Regional distribution of glycogen, glucose and phosphorylated sugars in rat brain after intoxicating doses of ethanol

~ ) Pergamon

Neurochem. Int. Vol. 25, No. 2, pp. 175-181, 1994

01~7-01S(,(~)EOO~t-Q

Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rightsreserved 0197-0186/94 $7.00+0.00

REGIONAL DISTRIBUTION OF GLYCOGEN, GLUCOSE A N D PHOSPHORYLATED SUGARS IN RAT BRAIN AFTER INTOXICATING DOSES OF ETHANOL J. GARRIGA1, M. Sus'r 2 a n d R. C u s s 6 ~* ~Unitat de Bioqulmica, Facultat de Medicina, Universitat de Barcelona, Barcelona 08028, Spain 2Unitat de Bioestadistica, Facultat de Medicina, Universitat de Barcelona, Barcelona 08028, Spain (Received 17 August 1993 ; accepted 24 January 1994)

Alntraet--Ethanol and anaesthetics increase glycogen levels in the brain. However, no data have been reported about the effect of ethanol on glycogen and glucose metabolism in specific brain regions. We have studied the concentrations of glycogen, glucose, glucose 6-P, glucose 1,6-P2 and fructose 2,6-P2 and the activities of glycogen synthase, glycogen phosphorylase and glycogen phosphorylase kinase in seven brain regions of starved rats following treatment with a single dose or several doses of ethanol. Our results show that: (1) the effect of ethanol on glucose metabolism depends on whether it is given in one single dose or in a series of doses; (2) glycogen concentration increases after a single dose of ethanol but not after long exposure; (3) glucose, glucose 6-P in some areas, and the bisphosphorylated sugar, fructose 2,6-P2 significantly increase after prolonged exposure to ethanol ; and (4) the enzymatic activities of glycogen metabolism are not modified after a long exposure to ethanol. In summary, these data show that ethanol may modify the use of glycogen, glucose and derivatives in brain. Moreover, the changes produced depend on the pattern of ethanol intake and the brain area considered.

Glucose is the main source of energy in brain (Sokoloff, 1981), but the rate at which it is metabolized depends on the region (Collins et al., 1987). The glycogen store in the brain is small but its turnover is as high as in other tissues (Lowry et al., 1964; Nahas and Abdul-Ghani, 1989). Consequently, the regional distribution of glycogen has also been shown to be varied (Sagar et al., 1987). Changes in blood glucose related to both chronic and acute administration of ethanol have been studied and it seems that when hepatic carbohydrate stores are sufficient, ethanol induces hyperglycaemia, whereas when they are low, it leads to hypoglycaemia (Reitz, 1979). In glucose metabolisation two bisphosphorylated hexoses, glucose 1,6-P2 (Glu 1,6-P2) and fructose 2,6P2 (Fru 2,6-P2), may modulate the activities of phosphofructokinase (PFK-1) and other key regulatory enzymes of carbohydrate metabolism (Andr6s et al., 1990; Beitner, 1985; Claus et aL, 1984; Hue and Rider, 1987). The distribution of Glu 1,6 P2 in mouse brain has been studied by Yip et al. (1985) and its synthesis by Rose et al. (1975, 1977). A specific Glu

1,6-P2

phosphatase has been identified in brain (Guha and Rose, 1983). The intake of ethanol modifies the intermediate metabolism in brain (Tejwani and Duruibe, 1985; Veloso et al., 1972; Wik et al., 1988). Alcohol affects the membranes and the activity of N a - K ATPase (J~irnefelt, 1961), the transport of glucose (Pentreath et al., 1982) and the phosphorylation of glucose by hexokinase (Kosow et al., 1973). Metabolites of the glycolytic pathway decrease with the consumption of ethanol and anaesthetics such as barbiturates (Theodore et al., 1986). Brain glycogen stores have been reported to increase (Delphia et al., 1978; Veloso et al., 1972) or decrease depending on the experimental conditions used (Wik et al., 1988 ; Delphia et al., 1978 ; Estler and Lachman 1976). The difficulty of quantitative determination of energy compounds such as glycogen and nucleotides in the different brain areas remained unsolved, owing to the drawbacks associated with dissecting frozen brain and the rapidity of metabolic change after death. However, the recent application of the microwave technique as a means of killing the animals facilitated the measurements of glycogen concentrations in the *Author to whom all correspondence should be addressed. different brain areas (Garriga and Cuss6, 1992 ; Sagar 175

176

J (i,x~l~Icia c / a /

et al., 1987) a n d the highest glycogen c o n c e n t r a t i o n s d e s c r i b e d to d a t e were r e p o r t e d . W e s t u d i e d the effect o f b o t h an a c u t e a n d a prol o n g e d a d m i n i s t r a t i o n o f e t h a n o l in different areas on the d i s t r i b u t i o n o f glycogen, glucose, glucose 6-P a n d b i s p h o s p h o r y l a t e d sugars in several b r a i n regions. w h e n b o t h b l o o d glucose utilization a n d b r a i n glyc o g e n stores are low o w i n g to s t a r v a t i o n . A t the s a m e time, the activities o f glycogen r e g u l a t o r y e n z y m e s in the s a m e r e g i o n s were analyzed. A f t e r e x p o s u r e to e t h a n o l , c h a n g e s in glycaemia a n d in glycogen utilization in the different b r a i n areas are expected. T h e effect o f e t h a n o l could be m o r e i m p o r t a n t in areas with high glucose m e t a b o l i s m .

EXPERIMENTAL PROCEDURES

Materials Radioactive substrates were from Amersham, U . K All enzymes were obtained from Boehringer Mannheim, IN, U.S.A. Other reagents were of analytical grade. Treatment o[animals Male Sprague-Dawley rats from Charles River (C.D) (300-500 g) and from our colony (350-450 g) were starved for 48 h prior to use. Ethanol administration was as described by Veloso et al. (1972). In experiments on the acute effects of ethanol, rats were injected i.p. with 7 M ethanol in 0.15 M NaC1 (0.75 ml ethanol/100 g animal weight) and killed 8 min later. In experiments on the effects of prolonged exposure to ethanol, rats were injected with 7 M ethanol in 0.15 M N aC1 as follows : 0.60 ml ethanol/100 g animal weight at 13 and 5 h and 0.2 ml ethanol/100 g animal weight at 3 and 1 h before death. Control rats received equal volumes of 0.15 M NaCh The animals were killed between 12 : 00 14.00 h. Tissue preparation CD rats were used to study the effects of ethanol on glycogen and sugar concentrations. The animals were killed using microwave irradiation (Garriga and Cuss6, 1992; Sagar et al., 1987). The oven was a Litton-System 70/50 with a power of 4.5 kW and the conditions applied were 2500 mHz and a power of 3.5 kW for 4s. After death the animals were decapitated. Brains were maintained at 4'C before dissection into areas according to Glowinski and Iverson (1966). To measure glycogen, glucose and glucose 6-P, samples from the different brain regions were homogenized with 0.03N HC1 in a Potter~ Elvehjem and immediately placed in a boiling water bath for l0 min. For the measurements of Glu 1,6-P2 and Fru 2,6-P2 about 30 mg of frozen sample were homogenized with a Potter-Elvehjem in 10 vol of 50 mM NaOH and kept at 90~C for 10 min. The insoluble material was removed by centrifugation and the supernatant was used for determination. Sprague Dawley rats were used to measure enzymatic activities. Rats were killed by decapitation and brains were quickly removed and maintained on ice during dissection into areas. The brain regions were also dissected according

to GIowinski and lverson (1966) and each region ~a~, immediately frozen in liquid nitrogen and stored ~t 40 (' until extractions were performed The enzyme extracts were prepared with 5 rot oi 50 mM Tris HCI, 4 mM EDTA, 50 mM KF and 30 mM /*-mcrcaptoethanol, pH 7.3. Homogenates were prepared in ~ Potter Elvehjem and centrifuged at I 1,000g for 211 rain at 4 C. Enzyme activities were determined in the supernatant A nalyt ical procedures Glycogen was assayed fluorimetrically in the homogenate by the method of Passonneau and Lauderdale (1974). Glycogen was degraded specifically by amylo :~-l,4-:~-l,6-glycosidase. The glucose formed in the reaction was determined with hexokinase and glucose 6-P dehydrogenase through the formation of NADPH. Glu 6-P was measured lollowing the addition of only glucose 6-P dehydrogenase to the acid homogenate. When amylo ct-l,4-~-1,6-glycosidase was not included the N A D P H formed in the second step was a measure of the endogenous glucose and glucose 6-P. To obtain glycogen concentration this value was subtracted from the value corresponding to the sample containing the amyloglycosidase. Glucose 1,6-P, was measured as a cofactor of phosphoglucomutase (Passonneau et al.. 1969). Fructose 2,6-Pz was determined following the method described by Van Schaftingen et al. (1982). Blood glucose was determined by the method of glucose oxidase (Werner et al., 1970) adapted to a Beckman Autoanalyzer Astra-4. Alcohol levels m blood were determined in a Perkin Elmer Gas Chromatograph (Column 60/80 carbopack B 5% carbowax 20%). Protein was determined according to the procedure of Lowry et al. (1951). Enzyme activities were measured as follows : glycogen synthase by the method described by Thomas et al. (1968), glycogen phosphorytase by the method ofGilboe et al., 1972) and glycogen phosphorylase kinase as described by Cohen (1973). The purified glycogen phosphorylase b used as substrate by phosphorylase kinase was prepared from rabbit muscle and purified according to Fisher and Krebs 11958). Slatistical analysis Differences among the seven brain areas within each experimental group were studied by the analysis of the 95% confidence intervals for the respective means. Differences between the same brain areas o f rats from different experimental groups were analysed by an Analysis of Variance (ANOVA), the appropriate a priori contrasts were analysed by the Student's t-test between the two particular groups of interest. In all tests we applied a significance level of P < 0;05.

RESULTS

E f f e c t s o b s e r v e d 8 m i n qf~er e x p o s u r e to ethanol

Eight m i n u t e s after e t h a n o l a d m i n i s t r a t i o n the c o n c e n t r a t i o n o f the toxic in b l o o d was a b o u t 46.4 m M . G l u c o s e c o n c e n t r a t i o n in b l o o d was the s a m e as c o n trol (4.6 m M ) (Table 1). G l y c o g e n c o n c e n t r a t i o n in b r a i n s o f c o n t r o l rats, w h i c h h a d been in s t a r v a t i o n ['or 48 h, was a b o u t 1/3 o f the c o n c e n t r a t i o n f o u n d in fed c o n d i t i o n s . Analysis

Ethanol and carbohydrate distribution in rat brain Table 1. Blood content of glucose and ethanol after intraperitoneal injection of ethanol Glucose (mM) Time after injection

Control

8 min (n = 7) 13 h (n = 8)

4.7-1-0.6 4.6-t-0.8 3.6+0.1 7.6___0.6

Ethanol (mM)

Ethanol

Control

Ethanol

---

46.4+ 5,8 73.9+%8

Values are mean + SEM.

of 95% of the confidence interval showed that glycogen levels in cerebellum and hippocampus were different and higher than those of striatum, cortex, hypothalamus and mid-brain. The highest glycogen level was found in the cerebellum in agreement with previous results (Garriga and Cuss6, 1992; Sagar et al., 1987) and the lowest was found in the cortex. Eight minutes after i.p. injection of ethanol, glycogen concentration increased [Fig. I(A)] in all the

177

areas. The analysis of 95% of confidence interval showed that the glycogen level in the cerebellum was still different from that of thalamus mid-brain and that of the cortex (with lowest glycogen concentration) differed from those of ports-medulla, hippocampus and cerebellum. ANOVA statistical analysis of glycogen concentration showed significant differences only in cerebellum. Initially glucose concentration was similar in all the areas studied [Fig. 1(B)]. After ethanol exposure the concentration decreased in all the areas except in thalamus-mid brain although the difference was not significant. Glu 6-P levels were similar in all the areas and ethanol injection produced significant decreases in striatum and ports-medulla [Fig. 1(C)]. In control rats the lowest Glu 1,6-P2 level was found in the cerebellum and pons-medulla and the highest in hippocampus and cortex. Exposure to a single dose of ethanol did not modify these levels (results not shown).

50"

. 40.

Z

v, ¢,

¢, -r,,,

"r

o. 4" l g,

o °~ 20. %

[

-~a-

Z f,

2-

¢,

m

c

1H

S

(A)

o

c

T

5-

Z

Z

~, 30.

H

S

Co

Hy

T-m

P-m

0

C

1

(B) OLUCOSE

Hy

to

Co

Hy

T-m

P-m

(c) OLUCOSE-6-P

GLYCOGEN

Co

I

T-m

P-m

C

H

S

Co

Hy

T-m

P-m

(D) FRUCTOSE 2,6-P=

Fig. 1. Effect in brain o f 8 min exposure to a unique dose o f ethanol. (A) effect o f glycogen concentration ; (B) effect on glucose concentration ; (C) effect on glucose 6-P concentration; (D) effect on fructose 2,6- P~ concentration. Unfilled bars (I-I) control rats (saline injected). Filled bars ([~) rats injected with ethanol. C, cerebellum; H, h i p p o c a m p u s ; S, striatum; Co, cortex ; Hy, h y p o t h a i a m u s ; T-m, thalamus mid-brain ; P-m, pons-medulla. Significant differences * P ~< 0.05. In all cases the n u m b e r o f control animals is 9 and the n u m b e r o f ethanol injected animals is 8.

178

,l. (.J,',RRI(I,\ cl a/,

The highest level of Fru 2,0-P: was found in the cerebellum and the lowest in hypothalamus. Acute intoxication by ethanol did not modify the Fru 2,6-P: concentrations [Fig. I(D)].

E~l~'c'ts obsen~ed aller 13 h ole_vposure to ethanol The concentration of ethanol in the blood after 13 h exposure was 74 mM. Glucose level rose to 7.6 mM, compared to 3.6 m M in controls. Before exposure to ethanol, the 95% confidence interval showed differences in glycogen concentration between cerebellum and striatum. After 13 h of ethanol administration (Fig. 2) glycogen concentration did not change significantly in any area [Fig. 2(A)] but the differences between them disappeared. In contrast administration of ethanol led to a significant rise in the levels of glucose (about 3fold) in all the areas. The analysis of 95% confidence interval showed no differences among the glucose lev-

els of the areas analysed [Fig. 2(B)]. as il happened before the ethanol administration. Glu 6-P values did not shox~ differences among areas and after ethanol exposure they increased in all the areas: this increase was signiticant in hippocampus, striatum and hypothalamus [Fig. 2(C)I. In control rats the Glu 1,6-P, concentration in cerebellum (the lowest of the areas studied) was different from that found in hippocampus, cortex and midbrain (areas with the highest concentrations of this metabolite). Alter ethanol administration there was no change in G 1,6-P~ levels but the differences among areas disappeared (results not shown). In control animals the analysis of 95% of the confidence interval showed differences in Fru 2,6-P- concentration among the areas. The highest value corresponded to cerebellum and this was different from the lowest values found in hippocampus, striatum and hypothalamus. 13 h exposure to ethanol 10-

70. 60.

8-

tr, 50. E

40 0

g 30 '5 213

lo.

i c

6.

g "6

4-

2"

H

S

Co

My

T-m

0

P-m

C

H

(A) GLYCOGEN

Co

Hy

T-rn

P-m

(C) GLUCOSE-6-P

50' o

o

o

o

4o.

H f/

o

,'i

& 3O-

//

"6 20.

z* 4, ~

,-/

~

f/ /i

"d 41

H

S

Co

My

(B) GLUCOSE

T-m

~,,j,

Z

cJ

F~

N c

o

,,,j

// Li // P-m

¢. ¢I ¢~

3'

N f.l

lO.

7.

,.-,

Z

¢-j

Z C

c

H

S

Co

Hy

T-m

P-m

(D) FRUCTOSE 2,6-P 2

Fig. 2. Effect in brain of 13 h exposure to several doses of ethanol. (A) effect on glycogen concentration; (B) effect on glucose concentration ; (C) effect on glucose 6-P concentration ; (D) effect on fructose 2,6- P: concentration. Unfilled bars (El) control rats (saline injected). Filled bars (g~) rats injected with ethanol. C, cerebellum ; H, hippocampus ; S, striatum ; Co, cortex ; Hy, hypothalamus ; T-m, thalamus mid-brain ; P-m, pons-medulla. Significant differences * P ~<0.05. In all cases the number of control animals is 9 and the number of ethanol injected animals is 8.

Ethanol and carbohydrate distribution in rat brain produced about 2-fold significant increase in all the areas, but there were still differences between the area with the lowest value (hypothalamus) and the ones with the highest (medulla, cortex, hippocampus and cerebellum) [Fig. 2(D)]. E n z y m e activities

The enzymes that regulate glycogen metabolism, glycogen synthase, glycogen phosphorylase and glycogen phosphorylase kinase, were determined in control rats and in rats after 13 h of exposure to ethanol. The activities of these enzymes after 48 h of starvation following by exposure to ethanol are shown in Table 2. The highest activities of glycogen synthase and glycogen phosphorylase in the different areas corresponded to the area with the highest glycogen concentration : medulla, cerebellum and striatum. Cortex and hypothalamus had the lowest activities. Ethanol intake did not modify the activities of the enzymes of glycogen metabolism. The proportion of glycogen synthase I was about 50% in control rats and decreased to 30% after 13 h of exposure to ethanol. The glycogen phosphorylase a activity was about 60% and it was not modified during alcohol exposure (data not shown). The high percentage found in both active forms was due to the time taken in brain dissection. The highest glycogen phosphorylase kinase activities were found in hypothalamus and hippocampus and they did not change with ethanol administration. DISCUSSION Exposure to a single dose of ethanol produced an increase in alcoholemia but alcohol still did not mod-

179

ify glucose concentration in blood. In such conditions, Veloso et al. (1972) reported a high alcohol concentration in brain. The administration of ethanol produced an increase in glycogen concentration in all areas, although the differences among them were maintained. In this case there was a general decrease in glucose and Glu 6-P, being significant for Glu 6-P only in two areas. This fact could be explained as a decrease in glycogen utilization produced by the direct inhibition that ethanol has on glycogen phosphorylase kinase at 5-50 mM ethanol concentration (Cuss6 et al., 1989). Another explanation could be the inhibitory effect that ethanol has on N a + - K + ATPase in the membrane (J~lrnefelt, 1961), which decreases the consumption of ATP and diminish the energy supply from glycogen. The bisphosphorylated sugars Glu 1,6-P2 and Fru 2,6-P2 were not modified during the short time of exposure to ethanol. In contrast, after 13 h of exposure to ethanol, glycaemia reached the normal values of a fed rat. In such conditions brain glycogen concentrations were not modified and there were no differences among areas. The increase in glycaemia was due to the intake of ethanol, which enables blood glucose to be used as the main energy supply again, without being restored by the synthesis of glycogen. The results found for glycogen in our case are different from those found by Veloso et al. (1972), who used rats that had been starved for 72 h. In our opinion differences could be due to the different periods of starvation. After 72 h starvation (conditions of Veloso et al., 1972), the resynthesis of glycogen is greater than at 48 h (Garriga

Table 2. Activitiesof the enzymesof glycogenmetabolismafter 13 h of ethanolexposure Glycogen synthase mU/mg Brain Region Cerebellum Cortex Striatum Hlppocamlms Hypothalamus Thalamus Mid-brain Pons..medulla

Glycogen phosphorylase U/mg

Glycogen phosphorylase kinase mU/mg

C

E

C

E

C

E

4.03 + 0.95 2.47 + 0.40 3.65 _ 1.19 3.33 ± 1.10 3.51 ± 1.48 2.93 ± 0.77 4.50 ± 1.57

3.62 _ 0.92 3.02 ± 0.87 3.42 _ 1.51 3.34 ± 0.81 3.37 ± 0.93 3.13 ± 0.94 4.69 ± 1.31

0.37 + 0.09 0.21 ± 0.06 0.26 ± 0.05 0.29 ± 0.10 0.46 ± 0.24 0.27 ± 0.05 0.37 ± 0.16

0.39 + 0.09 0.30 ± 0.10 0.22 ± 0.06 0.31 + 0.14 0.38 ± 0.11 0.27 ± 0.07 0.39 ± 0.09

1.64 ± 0.53 1.70 ± 0.51 1.50 ± 0.59 2.27 ± 0.65 3.13 + 0.75 2.02 ± 0.74 2.48 ± 1.06

1.05 ± 0.69 2.87 ± 1.18 1.61 ± 0.52 2.26 ± 0.75 2.22 ± 1.10 1.61 ± 0.39 1.62 ± 1.27

Values for total activitiesare mean+standard deviationand n = 7. In all cases activitiesarc referred to in mg of protein. C, control group; E, ethanol injected.

180

J. ( J,%R R [(i,', C{ ~11.

and Cuss6, 1992) and the response to a continuous exposure to ethanol could be an increase in glycogen levels. At the same time there could be an inhibition in the utilization of the carbohydrate polymer as happened after 8 min of ethanol exposure. Glucose and glucose 6-P increased significantly, about 3-fold in all the areas. These findings agree with the results ofVeloso et al. (1972). The increase in brain glucose is a consequence of the important increase in blood glucose that takes place after the long period of exposure to ethanol (hyperglycaemic effect). This effect might be due to the increase m glucose levels in the brain capillaries, but Passonneau et al. (1971), studying the glucose content in a single nerve cell showed clearly that this increase is intracellular, indicating that the glucose supply in nerve cells is dependent on the level of glycaemia. The bisphosphorylated sugar Glu 1,6-P2 did not change but Fru 2,6-P_, increased significantly in all the brain regions after the long period treatment. High Fru 2,6-P2 concentration has also been found in muscle of human chronic alcoholics (Cadefau et al., 1992). The variations in Fru 2,6-P2 concentration after alcohol intake could be related to the large availability of glucose supplied by blood and the increase in Fru 6P supply. The augment in glycaemia could induce the increase in the intake of glucose and the increase in Fru 2,6-P2 could promote glycolysis by activating P F K - I . This suggests that the concentration of this sugar increases when glycolytic flux must increase (Ambrosio et al., 1991). The existence in brain of a new bifunctional P F K - 2 isozyme has recently been described (Ventura et al., 1991). The activities of the enzymes of glycogen metabolism depend on the region (Garriga and Cuss& 1992 ; Knull and Khandelwal, 1982). The enzyme activities did not change after 13h of alcohol exposure. Our results suggest that the effect of ethanol on brain carbohydrate metabolism is dependent on the blood glucose availability and the effects of ethanol on liver enzymes" activities. Under our conditions, after 48 h of starvation, when liver has adapted to the production of ketone bodies (Ruderman et al., 1974) and other tissues, such as brain, to the low consumption of glucose, the response of liver to ethanol exposure is to increase the glycaemia. This may explain the increase in blood glucose that takes place after the long exposure to ethanol. Furthermore there is an increase in the intake of [~4C]deoxyglucose after chronic exposure to ethanol (Pietrzak et al., 1989). As a consequence of the glucose availability, brain may decrease the consumption of ketone bodies and partially restore the use of glucose.

The increase in the Fru 2.6-P: concentration can contribute to this fact as activating P F K - I promotes the glucose oxidation by glycolysis. Our results show that the glucose and I~ru 2,6-P~ metabolism are affected by the exposure to ethanol in the different areas depending on the dose of ethanol and the duration of exposure. A single dose of ethanol produces an increase in glycogen levels but the differences among its concentrations in the areas are maintained. Several doses of ethanol abolish the differences among areas. The fact that glucose and glycogen reach similar values in different areas of brain after 13 h exposure to ethanol could be explained as a reaction to maintain energy metabolism within minimal limits. This would avoid important lesions in cerebral structures, although the disappearance of initial differences among areas following ethanol ingestion may lead to transitory changes of certain brain functions

Acknowledgements--We are indebted to Dr Mario Salmona from the Instituto Mario Negri, Italy for introducing us to the use of the microwave oven and for his collaboration with rat treatment. We are indebted to Merc6 Vatiente and Esther Adanero for their technical assistance. This work has been supported by a Grant from C1RIT of the Generalitat de Catalunya.

REFERENCES

Ambrosio S., Ventura F., Rosa J. L. and Bartrons R. (1991) Fructose 2,6-bisphosphate in hypoglycaemic rat brain. J. Neurochem. 57, 200-203. Andr6s V., Carreras J. and Cusso R. (1990) Regulation of muscle phosphofructokinase by physiological concentrations of bisphosphorylated hexoses : effect of alkalinization. Biochem. biophys. Res. Commun. 172, 328 334. Beitner R. (1985) Glucose 1,6 bisphosphate. The regulator of carbohydrate metabolism. In : Regulation ~fCarbohydrate Metabolism (Beitner R., ed.), Vol. 1. CRC Press, Florida, U.S,A.

Cadefau J. A., Andr& V., Carreras J., Vernet M., Grau J. M., Urbano-Mfirquez A. and Cuss6 R. (1992) Glucose 1,6-bisphosphate and fructose 2,6-bisphosphate in muscle from healthy humans and chronic alcoholic patients. Alcohol and Alcoholism 27, 253 256. Claus T. H., EI-Maghrabi M. R., Regen D. M., Stewart H. B., McGrane M., Kountz P. D., Nyfeler F., Pilkis J. and Pilkis S. J. (1984) The role of fructose 2,6-bisphosphate in the regulation of carbohydrate metabolism. Curr, Topics Cell. Reg. 23, 57-86. Cohen P. (1973) The subunit structure of rabbit skeletal muscle phosphorylase kinase and the molecular basis of its activation reactions. Eur. J. Biochem. 34, 1 t4. Collins R. C., McCandless D. W. and Wagman I. L. (1987) Cerebral glucose utilization: comparison of [~4C]deoxyglucose and [6-~4C]glucose quantitative autoradiography. J. Neurochem. 49, 1564-1570.

Ethanol and carbohydrate distribution in rat brain Cuss6 R., Vernet M., Cadefau J. and Urbano-Mfirquez A. (1989) Effects of ethanol and acetaldehyde on the enzymes of glycogen metabolism. Alcohol and Alcoholism 24, 291297. Delphia J. M., Negulesco J. A. and Firran E. (1978) The effect of ethanol on cerebral glycogen levels in the chick embryo. Res. Commun Chem. path. Pharmac. 21,347-350. Estler C. J. and Lachmann V. (1976) Prevention by pyrazole of ethanol-induced decrease of brain glycogen and coenzyme A. J. Neurochem. 26, 653-654. Fischer E. H. and Krebs E. G. (1958) The isolation and crystallization of rabbit skeletal muscle phosphorylase b. J. biol. Chem. 231, 65-71. Garriga J. and Cuss6 R. (1992) Effect of starvation on glycogen and glucose metabolism in different areas of the rat brain. Brain Res. 591,277-282. Gilboe D. P., Larson K. L. and Nutall F. Q. (1972) Radioactive method for the assay of glycogen phosphorylases. Analyt. Biochem. 47, 20-27. Glowinski J. and Iversen L. C. (1966) Regional studies of catecholamines in rat brain. I. The disposition of [3H]norepinephrine, [3H] dopamine and [3H] DOPA in various regions of brain. J. Neurochem. 13, 655-669. Guha S. K. and Rose Z. B. (1983) Role of inosine 5'-phosphate in activating glucose-bisphosphatase. Biochemistry 22, 1356-1361. Hue L. and Rider M. H. (1987) Role of fructose 2,6-bisphosphate in the control of glycolysis in mammalian tissues. Biochem. J. 245, 313-324. J~mmefelt J. (1961) Inhibition of the brain microsomal adenosintriphosphatase by depolarizing agents. Biochim. biophys. Acta. 48, 111-116. Knull H. R. and Khandelwal R. L. (1982) Glycogen metabolizing enzymes in brain. Neurochem. Res. 7, 1307-1317. Kosow D. P., Oski F. A., Warms J. B. and Rose I. (1973) Regulation of mammalian hexokinase : regulatory differences between isoenzyme I and II. Archs. Biochem. Biophys. 157, 114-124. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. L. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Lowry O. H., Passonneau J. V., Hasselberger F. X. and Schulz D. W. (1964) Effect ofischemia on known substrate and cofactors of the glycolytic pathway in brain. J. biol. Chem. 239, 18-30. Nahas N. and Abdul-Ghani A. S. (1989) Species-directed variation and non-uniform distribution of glycogen in mammalian brains during starvation, diabetes and anaesthesia. Neurochem. Int. 14, 19-24. Passonneau J. V. and Lauderdale V. R. (1974) A comparison of three methods of glycogen measurement in tissues. Analyt. Biochem. 60, 405-412. Passonneau J. V., Lowry O. H., Schulz D. W. and Brown J. G. (1969) Glucose 1,6-diphosphate formation by phosphoglucomutase in mammalian tissues. J. biol. Chem. 244, 902-909. Passonneau J. V., Brunner E. A., Molstad C. and Passonneau R. J. (1971) Regional energy reserves in mouse brain and changes with ischaemia and anaesthesia. J. Neurochem. 18, 2317-2328.

181

Pentreath V. W., Seal L. H. and Kai-Kai M. A. (1982) Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues. Neuroscience 7, 759-767. Pietrzak E. R., Wilce P. A. and Shanley B. C. (1989) The effect of chronic ethanol consumption on [t4C]deoxyglucose uptake in rat brain in vivo. Neurosci. Lett. 100, 181-187. Reitz C. (1979) Effects of ethanol on the intermediary metabolism of liver and brain. In: Biochemistry and Pharmacology o f Ethanol (Majchrowicz E. and Noble E. P., eds), Vol. 1, pp. 353-382. Plenum Press, New York. Rose Z. B., Warms J. V. B. and Kaklij G. (1975) A specific enzyme for glucose 1,6 bisphosphate synthesis. J. biol. Chem. 250, 3466-3470. Rose Z. B., Warms J. V. B. and Wong L. J. (1977) Inhibitors of glucose 1,6-bisphosphate synthase. J. biol. Chem. 252, 4262-4268. Ruderman N. B., Ross P. S., Berger M. and Goodman M. N. (1974) Regulation of glucose and ketone-body metabolism in brain anaesthetized rats. Biochem. J. 138, 1-10. Sagar S. M., Sharp F. R. and Swanson R. A. (1987) The regional distribution on glycogen in rat brain fixed by microwave irradiation. Brain Res. 417, 172-174. Sokoloff L. (1981) Relationships among local functional activity, energy metabolism and blood flow in the central nervous system. Fedn. Proc. 40, 2311-2316. Tejwani G. A. and Duruibe V. A. (1985) Effect of ethanol on carbohydrate metabolism. In : Regulation o f Carbohydrate Metabolism (Beitner R. ed), Vol.l, pp. 67-92. CRC Press, Florida. Theodore W. H., DiChiro G., Margolin R., Fishbein D., Porter R. J. and Brooks R. A. (1986) Barbiturates reduce human cerebral glucose metabolism. Neurology 36, 60-64. Thomas S. A., Schlender K. K. and LarnmerJ. (1968) A rapid filter assay for UDP-glucose glucosyltransferase, including an improved biosynthesis of UDP-(141C)glucose. Analyt. Biochem. 25, 486-499. Van Schaftingen E., Lederer B., Bartrons R. and Hers H. G. (1982) A kinetic study of pyrophosphate : fructose 6phosphate phosphotransferase from potato tubers. Eur. J. Biochem. 129, 191-195. Veloso D., Passonneau J. V. and Veech R. L. (1972) The effects of intoxicating doses of ethanol upon intermediary metabolism in rat brain. J. Neurochem. 19, 2679-2686. Ventura F., Rosa J. L., Ambrosio S., Gil J. and Bartrons R. ( 1991) 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat brain. Biochem. J. 276, 455-460. Werner W. H., Rey U. H. and Wiltinger Z. (1970) Properties of a new chromogen for the determination of glucose in blood according to the GOD/POD (glucose oxidase-peroxidase) method. Fresenius'Z Anal. Chem. 252, 224-228. Wik G., Borg S., Sjtgren I., Wiesel F. A., Blomqvist G., Borg J., Greitz T., Nyb/ick H., Sedvall G., Stone-Elander S. and Widen L. (1988) PET determination of regional cerebral glucose metabolism in alcohol-dependent men and healthy controls using 1C-glucose. Acta Psychiatr. Scand. 78, 234-241. Yip V., Carter J. G., Dick E., Rose Z. B. and Lowry O. H (1985) Distribution of the glucose-l,6-bisphosphate and IMP-activated glucose bisphosphatase in brain and retina. J. Neurochem. 44, 1741-1746.