Biochimica et Biophysica Acta, 1180(1992) 207-214

207

© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

BBADIS 61212

Chronic effects of ethanol on muscle metabolism in the rat Elisabeth B. Cook

a

Lutfat A.Y. Adebiyi b Victor R. Preedy a Timothy J. Peters a and T. Norman Palmer c

a Department of Clinical Biochemistry, King's College School of Medicine and Dentistry, Bessemer Road, London (UK) b Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London (UK) and c Department of Biochemistry, University of Western Australia, Nedlands (Australia)

(Received 9 May 1992) (Revised manuscript received 15 August 1992)

Key words: Skeletal muscle; Glucose metabolism; Glycogen;Ethanol; Insulin Chronic ethanol feeding in the rat is associated with a skeletal myopathy involving primarily type-II muscle fibres, which is recognised to be mediated via a specific impairment in protein turnover. This paper investigates whether the cause of this myopathy may be related to abnormalities in carbohydrate and lipid metabolism in different muscles. [U-14C]Glucose metabolism was examined in two muscles with different fibre compositions, the extensor digitorum longus (EDL) muscle, which contains predominantly type-II muscle fibres, and the soleus muscle, which is composed primarily of type-I muscle fibres. Feeding on the ethanol-supplemented Lieber-DeCarli liquid diet for 2 or 6 weeks was associated with profound disturbances in glucose metabolism in both EDL and soleus muscles, particularly in relation 1o rates of glycogen and alanine formation. We discuss the importance of these metabolic changes in relation to the genesis of chronic alcoholic skeletal myopathy.

Introduction

Chronic ethanol consumption in man is associated with profound abnormalities in muscle structure and function [1]. Chronic abuse typically causes an alcoholic skeletal myopathy in up to two-thirds of alcoholic patients [2,3], which in its most severe form may involve the loss of 25% of the body's skeletal muscle mass. Histochemical analysis indicates that the basis for the myopathy is a selective decrease in type-II (fast twitch, glycolytic) muscle fibres [4-6]. The myopathy may be reproduced [7] in the rat by feeding on an ethanol-containing liquid diet (Lieber-DeCarli diet [7,8]). This myopathy, like that in man, is type-II-fibrespecific, occurs in the absence of nutritional deficiencies and may reduce whole-body skeletal muscle mass by more than 20% [7]. The pathogenesis of the skeletal myopathy is now recognised to involve a specific impairment in protein turnover [1,6]. Whole-body protein synthesis is acutely impaired by ethanol [10], and the rate of protein synthesis has been shown to be decreased in muscles from rats chronically [7] and acutely [11] administered ethanol, muscles containing predomi-

Correspondence to: T.N. Palmer, Department of Biochemistry,University of Western Australia, Nedlands, Western Australia 6009, Australia.

nantly type-II fibres being the most severely affected [1,6]. Aside from its myopathic action, ethanol may have other effects on muscle metabolism. In human volunteers, ethanol may acutely inhibit glucose uptake by leg muscles [12], reduce the stimulatory effects of prolonged exercise on glucose uptake by the exercising leg [13] and cause acute peripheral insulin resistance and diminished glucose oxidation [14]. Ethanol also decreases glucose utilization in fasted dogs [15] and impairs glucose uptake by the isolated rat diaphragm [16]. The implication is that ethanol may diminish peripheral glucose uptake and utilization via effects primarily on skeletal muscles. The question addressed in this paper is to what extent ethanol chronically compromises glucose and, to a lesser extent, fatty acid metabolism in isolated rat muscle preparations. A related question is whether the impairment in protein synthesis associated with chronic ethanol feeding is a specific lesion, or whether it arises secondary to an ethanol-related defect in glucose utilization. To establish whether the biochemical lesion(s) associated with ethanol feeding is intramuscular in location or is mediated by extracellular factors, muscle metabolism was studied in isolated muscle preparations. An important question concerns the extent to which fibre composition determines the effects of ethanol feeding on muscle metabolism. Two muscles

208 with different fibre compositions were accordingly selected for study: The extensor digitorum longus (EDL) contains predominantly type-II fibres, whereas the soleus muscle contains predominantly typed fibres [17,18]. Materials and Methods

Materials. [U- 14C]Glucose and [1-14C]palmitate were purchased from New England Nuclear (Stevenage, UK), Vitafood from Boots (Nottingham, UK) and Orovite 7 from Beecham (Brentford, UK). All other chemicals were obtained from Sigma (Poole, UK). Treatment of animals. Male Wistar rats were maintained at approx. 17°C in a 12 h-light/12 h-dark cycle (light started at 08.00 h) and were fed ad libitum on a standard laboratory chow diet (CRM diet: 57% digestible carbohydrate, 18% protein and 2.5% lipid, all by weight). In experiments designed to evaluate the in vitro effects of ethanol and its metabolites on muscle preparations, chow-fed rats (200 g) were anaesthetised under diethyl ether and their hind-limbs removed for the dissection of soleus and E D L muscles. Chronic ethanol feeding experiments were performed on male Wistar rats maintained as described above in grid-bottomed cages until their body weights reached 80-85 g. At this time, animals were paired on the basis of weight. Control rats were fed on a nutritionally-complete liquid diet comprising a commercial food drink (Vitafood) supplemented with glucose, casein and a vitamin mixture (Orovite 7) [7]. Ethanoltreated rats were given a similar diet ad libitum, in which glucose was replaced by isocaloric ethanol, such that ethanol comprised 23% of the total energy content of the diet on days 2 - 4 of feeding, and 35% thereafter [7]. Control rats were given identical volumes of diet to those consumed by their ethanol-treated paired animals on the previous day. Liquid diets were prepared fresh daily and given to the rats between 08:30 and 10:00 h. Rats were maintained on the liquid diets for either 2 or 6 weeks. Thereafter, animals were anaesthetised under diethyl ether, a blood sample taken from the left ventricle and the hind-legs removed for the preparation of soleus and E D L muscles. Animals were not provided with liquid diet on the morning of the experiment. In t'itro muscle preparations. The soleus and E D L muscles were rapidly dissected out of the hind-legs and divided in half longitudinally for muscles from rats fed for 6 weeks, or kept whole for muscles from rats fed on liquid diet for 2 weeks. Each muscle or half muscle was tied across a stainless-steel wire frame, under tension, and trimmed to 37 mg or less [19-22]. Muscles were incubated (pre-incubation) for 30 min at 37°C in 25 ml sealed conical flasks gassed continuously with O 2 / C O e (19 : 1) containing 4 ml Krebs-Ringer bicarbonate buffer

(pH 7.4), supplemented with 1% (w/v) bovine serum albumin (essentially fatty-acid free) and 5 mM glucose. After the preincubation, muscles were blotted dry and transferred to flasks containing 4 ml Krebs-Ringer bicarbonate buffer (pH 7.4), containing 1~ bovinc serum albumin (essentially fatty-acid free) and either 5 mM [U-lmC]glucose (1 p.Ci) or 1 mM [l-~4C]palmitatc (1 ~Ci) + 5 mM glucose. On occasions, the pre-incubation and incubation media were supplemented with 5(} mM ethanol or 250/~M acetaldehyde. Insulin sensitivity was measured by inclusion of 1 to 1(}000 ~units insulin per ml of incubation medium. The flasks, which contained empty upright Eppendorf tubes for the subsequent addition of Solvease, were sealed with rubber Subaseals and the muscles incubated at 37°( ` for 6(1 min with gassing with 0 2 / C 0 2 (19: 1) for the first 10 min. The incubations were terminated by the injection through the seal of 0.4 ml 60% (w/v) perchloric acid into the medium: ~4CO2 production was determined by injection of 0.5 ml Solvease to the empty Eppendorf tubes and gentle shaking for 60 min [23]. Muscle glycogen content was determined enzymically in tissue samples isolated by digestion in 30% (w/v) KOH at 100°C and ethanol precipitation [23] and glycogen synthesis was determined by 14C incorporation from [U-14C]glucose into glycogen [24]. [U14C]Glucose and [1-14C]palmitate oxidation were measured by 14C02 release and entrapment into Solvease. and lactate [25] and alanine [26] were measured by published spectrophotometric methods in the KOHneutralised perchloric acid-treated incubation media. Protein was determined by the method of Bradford [27]. Blood ethanol concentrations attained by the ethanol-fed rats were 10-55 mM [7]. Tissue triacylglycerol and glycogen. To determine the effects of ethanol feeding on muscle triacylglycerol and glycogen levels, rats were pair-fed with either the nutritionally complete liquid diet or the ethanol-supplemented liquid diet for 2 weeks. Diet was provided at 16.00-17.00 h daily. After 2 weeks, animals werc anaesthetised at 10.00-11.00 h under halothane anaesthesia and the individual muscles and liver were rapidly dissected out and freeze-clamped in aluminium clamps precooled in liquid nitrogen. Tissue glycogen was measured as described in Ref. 23, whereas triacylglycerols were extracted in chloroform/methanol (2 : 1) and the levels measured as triacylglycerol-glycerol [28]. The fibre compositions (fast oxidative glycolytic/fast glycolytic/slow oxidative) of the individual hindlimb skeletal muscles were tibialis anterior (66:32:2), E D L (59:38:3), soleus (16:0:84), plantaris (53:41:6) and gastrocnemius ( 3 7 : 5 8 : 4 ) [17,18]. Enzyme actirities. Glycogen phosphorylase (total and active as determined in the presence and absence of AMP) and glycogen synthase (total and active as determined in the presence and absence of glucose 6-phos-

209 phate) activities were determined by published methods [29,30] in homogenates of soleus and EDL muscles from ethanol-fed (2 weeks) and pair-fed controls. Muscles were assayed for enzyme activities after incubation in vitro in 4 ml Krebs-Ringer bicarbonate buffer (pH 7.4), containing 1% bovine serum albumin and 5 mM glucose (see above). Muscles were homogenised as described in Ref. 31. Statistics. Results are presented as means _+ S.E. for the numbers of rats shown in parentheses. Statistical analysis was by Student's paired t-test for comparisons between ethanol-fed animals and their pair-fed controls and otherwise by the Student's unpaired t-test.

feeding (P < 0.01; 3456 + 57 compared to 3769 + 38 mg/kg body weight). There was also a significant reduction in the relative weights of tibialis anterior muscles, which also contain predominantly type-II muscle fibres, in the ethanol-fed rats by 10% at 2 weeks feeding ( P < 0.001) and by 8% at 6 weeks feeding (P < 0.001) (results not shown). These findings, consistent with selective type-II atrophy, confirm previous reports [7,11]. The relationship between the selective myopathy and the growth abnormalities associated with chronic ethanol exposure remains obscure, although it may indicate that, rather than isovolumetric pair feeding, a more appropriate experimental protocol would be to adjust the volume of the control diet to achieve similar weight gain in the ethanol-treated and pair-fed control rats.

Results and Discussion

Muscle weights Ethanol feeding was associated with diminished growth and selective muscle atrophy. The body weights of the ethanol-fed rats after 2 weeks feeding on the liquid diet (74 _+ 1 g) were significantly less (by 14%: P < 0.001) than the pair-fed controls (86 _+ 1 g) [32]. The difference in body weight was increased to 18% (P < 0.001) after 6 weeks feeding (135 _+ 8 g compared to 165 _+ 10 g). Ethanol feeding was not associated with a statistically significant reduction in the weight of the soleus muscle expressed relative to total body weight. By contrast, the relative weights of the EDL muscles (expressed relative to total body weight) were significantly lower in the ethanol-fed rats by 8% after 2 weeks feeding (P < 0.01; 3392 _+ 45 compared to 3762 _+36 mg/kg body weight) and by 5% after 6 weeks

Tissue glycogen and triacylglycerol contents Chronic alcoholic myopathy in man [18,36-38] is reported to be associated with abnormal glycogen and lipid storage in affected muscles. It is important to establish whether there is a relationship between muscle-fibre composition and the response in individual muscles of glycogen and triacylglycerol stores to chronic ethanol feeding in the rat. The pattern of changes in the glycogen and triacylglycerol contents of individual skeletal muscles in response to chronic ethanol feeding for 2 weeks was complex. There was no correlation between muscle-fibre composition and abnormal glycogen or triacylglycerol storage. The glycogen contents of the EDL and quadratus lumborum muscles, both of which contain predominantly type-II muscle fibres,

TABLE I

Effects of chronic ethanol feeding on glycogen content and synthesis in isolated soleus and EDL muscles" Rats were pair-fed with a nutritionally complete diet containing isocaloric amounts of either glucose or ethanol. At the end of 6 weeks or 2 weeks, soleus and E D L muscles were dissected and incubated in Krebs-Ringer bicarbonate buffer, pH 7.4 containing 5 mM [U- t4C]glucose (see Materials and Methods). Glycogen synthesis was measured as 14C incorporation into glycogen from [U-~4C]glucose ( # m o l / g wet wt per h). Values are means-+ S.E. with numbers of determinations in parentheses. 6 weeks feeding

Soleus Glycogen content (~zmol as glucose / g wet wt) Glycogen synthesis ( / x m o l / g wet wt per h) EDL Glycogen content ( ~ m o l as glucose / g wet wt) Glycogen synthesis ( / ~ m o l / g wet wt per h)

2 weeks feeding

Control

Ethanol

Control

Ethanol

10.2_+0.4(18)

I0.I ,+0.4(18) a

8.9_+0.6(19)

7.7_+ 1.0(19)

0.6 _+0.1(18)

0.7 _+0.1(18)

0.7 _+0.1(21 )

0.5 ,+ 0.1(21) *

6.1 _+0.4(18)

5.3 _+0.3(18)

7.7_+0.6(21)

5.0_+0.5(21) * * *

0.6_+0.1(17)

0.6_+0.1(17) a

0.6_+0.1(21)

0.4_+0.1(21) **

* P < 0.05, * * P < 0.01 and * * * P < 0.001 for statistically significant effects of ethanol. P < 0.05 for statistically significant differences between 6 weeks and 2 weeks feeding.

210 were significantly lower in ethanol-fed rats (by 14%, P < 0.05:22.5 _+ 1.9 compared to 26.2 _+ 1.1 /zmol/g wet wt. and by 29%, P < 0.05:36.6 +_ 1.5 compared to 51.5 +_ 5.1 i z m o l / g wet wt., respectively). By contrast the glycogen contents of other type-II-fibre-rich muscles (viz., plantaris, tibialis anterior, gastrocnemius) and soleus muscle were not significantly altered by ethanol feeding (results not shown). Triacylglycerols, like glycogen, are recognised to be important sources of energy in skeletal muscles [33]. Whereas ethanol feeding resulted in increased levels of triacylglycerol in the gastrocnemius (by 123%, P < 0.05:34.5 +_ 4.9 compared to 15.5 + 3.4 /xmol/g protein) and plantaris (by 39%; P < 0.01:7.5 + 0.3 compared to 5.4 +_ 0.3 p~mol/g protein) muscles, there were no significant changes in the other muscles examined regardless of their fibre composition (results not shown). The implication is that muscle fibre composition is not the sole determinant of the effects of chronic ethanol feeding on muscle glycogen or triacylglycerol storage. The basis for these selective effects of ethanol feeding on muscle glycogen and lipid storage remains to be elucidated.

Chronic ethanol feeding for six weeks This paper is concerned with the mechanism(s) underlying the effects of chronic ethanol feeding on muscle. To identify the site of the biochemical lesion(s) specifically associated with chronic ethanol treatment, muscle metabolism was studied in isolated muscle preparations in vitro. An important element to the study was the selection of two skeletal muscles with different fibre compositions as models in which to examine the selectivity of the effects of chronic ethanol feeding: The E D L muscle contains predominantly type-II fibres, whereas the soleus contains predominantly type-I fibres [17].

Chronic ethanol feeding for 6 weeks was associated with abnormalities in glucose metabolism particularly in the isolated soleus muscle preparation. Whereas ethanol feeding had no significant effects on muscle glycogen content or rates of glycogen synthesis (measured as 14C incorporation into glycogen from [U14C]glucose [24]) (Table I) in both these muscles, in soleus muscle the rate of 14CO2 production (Table II) from 5 mM [U-14C]glucose was decreased by 17% ( P < 0.05) and the rates of lactate and alanine production were increased (by 37%, P < 0.001 and 61%, P < 0.05, respectively) by ethanol feeding. This reduction in glucose oxidation coupled with increased production of lactate and alanine may indicate a specific impairment in pyruvate oxidation. By contrast, the E D L muscle did not show a statistically significant reduction in ~4CO2 production from [U-~4C]glucose, although alanine, but not lactate, production was increased by 61% ( P < 0.0t ) (Table II).

Chronic ethanol feeding for two weeks The myopathic lesion in the rat is recognised to be fully developed after 6 weeks feeding on an ethanolsupplemented diet [9]. After 6 weeks feeding, it is difficult to distinguish between those biochemical events integral to the pathogenesis of the disorder and those that arise secondary to the end-stage skeletal myopathy. It is important, therefore, to study muscle metabolism during the active phase of the pathogenesis of the skeletal myopathy. To this end, muscle metabolism was examined after 2 weeks ethanol feeding. An important finding was that in contrast to the effects of 6 weeks ethanol feeding, rates of glycogen synthesis were significantly impaired in both the soleus and E D L muscles (by 29%, P < 0.05 and 33%, P < 0.01) and the glycogen content of E D L muscle was

T A B L E II

Effects of chronic ethanol feeding for 2 or 6 weeks on the metabolism of [ U- 14C]glttcose by isolated soleus and EDL muscles in t,itro Muscles w e r e i n c u b a t e d with 5 m M [U-14C]glucose. For e x p l a n a t i o n , see M a t e r i a l s and M e t h o d s and the l e g e n d to T a b l e 1. R a t e s Of m e t a b o l i t e p r o d u c t i o n ( / z m o l / g wet wt. p e r h) are the m e a n s + S.E. from at least 10 i n d i v i d u a l muscles. Muscle

Metabolic

6 weeks feeding

production ( / * m o l / g wet wt/h)

Control

Soleus

14CO2 lactate alanine

4.7 _+0.4(16) " 8.3 +_0.5(18) 4.4 + 0.8(10)

EDL

t4CO2 lactate alanine

7.0 _+0.5(15) ~' 17.1 +_0.6(18) b 3.6 _+0.5(10)

2 weeks feeding Ethanol

Control

Ethanol

3.9 + 0.4(16) * ,b 11.4 + 0.6(18) * * * 7.1 + 1.0(10)*

2.7 + 0.2(18) 10.8 _+0.9(34) 4.5 + 0.2(34)

2.0 + 0.3(18) 11.8 + 0.9(34) 5.3 + 0.3(34) * *

6.1 _+0.4(15) 16.0 +_0.6(18) 5.8 _+0.5(10) * *

5. l + 0.4(21 ) 21.8 +_0.9(33) 4.4 + 0.3(33)

4.8 +_0.5(21) 18.0 _+0.8(33) * * * 5.4 + 0.4(33 ) *

* P < 0.05, * * P < 0.01 and * * * P < 0.001 for statistically significant effects of ethanol. P < 0.01, b p < 0.001 for statistically significant d i f f e r e n c e s b e t w e e n 6 w e e k s and 2 w e e k s feeding.

211 T A B L E II1

Effect of chronic ethanol feeding on muscle glycogen synthase and glycogen phosphorylase activities Rats were pair-fed for 2 weeks on a nutritionally complete diet (controls) or isovolumetric amounts of a similar diet in which glucose was replaced by ethanol. Soleus and E D L muscles were dissected out and incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), containing 5 m M glucose (see Materials and Methods). Enzyme activities ( n m o l / m i n per mg wet wt) are expressed as m e a n s _+S.E. for 12 determinations. Enzyme

Soleus

activity

Ethanol-fed

Control

Glycogen synthase Total ( + glucose 6-phosphate) Active

0.7 + 0.06 0.2+0.04

0.6 + 0.03 0.2_+0.01

1.7 + 0.41 0.1 +0.02

1.2 + 0.31 0.l_+ 0.01

Glycogen phosphorylase Total ( + AMP) Active

2.7 _+0.49 0.5 _+0.11

1.9 + 0.35 0.4 + 0.09

73.9 + 7.1 2.3 + 0.46

68.1 +_ 13.0 1.9 _+ 0.60

reduced by 35% ( P < 0.001) (Table I) after 2 weeks feeding. In contrast to after 6 weeks feeding, the rate of 14CO2 production (Table II) from 5 mM [U-14C]glu cose by isolated soleus muscle, like that by isolated E D L muscle, was not affected by 2 weeks ethanol feeding. Rates of alanine production by soleus and E D L muscles, however, were increased by 18% ( P < 0.01) and 23% ( P < 0.05), respectively, in the ethanolfed rats. Ethanol feeding was also associated with a decrease (by 17%, P < 0.001) in the rate of lactate production by isolated EDL, but not soleus, muscles (Table II). It is noteworthy that rates of palmitate oxidation (measured as the rate of 14CO 2 production from [1~4C]palmitate) were similar in isolated E D L and soleus muscles ( 3 . 7 / x m o l / m i n per g wet wt compared to 3.4 /xmol/min per g wet wt) and were not altered by ethanol feeding for either 2 or 6 weeks (results not shown).

EDL Ethanol-fed

Control

Glycogen phosphorylase and glycogen synthase activities To establish whether the cause of the decreased rate of glycogen synthesis in muscles from ethanol-fed rats was alterations in the activities of glycogen metabolizing enzymes, the activities (total and active form) of glycogen synthase and phosphorylase were determined in soleus and E D L muscles from ethanol-fed and pairfed control rats. The determined enzyme activities, which were in reasonable agreement with published values [31], were not significantly affected by 2 weeks feeding on the ethanol-supplemented diet (Table III).

Insulin sensitivity The rate of glycogen synthesis in skeletal muscle is recognised to be stimulated by insulin in a concentration-dependent manner [19,20]. It is important to establish whether the impairment in glycogen synthesis after 2 weeks feeding on the ethanol-supplemented diet is accompanied by alterations in the responsiveness of the individual muscles to insulin. Ethanol feed-

(a) Soleus

(b) EDL 3

~

2

***

e.

eo

{

I

0 0

1

I

I

I

I

10 100 10(X) 10000 Insulin (l.tunits/ml)

0

1

10

100

1000

10000

Insufin ~units/ml)

Fig. 1. Effect of chronic ethanol feeding on the insulin sensitivity of glycogen synthesis in (a), soleus and (b), E D L in vitro. Soleus and E D L muscles from ethanol-fed rats ( l l ) and their pair-fed controls ( D ) were incubated in medium containing 5 m M [U-14C]glucose and insulin at different concentrations. Rates of glycogen synthesis are shown as means___ S.E. for at least 6 muscles. Statistically significant differences between ethanol-fed and control animals are denoted by * P < 0.05), * * P < 0.01, * * * P < 0.001. Basal rates (at 0 / z u n i t s / m l ) of glycogen synthesis were lower ( P < 0.05) in both soleus and E D L muscles from ethanol-fed rats compared to the pair-fed controls.

212 ing for 2 weeks had no effect on the insulin sensitivity of glycogen synthesis in either the isolated E D L or soleus muscles (Fig. 1). Whereas ethanol feeding, irrespective of insulin concentration, was consistently associated with reduced rates of glycogen synthesis (by 55 _+ 4.5%) in E D L muscles, the insulin concentration necessary to stimulate glycogen synthesis by 50% (C5~; approx. 40 ~ u n i t s / m l insulin) was similar in E D L and soleus muscles from ethanol-fed rats and their pair-fed controls. What is particularly noteworthy is that glycogen synthesis was less severely affected by ethanol feeding in isolated soleus as opposed to E D L muscle: ethanol feeding for 2 weeks was not associated with statistically significant reductions in the rates of glycogen synthesis in soleus muscles, except in the basal state (by 29%, P < 0.05), and at 10 /zunits/ml insulin (by 47%, P < 0.05). Since ethanol feeding was associated with the stimulation of alanine production by both isolated soleus and E D L muscles, an important question is whether this stimulatory effect is related to alterations in the sensitivity of alanine production to insulin in isolated muscles. The rate of alanine production by isolated soleus and E D L muscles, which was increased in muscles from rats fed ethanol for 2 weeks, was not changed to any significant extent by the addition of insulin, irrespective of its concentration (Fig. 2). In agreement with others [22], the rate of lactate production was increased in a concentration-dependent manner by insulin in both the isolated soleus and E D L muscles (results not shown). Ethanol feeding for 2 weeks was without effect on rates of lactate formation by isolated muscles, except that rates were decreased in the basal state (by 17%, P < 0.001: see also Table II) and at 1000

(a)

/xunits/ml (by 12%, P < 0.05) in EDL muscles from ethanol-fed rats.

Acute effects of ethanol and its metabolites on muscle metabolism The results above indicate that ethanol feeding for either 2 or 6 weeks results in biochemical lesions in both soleus and E D L muscles that perturb, albeit in different ways, glucose metabolism in vitro. One important question is whether these lesion(s) may be mimicked in vitro by acute exposure of isolated soleus and E D L muscles from chow-fed rats to ethanol and its metabolites. Ethanol is metabolised primarily in the liver by a pathway comprising the enzymes alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) [33]. This pathway oxidises ethanol to acetate via the intermediate acetaldehyde with the concomitant production of excess reducing equivalents as N A D H . Acetaldehyde is recognised to be implicated in the genesis of alcohol-related disorders [34,35]. The addition of either ethanol (50 mM) or acetaldehyde (250 txM) to E D L and soleus muscles taken from chow-fed rats and incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), containing 5 mM [U-J4C]glucose had no statistically significant effects on the rates of lactate production, glycogen synthesis (measured as 14C incorporation from [U-14C]glucose into glycogen) or alanine production (results not shown).

Pathophysiological implications This paper, which has focussed on the relationship between chronic ethanol feeding and compromised glucose metabolism in different muscle types, indicates unambiguously that type-II (EDL) and type-I (soleus)

(b) E D L

Soleus I

5' 4

.9

2

***

~

3

i

2 1

1

0 0

i

I

l

10

i

I

J

100 1000 10000

Insulin (launit/ml)

0 0

I

I

,

1

10

100

I

J

1000 10000

Insulin (Imnits/ml)

Fig. 2. Effects of chronic ethanol feeding on the insulin sensitivityof alanine release by isolated (a), soleus and (b), EDL in vitro. Soleus and EDL muscles from ethanol-fed rats (11) and their pair-fed controls (Q)were incubated in medium containing 5 mM [U-14Clglucoseand insulin at different concentrations. Rates of alanine released are shown as means+ S.E. for at least 6 muscles. Statistically significant differences between ethanol-fed and control animals are denoted by * P < 0.05, * * P < 0.01, * ** P < 0.001. Basal rates (at 0 ~zunits/ml) of alanine release were higher in both soleus (P < 0.01) and EDL ( P < 0.05) muscles from ethanol-fed rats compared to the pair-fed controls.

213 muscles are affected in a similar manner by ethanol feeding: Chronic ethanol feeding perturbs glucose metabolism but not fatty-acid oxidation in both soleus and EDL muscles, albeit in different ways and to different extent. The major metabolic effect after 2 weeks feeding was decreased glycogen synthesis. Although this effect was qualitatively similar in the two muscles, there were important quantitative differences, the primary one being that the EDL muscle is more severely affected. Since it is recognised that 2 weeks feeding coincides with the active phase in the pathogenesis of alcoholic myopathy [9], the differential impairment in glycogen synthesis in EDL muscle, which contains predominantly type-II muscle fibres, may be a factor in the pathogenesis of the myopathy. A second common feature of the response of both EDL and soleus muscles to ethanol feeding for 2 or 6 weeks was increases (by up to 61%) in the rate of alanine release. These increases were not necessarily accompanied by parallel increases in rates of lactate production, implying that the rate of alanine synthesis was not determined primarily by glycolytic flux and the availability of pyruvate. For example, whereas the rate of alanine synthesis was independent of insulin concentration, lactate production was markedly increased at elevated insulin concentrations. Chronic ethanol feeding is established to be associated with abnormalities in protein turnover [6] implying that the factor underlying the increase in alanine production may be enhanced protein catabolism, which would be expected to promote the transamination of pyruvate via increasing the availability of glutamate. Aside from its effects on glycogen synthesis and alanine release, ethanol feeding resulted in impairment in glycolysis and glucose oxidation in isolated muscle preparations. Ethanol feeding for 6 but not 2 weeks resulted in a selective reduction in ~4CO2 production from [U-~4C]glucose in the isolated soleus (but not EDL) muscle. Ethanol feeding for either 2 or 6 weeks was associated with increased rates of lactate production by isolated soleus but not EDL muscles (Table II). Whether this increase is caused by enhanced glycolytic flux or impaired pyruvate oxidation remains to be established. A paradoxical finding, which may indicate a decrease in glycolytic flux or diversion of lactate carbon into alanine synthesis, was that ethanol feeding for 2 but not 6 weeks resulted in decreased rates of lactate production by the isolated EDL muscles. An important question relates to the mechanism(s) underlying the effects of chronic ethanol feeding on glucose metabolism in isolated muscle preparations, particularly the abnormalities in glycogen synthesis and alanine release. Neither ethanol nor acetaldehyde had significant effects on rates of glycogen synthesis in isolated soleus and EDL muscle from chow-fed rats. Moreover, the lesion in glycogen synthesis associated

with ethanol feeding does not appear to involve abnormalities in the activities (total or % active form) of either glycogen synthase or glycogen phosphorylase (Table I). It remains to be established whether the lesion in glycogen synthesis relates to abnormalities in glucose uptake and phosphorylation.

Acknowledgements We thank the Brewers Society and the Central Research Fund of the University of London for financial support.

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Chronic effects of ethanol on muscle metabolism in the rat.

Chronic ethanol feeding in the rat is associated with a skeletal myopathy involving primarily type-II muscle fibres, which is recognised to be mediate...
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