Glucose metabolism during sleep and wakefulness

Pergamon Press

Life Sciences, pp . 291-296 Printed in the U.S .A .

GLUCOSE METABOLISM DURING SLEEP AND WAKEFULNESS James R . Macho and A. K . Sinha Department of Physiology and Biophysics College of Medicine and Dentistry of New Jersey Rutgers Medical School Piscataway, New Jersey 08854 (Received in final form December 10, 1979) Summary A radiorespirometric technique has been used to study glucose metabolism during sleep and wakefulness in the hamster . Using a small animal metabolic chamber, total C02 output and 14002 output from awake and sleeping hamsters were measured following I4C labelled glucose injection. During one hour of non-rapid-eye movement sleep 1400 2 increased by 143% . After 30 min. of sleep, no changes were observed in plasma glucose concentration or in plasma glucose specific activity . Brain slices obtained from hamsters during sleep yielded more 14 C02 from glucose than the brain slices from awake animals. The awake hamsters converted [1-140] glucose more rapidly than [6-140] glucoseto 14002 . It is concluded that there are quantitative and qualitative changes in glucose metabolism during sleep and wakefulness . There is a scarcity of data on metabolic alterations that occur during sleep and wakefulness . Although detailed studies are lacking there is evidence to suggest that there may be quantitative and qualitative changes in glucose metabolism between these two states . Van Den Noort and Brine (11) have reported an approximately 252 increase in brain glucose content and a 802 increase in glucose utilization in the sleeping rat brain. Anchors and Karnovsky (1) have described a four fold increase in phosphate labelling of glucose -6-phosphatase in rat brain during sleep. They suggest that an increase in glucose transport or metabolism may provide an explanation for this observation. Based on a number of indirect observations, Laborit (8) has proposed that the contribution of the pentose phosphate pathway to the metabolism of glucose increases during the sleeping state . Taberner et al . (10) have produced experimental evidence to suggest that pentose phosphate pathway activity increases during the hyponotic state produced by Y-hydroxybutyrate. They observed an increase in 140-1/140-6 ratios in the expired C02 from animals administered Y-hydroxybutyrate. Similar results were obtained in studies on incubated brain slices . In a series of experiments, using a radiorespirometric technique, we have determined the relative rates of glucose metabolism and its pathway orientation in awake and in naturally sleeping hamsters . Materials 6 Methods Adult male golden syrian hamsters weighing 100-115 g were used . The animals were placed on a 12 hr/12 hr light/dark cycle and were maintained on Purina laboratory chow . All radiochemicals were obtained from Sigma . 0024-3205/80/040291-06$02 .0010 Copyright (c) 1980 Pergamon Press Ltd

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Surgical : Four extradural stainless steel skull screws for recording EEG and two neck muscle electrodes for recording EMG were implanted in the hamsters under 80 mg/kg Pentobarbital anesthesia . Electrode leads were mount ed in an amphenol connector strip which was cemented to the skull with acrylic cement . At least one week was allowed for recovery from surgery . During thisperiod . the animals were acclimated to the metabolic chamber . State of arousal: EEG-EMG records were scored manually by 15 second epochs for wake, non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REK), following standard procedures . In vivo The C0 2 collection apparatus consisted of a snug-fitting, airtight acrylic chamber (with provision for EEG-EKG recording leads), connected to a series of 22 x 150 mm tubes containing 10 ml of a 1N NaOH solution through which the expired air was bubbled with a gas dispersion tube . Airflow through the system was maintained at a constant flow rate (2L/min) by a flow meter . A C02 trap consisting of barium hydroxide granules was placed at the inflow to the animal chamber . It was established that all of the incoming C02 was trapped by the barium hydroxide and that all of the C02 expired by the animal during the collection period was trapped by the NaOH solution. A final C02 trap was incorporated in the system to rule out the possibility of C02 spillover . A series of valves allowed collection tubes to be changed during the course of the experiment . All experiments were carried out at the same time of day to avoid possible circadian variations in glucose metabolism . In order to consolidate sleep and to enhance the chances of obtaining a successful experiment, the animals were kept awake for three hours prior to the experiments . This was accomplished by gentle tapping of the cages and by transferring the animal to a different cage . During this time, the animals had access to water but not to food . At the end of this period animals were randomly selected for sleep or wake experiments and placed in the metabolic chamber . EEG and EKG recording was begun at the onset of sleep and 2 .5pCi of [1-14 C] or [6-14 C] glucose were injected intraperitoneally . Collection of C02 was Once within the begun and collection tubes were changed every 10 minutes . chamber, the undisturbed animals fell asleep within 5-10 mins . The total period of collection was for 1 hour . In the parallel series of experiments the animals were kept awake for the collection period . The total C02 in each sample was determined by back titration of a 1 .0 ml aliquot with 0 .1 N HCl in a automatic burette (Radiometer) . A difference of Another aliquot 10 pl of titration volume could be measured by this apparatus . (0 .5 ml) of each sample was placed in 10 ml of triton/toluene/PPO cocktail and counted on a Beckman LS-150 liquid scintillation counter . To determine if the specific activity of plasma glucose was altered during sleep, the animals were injected with lOUCi of [U-14 C]-glucose at the beginning of the experimental period . After 30 minutes of sleep or wakefulness the ham sters were decapitated within 3 sec . of being disturbed, and heparinized blood samples were obtained . Plasma glucose was determined from this sample according to the method of Gatfield, et al . (4) . Specific activity of glucose was measured by the method of Costello and - Bourke (3) . Results During the fourth hour they were Hamsters were kept awake for 3 hours . either allowed to sleep or kept awake. During this last hour their EEG and EMG were recorded . Their NREM, REM and wake time is expressed as percent of (Table 1) . this fourth hour,

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TABLE 1 GROUP

NREM

REM

WARE

"Awake"

6 .0%

0.0%

94%

"Sleep"

87 .6%

0 .6%

11 .8%

After three hours of wakefulness it was found that the hamsters would sleep for 88% of a fourth hour, with most of the wakefulness confined to a period within the first 10 minutes of the hour . There was no significant amount of REM sleep. present during this time . The animals in the awake group were kept awake during the fourth hour by gentle tapping on the metabolic chamber . The "sleep time" in this group consisted of short (5-10 sec) episodes of slow wave activity in the EEG .

Oxidative Metabolism of Glucose It was found that the total C02 output from the hamster decreased 13 .4% during sleep, (Fig . 1) . In the awake hamsters, the output was 6 .48 _ + 0.17 -les/hr/100 g hamster (+ SEM) and in the sleeping hâmsters this v alue was 5 .61 _+ 0 .03 . The difference is significant with p < .001 . The output of 14C02 from [140-6] glucose was used to evaluate oxidative metabolism of glucose via Embden-Meyerhof and Krebs cycle pathways . An in-

mZL CO, OUTPUT

FIG . 1

04C-41 -D-1^ Nm w. a o

nE

FIG . 2

Fig . 1

Total C02 output from hamsters during sleep and wakefulness . Each graph is the mean of fourteen observations . S .E .M . is represented by vertical bars .

Fig . 2

Time course of 14 C02 elimination from awake and sleeping hamsters following intraperitoneal injection of [ 14 0-6]-D-glucose 14002 specific activity has been normalized for the dose of isotope-and the weight of the animal . Sleep points are the mean of six observations . Awake points are the mean of seven observation . The S .E .M . is indicated by vertical bars .

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The results creased amount of 14002 was eliminated during sleep, (Fig . 2) . are expressed as, specific activity of C02 normalized for isotope dose and the weight of the animals . The total change represents a 143% increase in specific activity of the C02 eliminated from the sleeping animals during the first hour . The awake experiments were repeated using animals that were not sleep deprived for 3 hours prior to the experiment to determine whether this imposed 3 hours of wakefulness before the ex eriment had an effect on the experimental results. It was found that the 1 CO 2 output with or without 3 hours of prior sleep deprivation was approximately equal (Fig . 3) . Specific activities of glucose were determined in plasma to resolve whether these differences were due to changes in plasma pool size . No changes were observed in plasma glucose specific activity (wake - 2 .53 _+ 0 .10 uCi/mole ; sleep = 2 .70 _+ 0 .05 uCi/mole) . Pathway of Glucose Metabolism In the whole body experiment the sleeping hamsters had a C-1/C-6 ratio that was close to 1.0 indicating insignificant contribution of the pentose phosphate pathway (Fig . 4) . . In the awake animals the ratio was close to 1 .7 .

Fig. 3

Time course of 14C02 elimination from awake hamsters following intraperitoueal injection of [140-6]-D-glucose. The deprivaThe other group tion group was kept awake for three hours . was not deprived of sleep . 14C02 specific activity has been normalized for the dose of isotope and the weight of the animal . Each point is the mean of seven observations . The S .E .M . is indicated by vertical bars .

Fig . 4

Ratio of recovery of 14002 from [140-1] glucose and [140-6] glucose from awake and sleeping hamsters following intraperEach point is the mean of itoneal adminstraiton of isotope . six observations .

Themean C-1/C-6 ratio for the sleeping group was 1 .08 + 0 .25 and for the awake The difference is significant with p < .01 . group was 1 .71 + 0.28 .

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Discussion In an _in vivo radioisotope study of sleep, a suitable animal should be small, should have long sleep cycles for detectable biochemical alterations to be established and should not be easily disturbed form sleep . The most common ly used animal in sleep studies, the cat, is not suitable for in vivo isotope studies because of its size . The other commonly used animal, the rat, has a short sleep cycle ( 10 min) and is easily aroused from sleep . Stringently imposed visual and acoustic isolation is required to study sleep in the rat . The hamster has been chosen as the experimental animal because it fulfills all three requirements . It is small. Its' sleep cycles are approximately equal to those of the cat and it sleeps well under minimal isolation conditions . The variable amounts of muscular activity during sleep and wakefulness may be a complicating factor in a metabolic study of sleep. To reduce muscular activity, both the awake and the asleep animals are enclosed in a closely fitting lucite cylinder during the period of the experiment . This burrow-like condition is probably more natural for the rodents and EMG recordings do not indicate much isometric muscular activity when the hamsters have been acclimated to the chamber. A large amount of EMG activity would be cause for discarding the results of a particular experiment . The sleep scoring data shows very little time spent in REM sleep . This is consistent with the aim of the study . Although it would be desirable to study the correlates of normally progressing sleep stages, it does not seem feasible to obtain metabolic data in such short time periods . Therefore, it was decided that changes between NREM sleep and wakefulness would be considered . The decrease in total C02 output during sleep is consistent with the previous findin s of studies on basal metabolic rate (7) . A significant increase in expired ltC02 specific activity during sleep was observed . However, no difference was noted between the awake group that was subjected to three hours of prior sleep deprivation and the group that was not deprived . This supports the contention that the method for the consolidation of sleep does not affect the results. The increase in expired 14C02 specific activity during sleep can have several explanations . The rate of 1400 2 elimination following administration of a labelled precursor depends not only on the rate of catabolism but also on other factors such as (a) the specific activity of the precursor in blood and tissue, (b) the rate of C0 2 elimination from the blood bicarbonate pool and (c) the rate of ventilation. By plotting the data as breath specific activity Rodden, et ( 14 C02/C02) any effect of alteration .in ventilation is avoided . _al. (9) have shown that in human subjects there is no difference in the rate of C02 elimination from the blood bicarbonate pool . In this study, it was determined that the plasma glucose specific activity did not change during sleep. These findings are consistent with a change in glucose catabolism or alteration in the specific activity of glucose in any one of the major metabolizing tissues of the body . In reference to whole body glucose metabolism via pentose phosphate pathway, a value of more than one for the C-1/C-6 ratio in expired C02 has been used as an index of pentose phosphate pathway activity (2,11) . Katz and Wood (5,6) pointed out the difficulties of using the C-1/C-6 ratio for this purpose . Although the method is not suitable for quantitating the number of glucose molecules traversing the pentose phosphate pathway, it is adequate for detecting a relative increase or decrease in the involvement of pentose phosphate pathway. The C-1/C-6 ratio of the sleeping animals is the same as the ratio obtained by Taberner et _ al . (10) for their control rats and shows a minimal involvement of pentose phosphate pathway while the C-1/C-6 ratios of the sleep deprived, awake animals have values more than one, indicating a significant involvement of pentose phosphate pathway . Taberner _et _al . have demonstrated increased involvement of pentose phosphate pathway following Y-hydroxybutyrate

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treatment in rats . It appears from our data that three hours of sleep deprivation in hamsters reorients glucose metabolism towards pentose phosphate pathway and if the animals are allowed to sleep, then the involvement of the pentose phosphate pathway returns to its normal low value . Acknowledgement This project was partly supported by a grant from the National Science Foundation, BNS 76-14945 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .

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