Epimerase-catalyzed glucose-galactose interconversion in the developing mouse brain

BRAIN RESEARCH

501

EPIMERASE-CATALYZED GLUCOSE-GALACTOSE I N T E R C O N V E R S I O N IN THE D E V E L O P I N G MOUSE BRAIN

ELIE A. S H N E O U R * AYD IDELL M. HANSEN

Department of Biolo,gieal Sciences, University of Utah, Salt Lake City, Utah 84112 (U.S.A.) (Accepted June 12th, 1969)

INTRODUCTION

Brain UDP-galactose-4-epimerase catalyzes the readily reversible interconversion of the uridyl glycoside forms of glucose and galactose in the presence of N A D 16,17. The sharp increase in the activity of this enzyme from developing mouse brain acetone powder extracts during the most active period of brain maturation, particularly including rnyelination, has been reported earlier 21. By contrast, no such peak of activity could be observed from acetone powder extracts of developing mouse liveffL The brain has been shown to exhibit a remarkable selectivity for glucose, while the uptake of galactose as well as other mono- and disaccharides is very slow compared to glucose ls,19. It was therefore suggested that the sharply increased brain requirement for galactose during active myelination could only be met by conversion in situ of dietary glucose from milk lactose, to galactose through the UDP-galactose-4epimerase pathway 2t. Since it is not possible to make an a priori statement about the steady state equilibrium of this interconversion reaction in vivo, it was necessary to test this hypothesis by a time-dependent analysis of galactose/glucose ratios in the brain during development. These experimental observations are presented in this report. A possible relationship between certain demyelination diseases of genetic origin and a defect in the UDP-galactose-4-epimerase pathway is also suggested. MATERIALS AND METHODS

Chemicals. [14C6]Glucose was obtained from New England Nuclear Corporation, Boston, Massachusetts, as a 10~ aqueous ethanol solution. The specific activity was standardized to 1 millicurie/millimole with glucose carrier and the purity checked immediately before use by paper chromatography. For use, the solution was distilled to dryness in vacuo and redissolved in sterile isotonic sodium chloride to yield a solution containing 100/~C/ml. Animals'. Swiss albino mice, maintained on a standard Purina Mouse Chow diet.

*Present address : Division of Neurosciences, City of Hope National Medical Center, Duarte, Calif. 91010, U.S.A. Brain Research, 16 (1969) 501 510

502

E. A. SHNEOUR AND I. M. HANSEN

Brain glucose and galactose determinations a. Free sugars. Mice were starved for 4 h prior to injection. Each mouse was

then injected intraperitoneally with 0.05 ml [14C6]glucose solution. Mice were sacrificed 1,2 and 4 h following injection and the rapidly dissected brains (30 sec maximum between decapitation and freezing) were collected in shaved ice, to yield 2 g of tissue for each experiment. To minimize postmortem changes, the following freeze-drying procedure was developed: the pooled brains were inserted into a 10 ml Virtis Screw Cap Vac Vial (No. 10-159) and crushed against the walls of the vial with a metal spatula to form a thin film, while the vial was partially immersed in a liquid nitrogen bath. The vial was then inserted into a Virtis vented adapter and freeze-dried to complete dryness with a Virtis mechanical freeze-drier. The resulting material was ground with an agate mortar to a fine homogeneous powder, and stored at --10°C. For each experiment, 200 mg of powder were homogenized with 2.0 ml of an ice-cold 1 0/o NaCI solution for 15 rain with a Virtis homogenizer at medium speed. The suspension was quantitatively transferred to a 12 ml Pyrex Sorvall centrifuge tube (No. 102A) with a Pasteur pipette, and the contents heated on a boiling water bath for 10 min. The denatured suspension was then centrifuged at 5000 ~ g for I0 min. The supernatant solution was decanted and the precipitate was resuspended in 2.0 ml 10/~oNaC1 solution. After homogenizing it again for 15 min, the suspension was centrifuged again at 5000 ~ g for 10 rain. The combined supernatant solutions were transferred to a 100 ml round-bottom flask and distilled in vacuo at 0°C to a volume of 0.5 ml. Five hundred milligrams of a I:1 mixture (by weight) of glucose and galactose were dissolved into a reduced volume of the supernatant solution. The solution was then desalted down to a 15 mA conductance level in a Baird and Tatlock desalter (Torbal-BIL Chromatographic Desalting Unit CD-I). Four 25 #I aliquots of the desalted solution were spotted on Whatman 3 M M paper with glucose and galactose standards, and developed for 72 h with a n-butanolpyridine-water (125:40:125; v/v) solvent system. The glucose and galactose spots were identified with a nondestructive spray indicator consisting of 40 mg bromcresol purple, 100 mg boric acid in 100 ml methanol to which 10 ml of a 1 ,~oaqueous solution of borax had been added. Each characterized yellow spot was cut into 1 mm wide strips, transferred into a 20 ml scintillation flask containing 0.5 ml water and allowed to stand at room temperature for 12 h. Nine and one-half ml of a scintillation solution having the following composition was then added to each flask: toluene 1 liter, p-dioxane 1 liter, absolute ethanol 0.6 liter, naphthalene 130 g, 2,5-diphenyloxazole (PPO) 13 g, 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene (dimethyl-POPOP) 0.25 g (ref. 22). Each flask was assayed for radioactivity with a Packard Tri-Carb scintillation spectrometer Model 3365, with appropriate blanks, standards and quenching calibration, and the quenching levels determined with an automatic external standard. The data were converted to disintegrations per minute (d.p.m.) to a confidence level of better than 95 ~ and in quadruplicate for each experimental point. b. Bound sugars. The I ~ NaC1 solution-washed and centrifuged pellet obtained by the above-described procedures was then treated as follows: Two ml of a 2 N Brain Research, 16 (1969) 501-510

GLUCOSE-GALACTOSEINTERCONVERSION

503

HC1 solution were added to each centrifuge tube, the pellet resuspended with a vibrating mixer and the tube heated on a boiling-water bath for 3 h. After centrifugation at 17,000 ~ g for 10 rain, the supernatant solution was decanted into a clean tube. The precipitate was washed with 2.0 ml 2 N HCI solution, resuspended with a vibrating mixer and centrifuged again at 17,000 x g. The combined supernatant solutions were transferred to a 100 ml round-bottom flask, neutralized with a 5 N Na2CO3 solution, and the volume reduced in vacuo to 0.5 ml. The same procedures as described for the analyses of free sugars were then used to assay for the bound sugars, namely addition of carriers, desalting, chromatography and scintillation spectrometry. All procedures described for these assays were verified for reliability by appropriate control experiments to determine completeness of recoveries and characterization ofeluates. For example, Bogoch I has advocated a much more drastic hydrolysis procedure to recover bound sugars. When the material to be hydrolyzed was exposed to 2 N HCI at 105°C for 24 h, the recovery of glucose and galactose remained within the standard error of the mean for identical samples treated for only 3 h as described at the beginning of this section. Experimental evidence in this laboratory suggests that the hydrolytic rates for the monosaccharides tested depend to a significant degree upon the prior handling and treatment history of the samples subjected to analysis. Therefore, while the hydrolyses of bound sugars did not seem to require longer and more drastic procedures, a significant uncertainty remains about the extent of the glucose and galactose recovery from these samples. Hexose degradation It is possible that the original injected [14C6]glucose could be degraded and resynthesized before recovery of both free and bound radioactive glucose and galactose. To eliminate this factor, carrier-diluted samples of original [14C6]glucose and samples of pooled reisolated glucose and galactose were each converted to potassium gluconate and oxidized with sodium metaperiodate to yield degraded carbons 1 and 6 according to Turner 24. The radioactivity ratio of carbons 1-6 for each sample was compared to that of the original [x4C6]glucose and found to be identical within experimental error. This confirms the observation reported by Burton et al. 4 and by Moser and Karnovsky 18 that the hexose carbon chain probably remains intact in this pathway. Since biological variations in the absolute amounts of sugar uptake and recoveries are inevitable and difficult to control adequately, the significant data obtained have been recorded~as galactose to glucose total radioactivity (d.p.m.) ratios. Preparation and radioassay o f brain subcellular )Cractions To determine the subcellular radiocarbon distribution in the brain from injected [14C~]glucose, the following procedure was used: Mice at 7, 15, 21 and 31 days of age were each injected i.p. with 5 ,uC [14C~]glucose and then sacrificed 24 h later. Brains were rapidly dissected as indicated above and pooled in crushed ice to a wet weight of 2 g. They were then homogenized and fractionated as described by Eichberg Brain Research, 16 (1969) 501-510

504

E. A. SHNEOUR AND I. M. HANSEN

TABLE 1 FREE GALACTOSE AND GLUCOSE IN BRAIN*

Age

Hours (post-injection)

Galactose d.p.rn,

Glucose d.p.m,

Gal/Glu d.p.m. ratio

2

1 2 4

539.9 ~ 61.7 91.2 j: 13.1 80.9 ! 14.0

590.6 -~ 15.3 167.1 3- 10.3 94.8 :~: 4.4

0.912 i 0.081 0.537 ~0.048 0.854 L 0.137

8

1 2 4

1801.7 4:159.7 181.7 :~: 20,4 109.3 ! 9.1

2082.4 :~.:63.1 102.7 :~::11.7 35.1 -~:: 2.3

0.865 :z: 0.060 1.736 :~: 0.217 3.115 ! 0.141

16

1 2 4

488.5-= 93.8 373.4 i 75.4 189.7 ! 46.0

54.9 ± 4.4 36.3 L 2.4 85.5 :~ 30.5

8.90 : 1.75 10.30 2.41 2.50 1.33

22

l 2 4

547.8 ! 36.9

35.0 -~ 4.5

15.89

i 2.10 i 0.25

32

1 2 4

39.0 j

2.0

16.8 :~+ 1.1

2.33

27.5 ~ 31.1 ! 33.3 :

4.7 8.7 5.1

15.7 ~ 3.1 18.7 L 3.1 11.6 ~ 2.5

1.82 i 054 1.64 t 031 3.03 i: 1.10

* Each d.p.m, value and ratio is based on a minimum of 4 samples, i: value is the standard error of the mean.

et al. 5 to yield the following p a r t i c u l a t e fractions: myelin fragments, ' s y n a p t o s o m e s ' ,

m i t o c h o n d r i a a n d ' m i c r o s o m e s ' . E a c h p a r t i c u l a t e fraction was r e s u s p e n d e d in its a p p r o p r i a t e sucrose s o l u t i o n a n d recentrifuged. These p a r t i c u l a t e s as well as the a q u e o u s soluble c y t o p l a s m i c fraction a n d the s u p e r n a t a n t s f r o m r e s u s p e n d e d particulates were each freeze-dried a n d the r a d i o a c t i v i t y o f these residues assayed. The r a d i o a s s a y o f each o f these samples was carried out as follows: E a c h fraction was t r a n s f e r r e d q u a n t i t a t i v e l y to a liquid scintillation vial. The s a m p l e was then c r u s h e d a n d p o w d e r e d with a glass rod. One d r o p o f water a n d 0.5 ml o f N C S solubilizing r e a g e n t ( N u c l e a r C h i c a g o Cat. No. 190620) were a d d e d a n d allowed to stand overnight at r o o m t e m p e r a t u r e . N i n e a n d o n e - h a l f milliliters o f scintillation solution No. 1 (5.0 g/liter P P O a n d 0.3 g/liter d i m e t h y l - P O P O P in toluene) were a d d e d , a n d the r a d i o a c t i v i t y d e t e r m i n e d in the m a n n e r d e s c r i b e d a b o v e for reisotated glucose and galactose. RESULTS AND DISCUSSION Glucose-galactose interconversion

E x p e r i m e n t s involving injection o f [14C6]glucose, followed by the r e i s o l a t i o n o f m o u s e b r a i n glucose a n d galactose at 1 , 2 a n d 4 h as a function o f age are r e p o r t e d in T a b l e I for the free sugars a n d in T a b l e I I for b o u n d sugars. I n i n t e r p r e t i n g the d a t a f r o m these tables, it is essential to keep in m i n d t h a t all mice in these e x p e r i m e n t s Brain Research, 16 (1969) 501-510

505

GLUCOSE-GALACTOSE INTERCONVERSION TABLE II BOUND GAEACTOSE AND GLUCOSE IN BRAIN

Age

Hours (post-injection)

Galactose d.p.m.

Glucosed.p.m.

Gal/Glud.p.m. ratio

2

1 2 4

183.4 t 20.8 199.5 % 38.3 148.6 I 19.0

246.5 ± 21.8 227.1% 14.9 137.5 ± 12.9

0.744 ± 0.159 0.890 + 0.233 1.078 ± 0 . 0 5 5

8

l 2 4

200.7 == 29.4 564.8 % 95.3 84.1 3 15.4

560.0 % 73.8 498.3 % 82.4 135.0% 15.4

0.361% 0.057 1.131 = 0.078 0.632 %0.159

16

1 2 4

20.2 2 1.6 47.0 % 7.4 123.7 % 33.0

15.1 ± 4.1 29.6 % 1.8 245.8 ± 42.5

1.468 %0.655 1.603 % 0.348 0.527 % 0.225

22

1 2 4

31.0% 5.8 133.6 % 77.4

23,7 % 4.5 50,9 % 8.7

1.346--0.369 2.775 - 1.993

l 2 4

10.9% 3.9 49.0 }~ 4.3 17.4 J 3.7

13.6 } 4.1 33,5 ~- 2.5 16.9% 2.2

0.883 ~0.534 1.462 ~=0.110 1.033 %0.239

32

were each injected with a constant 5 #C of radioglucose at a constant specific activity of I mC/mmole. However, between 2 and 32 days of age there was a 10-fold increase in mouse weight and a 4-fold increase in brain weight'~t These large changes cannot be effectively compensated for in the presentation of the data, and they are therefore reflected in a concomitant drop in the total radioactivity of the reisolated brain monosaccharides as a function of mouse age. It can be seen in Table I that the galactose to glucose ratio rises sharply at 16 days and reaches a peak at 22 days post-partum, then falls back to a low value at 32 days. The same overall pattern, at lower levels, can be seen fbr bound sugars in Table II. It can also be noted in the 1, 2 and 4 h post-injection data of Tables I and II that the free sugar radioactivity decreases with time, while that of the bound sugars generally tends to increase. Since the extent of the recovery of bound sugars remains in question as pointed out above, the data reported in Table II cannot be considered definitive. But they do provide a significant clue to the nature and kinetics of glucose-galactose incorporation in developing brain particulates. These data also confirm the observations of Moser and Karnovsky is for the 22 days post-partum galactose substrate peak in mouse brain. The UDPgalactose-4-epimerase activity, however, seems to reach a maximum 6 days earlier, at 16 days post-partum. This difference may reflect an accumulation of galactose in the pool between 16 and 22 days resulting from a decreasing rate of its utilization during this period. Galactose probably belongs to a large and varied brain metabolic pool which includes cholesterol, sphingosine and cerebroside fatty acids 4,2°. It is likely that this sugar is present in the pool in several forms, including free galactose, UDP-galactose and galactose sulfatides. McKhann and Ho 1~ have suggested that the Brain Research, 16 (1969) 501-510

506

E.A. SHNEOUR AND 1. M. HANSEN

availability of galactocerebrosides might be a limiting factor in the synthesis of galactosulfatides, and they have shown that the pattern of synthesis of these sulfatides in the developing brain follows closely that of neutral brain glycolipids4. The data reported from this laboratory are also in accord with these findings as they specifically concern the UDP-galactose-4-epimerase pathway and the dynamic state of galactose in brain cerebrosides 2°. It is not possible, however, to deduce from the available experimental evidence whether the several forms of galactose coexist in a large single pool, or whether a number of compartments develop during brain maturation, particularly between 16 and 22 days post-partum in mice. It is also not possible to determine what role the changing metabolic rate of injected radioglucose plays during this period s. While it is unlikely that these factors affect significantly the reported galactose-glucose ratios, they cannot be entirely dismissed in the interpretation of results. It is reasonable, however, to suggest that these results are consistent with the hypothesis that during brain development, the sharply increased synthetic requirements for galactose are met by in situ conversion of dietary glucose through the UDP-galactose-4-epimerase pathway, and that the sharp increase in this enzyme activity during myelination is directed to the production of UDP-galactose. The following sequence of enzyme reactions is proposed as the major pathway of glucose-galactose interconversion during this period*: Glucose 4- ATP ~ Glucose-6-P ~ ADP Glucose-6-P ~ Glucose- I-P Glucose-l-P + U T P ~ UDP-Glucose ! PP tO)

UDP-Glucose ~-UDP-Galactose 4,18

.

,

U DP-Galactose -~- 'microsomal acceptor lipid" -~ neutral galactohp~d ÷ UDP The enzymes (a) hexokinase 2,z~, (b) phosphoglucomutase 2, (c) UDP-glucose pyrophosphorylaseg, 11 and (d) UDP-galactose-4-epimerase 16 have been shown to occur in brain at substantially higher activities than those of the following reactions: Galactose ~ ATP ~ Galactose-l-P -~- ADP Galactose-l-P -~ UDP-Glucose ~ Glucose- I -P -~ UDP-Galactose Galactose-l-P ÷

UTP ~ UDP-Galactose -i- PP

The enzymes (e) galactokinase 7,1z,2~, (f) galactose-l-P uridyl transferase ~°,17 and (g) UDP-galactose pyrophosphorylase s appear to have very slow kinetics 3,1s, and their contribution during active myelination appear likely to be secondary.

* Abbreviations used: ATP, adenosine triphosphate; P, phosphate; PP, pyrophosphate; UTP, uridine triphosphate; ADP, adenosine diphosphate; UDP, uridine diphosphate; NAD. nicotinamide adenyl dinucleotide. Brain Research, 16 (1969) 501-510

e~

.q.

Myelin fraction Synaptosome fraction Mitochondria fraction Microsome fraction Soluble fraction Supernatant from myelin Supernatant from synaptosome Supernatant from mitochondria Supernatant from microsome

3,700 15,500 19,200 3,800 27,200 10,300 2,600 I, 100 23,600

26,300 280,000 446,400 118,800

6,400 10,200 12,100 6,100 5,100 4,600 1,300 160 473

A

A

B

16 days

8 days

151,100 34,600 257,900 206,400

B

4,300 11,400 10,300 3,800 11,400 1,200 222 156 2,000

A

22 days

--

136,700 134,800 215,100 128,900

B

3,200 3,500 2,300 1,400 2,200 1,200 621 55 447

A

32 days

45,200 15,400 57,100 42,500

B

A, Total d.p.m, in whole fraction; B, d.p.m, per g dry weight. All d.p.m, rounded off to nearest 100 except values less than 1,000 d.p.m.

R A D I O C A R B O N I N C O R P O R A T I O N IN BRAIN S U B C E L L U L A R FRACTIONS

TABLE 111

"--....I

L/3

508

F. A. SHNEOUR AN[) 1. M. HANSEN

Radiocarbon incorporation in brain subcellular Jractions In other experiments carried out in this laboratory, the UDP-galactose-4epimerase activity was assayed in a number of separate areas of the developing mouse brain, notably a comparison was made of the neocortex with the brain stem. Within experimental error, no difference in enzyme activity could be detected. These observations indicate that the enzyme is probably equally distributed throughout the brain, and therefore suggest that this activity is more likely to be associated with glia than with neuronslL This localization is consistent with a role for UDP-galactose4-epimerase in myelin biosynthesis. To assess such a possibility further, the radiocarbon incorporation from [taC6]glucose in brain subcellar fractions was determined, and the results are reported in Table 111. Radioglucose injections were held constant at 5/zC per mouse throughout these experiments. The data on the soluble cytoplasmic fractions and on the supernatant washings of the particulates are included in the table as a control of their radiocarbon levels: the magnitudes are sufficiently low to permit their elimination as a significant contamination factor in the particulate fractions. It can be seen that radiocarbon incorporation is maximal at 16 days post-partum in the myelin, mitochondrial and microsomal fractions, while the highest levels in the synaptosomes fraction is reached at 8 days post-partum. The subcellular fractionation technique used produced a myelin fraction contaminated with some mitochondria 14 but the pattern of radiocarbon incorporation is consistent with both the results on enzyme activity and substrate equilibria reported earlier. The microsomal fraction has been most directly implicated in the incorporation of galactose into neutral galactolipids 4 and the data support these findings. The decline in radiocarbon incorporation at 32 days in all particulate fractions, and in the mitochondrial fraction in particular, do not represent a decrease in their concentration, Klee and Sokoloff ~z, for example, have shown that while rat brain /:~-hydroxybutyric dehydrogenase and cytochrome oxidase activities rise together and reach an optimum, the cytochrome oxidase levels remain high, while the former activity drops to low levels at maturity in the same manner as it has been described for UDP-galactose-4epimerase. Thus, the rise and fall in these activities is probably not attributable simply 1o a change in the subcellular particle content of the brain, but rather to a change in their composition or function during maturation ~z. Radin et al. 2° have reported evidence for the presence in the developing rat brain of a protein-bound galactolipid and a rapidly metabolizing galactolipid. They have also reported that the strandin fraction galactose in adults has a half life of the order of 8 days. These results are reported to be consistent with the observed disappearance of galactolipids in Wallerian degeneration and in certain demyelination diseases. In preliminary experiments in this laboratory with some neurological mutants of the mouse which exhibit progressing CNS demyelination both the UDPgalactose-4-epimerase activity rise and the galactose surges at 16 days post-partum observed with Swiss albino mice appear to be lacking in some of these animals. The possibility of a relationship between certain CNS demyelination syndromes and a defect in the UDP-galactose-4-epimerase pathway appears to warrant further

investigation. Brain Research, 16 (1969) 501-510

GLUCOSE-GALACTOSE INTERCONVERSION

509

SUMMARY

Mice aged f r o m 2 to 32 days were injected i.p. with [14C6]glucose a n d sacrificed at I, 2 a n d 4 h post-injection. Free and b o u n d glucose and galactose were reisolated from brain, and their r a d i o a c t i v i t y was determined, as well as the integrity o f the hexose c a r b o n chain. A 10-15-fold increase in the free galactose to free glucose ratio was observed between 16 a n d 22 days post-partum. Lower values were observed for b o u n d sugar ratios. These changes coincide with increased brain U D P - g a l a c t o s e 4-epimerase activity and with active myelination. The i n c o r p o r a t i o n o f r a d i o c a r b o n in myelin was d e t e r m i n e d by subcellular f r a c t i o n a t i o n o f brain 24 h after i.p. injection o f [~4C6]glucose, O p t i m a l i n c o r p o r a t i o n o f r a d i o c a r b o n was o b s e r v e d at 16 days p o s t - p a r t u m and is consistent with a p r i m a r y role for U D P - g a l a c t o s e - 4 - e p i m e r a s e in myelin biosynthesis. These results also s u p p o r t the hypothesis that the sharply increased brain requirements for galactose d u r i n g myelination are met by in situ conversion o f dietary milk glucose to galactose t h r o u g h the U D P - g a l a c t o s e - 4 epimerase pathway. ACKNOWLEDGEMENTS

The a u t h o r s gratefully a c k n o w l e d g e the advice o f Dr. Abel Lajtha and Dr. Eugene Roberts. This w o r k was s u p p o r t e d , in part, by research grants from the N a t i o n a l A e r o nautics and Space A d m i n i s t r a t i o n and the U.S. Public Health Service.

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Brain Research, 16 (1969) 501-510