Involvement of Hormones in the Swift Increase in Alcohol Metabolism*f

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1 ANNALS O F C L IN IC A L A N D LABORATORY S C IE N C E, Vol. 18, No. 4 Copyright 1988, Institute for Clinical Science, Inc. Involvement of Hormones in the Swift Increase in Alcohol Metabolism*f DONALD T. FORMAN, Ph.D.,î BLAIR U. BRADFORD, 1 JEFFREY A. HANDLER, Ph.D., I EDWARD B. GLASSMAN, Ph.D., and RONALD G. THURMAN, Ph.D." Departments of tpathology, 1Pharmacology, and Biochemistry, University of North Carolina School of Medicine Chapel Hill, NC ABSTRACT The purpose of this study was to determ ine the time course of changes in blood levels of various hormones in C57BL/6J mice during exposure to ethanol vapor. Groups of adult male mice were given 2.0 g per kg ethanol intraperitoneally or as continuous vapor for four hours and rates of ethanol elimination were measured. In parallel, blood samples were collected at timed intervals over 5.5 hours during and following exposure to ethanol. Blood levels of epinephrine, norepinephrine, corticosterone, and glucagon were elevated two- to four-fold during ethanol treatm ent and declined to basal values within one hour following termination of treatment. Elevated blood levels of epinephrine, norepinephrine and corticosterone were highly correlated with higher rates of ethanol elimination (r = 0.80, 0.78, and 0.72, respectively). In contrast, thyroxine and insulin levels were not affected by ethanol. These findings are consistent with the idea that acute administration of ethanol causes the release of glycogenolytic hormones which in turn increase rates of ethanol metabolism. Introduction comes from studies with inbred strains of rat. These studies have shown that castration or treatm en t w ith estradiol in males raises both the rate of ethanol metabolism and the specific activity of alcohol dehydrogenase.2,14 Testosterone appears to have an opposite effect in fe m a le s.17 In ad d itio n, it has b e e n observed that thyroidectomy leads to an increase in hepatic alcohol dehydrogenase (ADH) activity without any corresponding increase in the rate of ethanol metabolism,15 while rats acclimatized to cold conditions are reported to metabo /88/ $01.20 Institute for Clinical Science, Inc. Evidence relevant to the question of w hether or not hormones are involved in the regulation of ethanol m etabolism * This work was supported, in part, by grants AA03624 from the National Institute on Alcohol Abuse and Alcoholism and ARA8907, from the North Carolina Alcoholism Research Authority. Jeffrey A. Handler was the recipient of predoctoral fellowship (ARA8512) from the N orth C arolina Alcoholism Research Authority. t Address reprint requests to: Donald T. Forman, Ph.D.

2 HORMONES AND ETHANOL METABOLISM lize ethanol m ore quickly, despite a small reduction in liver (ADH) activity.21 In a previous study, Yuki and Thurm an,24 using th e p erfu sed rat liver model, showed that ethanol uptake was nearly doubled 2.5 hours after treatm ent with ethanol. They also dem onstrated that epinephrine given one hour prior to liver perfusion increased hepatic oxygen consum ption by about 40 percent. In addition, the effects of ethanol and epinephrine were not additive, suggesting that they may have common mechanisms of action. The increase in the rate of ethanol metabolism was inhibited largely by 4-methylpyrazole, an inhibitor of ADH, indicating that ADH is involved in the Swift Increase in Alcohol M etabolism (SIAM).25 Conclusive evidence in support of this hypothesis has come from studies comparing ADH-positive and negative deerm ice. At any given dose of ethanol, vapor treatm ent with ethanol increased rates of ethanol metabolism in ADH-positive, but not ADH-negative deerm ice.8 Additional studies using alpha and beta adrenergic blocking agents dem onstrated a decrease in hepatic oxygen consumption and a significant reduction in the rate of ethanol elimination in perfused rat liver indicating that adrenergic hormones play an important role in regulation of alcohol elimination. The SIAM phenomenon has been dem onstrated in vivo in rats,19 in several inbred strains of m ice,20 the ADH-positive deerm ouse,8 and humans.1 Based on previous observations, it is reasonable to propose that an early key element in the sequence of events leading to SIAM may be the release of horm ones by ethanol. In support of this hypothesis, SIAM can be obtained by the administration of adrenergic hormones and blocked by alpha and beta antagonists, adrenalectom y and hypophysectomy.25 While several studies have been performed to examine the effects of hormones administered over several days on enzymes involved in ethanol metabolism and rates of ethanol elimination,1317 little is know n co ncern in g sh o rt-te rm effects of ethanol treatm en t on blood horm one levels. T he purpose of this study was to investigate the effect of ethanol administration on the time course of changes in circulating levels of adrenergic and glycogenolytic hormones, and to d e te r m in e p o s s ib le r e la tio n s h ip s between circulating hormone levels and rates of ethanol elimination. Preliminary accounts of this work have appeared elsewhere.7 Materials and Methods A n im a l s Male inbred m ice (C57BL/6J) w ere obtained from the Jackson Laboratory.* Animals were well fed adults betw een 25 to 38 g and eight to 12 weeks of age. Mice w ere maintained on a lab chow diet and given water ad libitum. D e t e r m in a t io n o f B r e a t h E t h a n o l C o n c e n t r a t io n s Blood ethanol concentration were calculated from m easurem ents of breath ethanol as d escrib ed in d etail e lse w here.22 Individual mice were forced to breathe for 15 to 20 seconds into a 2.75 ml closed vessel maintained at 37 C in order to ensure complete equilibration betw een breath and cham ber vapor. A 1.0 ml sam ple of cham ber vapor was in je c te d in to a gas c h ro m a to g ra p h (Model 5720)t equipped with a flam e ionization detector. Ethanol was separated on a carbowax 60/80 column (6 feet * Bar Harbor, ME. t Hewlett-Packard Co, Palo Alto, CA

3 32 0 FORMAN, BRADFORD, HANDLER, GLASSMAN AND THURMAN long by Vi inch). Operating parameters for all eth an o l m easu rem en ts w ere: oven 110 C; detecto r 315 C; injection port 300 C; and gas flow 80 ml p e r m in. E th anol co n cen tratio n s in blood correlated well with breath ethanol (data not shown). Rates of ethanol elimination were calculated from the linear decline in blood ethanol concentrations by the method of W idmark.23 T r e a t m e n t W it h E t h a n o l Mice received two g per kg ethanol in saline by intraperitoneal injection, or were exposed for four hours to 15 to 30 mg per 1 ethanol vapor in a Plexiglass inhalation chamber (50 X 50 X 40 cm). After treatment, rates of ethanol elimination w ere determ ined. Four hours of vapor treatm ent was chosen to achieve maximal increases in ethanol metabolism over basal values.24 A n a l y sis o f P l a s m a H o r m o n e s A nim als w ere d ecapitated rapidly w ithin 30 seconds after removal from their respective cham bers. Blood was collected in heparinized tubes and centrifuged immediately at 2000 X g for 15 minutes at 4 C. Epinephrine and norepinephrine in plasma were assayed in triplicate employing a radioenzymatic procedure,4 and plasma levels of thyroxine, corticosterone, glucagon and insulin were quantitated in duplicate using specific radioimmunoassay techniques.5 S t a t is t ic a l A n a l y sis Correlations between rates of ethanol elimination and plasma hormone levels w ere analyzed by a m odified linear regression method.3 Data from the animal studies w ere further analyzed by Student s paired-t test. Results A typical experim ent describing the protocol used in this study is illustrated in figure 1. Mice were placed in a Plexiglass cham ber and exposed to ethanol for four hours as described in Materials and Methods. At various times, animals were removed and breath samples were taken. Blood ethanol concentrations typically ranged from 5 to 15 mmol per 1 for the first 2.5 hours of vapor treatm ent and increased to about 27 mmol per 1 after four hours of exposure (figure 1). Breath samples w ere taken over the next 1.5 hours and rates of ethanol elimination were calculated. Rates of ethanol elimination ranged from 5 to 18 mmol per kg per h and were consistent with previous d a ta o b ta in e d w ith th e C 5 7B L /6 J mouse.20 Circulating levels of plasma epineph Ethanol Vapor HOURS F ig u r e 1. Blood Ethanol Concentrations During and Following Exposure to Ethanol Vapor. C57BL/6J m ice w ere placed in Plexiglass ch am b ers and exposed to ethanol vapor (15 to 30 mg per 1) for four hours. Breath samples were taken at the time intervals noted in the figure and analyzed for ethanol. After four hours of ethanol treatment, vapor exposure was discontinued and breath sam ples w ere taken every 30 minutes for 1.5 hours. Rates of ethanol elim ination were calculated from the linear decline in blood ethanol concentrations.21

4 rine increased from six to 18 ng per ml during the first two hours of ethanol treatm ent (figure 2). This represented an increase of about 300 percent compared to the basal state. Control mice placed in Plexiglass cham bers and handled in a similar manner, but not exposed to ethanol, showed only a slight increase in circulating epinephrine of approximately 30 percent, presum ably related to the stress of handling. After two hours of ethanol treatm ent in a vapor chamber, plasma epinephrine values declined toward basal levels and continued to decline for 1.5 hours after the vapor treatm ent was discontinued (figure 2). Similar results were obtained for norepinephrine (not shown). Plasma norepinephrine was elevated to eight ng per ml from a basal level of three ng per ml w ithin one hour after exposure to ethanol and rem ained elev ated until exposure to ethanol vapor was discontinued. Plasma corticosterone levels were in creased th ree-fo ld d u rin g ethanol administration, reaching peak values in Ethanol Vapor HORMONES AND ETHANOL METABOLISM 32 1 less than 60 m inutes and declining to basal values after ethanol exposure was interrupted (figure 3). In contrast, concentrations of thyroxine and insulin were unaffected by ethanol treatm en t. In table I are illustrated the average basal hormone values and maximum increases observed during acute ethanol tre a t ment. Plasma epinephrine, norepinephrine, corticosterone, and glucagon concentrations were increased significantly from control levels (up to 400 percent after exposure to ethanol vapor. The statistically significant correlations between maximal changes in plasma epinephrine, norepinephrine, corticosterone and glucagon levels and rates of ethanol elim in atio n (r = 0.80, P < 0.005, 0.78, P < 0.001, 0.72, P < 0.001, 0.52, P < 0.05, respectively) support the hypothesis that adrenergic and glycogenolytic hormones may play a role in SIAM (table I, figure 4). Discussion D ata p resen ted h e re indicate that after four hours of vapor treatm ent with HOURS F ig u r e 2. Plasma Epinephrine Concentrations D uring and Following T reatm ent with Ethanol Vapor. C57BL/6J mice were exposed to ethanol vapor (15 to 30 mg per 1) as indicated by the solid line. Control mice exposed to air are denoted graphically by a dashed line. Each point represents the mean of duplicate analysis. HOURS F ig u r e 3. Plasma Corticosterone Concentrations During and Following Treatment of Mice with Ethanol Vapor. Conditions as in figure 2. Corticosterone was measured by a standard radioimmunoassay procedure. Data represents averages of two mice per time point.

5 32 2 FORMAN, BRADFORD, HANDLER, GLASSMAN AND THURMAN ethanol or intraperitoneal injection of ethanol plasma concentrations of epinephrine, norepinephrine, corticoste-.o rone and glucagon were elevated two- to o four-fold above basal values. This treat- g m ent also increased rates of ethanol elim ination significantly in C57BL/6J _ mice. c It is well known that alcohol causes a release of epinephrine and norepineph- W rine from the adrenal medulla.16 It is also O well established that epinephrine and glucagon cause glycogenolysis and elevate blood glucose.6 As reported by Yuki and Thurm an,25 the acute administration of ethanol to mice leads to decreased rates of glycolysis, increased glucose outp u t, and in c re a se d rates of oxygen uptake. M oreover, alcohol treatm ent elevated levels of circulating catecholam in es at tim e s (2.5 h) w hen th e enhanced effects on oxygen uptake were observed. T herefore, adrenergic and glycogenolytic hormones may m ediate the sequence of events reponsible for the increased rate of ethanol metabolism. A possible explanation is that horm onestimulated glycogenolysis leads to depletion of tissue carbohydrate reserves (glycogen), which in turn causes glycolysis to decline. Glycolysis is an adenosine-tri- ^ Plasm a Epinephrine (n g /m l) F ig u re 4. Correlation Between Maximal Concentrations of Epinephrine in Plasma and Rates of Ethanol Elimination. Maximal values of plasma epinephrine levels were plotted against rates of ethanol elim ination and a correlation was derived using a least-squares linear regression analysis. phosphate (ATP)-producing reaction, so liver adenosine diphosphate (ADP) not phosphorylated via glycolysis enters the mitochondria and is phosphorylated by the electron transport chain with a concom itant increase in oxygen consum p tion. As a consequence, reduced nicotin- T A B L E I Maximal Increases in Plasma Hormone Levels During Acute Ethanol Treatment Hormone B a sa l Plasm a* Hormone C o n c e n tr a tio n M aximal Plasm a Hormone C o n c e n tr a tio n Percent I n c r e a s e C o r r e la tio n w ith E th a n o l E lim in a tio n r P Epinephrine 4.4 ng/ml 17.2 ng/ml <0.005 Norepinephrine 3.1 ng/ml 7.8 ng/ml <0.001 Corticosterone ng/ml ng/ml <0.001 Thyroxine 5.7 ng/ml 6.0 ng/ml NS Glucagon pg/ml pg/ml <0.05 Insulin 19.3 yu/ml 15.2 pu/ral < NS *3efore ethanol treatment NS = Not 'significant C57BL/6J mice were exposed to ethanol vapor (15 to 30 mg/1) for four hours, and rates of ethanol elimination were determined following withdrawal of vapor treatment as described in the Materials and Methods section. Maximal concentrations of plasma hormones were correlated with rates of ethanol elimination using a least-squares linear regression analysis. Six points per hormone were used for the regression calculation.

6 amide-adenine dinucleotide (NADH) is reoxidized at a faster rate, thereby providing more nicotinamide-adenine dinucleotide+ (NAD + ) for the alcohol dehyd ro g e n a se (A D H ) re a c tio n and an enhanced rate of ethanol elimination.26 As illustrated in figure 5, this normally re q u ire s th e m ediation of hydrogen sh u ttles capable of m oving reducing equivalents from cytosol to mitochondria and can also be influenced by hormones, enzymes, and other factors involved in the regulation of ethanol oxidation. Acute ethanol administration appears to stimulate glucagon release (Table I). It has been proposed that the glycogenolytic effect of glucagon is similar to that produced by the catecholamines with a concomitant increase of liver phosphorylase activity and rapid decrease in liver glycogen c o n te n t.11 T hus, e lev a te d plasma levels of glucagon may contribute to the depletion of hepatic glycogen and activation of the SIAM response. In contrast, other hormones which affect glycogen utilization directly such as insulin, or indirectly as thyroxine, were unaffected HORMONES AND ETHANOL METABOLISM 32 3 F ig ure 5. P rim a ry P a th w a y s o f E th a n o l M etab o lism. P ro posed m echanisms of hormonal (glucagon) stimulation of ethanol oxidation via alcohol dehydrogenase as well as stimulation of hydrogen peroxide generation from increased fatty acid levels caused by ep in ep h rin e and n orep ineph rin e and subsequent peroxidation of eth an o l via catalaseby ethanol treatm ent. Thus, these latter tw o horm ones do n o t a p p e a r to be involved in SIAM. Plasma corticosterone levels were elevated rapidly and significantly by ethanol treatm ent (figure 3) and dem onstrate an immediate reponse to alcohol-induced stress.12 It is not clear what role corticosterone might play in SIAM. A drenergic and glycogenolytic hormones may also activate peripheral lipolysis of adipose stores leading to elevation of c irc u la tin g fre e fatty acid s and enhancement of the peroxidatic reaction of catalase for ethanol.18 It has been demonstrated recently that peroxisomal (3-oxidation of fatty acids provides H20 2 for the peroxidation of ethanol by catalase-h H andler et al10 dem onstrated elegantly that rates of H 2Oa generatio n in th e p erfu sed livers from ADH-negative deermice were sufficient to support ethanol metabolism via catalase at rates up to 60 xmol per g per h, about three-fourths of the rate in perfused livers from A D H -positive d e e r mice. They concluded th at catalase- N E, n o r e p in e p h rin e ; FFA, free fatty acids; FA- FAB P, fatty acid-fatty acid binding protein; CAT, catalase; ADH, alcohol dehydrogenase; phos a, phosphorylase a) L iver Cell

7 32 4 FORMAN, BRADFORD, HANDLER, GLASSMAN AND THURMAN d ep en d en t ethanol m etabolism could occur significantly in perfused livers if adequate substrate for H2Oa generation (i.e., albumin-bound fatty acids for peroxisom al 3-oxidation) was provided. Thus, it is conceivable that a portion of the SIAM response is attributable to increased peroxidation of ethanol via catalase caused by higher rates of H2Oz generation from elevated levels of fatty acids released from adipocytes by lipogenic hormones (figure 5). Since specific adrenergic and glycogenolytic horm ones are elev ated by acute ethanol treatm en t w ith a tim e course sim ilar to SIAM, sh o rt-term increases in rates of ethanol metabolism may be a consequen ce of horm oneinduced changes in liver interm ediary m etabolism. The m arked changes in plasm a horm ones over tim e may also explain why the dose and time relationships in SIAM are so complex. For examp le, if p la sm a e p in e p h r in e le v e ls increase slowly after exposure to e th anol, a longer period of tim e will be needed to achieve the maximum SIAM response. In contrast, plasma epinephrine and corticosterone levels increased significantly within one to two hours follow ing exposure to ethanol and then declined rapidly, resulting in a sharp increase followed by a swift decrease in ethanol elimination to basal values. The differential alterations in circulating horm one levels may explain th e varied SIAM responses seen in rats25 and different inbred strains of mice.20 Acknowledgment The authors wish to thank Dr. Cynthia Kuhn of Duke University Medical Center, Durham, NC for her technical assistance and advice with the determinations of plasma epinephrine and norepinephrine. References 1. C h e r e n, I., G l a s s m a n, E., a n d T h u r m a n, R. G.: T h e sw ift in cre a se in alcohol m etab o lism in h u m a n s : d o s e -re s p o n s e r e la tio n s. A lc o h o l 2:168, C ic e r o, T. J., B e r n a r d, J. D., and N e w m a n, K. : Effects of castration and chronic m orphine administration on liver alcohol dehydrogenase and the metabolism of ethanol in the male Sprague-Dawley rat. J. Pharmacol. Exper. Therap. 225: , C o r n b l e e t, P. J. and G OCHM AN, N.: Incorrect least-squares regression coefficients in methodcomparison analysis. Clin. Chem. 25: , C o y l e, J. T. and H enry, D.: Catecholamine in fetal and new born rat brain. J. N eurochem. 22:61-67, E kins, R. P.: Radioimmunoassay and saturation analysis: Basic principles and theory. Brit. Med. Bull. 30:3-15, E x t o n, J. H., R o b i n s o n, G. A., S u t h e r l a n d, E. W., and PARK, C. R.: Studies on the role of adenosine 3',5'-m onophosphate in the hepatic actions of glucagon and catecholamines. J. Biol. Chem. 246: , F o r m a n, D. T., B r a d f o r d, B. U., K u h n, C., G l a ssm a n, E. and T h u r m a n, R. G.: Plasm a hormone levels in mice after ethanol treatm ent. Ann. Clin. Lab. Sci. 24:331, G l a ssm a n, E. B., M c L a u g h l in, G. A., F o r m a n, D. T., F e l d e r, M. R., and T h u r m a n, R. G. : Role of alcohol dehydrogenase in the swift increase in alcohol m etabolism (SIAM): Studies with deermice deficient in alcohol dehydrogenase. Biochem. Pharmacol. 34: , H a n d l e r, J. A. and T h u r m a n, R. G.: Rates of H20 2 generation from peroxisomal beta-oxidation are sufficient to account for fatty acid-stimulated ethanol metabolism in perfused rat liver. Alcohol 4: , H a n d l e r, J. A., B r a d f o r d, B. U., G l a s s m a n, E. B., F o r m a n, D. T., and Thurm an, R. G.: Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose. Biochem. J. 248: , Latner, A. L.: Endocrine influences in carbohydrate metabolism. Clinical Biochemistry, 7th ed. Philadelphia, W.B. Saunders C o., 1975, pp Latner, A. L. Endocrine influences in carbohydrate metabolism. Clinical Biochemistry, 7th ed. Philadelphia, W.B. Saunders Co., 1975, pp M ezey, E., P o t ter, ]. J., and K vetn ansk y, R.: Effects of stress by repeated immobilization on hepatic alcohol dehydrogenase activity and ethanol metabolism. Biochem. Pharmacol. 28: , Mezey, E., Potter, J. J., Harm on, S. M., and T sito u r a s, P. D.: Effects of castration and testosterone adm inistration on rat liver alcohol dehydrogenase activity. Biochem. Pharmacol. 29: , M ezey, E. and P o t ter, J. J. : Effects of thyroidectomy and triliodothyronine administration on rat liver alcohol dehydrogenase. Gastroenterology 80: , 1981.

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