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Effect of chronic alcohol administration on the hormonal sensitivity of isolated perfused rat liver
Life Sciences, Vol. 29, pp. 135-141 Printed in the U.S.A.
Pergamon Press
EFFECT OF CHRONIC ALCOHOL ADMINISTRATION ON THE HORMONAL SENSITIVITY OF ISOLATED PERFUSED RAT LIVER* Hung Lee and E.A. Hosein Department of Biochemistry, McGill University, Montreal, Quebec, Canada (Received in final form May 6, 1981)
Summary Acute or chronic alcohol administration (37% t o t a l l y l i q u i d lowf a t Metrecal d i e t ) to rats does not a f f e c t the normal rate of lactate gluconeogenesis in the isolated perfused l i v e r . However, under challenging doses of e i t h e r epinephrine (IO-6M) or glucagon (2XIO-SM), the isolated perfused alcoholic l i v e r s showed subnormal percentage stimulation in the rate of gluconeogenesis when compared to the controls. The a c t i v i t i e s of two key hepatic gluconeogenesic enzymes in the cytosol PEPCK and FDPase, were not appreciably altered by chronic alcohol feeding. These results suggest another aspect of membrane involvement as a consequence of the chronic alcohol feeding in the observed depression of hormonal s e n s i t i v i t y . Alcohol is known to exert nonspecific physico-chemical effects on biological membranes ( I ) . These include expansion, disorganization and d i s o r i e n t a t i o n of the l i p i d b i l a y e r (2). Chronic alcohol administration affects the membrane s t i l l f u r t h e r by changing i t s l i p i d composition. This has been demonstrated in the rat l i v e r microsomal, inner and outer mitochondrial membranes (3,4). Presumably, i f this compositional change should extend to the l i v e r plasma membrane as w e l l , though this has not yet been documented, normal plasma membrane functions w i l l undoubtedly be disturbed. A number of amines and peptide hormones are known to exert t h e i r control on metabolism by acting through membrane-bound receptors (5). The "binding" of these membrane-active hormones to t h e i r receptors then i n i t i a t e s the chain of events leading to generation of the second messenger and subsequent physiological responses. In the rat l i v e r , the second messenger for glucagon and ~-adrenergic agonists is believed to be cAMP while that for ~-adrenergic agonists is not yet c l e a r l y established (6). In e i t h e r case, the "coupling" between hormone receptor binding and generation of the second messenger is a complex process which is incompletely understood. I t is known, however, that the actions of these two classes of membrane-active hormones are highly dependent on the i n t e g r i t y and proper functional state of the plasma membrane (5). I t is thus conceivable that the chronic e f f e c t s of ethanol on membrane structure and function could influence the action of membrane-active hormones. * This work was presented in part at the Canadian Physiology Society meeting held at Banff, Alberta, January 23-25, 1980. 0024-3205/81/020135-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Israel et al (7) previously observed that administration of ethanol for 2 weeks induced an increase of synaptosomal adenylate cyclase a c t i v i t y and a loss of the responsiveness of adenylate cyclase to f u r t h e r stimulation by norepinephrine in the r a t cerebral cortex slices in v i t r o . French et al (8) found that adenylate cyclase in l i v e r mitochondria of ethanol-withdrawn rats was hypersensitive to norepinephrine stimulation when compared to controls. Morland (9) also reported a diminished response to glucagon-stimulated induction of tyrosine aminotransferase in isolated perfused l i v e r of c h r o n i c a l l y alcoholic rats. More r e c e n t l y , Banerjee et al (I0) demonstrated decreased s p e c i f i c binding of 3H-dihydroalprenolol, a B-adrenergic antagonist to both r a t heart and brain p a r t i c u l a t e f r a c t i o n s a f t e r chronic ethanol administration. Upon withdrawal from alcohol, binding increased gradually, an e f f e c t a t t r i b u t a b l e to an increase in the number of binding s i t e s . In this communication, the s e n s i t i v i t y of the i n t a c t , perfused alcoholic rat l i v e r to stimulation by epinephrine and glucagon is examined. The maximal rate of gluconeogenesis supported by L - l a c t a t e was used as the index of in v i t r o physiological response, This system allows independent assessment of the e f f e c t of d i f f e r e n t hormones without the inherent complexity of the in vivo s i t u a t i o n where inputs from other groups may d i s t o r t i n t e r p r e t a t i o n of the r e s u l t s . Methods Male Sprague Dawley rats were used in all studies. Chronic alcohol administ r a t i o n in a t o t a l l y l i q u i d low f a t Metrecal d i e t was as previously described in detail (11,12) such that the experimental animals received 37% of t h e i r t o t a l c a l o r i c intake as alcohol while with the pair fed control animals, this was i s o c a l o r i c a l l y replaced by sucrose. Rats were maintained on this regimen for a minimum of 5 weeks before s a c r i f i c e . Acute alcohol administration was achieved by intubating 3 gm alcohol per kg body weight to control rats in a 20% (v/v) solution 2 hr before s a c r i f i c e . Control and alcoholic rats were fasted for 20-24 hr in order to deplete hepatic glycogen, Livers were isolated and perfused with r e c i r c u l a t i o n at 37°C. The medium (70 ± 2 ml) consisted of Krebs-Henseleit bicarbonate buffer pH 7.357.40; aged human RBC (25 to 45 days) hematocrit 20-24% and bovine serum albumin (Cohn Fraction V) at 2.5 gm%. The medium was e q u i l i b r a t e d with 95% 02 and 5% C02 using the s i l a s t i c tubing method (13) p r i o r to r e - e n t r y into the l i v e r via the portal vein. All l i v e r s were r o u t i n e l y allowed 60 min to recover from the trauma of surgical i s o l a t i o n p r i o r to substrate or hormone addition. Perfusion was terminated at 180 min. Neutralized L - l a c t a t e (20 mM) was added as a single dose into the reservoir. Concentrated solutions of epinephrine and glucagon were infused (Harvard Infusion Pump) continuously into the r e s e r v o i r for 50 min (13). Aliquots of the perfusate were taken from the reservoir every 15 min to follow the rate of glucose output a f t e r L - l a c t a t e addition and they were immediately replaced by equal volumes of fresh perfusate so as to maintain the volume and hematocrit in the system (15). The samples were immediately deproteinized with 7 volumes of ice cold 0.6 N perchloric acid. Precipitated protein was removed by c e n t r i f u g a t i o n and the supernatant used for metabolite analysis. Glucose, l a c t a t e and pyruvate were determined by established enzymatic procedures (16). For enzyme studies, a f t e r the recovery period, a small piece of the perfused l i v e r (2 1 g) was excised and q u i c k l y homogenized in 20 volumes of 0.25 M sucrose containing 1 mM Tris-HCl, pH 7.4. 15 min a f t e r hormone infusion,
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another piece of the l i v e r was excised and treated the same way. A f t e r c e n t r i fugation for 1 hr at I00,000 X g at O°C, the supernatants were used for determination of phosphoenolpyruvate carboxykinase (PEPCK) and Fructose diphosphatase (FDPase) a c t i v i t i e s (17,18). Glucagon, epinephrine, L - l a c t a t e acid and a l l the reagents used for the enzyme assays were obtained from e i t h e r Sigma Chemicals or Boehringer Mannheim Canada. Metrecal was obtained from Mead Johnson, Division of Bristol-Myers, Canada. Human whole blood was kindly supplied by the Canadian Red Cross. Results Rats maintained on the 37% low f a t alcohol d i e t gained weight at a slower rate than t h e i r sucrose pair fed controls (12). Upon withdrawal from t h e i r alcohol i c d i e t , these rats usually displayed v i v i d signs of tremor, r i g i d i t y and generalized h y p e r e x c i t a b i l i t y p r i o r to s a c r i f i c e . The c a l o r i c intake of the rats was usually between 80-90 calories per day per rat. The alcoholic rats r e g u l a r l y consumed 15-18 gm alcohol per Kg body weight per day. The average body weight and the wet l i v e r weight a f t e r 3 hours perfusion are summarized in Table I. TABLE I
Control Average body weight at time of s a c r i f i c e (gm)
Alcoholic
390 ± I0 (16)
310 ± I0 (16)*
Wet l i v e r weight a f t e r 3 hr perfusion (gm)
10.3 ± 0.3 (16)
9.0 ± 0.2 (16)t
Liver water content a f t e r 3 hr perfusion (%)
70.3 ± 0.3 (16)
70.1 ± 0.5 (16)
Perfusion rate (ml/min/gm wet l i v e r )
1.41 ± 0.06 (18)
1.48 ± 0.08 (17)
330 ± 20 (19)
320 ± I0 (19)
Hepatic Protein Content (mg/9 m wet l i v e r )
Comparison of hepatic parameters of control and alcoholic rat. The values are means ± S.E.M. together with the number of observations in parentheses. Significance as compared to the control value:
* t
p < 0.001 0.01 > p > 0.001
I n i t i a l experiments were performed to assess the v i a b i l i t y and s t a b i l i t y of the r a t l i v e r s during perfusion. In these experiments, rats were not fasted and no substrate was added during perfusion. The parameters being followed included rate of b i l e secretion; synthesis of t r i g l y c e r i d e , urea and the stab i l i t y of metabolites (glucose, l a c t a t e , pyruvate, 3-hydroxybutyrate and aceto-
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acetate) in the perfusate. These were found to conform to p r e v i o u s l y published values (19,20) f o r both the control and a l c o h o l i c l i v e r s . In some experiments, small doses of ethanol (I0 mM) were added repeatedly i n t o the perfusate. The well characterized redox s h i f t s in both the cytoplasm and mitochondria as measured by changes in the l a c t a t e / p y r u v a t e and 3-hydroxybutyrate/acetoacetate r a t i o s were repeatedly observed. There was no s i g n i f i c a n t d i f f e r e n c e in the response of the control and a l c o h o l i c l i v e r s . In the absence of added s u b s t r a t e , n e g l i g i b l e amounts of glucose were released i n t o the medium by the fasted control and a l c o h o l i c l i v e r s , in agreement with other published data (21). This indicated t h a t the hepatic glycogen l e v e l s were extremely low under the fasted c o n d i t i o n s . TABLE I I
Substrate or Hormone Added L-lactate
Control Rate
Rate A f t e r Alcohol Treatment Chronic Acute
51 + 2 (6)
47 + 1 (5)
53 + 2 (4)
100%
100%
100%
L - l a c t a t e (20 mM) + Epinephrine (IO-~M)
85 ± 4 (5) 167%
65 ~ 3 (5)* 138%
68 + 2 (4)* 128%
L - l a c t a t e (20 mM) + Glucagon (2xlO-SM)
81 ± 3 (5) 159%
64 + 5 (5)# 136%
83 + 1 (4) 157%
(20 mM)
Maximal rates of gluconeogenesis (~moles glucose/hr/g l i v e r ) from 20 mM L - l a c t a t e in i s o l a t e d , perfused l i v e r s of control and alcohol treated r a t s . A l l a d d i t i o n s were made a f t e r 60 min recovery period. L - l a c t a t e was added as a s i n g l e dose. Epinephrine and glucagon were infused cont i n u o u s l y from concentrated s o l u t i o n s f o r 50 min. Values represent mean ± S.E.M. f o r the number of observations made as indicated in the brackets. * t
Value is s i g n i f i c a n t l y O. OO1 ) Value i s s i g n i f i c a n t l y 0.01)
different
from the control value (0.01 > p >
different
from the control value (0.02 > p >
Addition of L - l a c t a t e (20 mM) i n t o the medium r e s u l t e d in a s i g n i f i c a n t enhancement of gluconeogenesis which occurred in a manner s i m i l a r to t h a t described p r e v i o u s l y by others (21,22,23). Much of t h i s increased glucose output can be accounted f o r by the concomitant disappearance of L - l a c t a t e from the medium. The rate o f t h i s increased glucose production u s u a l l y remained l i n e a r f o r at l e a s t 30 min a f t e r a short lag period. This l i n e a r segment was used to c a l c u l a t e the r a t e of gluconeogenesis. The average r a t e obtained f o r the control l i v e r s (51 ± 2 um~l~r/gm wet l i v e r ) was in good agreement with prev i o u s l y reported values (21,22,23). E i t h e r in v i v o acute or chronic alcohol treatments has no e f f e c t on t h i s L - l a c t a t e supported gluconeogenic capacity (Table I I ) , when evaluated with the student's t - t e s t .
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Infusion of gluca9on ( f i n a l concentration 2 X IO-SM) or epinephrine ( f i n a l concentration IO-SM) into the l a c t a t e containing medium f o r 50 min, led to a s i g n i f i c a n t stimulation of the rate of l a c t a t e gluconeogenesis in a l l (cont r o l , acutely or c h r o n i c a l l y treated) l i v e r s tested. As shown in Table I I , acute alcohol treatment s i g n i f i c a n t l y depressed the rate of epinephrine s t i mulated gluconeogenesis while having no e f f e c t on the glucagon stimulated rate. Chronic alcohol treatment however led to a s i g n i f i c a n t reduction in both the epinephrine and glucagon stimulated rates of gluconeogenesis. More r e c e n t l y , p r e l i m i n a r y studies carried out with isolated r a t hepatocytes have shown that chronic alcohol administration led to c o n s i s t e n t l y depressed percentage stimulation of the rate of l a c t a t e gluconeogenesis from 20 mM l a c t a t e over a range of epinephrine (10 -9 to lO-SM) and glucagon (10 -11 to IO-TM) concentrations (Lee and Hosein, unpublished). The a c t i v i t y of two key enzymes in gluconeogenesis PEPCK and FDPase, were also measured. No s i g n i f i c a n t differences in t h e i r basal a c t i v i t i e s were found between the control and a l coholic l i v e r (Table I I I ) . Neither PEPCK nor FDPase a c t i v i t y was s i g n i f i c a n t l y stimulated a f t e r infusion of epinephrine or glucagon. This may be due to very high basal a c t i v i t i e s of these c y t o s o l i c enzymes subsequent to short term s t a r v a t i o n (24). TABLE I I I
PEPCK A c t i v i t y
FDPase A c t i v i t y
Control
Alcoholic
Control
Alcoholic
Basal
3.1 ± 0.I (6)
3.05 ± 0.09 (6)
30 ± 2 (6)
32 ± 1 (6)
Epinephrine Stimulated
2.9 + 0.3 (3)
2 8 + 0.I (3) •
31 + 5 (3) -
36 + 2 (3) -
Glucagon Stimulated
2 7 + 0.3 (3) . .
3 2 + O.l (3) . .
31 + 4 (3) .
33 + 2 (3)
Gluconeogenic enzyme a c t i v i t i e s in perfused l i v e r s from control and a l coholic rats. PEPCK a c t i v i t y is expressed as nmol NADH converted/min/ mg protein while FDPase a c t i v i t y is nmol NADPH formed/min/mg protein. Values represent means ± S.E.M. f o r the number of observations in the parentheses.
Discussion Morland (9) has previously shown that the basal a c t i v i t y of tyrosine aminotransferase in the a l c o h o l i c r a t l i v e r was s i m i l a r to that found in the controls. However, in the perfused a l c o h o l i c l i v e r , the glucagon-stimulated induction of t h i s enzyme was g r e a t l y depressed, presumably due to a lower rate of enzyme synthesis. These r e s u l t s c l o s e l y p a r a l l e l those described in t h i s communication in that only the hormone (glucagon and epinephrine) stimulated l a c t a t e gluconeogenesis was d r a m a t i c a l l y diminished in the c h r o n i c a l l y a l c o h o l i c l i v e r . The f a c t that acute in vivo alcohol treatment s i g n i f i c a n t l y
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depressed only the epinephrine but not glucagon stimulated gluconeogenesis suggests that the acute e f f e c t s may r e f l e c t desensitization to epinephrine subsequent to the short term stress from intubation. These results have important implications in view of the roles of both epinephrine and glucagon in long and short term physiological stress (6). Previous work (1,2) has shown that chronic alcohol administration can a f f e c t biological membrane profoundly. The most drastic and long-lasting consequence involves changes in l i p i d composition. This has been shown to influence membrane l i p i d f l u i d i t y (1,25,26) and may underlie the a l t e r a t i o n s observed in many membrane-related phenomena including hormone receptor function (10,27) and the s e n s i t i v i t y of adenyl cyclase (7,28). A l t e r i n g the f l u i d i t y state of the plasma membrane e i t h e r by changing the l i p i d composition or by the local anesthetic benzyl alcohol has been shown to modulate the a c t i v i t y of glucagonstimulated adenyl cyclase (29,30). Both epinephrine and glucagon are membrane-active hormones and d i f f e r e n t mechanisms are believed to be involved in mediating t h e i r actions (6,31). Their a c t i v i t i e s are also known to be associated with changes in membrane l i p i d f l u i d i t y (32), Thus the generalized f l u i d i z i n g actions of chronic a l cohol feeding may have induced changes in the membrane l i p i d s such that the ethanol-adapted membrane could not be f l u i d i z e d f u r t h e r on stimulation by e i t h e r epinephrine or glucagon, leading to the diminished hormonal response observed. Our data showing that the a c t i v i t i e s of cytosolic enzymes PEPCK and FDPase remained unaltered by chronic alcohol feeding, suggest that they were not r a t e - l i m i t i n g in the overall process from hormone a c t i v a t i o n to glucose output. These data, therefore are supportive of membrane involvement in the observed depression of hormonal s e n s i t i v i t y . The ethanol-adapted membrane could a f f e c t the hormone-response system at several sites including augmentation of r e c e p t o r - f u n c t i o n , coupling, the catal y t i c unit and possibly transport of L - l a c t a t e across the plasma membrane. Any or all of these processes can conceivably be influenced by adaptive changes in membrane l i p i d f l u i d i t y following chronic alcohol administration and they are c u r r e n t l y being studied in t h i s laboratory. Acknowledgements Human RBC were kindly donated by the Canadian Red Cross. This work was supported by MRC and the RODA d i v i s i o n of National Health and Welfare, Canada. HL is the holder of an MRC Studentship. We thank the late Dr. D. Rubinstein for advice and loan of equipment; Mr. B. Rovinski for expert technical assistance and Ms. D. lasenza for s k i l l f u l typing of this manuscript. References I. 2. 3. 4. 5. 6.
G. Freund, Cancer Research 39, 2899-2901 (1979). W.A. Hunt, The Effects of A l i p h a t i c Alcohols on the Biophysical and Biochemical Correlates of Membrane Function in Biochemical Pharmacology of Ethanol, Ed. E. Majchrowicz, p. 195-'210 (1975). J.N. M i c e l l i and W.J. F e r r e l l , Lipids 8, 722-727 (1973). S.W. French, T.J. l h r i g , G.P. Shaw, T.T. Tanaka and M.L. Norum, Research Communications Chem. Path. Pharmacol. 2, 567-585 (1971). P.A. Insel, Membrane-Active Hormones in Biochemistry and Mode of Action of Hormones I I , Eds. H.L. Kornberg and D.C. P h i l l i p s ; University Park Press, Baltimore, p. 2-43 (1978). D.A. Hems, Clino Science 56, 197-202 (1979).
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7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32.
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M . A . Israel, H. Kimura and K. Kuriyama, Experientia 28, 1322-1323 (1972). S.W. French, D.S. Palmer and M. Narad, Amer. J. Patho-T. 74, 67a (1974). J. Morland, Biochem. Pharmacol. 23, 3239-3246 (1974). S.P. Banerjee, V.K. Sharma and J.M. Khanna, Nature 276, 407-408 (1978). I. Hofmann and E.A. Hosein, Biochem. Pharmacol. 27, 457-463 (1978). E.A. Hosein, H. Lee and I. Hofmann, Can. J. Biochem. 58, 1147-1155 (1980). R.L. Hamilton, M.N. Berry, M.C. Williams and E.M. Severinghaus, J. Lipid Res. 15, 182-186 (1974). L.E. Mallette, J.H. Exton and C.R. Park, J. Biol. Chem. 244, 5713-5723 (1969). E.R. Gordon, Biochem. Pharmacol. 21, 2991-3004 (1972). H.V. Bergmeyer, Methods of Enzymatic Analysis, Academic Press, New York, p. 1196-1201, 1446-1451, 1464-1468 (1974). I. Petrescu, O. Bojan, M. Saied, O. BSrzu, F. Schmidt and H.F. Kuhnle, Anal. Biochem. 96, 279-281 (1979). T. Chatterjee and A.G. Datta, Biochem. Biophys. Res. Comm. 84, 950-956 (1978). G. Signurdsson, S.P. Noel and R.J. Havel, J. Lipid Res. 19, 628-634 (1978). T. Sugano, K. Suda, M. Shimada and N. Oshino, J. Biochem. (Japan) 83, 995-1007 (1978). J.H. Exton and C.R. Park, J. Biol. Chem. 242, 2622-2636 (1967). R. Hems, B.D. Ross, M.N. Berry and H.A. Krebs, Biochem. J. I01, 284-292 (1966). D.L. Bloxam, B r i t . J. Nutrition 26, 393-422 (1971). J.H. Exton, Hormonal Control o f Gluconeogenes!s in Hormones and Energy Metabolism, Eds. D.M. Klachko, R.R. Anderson and M. Heimberg; Plenum Press, p. 125-157 (1976). J.H. Chin, D.B. Goldstein and L.M. Parsons, Alcoholism, Clin. Exp. Res. 3, 47-49 (1979). D.B. Goldstein, Speculations on Membrane Lipid Adaptation as a Mechanism for Drug Tolerance and Dependence in Membrane Mechanisms of Drugs of Abuse, Eds. C.W. Sharp and L.G. Abood; Alan R. Liss Inc., New York, p. 151-166 (1979). F.R. Ciofalo, Proc. West. Pharmacol. Soc. 22, 367-369 (1979). K. Kuriyama and M.A. Israel, Biochem. Pharmacol. 22, 2919-2922 (1973). M.D. Housley, T.R. Hesketh, G.A. Smith, G.B. Warren and J.C. Metcalfe, Biochim. Biophys. Acta 436, 495-504 (1975). I. Dipple and M.D. Housley, Biochem. J. 174, 179-190 (1978). J.H. Exton, Amer. J. Physiol. 238, E3-EI2-~-I980). E.M. DeMelian, E.M. Massa, R.D. Moreno and R.N. Farias, FEBS Lett. 92, 143-146 (1978).
Pergamon Press
EFFECT OF CHRONIC ALCOHOL ADMINISTRATION ON THE HORMONAL SENSITIVITY OF ISOLATED PERFUSED RAT LIVER* Hung Lee and E.A. Hosein Department of Biochemistry, McGill University, Montreal, Quebec, Canada (Received in final form May 6, 1981)
Summary Acute or chronic alcohol administration (37% t o t a l l y l i q u i d lowf a t Metrecal d i e t ) to rats does not a f f e c t the normal rate of lactate gluconeogenesis in the isolated perfused l i v e r . However, under challenging doses of e i t h e r epinephrine (IO-6M) or glucagon (2XIO-SM), the isolated perfused alcoholic l i v e r s showed subnormal percentage stimulation in the rate of gluconeogenesis when compared to the controls. The a c t i v i t i e s of two key hepatic gluconeogenesic enzymes in the cytosol PEPCK and FDPase, were not appreciably altered by chronic alcohol feeding. These results suggest another aspect of membrane involvement as a consequence of the chronic alcohol feeding in the observed depression of hormonal s e n s i t i v i t y . Alcohol is known to exert nonspecific physico-chemical effects on biological membranes ( I ) . These include expansion, disorganization and d i s o r i e n t a t i o n of the l i p i d b i l a y e r (2). Chronic alcohol administration affects the membrane s t i l l f u r t h e r by changing i t s l i p i d composition. This has been demonstrated in the rat l i v e r microsomal, inner and outer mitochondrial membranes (3,4). Presumably, i f this compositional change should extend to the l i v e r plasma membrane as w e l l , though this has not yet been documented, normal plasma membrane functions w i l l undoubtedly be disturbed. A number of amines and peptide hormones are known to exert t h e i r control on metabolism by acting through membrane-bound receptors (5). The "binding" of these membrane-active hormones to t h e i r receptors then i n i t i a t e s the chain of events leading to generation of the second messenger and subsequent physiological responses. In the rat l i v e r , the second messenger for glucagon and ~-adrenergic agonists is believed to be cAMP while that for ~-adrenergic agonists is not yet c l e a r l y established (6). In e i t h e r case, the "coupling" between hormone receptor binding and generation of the second messenger is a complex process which is incompletely understood. I t is known, however, that the actions of these two classes of membrane-active hormones are highly dependent on the i n t e g r i t y and proper functional state of the plasma membrane (5). I t is thus conceivable that the chronic e f f e c t s of ethanol on membrane structure and function could influence the action of membrane-active hormones. * This work was presented in part at the Canadian Physiology Society meeting held at Banff, Alberta, January 23-25, 1980. 0024-3205/81/020135-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Israel et al (7) previously observed that administration of ethanol for 2 weeks induced an increase of synaptosomal adenylate cyclase a c t i v i t y and a loss of the responsiveness of adenylate cyclase to f u r t h e r stimulation by norepinephrine in the r a t cerebral cortex slices in v i t r o . French et al (8) found that adenylate cyclase in l i v e r mitochondria of ethanol-withdrawn rats was hypersensitive to norepinephrine stimulation when compared to controls. Morland (9) also reported a diminished response to glucagon-stimulated induction of tyrosine aminotransferase in isolated perfused l i v e r of c h r o n i c a l l y alcoholic rats. More r e c e n t l y , Banerjee et al (I0) demonstrated decreased s p e c i f i c binding of 3H-dihydroalprenolol, a B-adrenergic antagonist to both r a t heart and brain p a r t i c u l a t e f r a c t i o n s a f t e r chronic ethanol administration. Upon withdrawal from alcohol, binding increased gradually, an e f f e c t a t t r i b u t a b l e to an increase in the number of binding s i t e s . In this communication, the s e n s i t i v i t y of the i n t a c t , perfused alcoholic rat l i v e r to stimulation by epinephrine and glucagon is examined. The maximal rate of gluconeogenesis supported by L - l a c t a t e was used as the index of in v i t r o physiological response, This system allows independent assessment of the e f f e c t of d i f f e r e n t hormones without the inherent complexity of the in vivo s i t u a t i o n where inputs from other groups may d i s t o r t i n t e r p r e t a t i o n of the r e s u l t s . Methods Male Sprague Dawley rats were used in all studies. Chronic alcohol administ r a t i o n in a t o t a l l y l i q u i d low f a t Metrecal d i e t was as previously described in detail (11,12) such that the experimental animals received 37% of t h e i r t o t a l c a l o r i c intake as alcohol while with the pair fed control animals, this was i s o c a l o r i c a l l y replaced by sucrose. Rats were maintained on this regimen for a minimum of 5 weeks before s a c r i f i c e . Acute alcohol administration was achieved by intubating 3 gm alcohol per kg body weight to control rats in a 20% (v/v) solution 2 hr before s a c r i f i c e . Control and alcoholic rats were fasted for 20-24 hr in order to deplete hepatic glycogen, Livers were isolated and perfused with r e c i r c u l a t i o n at 37°C. The medium (70 ± 2 ml) consisted of Krebs-Henseleit bicarbonate buffer pH 7.357.40; aged human RBC (25 to 45 days) hematocrit 20-24% and bovine serum albumin (Cohn Fraction V) at 2.5 gm%. The medium was e q u i l i b r a t e d with 95% 02 and 5% C02 using the s i l a s t i c tubing method (13) p r i o r to r e - e n t r y into the l i v e r via the portal vein. All l i v e r s were r o u t i n e l y allowed 60 min to recover from the trauma of surgical i s o l a t i o n p r i o r to substrate or hormone addition. Perfusion was terminated at 180 min. Neutralized L - l a c t a t e (20 mM) was added as a single dose into the reservoir. Concentrated solutions of epinephrine and glucagon were infused (Harvard Infusion Pump) continuously into the r e s e r v o i r for 50 min (13). Aliquots of the perfusate were taken from the reservoir every 15 min to follow the rate of glucose output a f t e r L - l a c t a t e addition and they were immediately replaced by equal volumes of fresh perfusate so as to maintain the volume and hematocrit in the system (15). The samples were immediately deproteinized with 7 volumes of ice cold 0.6 N perchloric acid. Precipitated protein was removed by c e n t r i f u g a t i o n and the supernatant used for metabolite analysis. Glucose, l a c t a t e and pyruvate were determined by established enzymatic procedures (16). For enzyme studies, a f t e r the recovery period, a small piece of the perfused l i v e r (2 1 g) was excised and q u i c k l y homogenized in 20 volumes of 0.25 M sucrose containing 1 mM Tris-HCl, pH 7.4. 15 min a f t e r hormone infusion,
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another piece of the l i v e r was excised and treated the same way. A f t e r c e n t r i fugation for 1 hr at I00,000 X g at O°C, the supernatants were used for determination of phosphoenolpyruvate carboxykinase (PEPCK) and Fructose diphosphatase (FDPase) a c t i v i t i e s (17,18). Glucagon, epinephrine, L - l a c t a t e acid and a l l the reagents used for the enzyme assays were obtained from e i t h e r Sigma Chemicals or Boehringer Mannheim Canada. Metrecal was obtained from Mead Johnson, Division of Bristol-Myers, Canada. Human whole blood was kindly supplied by the Canadian Red Cross. Results Rats maintained on the 37% low f a t alcohol d i e t gained weight at a slower rate than t h e i r sucrose pair fed controls (12). Upon withdrawal from t h e i r alcohol i c d i e t , these rats usually displayed v i v i d signs of tremor, r i g i d i t y and generalized h y p e r e x c i t a b i l i t y p r i o r to s a c r i f i c e . The c a l o r i c intake of the rats was usually between 80-90 calories per day per rat. The alcoholic rats r e g u l a r l y consumed 15-18 gm alcohol per Kg body weight per day. The average body weight and the wet l i v e r weight a f t e r 3 hours perfusion are summarized in Table I. TABLE I
Control Average body weight at time of s a c r i f i c e (gm)
Alcoholic
390 ± I0 (16)
310 ± I0 (16)*
Wet l i v e r weight a f t e r 3 hr perfusion (gm)
10.3 ± 0.3 (16)
9.0 ± 0.2 (16)t
Liver water content a f t e r 3 hr perfusion (%)
70.3 ± 0.3 (16)
70.1 ± 0.5 (16)
Perfusion rate (ml/min/gm wet l i v e r )
1.41 ± 0.06 (18)
1.48 ± 0.08 (17)
330 ± 20 (19)
320 ± I0 (19)
Hepatic Protein Content (mg/9 m wet l i v e r )
Comparison of hepatic parameters of control and alcoholic rat. The values are means ± S.E.M. together with the number of observations in parentheses. Significance as compared to the control value:
* t
p < 0.001 0.01 > p > 0.001
I n i t i a l experiments were performed to assess the v i a b i l i t y and s t a b i l i t y of the r a t l i v e r s during perfusion. In these experiments, rats were not fasted and no substrate was added during perfusion. The parameters being followed included rate of b i l e secretion; synthesis of t r i g l y c e r i d e , urea and the stab i l i t y of metabolites (glucose, l a c t a t e , pyruvate, 3-hydroxybutyrate and aceto-
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acetate) in the perfusate. These were found to conform to p r e v i o u s l y published values (19,20) f o r both the control and a l c o h o l i c l i v e r s . In some experiments, small doses of ethanol (I0 mM) were added repeatedly i n t o the perfusate. The well characterized redox s h i f t s in both the cytoplasm and mitochondria as measured by changes in the l a c t a t e / p y r u v a t e and 3-hydroxybutyrate/acetoacetate r a t i o s were repeatedly observed. There was no s i g n i f i c a n t d i f f e r e n c e in the response of the control and a l c o h o l i c l i v e r s . In the absence of added s u b s t r a t e , n e g l i g i b l e amounts of glucose were released i n t o the medium by the fasted control and a l c o h o l i c l i v e r s , in agreement with other published data (21). This indicated t h a t the hepatic glycogen l e v e l s were extremely low under the fasted c o n d i t i o n s . TABLE I I
Substrate or Hormone Added L-lactate
Control Rate
Rate A f t e r Alcohol Treatment Chronic Acute
51 + 2 (6)
47 + 1 (5)
53 + 2 (4)
100%
100%
100%
L - l a c t a t e (20 mM) + Epinephrine (IO-~M)
85 ± 4 (5) 167%
65 ~ 3 (5)* 138%
68 + 2 (4)* 128%
L - l a c t a t e (20 mM) + Glucagon (2xlO-SM)
81 ± 3 (5) 159%
64 + 5 (5)# 136%
83 + 1 (4) 157%
(20 mM)
Maximal rates of gluconeogenesis (~moles glucose/hr/g l i v e r ) from 20 mM L - l a c t a t e in i s o l a t e d , perfused l i v e r s of control and alcohol treated r a t s . A l l a d d i t i o n s were made a f t e r 60 min recovery period. L - l a c t a t e was added as a s i n g l e dose. Epinephrine and glucagon were infused cont i n u o u s l y from concentrated s o l u t i o n s f o r 50 min. Values represent mean ± S.E.M. f o r the number of observations made as indicated in the brackets. * t
Value is s i g n i f i c a n t l y O. OO1 ) Value i s s i g n i f i c a n t l y 0.01)
different
from the control value (0.01 > p >
different
from the control value (0.02 > p >
Addition of L - l a c t a t e (20 mM) i n t o the medium r e s u l t e d in a s i g n i f i c a n t enhancement of gluconeogenesis which occurred in a manner s i m i l a r to t h a t described p r e v i o u s l y by others (21,22,23). Much of t h i s increased glucose output can be accounted f o r by the concomitant disappearance of L - l a c t a t e from the medium. The rate o f t h i s increased glucose production u s u a l l y remained l i n e a r f o r at l e a s t 30 min a f t e r a short lag period. This l i n e a r segment was used to c a l c u l a t e the r a t e of gluconeogenesis. The average r a t e obtained f o r the control l i v e r s (51 ± 2 um~l~r/gm wet l i v e r ) was in good agreement with prev i o u s l y reported values (21,22,23). E i t h e r in v i v o acute or chronic alcohol treatments has no e f f e c t on t h i s L - l a c t a t e supported gluconeogenic capacity (Table I I ) , when evaluated with the student's t - t e s t .
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Infusion of gluca9on ( f i n a l concentration 2 X IO-SM) or epinephrine ( f i n a l concentration IO-SM) into the l a c t a t e containing medium f o r 50 min, led to a s i g n i f i c a n t stimulation of the rate of l a c t a t e gluconeogenesis in a l l (cont r o l , acutely or c h r o n i c a l l y treated) l i v e r s tested. As shown in Table I I , acute alcohol treatment s i g n i f i c a n t l y depressed the rate of epinephrine s t i mulated gluconeogenesis while having no e f f e c t on the glucagon stimulated rate. Chronic alcohol treatment however led to a s i g n i f i c a n t reduction in both the epinephrine and glucagon stimulated rates of gluconeogenesis. More r e c e n t l y , p r e l i m i n a r y studies carried out with isolated r a t hepatocytes have shown that chronic alcohol administration led to c o n s i s t e n t l y depressed percentage stimulation of the rate of l a c t a t e gluconeogenesis from 20 mM l a c t a t e over a range of epinephrine (10 -9 to lO-SM) and glucagon (10 -11 to IO-TM) concentrations (Lee and Hosein, unpublished). The a c t i v i t y of two key enzymes in gluconeogenesis PEPCK and FDPase, were also measured. No s i g n i f i c a n t differences in t h e i r basal a c t i v i t i e s were found between the control and a l coholic l i v e r (Table I I I ) . Neither PEPCK nor FDPase a c t i v i t y was s i g n i f i c a n t l y stimulated a f t e r infusion of epinephrine or glucagon. This may be due to very high basal a c t i v i t i e s of these c y t o s o l i c enzymes subsequent to short term s t a r v a t i o n (24). TABLE I I I
PEPCK A c t i v i t y
FDPase A c t i v i t y
Control
Alcoholic
Control
Alcoholic
Basal
3.1 ± 0.I (6)
3.05 ± 0.09 (6)
30 ± 2 (6)
32 ± 1 (6)
Epinephrine Stimulated
2.9 + 0.3 (3)
2 8 + 0.I (3) •
31 + 5 (3) -
36 + 2 (3) -
Glucagon Stimulated
2 7 + 0.3 (3) . .
3 2 + O.l (3) . .
31 + 4 (3) .
33 + 2 (3)
Gluconeogenic enzyme a c t i v i t i e s in perfused l i v e r s from control and a l coholic rats. PEPCK a c t i v i t y is expressed as nmol NADH converted/min/ mg protein while FDPase a c t i v i t y is nmol NADPH formed/min/mg protein. Values represent means ± S.E.M. f o r the number of observations in the parentheses.
Discussion Morland (9) has previously shown that the basal a c t i v i t y of tyrosine aminotransferase in the a l c o h o l i c r a t l i v e r was s i m i l a r to that found in the controls. However, in the perfused a l c o h o l i c l i v e r , the glucagon-stimulated induction of t h i s enzyme was g r e a t l y depressed, presumably due to a lower rate of enzyme synthesis. These r e s u l t s c l o s e l y p a r a l l e l those described in t h i s communication in that only the hormone (glucagon and epinephrine) stimulated l a c t a t e gluconeogenesis was d r a m a t i c a l l y diminished in the c h r o n i c a l l y a l c o h o l i c l i v e r . The f a c t that acute in vivo alcohol treatment s i g n i f i c a n t l y
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depressed only the epinephrine but not glucagon stimulated gluconeogenesis suggests that the acute e f f e c t s may r e f l e c t desensitization to epinephrine subsequent to the short term stress from intubation. These results have important implications in view of the roles of both epinephrine and glucagon in long and short term physiological stress (6). Previous work (1,2) has shown that chronic alcohol administration can a f f e c t biological membrane profoundly. The most drastic and long-lasting consequence involves changes in l i p i d composition. This has been shown to influence membrane l i p i d f l u i d i t y (1,25,26) and may underlie the a l t e r a t i o n s observed in many membrane-related phenomena including hormone receptor function (10,27) and the s e n s i t i v i t y of adenyl cyclase (7,28). A l t e r i n g the f l u i d i t y state of the plasma membrane e i t h e r by changing the l i p i d composition or by the local anesthetic benzyl alcohol has been shown to modulate the a c t i v i t y of glucagonstimulated adenyl cyclase (29,30). Both epinephrine and glucagon are membrane-active hormones and d i f f e r e n t mechanisms are believed to be involved in mediating t h e i r actions (6,31). Their a c t i v i t i e s are also known to be associated with changes in membrane l i p i d f l u i d i t y (32), Thus the generalized f l u i d i z i n g actions of chronic a l cohol feeding may have induced changes in the membrane l i p i d s such that the ethanol-adapted membrane could not be f l u i d i z e d f u r t h e r on stimulation by e i t h e r epinephrine or glucagon, leading to the diminished hormonal response observed. Our data showing that the a c t i v i t i e s of cytosolic enzymes PEPCK and FDPase remained unaltered by chronic alcohol feeding, suggest that they were not r a t e - l i m i t i n g in the overall process from hormone a c t i v a t i o n to glucose output. These data, therefore are supportive of membrane involvement in the observed depression of hormonal s e n s i t i v i t y . The ethanol-adapted membrane could a f f e c t the hormone-response system at several sites including augmentation of r e c e p t o r - f u n c t i o n , coupling, the catal y t i c unit and possibly transport of L - l a c t a t e across the plasma membrane. Any or all of these processes can conceivably be influenced by adaptive changes in membrane l i p i d f l u i d i t y following chronic alcohol administration and they are c u r r e n t l y being studied in t h i s laboratory. Acknowledgements Human RBC were kindly donated by the Canadian Red Cross. This work was supported by MRC and the RODA d i v i s i o n of National Health and Welfare, Canada. HL is the holder of an MRC Studentship. We thank the late Dr. D. Rubinstein for advice and loan of equipment; Mr. B. Rovinski for expert technical assistance and Ms. D. lasenza for s k i l l f u l typing of this manuscript. References I. 2. 3. 4. 5. 6.
G. Freund, Cancer Research 39, 2899-2901 (1979). W.A. Hunt, The Effects of A l i p h a t i c Alcohols on the Biophysical and Biochemical Correlates of Membrane Function in Biochemical Pharmacology of Ethanol, Ed. E. Majchrowicz, p. 195-'210 (1975). J.N. M i c e l l i and W.J. F e r r e l l , Lipids 8, 722-727 (1973). S.W. French, T.J. l h r i g , G.P. Shaw, T.T. Tanaka and M.L. Norum, Research Communications Chem. Path. Pharmacol. 2, 567-585 (1971). P.A. Insel, Membrane-Active Hormones in Biochemistry and Mode of Action of Hormones I I , Eds. H.L. Kornberg and D.C. P h i l l i p s ; University Park Press, Baltimore, p. 2-43 (1978). D.A. Hems, Clino Science 56, 197-202 (1979).
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7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
27. 28. 29. 30. 31. 32.
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M . A . Israel, H. Kimura and K. Kuriyama, Experientia 28, 1322-1323 (1972). S.W. French, D.S. Palmer and M. Narad, Amer. J. Patho-T. 74, 67a (1974). J. Morland, Biochem. Pharmacol. 23, 3239-3246 (1974). S.P. Banerjee, V.K. Sharma and J.M. Khanna, Nature 276, 407-408 (1978). I. Hofmann and E.A. Hosein, Biochem. Pharmacol. 27, 457-463 (1978). E.A. Hosein, H. Lee and I. Hofmann, Can. J. Biochem. 58, 1147-1155 (1980). R.L. Hamilton, M.N. Berry, M.C. Williams and E.M. Severinghaus, J. Lipid Res. 15, 182-186 (1974). L.E. Mallette, J.H. Exton and C.R. Park, J. Biol. Chem. 244, 5713-5723 (1969). E.R. Gordon, Biochem. Pharmacol. 21, 2991-3004 (1972). H.V. Bergmeyer, Methods of Enzymatic Analysis, Academic Press, New York, p. 1196-1201, 1446-1451, 1464-1468 (1974). I. Petrescu, O. Bojan, M. Saied, O. BSrzu, F. Schmidt and H.F. Kuhnle, Anal. Biochem. 96, 279-281 (1979). T. Chatterjee and A.G. Datta, Biochem. Biophys. Res. Comm. 84, 950-956 (1978). G. Signurdsson, S.P. Noel and R.J. Havel, J. Lipid Res. 19, 628-634 (1978). T. Sugano, K. Suda, M. Shimada and N. Oshino, J. Biochem. (Japan) 83, 995-1007 (1978). J.H. Exton and C.R. Park, J. Biol. Chem. 242, 2622-2636 (1967). R. Hems, B.D. Ross, M.N. Berry and H.A. Krebs, Biochem. J. I01, 284-292 (1966). D.L. Bloxam, B r i t . J. Nutrition 26, 393-422 (1971). J.H. Exton, Hormonal Control o f Gluconeogenes!s in Hormones and Energy Metabolism, Eds. D.M. Klachko, R.R. Anderson and M. Heimberg; Plenum Press, p. 125-157 (1976). J.H. Chin, D.B. Goldstein and L.M. Parsons, Alcoholism, Clin. Exp. Res. 3, 47-49 (1979). D.B. Goldstein, Speculations on Membrane Lipid Adaptation as a Mechanism for Drug Tolerance and Dependence in Membrane Mechanisms of Drugs of Abuse, Eds. C.W. Sharp and L.G. Abood; Alan R. Liss Inc., New York, p. 151-166 (1979). F.R. Ciofalo, Proc. West. Pharmacol. Soc. 22, 367-369 (1979). K. Kuriyama and M.A. Israel, Biochem. Pharmacol. 22, 2919-2922 (1973). M.D. Housley, T.R. Hesketh, G.A. Smith, G.B. Warren and J.C. Metcalfe, Biochim. Biophys. Acta 436, 495-504 (1975). I. Dipple and M.D. Housley, Biochem. J. 174, 179-190 (1978). J.H. Exton, Amer. J. Physiol. 238, E3-EI2-~-I980). E.M. DeMelian, E.M. Massa, R.D. Moreno and R.N. Farias, FEBS Lett. 92, 143-146 (1978).