Laboratory of Physiology, Department of Biology, Morgan State University, Baltimore, Maryland
Submitted 11 November 2004 ; accepted in final form 14 February 2005
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ABSTRACT |
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rapid eye movement; oxygen consumption; uncoupling protein-1
REM-SD is achieved simply and effectively with the platform (i.e., flowerpot) method. The setup has a rat on a small platform (e.g., inverted flowerpot) surrounded by water. Enforcement takes advantage of muscle atonia during REM sleep, resulting in making contact with or falling into the water; the rat abruptly awakens and repeats the cycle. Electroencephalography of platform rats confirms that one-half to all of REM sleep and varying amounts of non-REM sleep are lost (20, 23, 27, 28), therefore validating its utility.
Comparisons between the two methods show that some syndromes are similar: hyperphagia and weight loss (5, 30, 31, 34, 44, 49) and decreased plasma thyroid hormones (3, 13, 31) and leptin (10, 21; this study). Importantly, because many serious pathologies manifest after more than 1 wk (34), whether or not other DOW syndromes come about with the platform method remain unanswered, because almost all of the studies involving the latter have been short term, typically with a time course of 96 h or less (1, 5, 7, 30, 31, 4043, 49).
Regardless of enforcement method, sleep-deprived rats exhibit hyperphagia with concomitant loss of body weight, two pathologies that cannot be explained by changes in digestive absorption efficiency (3) or development of diabetes (9). The only way to account for the ensuing state of negative energy balance is for metabolism to be elevated, and yet, the most commonly used and accepted procedure to measure metabolic rate, oxygen consumption, has never been employed for SD studies. To gain further understanding of some of these phenomena, we wanted to elucidate the relationship between REM-SD and increased metabolism. No inquiry has addressed this problem, but even a cursory review of mammalian metabolic physiology points to brown adipose tissue (BAT) as a logical place to begin. BAT is the primary site of regulatory nonshivering thermogenesis in rodents (15), and heat production is mediated by upregulation of uncoupling protein-1 (UCP1; reviewed comprehensively in Ref. 6). For this study, our objectives were to test the hypotheses that 1) chronic REM-SD of rats leads to progressively elevated metabolic rate with a time course corresponding to increased gene expression of UCP1 in BAT and 2) leptin, which acts centrally as a satiety signal to blunt appetite, would be depressed, permitting the development of hyperphagia.
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MATERIALS AND METHODS |
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The REM-SD paradigm.
Two Plexiglas REM-SD tanks were each divided into five compartments of 30 x 30 x 40 cm. Compartments had a 10-cm-high column onto which platforms of different diameters can be interchangeably attached. Ten-centimeter platforms were used for REM-SD and 15-cm ones for tank controls; rats in home cages were additional controls. Smaller platforms (67 cm) are typically used for most REM-SD studies of 96 h duration (1, 5, 7, 30, 31, 4043, 49), but our observation has been that rats become completely exhausted beyond 96 h. To minimize confinement as a restraint-like stressor, 10-cm platforms were used to provide more space and mobility.
Inlet and outlet ports on opposite ends allow continuous water flow to carry away waste and debris and flood each chamber to 1 cm below the platform surface. Rats increasingly experience water immersions after 2 wk, so water temperature was adjusted to
30°C to lessen cooling effects. Food and water are easily accessible; rats can groom and rest by lying down. When rats on 10-cm platforms lapse into REM sleep, they lose muscle tone, make facial contact with or fall into the surrounding water, abruptly awaken, and the cycle is repeated. Thus the platform method is selective for abolishing REM sleep. The 15-cm platform is large enough so that, within a day or two, rats learn to position themselves to sleep with minimal or no water immersions. Tank control rats usually remain dry even after 20 days; however, there can still be loss of REM sleep (27, 28), although adaptation may eventually occur (28).
Experimental design.
For 2 wk, rats were accustomed to routine handling and the novel environment of the REM-SD tanks by placement onto the platforms for 1 h each day. Body weights were obtained, and 24-h food consumption was estimated by weighing leftover chow. These data were gathered daily between 0800 and 0900 during baseline, 20 days of experiments, and through recovery. Food intake was normalized as grams per day per kilogram of body weight taken to the 0.67 power to compensate for differences in metabolic rate as a function of body mass (22). No corrections were made for scattered crumbs because they were washed out. For metabolism experiments, the numbers of rats (n = 16) and available platforms (n = 10) necessitated a staggered schedule. First, five rats were placed on 10-cm platforms; one day later, another five rats were put on 15-cm platforms. Beginning with the 4th day, respirometry was measured every 48 h. Respirometry for tank controls after day 10 included only days 16 and 20 because no change was apparent. An additional six rats were then REM-sleep deprived for respirometry. All rats were returned to their home cages after day 20 for recovery. The recovery period was unremarkable, and there were no mortalities during the experiments.
Measurement of metabolic rate.
Metabolic rate was determined by indirect calorimetry as O2 consumption (O2) and CO2 production (
CO2) using the open-circuit Eco-Oxymax system (Columbus Instruments) running OxymaxWin software, version 2.42. After morning data collection, a rat was transferred to the respirometry chamber, a Plexiglas box virtually identical to a REM-SD tank compartment. Water surrounded the platform but without a flow-through system. Sample air was dehydrated using two in-series columns of Aquasorb [Mg(ClO4)2]; water evaporation was minimized by floating plastic balls (19 mm diameter) that completely covered the water surface. Before use in our experiments, the chamber was validated for respirometry of rats by Columbus Instruments.
Before each day's respirometry runs, the instrument was calibrated with a certified standard gas mixture of 20.40% O2-0.492% CO2. Data were collected every 60 s; respiratory quotient (RQ) was calculated as the molar ratio of O2 to
CO2. Three or four measurements of resting metabolic rate of each rat were recorded over several days as baseline data before initiation of experiments. Resting metabolism was defined as the periods when a rat was alert but resting quietly on the platform. Immediately following respirometry, rats were returned to the REM-SD tanks. System performance was checked regularly as recommended by the manufacturer and by fasting two rats for 48 h for RQ determination.
The units for respirometry were milliliters of O2 or CO2 per hour per kilogram body weight taken to the 0.67 power. The allometric equation for resting metabolic rate is RMR = aMb, where a is the scaling constant (intercept), M is body mass, and b is the scaling exponent. For b, 0.75 is typically used across broad taxa from unicellular organisms to plants and animals (24). The quantity of the exponent remains a topic of debate (46), but cogent arguments were recently made in favor of geometric scaling with an exponent of 0.67 for mammals (47), which we employ herein.
Semiquantitative RT-PCR of BAT UCP1 mRNA. A separate cohort of rats (n = 15) was REM-sleep deprived as described above, and after 5, 10, and 20 days, squads of five rats were killed by CO2 inhalation; controls were rats in home cages (n = 5). Excised interscapular BAT pads were freeze-clamped and stored at 75°C. Total RNA was extracted with Tri Reagent (Molecular Research Center), and sample integrity was confirmed by visual inspection of ethidium bromide-stained rRNA after denaturing (formaldehyde) gel electrophoresis. Two micrograms of input RNA was reverse transcribed (Superscript First Strand Synthesis System, Invitrogen Life Technologies) and then PCR amplified with the primers illustrated in Table 1 (Integrated DNA Technologies).
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Western blotting of BAT UCP1 protein.
BAT was homogenized [25 mM Tris·HCl, 1 mM EDTA, 1% Triton X-100, 0.5% Na-deoxycholate, pH 7.4, 50 µl/g protease inhibitor cocktail (Sigma, P8340), 1 mM PMSF], sonicated (5 W, 5 s), and centrifuged (1,000 g, 10 min). Lipid-free supernatant was collected and stored at 75°C. Protein content was determined in triplicate with the bicinchoninic acid assay (Pierce). BAT protein (20 µg) was electrophoresed in SDS-10% polyacrylamide minigels and electroblotted onto nitrocellulose (NitroBind, Osmonics). In addition, BAT and liver protein (20 µg) from cold-acclimated rats (9 days at 5°C) were run as positive and negative controls, respectively. Blocked (5% nonfat dry milk) nitrocellulose was probed with rabbit anti-UCP1 antiserum (Calbiochem, cat. no. 662045; no cross-reactivity to UCP2 or UCP3) at 1:1,000 dilution (2.1 µg IgG/ml). Secondary antibody was horseradish peroxidase-conjugated mouse anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, cat. no. 211035-109) at 1:20,000 (20 ng/ml). All incubations were for 1 h at room temperature; immune complexes were detected by chemiluminescent (SuperSignal West Pico, Pierce) exposure to RX-B blue X-ray film (Daigger). Images were digitally acquired and analyzed as with RT-PCR described above.
Leptin RIA. Trunk blood was collected by cardiac puncture from rats killed by CO2 inhalation (as part of the BAT UCP1 study), and serum was harvested. Also included were archived serum samples of control and REM-sleep-deprived rats (n = 2 per time point) from a pilot study. All samples (total n = 7 per time point) were assayed in duplicate in a single run using the Linco rat leptin RIA kit; coefficient of variation between duplicates averaged 4.7%.
Data analyses. All data (means ± SE) are presented as percentages of baseline or cage controls, where those quantities were assigned a value of 100%. Parametric one-way ANOVA was used with Dunnett's posttest, but if standard deviations were significant (Bartlett test), nonparametric Kurskal-Wallis (KW) testing was done with Dunn's posttest. Analyses were conducted using GraphPad InStat, version 3.06, and GraphPad Prism, version 4.2 (GraphPad Software).
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RESULTS |
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REM-SD increases metabolic rate.
O2 was measured to determine the effects of chronic REM-SD on resting metabolic rate. Figure 2 (n = 11;
) shows that it steadily increased, peaking at 166% of baseline by day 20 (1,137 ± 15 to 1,884 ± 89 ml O2/h per kg0.67; KW = 95.38, P < 0.0001). When rats were in recovery, it quickly fell to baseline levels within 48 h. Metabolism of tank control rats (n = 5;
) showed no changes compared with baseline. The relation between increased metabolic rate and decreased body weight of REM-sleep-deprived rats is presented in Fig. 3, showing a strong correlation (Pearson r = 0.949, P < 0.0001).
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Gene expression of UCP1 in BAT is upregulated during REM-SD.
With a major goal being to determine the effects of REM-SD on UCP1 gene expression in BAT, we first performed RT-PCR. Abundance of mRNA is shown in Fig. 4A of pooled UCP1 and 36B4 amplicons (n = 5 per time point). Cage control rats constitutively had low amounts of UCP1 mRNA, but it tripled by day 5. After 10 and 20 days, UCP1 mRNA rose more than 7- and 11-fold, respectively [F(3,16) = 316.98, P < 0.0001]. Levels of 36B4 mRNA remained consistent; however, mRNA for GAPDH declined modestly, and for -actin it increased between days 10 and 20 (data not shown). To eliminate any question of the increases in UCP1 mRNA being the result of a nonspecific response, the average ratios of UCP1 to each reference gene were taken, and this normalization was used as a more stringent evaluation of the index of change. Averaged data (n = 5 per time point) are summarized in Fig. 4B for the UCP1-to-36B4 ratio. The principal finding is that normalized UCP1 mRNA levels markedly increased with time of REM-SD (F3,16 = 137.07, P < 0.0001). Highly significant changes were also found when UCP1 was normalized to GAPDH and
-actin (data not shown). Fig. 4B, inset, shows the relation between resting metabolic rate and normalized UCP1 mRNA. The obvious feature of this correlation is that as resting metabolic rate increased there was a concurrent increase in UCP1 mRNA abundance. Next, to confirm that changes in mRNA are reflected in protein levels, we performed Western blot analysis. As Fig. 5 illustrates, rats had very high levels of the 32-kDa UCP1 protein in BAT from day 5 up to day 20. To highlight the specificity of the response (and that of the antibody) additional controls were included. That is, because immunoreactive UCP1 is strongly detected in BAT following cold adaptation (see DISCUSSION), BAT protein from rats acclimated to 4°C for 9 days was run as a positive control; also, because UCP1 expression occurs exclusively in BAT, liver protein served as a negative control. The results were unequivocal: there was abundant immunoreactive UCP1 in BAT of cold-acclimated rats, but none was found in liver.
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DISCUSSION |
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An issue arises in that the platform method activates the hypothalamic-pituitary-adrenal (HPA) axis (1, 30, 4044, 49), with nonspecific stress as a potentially confounding factor. Interestingly, despite many sleep deprivation pathologies being severe, an equivalent HPA response does not occur with the DOW method (34). A confounding stress factor cannot be ignored, but it is intriguing that, HPA stimulation or not, the methods have similar outcomes: rats are hyperphagic and lose body weight (5, 30, 31, 34, 40, 44, 49), they have increased energy expenditure (3, 9, 26) or elevated resting O2 (this study), and they are hypothyroxemic (3, 13, 31) with low leptin (10, 21; this study).
Our results are consistent with current understanding of thermogenesis. UCP1 is a unique 32-kDa inner mitochondrial membrane protein that allows proton leakage and short-circuits cellular respiration. As a consequence, the thermodynamic energy of the proton-motive force is dissipated as heat rather than being conserved by ATP formation (6). Accordingly, conditions that mandate augmented thermogenesis will increase UCP1 gene expression in BAT, as we demonstrate here for REM-SD. In control rats, constitutive levels of UCP1 mRNA were low, which is expected for normothermic, nonstressed rodents (6). As time of REM-SD lengthened, however, mRNA abundance rose with corresponding increases in metabolic rate, but an unexpected outcome was the disproportionately enormous change in UCP1 protein compared with the approximately threefold rise in its mRNA at day 5. Early on, even as gene transcription increases, synthesis of the protein occurs much more briskly, and these events probably cause metabolism and thermogenesis to rapidly ramp upward.
Other stress modalities can affect UCP1 expression, and two pertinent examples are given. When small mammals are challenged by cold exposure, UCP1 is vigorously induced (6). Recalling that REM-SD enforcement is by water contact, acute cold stress may have occurred despite warm water flowing through the tanks. This scenario is unlikely, however, because by day 10, when both metabolic rate and UCP1 gene expression were already significantly elevated, rats remained dry. In fact, they did not experience frequent immersions until the last quarter of the experiment. The second example is immobilization, a potent psychological stressor. When applied acutely, UCP1 mRNA does not change (36) but repeated and chronic episodes improve cold tolerance (25) with increased UCP1 mRNA and protein (18). It seems, then, that chronic REM-SD causes rats to respond as if they were adapting to cold but without a temperature effect being present. In addition, the similarities of REM-SD to these examples denote that it probably has both physical and psychological attributes as a stressor.
Hypermetabolism during REM-SD is not a process running out of control. If it were, O2 could not have declined so rapidly to baseline levels (<48 h) when rats were in recovery (also see Ref. 11). The implication is that metabolic regulation remains intact, and some of its mechanistic features may be gleaned by examining hormonal profiles. It is well established that increased norepinephrine (NE) and thyroid hormones, during cold adaptation, for example, are responsible for upregulating UCP1 (37; reviewed in Ref. 6). Chronically sleep-deprived rats present differently, however, in that circulating catecholamines are high (3, 11, 32) but they are profoundly hypothyroxemic (3, 13, 31). It is noteworthy that a compensatory increase in plasma epinephrine occurs when NE release is blocked by guanethidine (32), suggesting that maintaining high plasma catecholamines is vital. Perhaps more importantly, hypothyroxemia is offset by a substantial increase in activity of the type II deiodinase in BAT (2), and presumably, this adaptive response maintains appropriate levels of triiodothyronine to ensure persistent, elevated thermogenesis. Further understanding of the regulatory processes again comes from cold-adapted mammals, where changes in sympathetic activity promptly stimulate heat production during acute cold exposure or cause its inhibition with warmth (48). Hence, elevated plasma catecholamines found in sleep deprivation (3, 11, 32) resemble those of cold-adapted rats (6) and hint that the rapidly triggered metabolic "on" and "off" switch may be sympathetically regulated. Substantiating this view is that, following relief from sleep deprivation,
O2 (this study) and energy expenditure (11) expeditiously returned to baseline levels and that most anabolic hormones are suppressed (10).
We expected that RQ would trend toward 0.7, an indicator of fat oxidation, because body weight declines with depletion of fat depots (9, 26, 30) and there is rapid mobilization of tissue glycogen (24a). An operational problem with the calorimetry system was ruled out because fasted rats had values of 0.7. With average RQ being
0.89, rats were oxidizing a mixture of fuels, but mostly carbohydrate. To explain our results, a closer examination of the hyperphagic response may be instructive. Clearly, hyperphagia was insufficient to sustain increased metabolism, but, as Suchecki et al. (40) propose, it may be that REM-SD brings about a need to augment gluconeogenesis to satisfy growing demands for fuel. Their argument is appealing because, if fat were mobilized, not for oxidation, but rather for gluconeogenesis with attendant carbohydrate oxidation, RQ values would probably be much above 0.8. Another consideration is the caloric content of the chow fed to our rats. About 58% of its metabolizable energy is carbohydrate. Hence, increased carbohydrate ingestion, entirely as a function of hyperphagia, may account for the high RQ values.
As shown in the present study and elsewhere (10, 21), leptin levels declined within a few days of sleep deprivation. Suppression of leptin might be viewed as an appropriate response to obviate its anorectic effect, but the changes occurred before there were any significant alterations in food consumption, and even though leptin secretion is correlated to adiposity (16), the pace of fat depletion cannot account for the low levels found early in the regimen. Instead, it is more likely that increased sympathetic activity to white adipose tissue (19), which downregulates the leptin gene (33), allowed increased food intake.
Finally, it is important to note that the sleep deprivation effects discussed so far are not unique to rodent models. In studies of human sleep deprivation or sleep disorders (e.g., apnea, insomnia), the pathophysiological consequences have many similarities to those of rats. For instance, there is heightened sympathetic tone and altered carbohydrate metabolism (38); food intake increases, coincident with elevated ghrelin (an orexigen) and decreased leptin in the circulation (39); immune functions decline (8); and O2 is elevated (4). Sleep loss can also exacerbate morbidity in critically ill patients (17), contribute to health problems of persons of low socioeconomic status (45), and may even lead to premature mortality (29).
In summary and conclusion, our results show that 20 days of REM-SD brings about progressive increases in resting metabolic rate and hyperphagia while body weight is lost. We provide evidence that one of the mechanisms mediating hypermetabolism is a pronounced increase in thermogenic capacity, as illustrated by robust gene expression of UCP1 in BAT. Finally, we show that leptin decreases markedly, consistent with elevated sympathetic activity and the need to maintain hyperphagia. Apparently, the increased metabolic rate is a necessary (although curious) outcome of REM-SD that must be strongly defended, but its physiological advantage, if any, awaits clarification.
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GRANTS |
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ACKNOWLEDGMENTS |
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The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of National Institutes of Health.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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