J Appl Physiol 95: 2350-2354, 2003.
First published August 15, 2003; doi:10.1152/japplphysiol.00287.2003
8750-7587/03 $5.00
Effects of hypergravity on mammary metabolic function: gravity acts as a continuum
K. Plaut,1
R. L. Maple,1
C. E. Wade,2
L. A. Baer,2 and
A. E. Ronca2
1University of Vermont, Burlington, Vermont 05405; and 2National Aeronautics and Space Administration-Ames Research Center, Moffett Field, California 94035
Submitted 19 March 2003
; accepted in final form 6 August 2003
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ABSTRACT
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Mammary metabolic activity in pregnant rats is significantly increased in response to spaceflight. To determine whether changes in mammary metabolism are related to gravity load, we exposed pregnant rats to hypergravity and measured mammary metabolic activity. From days 11–20 of gestation (G), animals were centrifuged (20 rpm; 1.5, 1.75, or 2.0x gravity) or were maintained at 1 G. On G20, five rats from each group were removed from the centrifuge and euthanized. The remaining dams (n = 5/treatment) were housed at 1 G until parturition. After 2 h of nursing by the pups, the postpartum dams were euthanized (G22). Glucose oxidation to CO2 and incorporation into lipids was measured. Mammary glands from dams euthanized on G20 revealed a strong negative correlation between metabolic rate and increased G load. Approximately 98% of the variation in glucose oxidation and 94% of the variation in glucose incorporation into lipids can be accounted for by differences in G load. Differences in metabolic activity disappeared in the postpartum dams. When we combined previous data from the microgravity with hypergravity environments and plotted the ratio of mammary metabolic rate vs. G load, there was a significant exponential relationship (r2 = 0.99). These data demonstrate a remarkable continuum of response across the microgravity and hypergravity environments and support the concept that gravitational load influences mammary tissue metabolism.
gravity; mammary gland; rat
AS PART OF THE EMPHASIS BY the National Aeronautics and Space Administration (NASA) on reproduction and development, we conducted studies on mammalian pregnancy and fetal development in space. Our studies of 10 pregnant rats flown on two jointly sponsored spaceflight missions comprised the first detailed examination of the effects of microgravity on mammalian pregnancy, birth, lactation, and early development (11, 12). One major finding of these studies was that, after exposure to microgravity during the second half of pregnancy, followed by readaptation to a 1-G environment, dams gave birth normally (12). However, there were large differences in mammary gland metabolism during gestation in dams exposed to microgravity. Specifically, exposure to microgravity raised mammary tissue metabolism (11).
Successful reproduction in mammals requires a wide range of adaptations to occur in the mother during pregnancy and lactation. Changes in maternal metabolic, physiological, and physical demands during these periods are controlled by homeostatic and homeorhetic regulators (1). Homeostasis is the maintenance of physiological equilibrium, whereas homeorhesis is the coordinated control in metabolism of body tissues necessary to support a physiological state (1). Adaptive changes in mothers are regulated by neuroendocrine responses to pregnancy and lactation and not only stimulate mammary growth and development but also drive maternal behavior, feeding, and appetite to enable nourishment of the neonate (5). Changes in mammary metabolism indicate that maternal homeorhesis in pregnant dams is affected by the environment of space. These changes may indicate maternal adaptation to microgravity or a lack of maternal homeostasis that could be detrimental to dams as well as developing fetuses.
We hypothesized that gravity affects maternal homeorhetic regulators during pregnancy, resulting in changes in mammary metabolism. To begin to test this hypothesis, we examined the effect of hypergravity on dam mammary metabolic activity and body weight. We found that there was a significant relationship between gravitational load and mammary metabolic activity, as well as dam and fetal body mass.
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MATERIALS AND METHODS
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Animals and treatments. All procedures were reviewed and conducted under NASA Ames Research Center and The University of Vermont Institutional Animal Care and Use guidelines. Pregnant Sprague-Dawley rats were obtained from Taconic Laboratories (Germantown, NY). On day 11 of gestation (G), 30 pregnant rats (n = 10 per condition) were matched by mass and centrifuged at 20 rpm in a 24-ft. centrifuge (NASA Ames Research Center, Moffett Field, CA) to produce either 1.5, 1.75, or 2 G. Ten rats served as stationary controls and were maintained under identical environmental conditions as the centrifugation groups, except that they remained at 1 G for the duration of the experiment. On G20, the animals were removed from the centrifuge. Within 2 h, the animals were euthanized, and abdominal mammary glands were removed from five animals exposed to each hypergravity level, thereby limiting readaptation to 1 G. The remaining animals were placed in stationary cages and allowed to readapt at 1 G and gave birth 
48–72 h later (G22). After parturition, the pups nursed for 2–3 h, the dams were then euthanized, and the mammary tissue was removed for measurement of metabolic activity.
Measurement of mammary metabolic activity. Glucose oxidation to CO2 and incorporation into lipid (metabolic activity) was measured by using U-14C-labeled glucose, as described by Bauman et al. (2). Briefly, sections of mammary tissue, weighing between 140 and 180 mg, were placed in Ehrlenmeyer flasks containing Krebs Ringer bicarbonate buffer with 1 µg/ml insulin and [U-14C]labeled glucose (1 µCi/flask). Each flask contained a rubber stopper with a center well and a piece of filter paper. The flasks were placed in a shaking water bath for 3 h at 37°C. After the 3-h incubation, the tissues were injected with 0.5 M H2SO4 to stop metabolic activity, and CO2 was trapped as bicarbonate by adding 1 M hyamine hydroxide to the filter paper in the center well. The filter papers were removed and counted in aqueous scintillation cocktail (Bio-Safe II Counting Cocktail, Research Products International, Mt. Prospect, IL) in a scintillation counter (LS6500 Multipurpose Scintillation Counter, Beckman Instruments, Fullerton, CA) to calculate glucose oxidation to CO2. Mammary tissues were saponified in 5 M NaOH by heating at 90°C for 4 h, and then lipids were extracted with petroleum ether. Two milliliters of the ether extracts were added to an 8-ml scintillation cocktail for organics (Bio-Safe NA Counting Cocktail, Research Products International, Mt. Prospect, IL) and counted in the scintillation counter to calculate glucose incorporation into lipids.
Measurement of maternal and fetal body mass. Dams were weighed before the start of the study on G11 and again when they were removed from the centrifuge on G20. For dams that were euthanized at G20, each fetus was removed from the uterine horn and placenta and weighed, and the sex was determined. Dams that were readapted to 1 G were weighed daily from G20 to parturition. Pups were weighed and sexed just before euthanizing the dams.
Statistical analysis. Data were analyzed by using analysis of variance with G load as the main effect. Bonferroni t-tests for multiple comparisons were performed when the overall F statistic was significant (Statistical Analysis Systems Software, Cary, NC). Regression analyses, both linear and exponential, were used to determine the relationship between G load and mammary metabolic activity.
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RESULTS
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Mammary metabolic activity at G20 decreased significantly (P < 0.01) in response to increased gravitational load, as measured by glucose oxidation to CO2 and incorporation into lipid compared with controls. Glucose oxidation to CO2 decreased 13, 28, and 23% at 1.5, 1.75, and 2 G, respectively, in animals exposed to hypergravity compared with 1-G controls (P < 0.01). Glucose incorporation into lipid decreased 43, 58, and 63% at 1.5, 1.75, and 2 G, respectively, compared with 1-G controls (P < 0.01; Fig. 1). Linear regression of mammary metabolism vs. G load revealed a correlation coefficient (r2) of 0.98 for glucose oxidation to CO2 and 0.94 for glucose incorporation into lipid.
At G22, the absolute values of mammary metabolic activity increased in all treatments to compensate for lactation. The dams that were removed from the centrifuge at G20 and then readapted at1G48–72 h until parturition and nursing showed no significant differences in mammary metabolic activity relative to control animals (Fig. 2). Furthermore, when metabolic rate was regressed against G load for these animals, there was no relationship. At G20, but not G22 (parturition), mammary metabolic activity was significantly different between 1-G control dams and all hypergravity treatments. However, mammary metabolic activities among hypergravity-treated dams were not significantly different, perhaps due to the limited power of multiple pairwise comparisons.
There was no significant difference in dam body mass at G11 before centrifugation. At the time of removal from the centrifuge on G20, the 1-G stationary control animals weighed significantly more than the animals exposed to hypergravity (Fig. 3). Fetal mass was also significantly different at G20 (P < 0.01), with all fetuses exposed to hypergravity having a smaller mass than the stationary control fetuses (Fig. 4). Animals that were readapted at 1 G gained body mass after they were removed from the centrifuge, such that, by the time they gave birth, there was no significant difference in body mass between controls and animals exposed to hypergravity (Fig. 3). Neonatal body mass was not significantly different between treatments and was very variable. There was no significant difference in litter size (average litter size = 12.5) among any of the treatments.
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Fig. 3. Body mass (g) of pregnant rat dams on gestation days 11, 20, 21, and 22. The dams were matched by mass on gestation day 11 and centrifuged at 1, 1.5, 1.75, or 2 G until gestation day 20, at which time one-half of the dams were processed for mammary metabolic activity. The remaining dams were housed at 1 G until parturition and then processed for metabolic activity. Values are means ± 5 SE; n = 5 for each G load. P < 0.01.
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Fig. 4. Average fetal mass (g) on gestation day 20 after exposure to 1, 1.5, 1.75, or 2 G. The fetuses were removed from the dams immediately after the dams were removed from the centrifuge. Values are means ± 0.1 SE. Average size of litter = 12.5 pups for each G load. P < 0.01.
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DISCUSSION
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Mammary metabolic rate seems to be intricately tied to gravitational force. In this study, we observed a steady decline in mammary metabolic rate as G load increased. In our laboratory's previous study (11), in which we measured mammary metabolic rate in response to microgravity, glucose oxidation to CO2 and incorporation into lipid increased 43 and 300%, respectively, compared with 1-G controls at G20. When the data from both the microgravity and hypergravity studies were combined, we were able to regress mammary metabolic rate (normalized for differences at 1 G) against gravitational load over five gravitational loads using an exponential rate equation. There was a highly significant relationship between mammary metabolic rate and G load, indicating that the metabolic rate change is constant as gravity load is altered from 0 to 2 G (Fig. 5). An astounding 99% of the variation in metabolic activity for both CO2 and lipid at G20 could be accounted for by differences in the G load. This indicates that animals exposed to altered gravitational force exhibit clear, dramatic changes in mammary metabolism during late gestation in direct response to gravitational force.
Whereas hypergravity decreased the dam's body mass and fetal mass, microgravity did not alter the body mass of the dam. Unfortunately, fetal mass was not recorded in animals exposed to microgravity. The effect observed in hypergravity may be due to changes in energy balance. Kobayashi et al. (6) centrifuged rats at 4.13 G for >1 yr and observed that, even though the rats ate more, they had a lower body mass than animals exposed to the 1-G environment, indicating that there is an energetic cost due to hypergravity. The question remains whether these energetic costs affected mammary tissue metabolism. We do know that, during starvation, basal metabolic rate decreases (7). However, mammary metabolism increased in hypogravity and decreased in hypergravity environments, indicating that energy balance is not the cause of changes in metabolic rate; rather changes in metabolic rate result from changes in G load.
Animals showed marked readaptation to the 1-G environment, such that all changes in mammary metabolic rate, due to hyper- or microgravity, disappeared during the period when animals were kept at 1 G after being exposed to hypergravity. In addition, body mass of the dam recovered over the 48- to 72-h time period between removal of the animals from the centrifuge and delivery of their pups. Similar results were obtained by Wade et al. (15), who centrifuged growing male rats for 16 days at 2 G. He observed a marked decrease in body mass followed by compensatory gains to attain the same body mass as the control within 48 h after centrifugation ceased. In addition, whereas fetal mass, which was measured immediately after centrifugation, was significantly decreased by hypergravity, neonatal mass was not different from 1 G after a 24- to 48-h readaptation period. Therefore, it is likely that adaptation to changes in G force after short-term exposure to altered gravitational force can be rapidly ameliorated.
There is a strong relationship between other biological responses and gravity; however, few studies have been conducted across multiple G loads. Studies by Engelmann et al. (4) showed that the percentage of linearly motile spermatozoa increased under microgravity conditions compared with 1 G. Shellenberger et al. (14) examined growth hormone-secreting cells from the anterior pituitary of rats exposed to various G loads. In both spaceflight and hypergravity, the ability of the cells to release bioactive growth hormone was compromised. In contrast, prolactin (Prl)-secreting pituitary cells were not altered in response to either spaceflight or hypergravity. However, gravity-induced declines in serum Prl concentrations in vivo have been observed in lactating animals exposed to hypergravity (8, 9).
Megory and Oyama (8, 9) reported significant reductions in plasma Prl levels in animals exposed to hypergravity during the periparturient period. They observed that mothers that did not nurse their pups did not exhibit the increase in Prl normally associated with delivery. This is consistent with the studies by Shaar and Clemens (13) in which they demonstrated that inhibition of Prl secretion during pregnancy and lactation in the rat was associated with poor weight gain and reduced mammary tissue in rats. Another possibility is that Prl receptors in the mammary gland are altered. When Prl action was blocked by administering a Prl receptor antiserum to pregnant rats, the rats exhibited reduced pup growth, indicative of poor mammary metabolic activity (3). Furthermore, heterozygous mice carrying a germ-line null mutation of the Prl receptor showed almost complete failure of lactation, indicating that even a partial reduction in receptor number affects lactation (10).
Our findings demonstrate that gravity affects mammary metabolism and dam and fetal body weight during gestation. It is important to determine whether these are adaptive responses of the dam to a different environment or indicative of an attempt to maintain homeostasis at a time when pregnancy is adding demand to the dam. We hypothesize that these changes in mammary metabolic activity induced by changes in G load during pregnancy are driven by the hormone Prl. We are presently pursuing additional studies to determine whether there is a relationship between G load and Prl regulation of homeorhesis during pregnancy.
Conclusion. Mammary metabolic rate exhibited a clear continuum of response across five different G loads, from microgravity through hypergravity (0–2 G) environment. Maternal and fetal masses were also decreased by the hypergravity environment but did not exhibit the clear continuum of response observed in the mammary gland. After a period of readaptation to 1 G, all biological responses returned to normal. These studies lend support to the concept that gravity is an important environmental cue that has shaped biological responses. Further studies are necessary to determine the role of hormones in modulating this response.
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DISCLOSURES
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The work was supported by NASA Grants NCC2-870, NAS121-1040, and NCC2-8064.
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ACKNOWLEDGMENTS
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We acknowledge the help and support of National Aeronautics and Space Administration (NASA) Ames Research Center Acceleration Facility staff.
We express our appreciation to the late Dave Brown, NASA astronaut, STS-107 Columbia, who was instrumental in fostering the pursuit of biological research in space.
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FOOTNOTES
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Address for reprint requests and other correspondence: K. Plaut, Univ. of Vermont, 570 Main St., Burlington, VT 05405 (E-mail: kplaut{at}zoo.uvm.edu).
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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Copyright © 2003 by the American Physiological Society.
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ABSTRACT
MATERIALS AND METHODS
) and incorporation into lipid (
). Metabolic activity was regressed across G load on day 20 to develop a linear rate equation. Values are means ± 26 SE for CO2 and means ± 20 SE for lipid; n = 5 for each G load. P < 0.01.
