase activity was then calculated from the molecular extinction coefficient 6.22 cm*/pmol for NADH (19). In thyroidectomized and sham-operated animals not receiving hormone injections, alcohol dehydrogenase was also determined at more physiologic conditions of 37’C and pH 7.2 by the method of Crow et al. (20), and in the reductive direction with 3.5 mM acetaldehyde as a substrate by the method of Krebs et al. (21). The microsomal ethanol-oxidizing activity was determined as described by Lieber and DeCarli (22). Protein concentration was determined by the method of Lowry et al. (23) with bovine serum albumin used as a standard. In the case of liver alcohol dehydrogenase, MichaelisMenten constants for ethanol and NAD’ were determined from Lineweaver-Burk plots obtained from assay of the enzyme at nonsaturating ethanol or NAD+ concentrations,

Effect of Triiodothyronine Dehydrogenase

on Liver Alcohol

Varying concentrations of triiodothyronine were added in a volume of 0.1 ml to the reaction mixture used in the measurement of the enzyme activity immediately before the addition of ethanol. At appropriate inhibitory concentrations of triiodothyronine the kinetics of inhibition were determined with regard to ethanol and NAD+ using Lineweaver-Burk plots.

Jsoenzymes

of Alcohol

567

Dehydrogenase

Liver samples were homogenized in 2 vol of water and then were centrifuged at 9000 g for 10 min. The resulting precipitate was discarded and the supernatant centrifuged at 106,000 g for 60 min. Starch gel electrophoresis of the 106,000-g supernatant was carried out at pH 8.6 and 4“C on horizontal 10.4% starch gel (Electrostarch Co., Madison, Wis.) for 18 h as described by Smith et al. (24). At the end of the electrophoresis the gels were stained for alcohol dehydrogenase activity of incubating them for 1 h at 37’C in 0.05 M Tris-HCl buffer, pH 8.6, containing (per 100 ml): NAD’, 80 mg; nitro blue tetrazolium, 40 mg; phenazine methosulfate, 8 mg; and 100% ethanol, 1.2 ml. Control gels were incubated in the absence of ethanol.

Rates

of Ethanol

Disappearance

Rates of ethanol disappearance were determined in thyroidectomized and sham-operated animals 15 days after surgery. Ethanol (2.0 g/kg of body wt) was injected intraperitoneally as a 10% solution (wt/vol) in water. Blood was obtained from the retroorbital plexus of each animal with a heparinized capillary tube starting at 1 h after ethanol administration and at 30 min intervals thereafter for 3 h. After centrifugation at 2000 g for 10 min the separated plasma samples were analyzed for ethanol concentration by gas-liquid chromatography (25). Methanol was used as an ihternal standard for each sample. Ethanol concentrations in the plasma, when plotted against time, followed a linear function. The rate of ethanol disappearance from the plasma was obtained from the slope of the regression line calculated by the method of the least squares. The ethanol degradation rate expressed in mmoles/kilogram of body weight/hour was obtained by first calculating Widmark factor, r, and then multiplying it by 10 times the rate of ethanol disappearance from the blood.

Hepatic

NAD+/NADH

Ratio

Ethanol (2.0 g/kg of body wt) as a 10% solution (wt/vol) or an isovolumetric amount of saline was injected intraperitoneally to thyroidectomized and sham-operated animals 15 days after surgery. The animals were killed, by cervical dislocation, 2 h after the injections. The livers were removed, freeze-clamped with aluminum plates as described by Wollenberger et al. (26) and dropped in liquid nitrogen. Less than 10 s elapsed between the blow on the neck and freezing of the liver. Thereafter the frozen liver was pulverized in a precooled mortar with the addition of liquid nitrogen, transferred to a preweighed container containing 6% (wt/vol) perchloric acid, homogenized, and the weight of the liver was recorded. Further treatment of the tissue consisted of centrifugation to separate precipitated protein, adjustment of the supernatant to pH 6.0 with 20% (wt/vol) KOH, followed by centrifugation to remove KClO,, and treatment with Florisil (Floridin Co., Hancock, W. Va.) as described by Williamson et al. (27). The supernatant was used for the determination of metabolites. Lactate was determined by the method of Hohorst (28) and pyruvate by the method of Biicher et al (29). ,&-

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MEZEY AND POTTER

Table

1.

GASTROENTEROLOGY

Body Weight, Liver Weight, Cytosolic, and Microsomal

Protein Concentrations

in the Various

Vol. 60, No. 3

Experimental

Groups” Control

Determinations

Thyroidectomy

228.5 f 8.58 9.2 f 0.50

Body weight (g) Liver weight (g)

180.3 f 6.8 f 3.8 f 84.7 f 24.0 k

3.9 f 0.009

(g/loo g body wt)

Cytosolic protein (mg/g) Microsomal protein (mg/g)

84.9 f 2.48 23.3 f 1.08

7.22b 0.35b 0.09 2.55 0.46

Control + T3 213.9 f 7.4 f 3.5 f 89.9 f 27.2 f

Thyroidectomy +T3

3.70 O.lab O.Oab 2.2gb 0.4gb

168.4 + 5.0 f 3.0 + 92.4 f 24.0 f

Thyroidectomy +GH

9.28’ 0.78’ O.llCJJ 2.02e 0.55

174.1 f 6.0 f 3.4 f 85.4 f 24.4 f

3.48 0.31’ 0.14 3.24 0.42

a All values are expressed as means f SEM of eight animals. T3 denotes triiodothyronine; GH, growth hormone. b Significantly different from control at p < 0.01. ’ Significantly different from control at p < 0.001. d Significantly different from thyroidectomy alone at p < 0.001. e Significantly different from thyroidectomy alone at p < 0.65.

Hydroxybutarate was determined by the method of Mellanby and Williamson (SO), and acetoacetate by the method of Williamson et al. (31). The cytoplasmic-free NAD+/NADH ratio was calculated from the lactate dehydrogenase reaction (32), while the mitochondrial-free NAD+/NADH was calculated from the &hydroxybutarate dehydrogenase reaction (27). The results are expressed as the mean f SE. Statistical significance was determined by the Student’s t-test.

Results The thyroidectomized animals had a decreased body weight and liver weight as compared with the sham-operated control animals (Table 1). The liver/body weight ratio and the concentrations of protein in the cytosol and microsomes were similar in thyroidectomized and control animals. Administration of triiodothyronine to control animals did not change body weight but resulted in a decrease in liver weight and liver/body weight ratio and an increase in the concentrations of protein in the cytosol and microsomes. Administration of triiodothyronine to thyroidectomized animals resulted in no significant changes in body or liver weight, but caused a decrease in liver/body weight ratio and an increase in cytosolic protein. By contrast, administration of growth hormone to the thyroidectomized animals

Table 2.

had no significant effect on body weight, liver weight, or protein concentration. Thyroidectomy resulted in a marked increase in liver alcohol dehydrogenase activity at p < 0.001 whether expressed per milligram of protein, per gram of wet liver weight, per kilogram of body weight (not shown), or per total animal (Table 2). Measurement of the enzyme activity at more physiologic conditions of 37’C and pH 7.2 revealed activities of 3.18 t 0.142 pmol/mg/h in thyroidectomized as compared with 1.84 + 0.108 pmol/mg/h in controls (p < 0.001). The increase in the enzyme activity was also demonstrated in the reductive direction with acetaldehyde as a substrate; the mean activity in the thyroidectomized animals was 14.78 f 0.823 as compared with 8.15 f 0.431 pmol/mg/h in the controls (p < 0.001). Triiodothyronine administration resulted in a decrease in liver alcohol dehydrogenase in sham-operated and in a suppression of the enhanced enzyme activity in the thyroidectomized animals. The enzyme activity in the thyroidectomized animals after the administration of triiodothyronine was lower than the control value, but higher than the value obtained after the administration of the same dose of triiodothyronine to the control animals. The administration of growth hormone had no sig-

Effect of Thyroidectomy and Triiodothyronine Administration on Liver Alcohol

Dehydrogenase

Activity”

Alcohol dehydrogenase Treatment Control Thyroidectomy Control + T3 Thyroidectomy Thyroidectomy

(pmol/mg protein/h)

+ T3 + GH

1.74 f 2.99 f 0.86 f 1.36 f 3.51 f

0.098 0.130b 0.115b 0.085Ctd 0.208b

(pmol/g liver/h) 146.2 f 277.3 f 80.8 * 144.3 f 338.7 f

9.00 20.81b 10.95b 9.2ab 36.10b

(mmoI/rat/h) 1.33 f: 0.065 1.83 f 0.074b 0.60 * O.fJaab 0.71 f 0.048bsC 1.95 f 0.086b

a The values are expressed as means f SEM of eight animals. T3 denotes triiodothyronine; GH, growth hormone. b Significantly different from control at p < 0.001. c Significantly different from thyroidectomy alone at p < 0.001. d Significantly different from control at p < 0.05.

THYROIDECTOMY

March 1981

AND LIVER ALCOHOL DEHYDROGENASE

Table 3. Effect of Thyroidectomy and Triiodothyronine Administration on the Activity of the Microsomal Oxidizing

Ethanol

System” Microsomal (nmoJ/mg protein/min)

Treatment Control Thyroidectomy Control + T3 Thyroidectomy Thyroidectomy

569

+ T3 + GH

ethanol oxidizing system (pmol/rat/h)

(nmol/g Jiver/min)

8.70 f 0.945 9.58 f 0.437 9.30 f 0.599 13.00 f 0.972b.C 9.62 ZIZ 1.080

244.0 f 299.9 f 252.2 f 309.8 + 234.5 +

24.75 10.48 12.44 19.67 25.63

1.96 f 1.39 f 1.85 f 1.55 f 1.33 f

o The values are expressed as means f SEM of eight animals. T3 denotes triiodothyronine; GH, growth hormone. ent from thyroidectomy alone at p < 0.01. ’ Significantly different from control at p < 0.05.

nificant effect on the enhanced liver alcohol dehydrogenase in the thyroidectomized animals. The hepatic activity of the microsomal ethanol oxidizing system was not affected by thyroidectomy or by the administration of triiodothyronine to control animals (Table 3). Triiodothyronine administration in thyroidectomized animals increased the activity of microsomal ethanol oxidizing systems per milligram of microsomal protein, but not per gram of liver, or per total animal. Thyroidectomy and the administration of triiodothyronine did not affect the K, of alcohol dehydrogenase for ethanol and NAD’ in control and thyroidectomized animals. Also the administration of growth hormone had no significant effect on either K, in thyroidectomized animals. Triiodothyronine inhibited alcohol dehydrogenase in vitro in both the control and thyroidectomized animals (Table 4). Kinetic studies revealed that the inhibition of alcohol dehydrogenase was competitive with respect to NAD’ (Figure 1) and uncompetitive with respect to ethanol (Figure 2) in both the control and thyroidectomized animals. Starch gel electrophoresis of the liver supernatant fraction revealed three bands of alcohol dehydrogenase activity migrating toward the cathode in thyroidectomized animals, as compared with two bands in the sham-operated control animals (Figure 3). Thyroidectomy did not result in any significant changes in the rate of ethanol elimination. The rates of ethanol elimination were 7.33 f 0.335 mmol/kg body wt/h (1.18 + 0.090 mmol/rat/h) in controls as compared with 6.50 + 0.272 mmol/kg body wt/h (1.01 k 0.052 mmol/rat/h) in thyroidectomized animals. The cytosolic-free NAD+/NADH ratio was similar after the administration of saline in control and thyroidectomized animals (Table 5). Free hepatic concentrations of acetoacetate and P-hydroxybutarate were higher in thyroidectomized than control animals after the administration of saline, but

0.202 0.090 0.058 0.146 0.166

b Significantly

differ-

the calculated mitochondrial-free NAD+/NADH ratio was not significantly different. Two hours after ethanol administration the cytosolic-free NAD+/ NADH ratio decreased in both the control and thyroidectomized animals, while the mitochrondrial-free NAD+/NADH ratio decreased in the control but not in the thyroidectomized animals.

Discussion This study shows that thyroidectomy in the rat increases the activity of liver alcohol dehydrogenase. This agrees with an earlier observation of Susuki et al. (5), who found an increase in the activity of the enzyme expressed per milligram of cytosol protein and measured in the reductive direction with acetaldehyde as a substrate. Because the concentration of cytosol protein in the liver, liver weight, or body weight were not provided in that study, it was unclear whether or not thyroidectomy resulted in an increase in total activity of the enzyme. This study shows that the increase can be demonstrated with both ethanol and acetaldehyde as substrates when expressed per milligram of cytosol protein, and that there is indeed an increase in

Table

4.

Effect of Triiodothyronine Dehydrogenase In Vitro”

on Liver Alcohol

Activity (%) Triiodothyronine (Gw 0 0.1

5.0 50.0 125.0 370.0

Control 100.0 92.4

89.8 84.4 72.8 48.0

Thyroidectomized 100.0

85.7 88.9 86.3 74.9 44.8

o Triiodothyronine was added to the reaction mixture containing the liver supernatant. The reaction was started by the addition of ethanol. The enzyme activities are expressed as a percentage of the value in the absence of triiodothyronine.

570

GASTROENTEROLOGY

MEZEY AND POTTER

THYROIDECTOMIZED

CONTROL 350

-/

250

-

Vol. 80, No. 3

-0.1 “[NAD+]

“[NAD+]

(PM 1

(pM 1

Figure I Lineweaver-Burk plot of the inhibition of liver alcohol dehydrogenase of the control and thyroidectomized animals by triiodothyronine at varying concentrations of NAD+ in the reaction mixture. The concentrations of triiodothyronine in the reaction mixture were: O-0-0, 0; O-O-O, 100 pm; and W, 370 PM.

total liver alcohol dehydrogenase. In another study, the administration of propylthiouracil for a period of 6 wk, which was assumed to result in a hypothyroid state, also resulted in an increase in total liver alcohol dehydrogenase activity (6). The effect of thyroidectomy in increasing the activity of liver alcohol dehydrogenase joins previous observations showing increases of the enzyme after removal of other endocrine organs such as after hypophysectomy (2) and castration (4). The unique feature of the increase in the enzyme activity after thyroidectomy is the appearance of an additional band of alcohol dehydrogenase activity migrating toward the cathode on starch gel electrophoresis. We are currently initiating studies to purify and characterize this isoenzyme. The removal of a repressive effect of thyroid hormones on the enzyme after thyroidectomy is a possible cause for the appearance of an additional isoenzyme of alcohol dehydrogenase. The effect of triiodothyronine in decreasing hepatic lactate dehydrogenase activity with elimination of the LDH-5 isoenzyme in the rabbit is of interest in this respect (33). The increased activity of liver alcohol dehydrogenase after thyroidectomy was readily

suppressed by the administration of triiodothyronine, much in the same way that the increased enzyme activities in the hypophysectomized (2) and castrated (4) animals were suppressed by growth hormone and testosterone, respectively. The adminTHYROlDECTOMlZED

CONTROL 200

200 -

1

H I.0

-1.0

‘/ [ETHANOL]

IBM)

20

3.0

1.0

-1.0 ‘/[ETHANOL]

2.0

3.0

(mu)

Figure 2. Lineweaver-Burk plot of the inhibition of liver alcohol dehydrogenase of the control and thyroidectomized animals at varying concentrations of ethanol in the reaction mixture. The concentrations of triiodothyronine in the reaction mixture were: O-O-0,0; O-O-O, 100 PM; and W-B-H, 370 PM.

THYROIDECTOMY

March 1961

AND LIVER ALCOHOL

DEHYDROGENASE

571

0+ .

THYROlDECTUMtZEP

CONTROL

Figure 3. Starch gel electrophoresis of liver alcohol dehydrogenase from thyroidectomized and sham-operated control rats. Samples of the 106,00-g supernatants of rat liver homogenate were used. The electrophoresis was performed with 10.4% starch gel at 7 V/ cm for 8 h at 4’C with 25 mM Tris-HCl (pH 8.6) gel buffer containing 40 mM NAD+ and 0.3 M Tris-HCI (pH 8.6) as the bridge buffer.

istration of growth hormone had no effect on the enzyme activity in the thyroidectomized animals. Thyroidectomy in rats results in a decrease in the pituitary and serum content of growth hormone (7). Hence, the lack of suppression of the enhanced enzyme activity in the thyroidectomized animals by growth hormone suggest that the increase in the enzyme was not mediated by a decrease in growth hormone alone. The inhibitory effect of triiodothyronine on the enzyme demonstrated after its administration in vivo and in vitro by its addition to the liver cytosol is in agreement with prior observations in the rat in

vivo (6,10,11,15) and with horse liver enzyme in vitro (8,9). It suggests that removal of an inhibitory effect of thyroid hormone is responsible for the increase in the enzyme activity found after thyroidectomy. The competitive inhibition of the rat liver enzyme by triiodothyronine with respect to NAD’ is similar to the observations made with the horse liver enzyme by Gilleland and Shore (9), who suggested that triiodothyronine interfered with coenzyme binding by blocking the binding site of the ADP-ribose portion of the coenzyme. In addition, triiodothyronine was found to be a noncompetitive inhibitor of the rat enzyme with respect to ethanol, which differs from the

572

MEZEY AND POTTER

Table

5.

GASTROENTEROLOGY

Effect of Acute Ethanol Administration

on the Concentration

of Metabolites

Vol. 80, No. 3

and on the Redox State in the

Freeze-Clamped Liver” Thyroidectomized

Control Determination Pyruvate (woI/g) Lactate @mol/g) [NAD+]/[NADH] Cytosol Acetoacetate @mol/g) P-Hydroxybutyrate &mol/g) [NAD+]/[NADH] Mitochondria

Saline 0.093 f 0.807 f 1035.2 f 0.025 f 0.146 f 4.13 f

0.0139 0.0913 115.76 0.0052 0.0233 0.508

Ethanol 0.019 f 0.762 f 255.8 f 0.025 f 0.280 f 2.54 f

0.0031b 0.0876 47.3gb 0.0020 0.0823 0.538b

Saline 0.083 + 0.987 f 878.3 k 0.044 f 0.264 f 3.90 +

0.0132 0.131 106.32 0.0043b 0.0441b 0.597

Ethanol 0.019 f 0.746 f 304.9 f 0.023 + 0.202 * 2.91 *

o.0047c 0.0813 49.08’ 0~3037~ 0.0395 0.540

a All values are expressed as means f SEM of eight animals in each group. b Significantly different from control plus saline at p < 0.05. c Significantly different from thyroidectomized plus saline at p < 0.001. d Significantly different from thyroidectomized plus saline at p < 0.01.

uncompetitive inhibition with respect to substrate demonstrated for the horse enzyme (9). Ethanol is also oxidized by a microsomal ethanol oxidizing system (34). It is estimated that this enzyme system accounts for only a small portion of ethanol metabolism in vivo (35), mostly at high ethanol concentrations (36,37). A recent study revealed an increase in the microsomal ethanol-oxidizing activity expressed per milligram of microsomal protein after the administration of either thyroxine or triiodothyronine to normal female rats (11). The authors suggested that an increase in the activity of this microsomal system may be a mechanism for increased in vivo ethanol elimination in hyperthyroid states. However, no data was provided to determine whether or not there was an increase in the total activity of this enzyme system, which is necessary to make any assumptions about its role in vivo. Indeed in the present study increases in the activity of the microsomal ethanol oxidizing activity were found after the administration of triiodothyronine to the thyroidectomized, but not the control animals, only when expressed per milligram of microsomal protein, but not per gram of liver or per total rat. The increase in microsomal protein concentration per gram of liver after triiodothyronine administration in the sham-operated, but not in the thyroidectomized animals, is probably due to the greater effect of triiodothyronine in stimulating microsomal protein synthesis in intact as compared with thyroidectomized animals (38). The increased activity of liver alcohol dehydrogenase after thyroidectomy was not associated with a change in the rate of ethanol elimination. This is similar to the previous finding of an enhanced liver alcohol dehydrogenase activity in association with a normal rate of ethanol elimination in experimental uremia (39). On the other hand, the decreases in liver alcohol dehydrogenase after thyrox(6,10,11) are ine and triiodothyronine administration

associated with either increases (10) or no changes (6) in the rate of ethanol elimination. It suggests that under these circumstances factors other than total alcohol dehydrogenase activity are rate-limiting in ethanol oxidation. This is in contrast to the findings of an association between increases in liver alcohol dehydrogenase activity and increased rates of ethanol elimination after stress (1) and castration (4) and the parallel decrease in both after fasting (40) in which alcohol dehydrogenase appears to be ratelimiting. The most likely factor limiting ethanol oxidation by alcohol dehydrogenase in the thyroidectomized animal is the rate of reoxidation of NADH. The reoxidation of NADH could be limited by the transfer of reducing equivalents into the mitochondria or their oxidation by the mitochondrial respiratory chain. A defect in the transport of reducing equivalents into the mitochondria of the thyroidectomized animals is suggested by a decrease in the cytosol but not the mitochondrial-free NAD+/NADH ratio after ethanol administration, as compared with the parallel decrease in both ratios in the control animals. The defect in transport of reducing equivalents is most likely related to the dependence of mitochondrial a-glycerophosphate dehydrogenase and hence of the a-glycerophosphate shuttle on thyroid hormones, being increased after thyroid hormone administration and decreased after thyroidectomy (41). In addition, decreases in mitochondrial function and oxygen consumption demonstrated in thyroidectomized animals (42-44) suggest that the reoxidation of NADH by the respiratory chain may also be decreased. It is of note that epinephrine administration increases oxygen consumption by liver slices of intact but not thyroidectomized animals, and that chronic ethanol administration leads to a much lesser increase in oxygen consumption by liver slices of thyroidectomized as compared with those of intact animals (45). The increased free hepatic concentrations of acetoacetate and P-hydroxy-

March 1961

THYROIDECTOMY

animals are most butarate in the thyroidectomized likely the result of decreases in Krebs cycle activity in these animals (44). Increased formation of ketones was demonstrated in the perfused livers of hypothyroid animals in association with decreased gluconeogenesis (44). In conclusion, the increase in liver alcohol dehydrogenase and the unique appearance of a new isoenzyme in the thyroidectomized rat suggest that the thyroid is a repressor of the enzyme. Thyroidectomy, however, did not increase the rate of ethanol elimination because it decreases mitochondrial transport and reoxidation of reducing equivalents, and hence the availability of NAD’ necessary for ethanol metabolism by alcohol dehydrogenase in vivo. The physiologic significance and mechanism for the effects of the thyroid on liver alcohol dehydrogenase remain to be elucidated.

References 1. Mezey

E, Potter JJ, Kvetnansky R. Effect of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism. Biochem Pharmacol 1979;26:657-63. 2. Mezey E, Potter JJ. Rat liver alcohol dehydrogenase activity: effects of growth hormone and hypophysectomy. Endocrinology 1979;104:1667-73. 3. Rachamin G, MacDonald JA, Wahid S. et al. Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Effect of chronic ethanol administration. Biochem J 1980;186:463-90. 4. Mezey E, Potter JJ, Tsitouras PD. Effects of castration on liver alcohol dehydrogenase and ethanol elimination (abstr). Clin Res 1969;26:464A. 5. Suzuki M. Imai K, Ito A, et al. Effects of thyroidectomy and triiodothyronine administration on oxidative enzymes in rat liver microsomes. J Biochem lQ67;62:447-55. 6. Hillbom ME, Pikkarainen PH. Liver alcohol and Sorbitol dehydrogenase activities in hypo- and hyperthyroid rats. Biothem Pharmacol 1970;19:2097-10% 7. Coiro V, Braverman LE, Christianson D, et al. Effects of hypothyroidism and thyroxine replacement on growth hormone in the rat. Endocrinology 1979:105&U-6. 6. McCarthy K, Lovenberg W, Sjoerdsma A. The mechanism of inhibition of horse liver alcohol dehydrogenase by throxine and related compounds. J Biol Chem 1966;246:2754-60. 9. Gilleland MJ, Shore JD. Inhibition of horse liver alcohol dehydrogenase by L-3,3’,5_triiodothyronine. J Biol Chem 1969; 244:5357-m. 10. Israel Y, Videla L, Fernandez-Videla V, et al. Effects of chronic ethanol treatment and thyroxine administration on ethanol metabolism and liver oxidative capacity. J Pharmacol Exp Ther 1975;192:565-74. 11. Moreno F, Teschke R, Strohmeyer G. Effect of thyroid hormones on the activity of hepatic alcohol metabolizing enzymes. Biochem Biophys Res Commun 1979;69:696-12. 12. Ugarte G, Peresa T. Influence of hyperthyroidism on the rate of ethanol metabolism in man. Nutr Metab 1978;22:113-18. 13. Ylikahri RH, Maenpaa PH. Rate of ethanol metabolism in fed and starved rats after thyroxine treatment. Acta Chem Stand 1968;22:1707-9. 14. Goldberg M. Hehir R, Hurowitz M. Intravenous triiodo-

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