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The effect of maternal malnutrition during pregnancy in the rat on the offspring’s weight, glucose uptake, glucose transporter protein levels and behaviors
Nutrition Research 21 (2001) 755–769 www.elsevier.com/locate/nutres
The effect of maternal malnutrition during pregnancy in the rat on the offspring’s weight, glucose uptake, glucose transporter protein levels and behaviors Sam N. Penningtonb,*, J. Sue Penningtonc, Lisa D. Ellingtona, F. Melinda Carverb, Ivan A. Shibley Jrd, Neil Jeansonneb, Steven A. Lynchb, Lori A. Robersona, Debbie S. Milesb, Elaine P. Wormingtona, Larry W. Meansa a
Department of Psychology, College of Arts and Sciences, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA b Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA c Department of Comparative Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA d Department of Chemistry, Penn State Berks Campus, Box 7009 Reading, PA 19610, USA Received 17 January 2000; received in revised form; 30 July 2000; accepted 31 July 2000
Abstract Intrauterine growth retardation (IUGR) is associated with multiple molecular and physiological changes in the offspring. In the current study, offspring of dams acutely fasted on days 17–19 were compared to the offspring of non-food deprived dams with respect to postnatal growth, glucose uptake, brain glucose transporter Glut 1 protein levels and age-specific behaviors. Further, in a chronic study, control mothers were given ad lib. access to a nutritionally balanced liquid diet from day 0 until day 18 or day 20 of pregnancy. These offspring were compared to the offspring of mothers who consumed 90% of the ad lib mothers’ calories. Overall, the malnourished offspring had: 1) IUGR, 2) small changes in brain glucose uptake and in Glut 1 transport protein content and, 3) minor alterations in their behavior. In both models, pups whose dams were malnourished weighed less than control offspring at birth and the growth retardation was sustained through six months of age. In the acute model, behavioral comparisons revealed that rats from acutely malnourished litters were impaired only on incline descent, requiring more time to descend than did the non-malnourished control pups. Brain glucose uptake showed only modest differences. Western blotting of glucose transporter
* Corresponding author. Tel.: ⫹1-252-816-2076; fax: ⫹1-252-816-3260. E-mail address: [email protected] (S.N. Pennington). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 7 0 - 6
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proteins indicated that there was little correlation between brain glucose uptake and Glut 1 protein levels in pups from either malnourished or control litters. At 60 days, malnourished offspring were glucose intolerant relative to offspring from control litters. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Malnutrition; Glucose uptake; Glucose transporters; Brain; Behavior; Growth suppression
1. Introduction Poor maternal nutrition during gestation leads to intrauterine fetal growth retardation (IUGR) of the developing organism and is associated with a number of unfavorable outcomes for the offspring. Both human and animal studies have shown that intrauterine growth retardation is associated with behavioral, metabolic and physiological abnormalities in the progeny that may not fully appear until adult life [1– 4]. Furthermore, studies examining the effects of pre- and postnatal malnutrition on brain anatomy and physiology have shown that gestation is a highly vulnerable period [5,6]. Malnutrition throughout gestation in rats has many documented behavioral consequences in the offspring. For example, chronic protein/ calorie under nutrition throughout pregnancy increases exploratory activity [7,8], open arm entries in a plus maze [8,9], and produces deficits in the differential reinforcement of low rates of responding at 18 second (DRL 18r) acquisition [10], brightness discrimination [11,12] and reversal [12], water maze reversal [13] and repeated reversal learning [12]. In contrast, prenatal malnutrition has not been found to significantly impair inhibitory avoidance [8,11,12] or performance on working memory tasks [7,8,11,12,14]. In the current studies, behavioral comparisons were made of one male pup and one female pup from each acutely malnourished and control litter on righting reflex, elevation of shoulders, elevation of head, geotactic reaction, open field activity, wire mounting, narrow bridge crossing, and incline descent at ages 1 through 21 days, response in an elevated plus maze at ages 29 and 61 days, repeated spatial reversal at ages 54 –58 and 89 –93 days, and response to a novel alley at age 113 days. Retrospective studies in humans [15–17] as well as prospective studies in animal models [18] have shown that significant correlation also exists between IUGR and molecular alterations in glucose uptake/metabolism. Cell division and fetal growth require a complex series of biochemical events including a concurrent increase in energy metabolism. One of the earliest consequences of entry into the cell cycle is an increase in the uptake of glucose. The focal role of glucose uptake in growth and metabolism has led to a substantial literature describing the glucose transporter (Glut) proteins [19]. Glut 1 is expressed in most tissues, in most species and is the major transporter in brain and in the developing fetus [20 –24]. In the brain, the Glut 1 55 kilodalton (kD) isoform is found at the blood brain barrier while the 45kD isoform is associated primarily with neurons and glial cells [19]. In the present studies, brain tissue glucose uptake was determined and brain Glut 1 protein levels (45kD and 55kD isoforms) were assayed in the total membrane fraction of brains isolated from young and adult rat offspring from control and acutely malnourished pregnancies.
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2. Materials and methods 2.1. Animal model (acute). Twelve timed-pregnant rats (Harlan Sprague-Dawley, primigravida) were purchased from Harlan (Indianapolis, IN). The animals were individually housed and on day 17 of their pregnancy, randomly divided into two groups. Group one (controls) continued to have access to chow ad lib. while group two (IUGR) continued to have access to water but was fasted for 72 hours (from 08:00 hours of day 17 through 08:00 hours of day 20). On the morning of day 20, the starved animals were returned to ad lib. chow feeding and all pregnancies (6 controls and 6 IUGR animals) were allowed to go to term. The offspring from these litters were behaviorally tested as described below. In addition, one male and one female offspring from each of these litters were killed at 2, 7, 28, 42 and 112 days and their brains assayed for glucose uptake and glucose transporter protein content, also described below. 2.2. Animal model (chronic). Virgin Sprague Dawley female rats (200 –225 gm, Harlan) were given access to rat chow and a nutritionally balanced liquid diet (BioServe) for one week prior to mating. The diet is designed for pregnant mothers and contains maltose dextrin. Each female was placed with a fertile male during the dark portion of the light cycle. At the start of pregnancy (vaginal plug ⫽ day 0), each female was randomly assigned to one of two dietary groups. Group 1 (Controls) were given free access only to the maltose dextrincontaining liquid diet through out their pregnancy. Group 2 (Experimental) received a reduced amount of the liquid diet (based on kcal/kg) calculated to provide 90% of the calories consumed by the paired Control female on the previous day. On day 20 of their pregnancies, all mothers was returned to lab chow until the birth of their pups. Within 48 hours of parturition, the neonates were culled to 4 males and 4 females per litter (whenever possible) and each litter of pups was surrogate fostered to a nursing female who had received free access to only lab chow through out her pregnancy. All pups were weaned at 21 days of age and received free access to lab chow. 2.3. Maturation: behavioral milestones. During the first 21 postnatal days, one male and one female pup from each litter were tested for developmental level using several behaviors (see Table 1). Pups were tested individually for righting on a surface (time to turn from back on to all four paws), geotaxic reaction (time to turn head 1800 to upward orientation when placed facing down on an incline), elevation of shoulders (duration that shoulders were raised above the supporting surface), elevation of head (duration that the head was held above the supporting surface), hind limb support (ability to grasp a 2-mm diameter wire with one or both hind paws after being suspended from the wire by front paws), traversing a 1.2-mm wide 60-cm long wooden platform, descending a 450 wire mesh surface, and number of squares entered in an open field. The pups were also observed for the first appearance of walking in the open field and examined for day of eye opening. For all behavioral measurements, the operator was blinded as to the animals in utero nutritional treatment. 2.4. Elevated plus maze. One male and one female pup from each control and experimental litter were tested at both 29 and 61 days of age. The maze had two open arms, located
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Table 1 Behavioral tests given to acutely malnourished and control pups during first 21 postnatal days Behavior1
Normal age2
Ages of testing (days)
Dependent variables
Righting on a surface Geotaxic reaction Elevation of shoulders
13 2⫹3 64
Latency Latency Duration, % of time
Elevation of head Open field activity, walking
114 12–135
Eye opening Hind limb support Traversing 1.2 cm path Descending on wire mesh
14–155 153 173 183
1–9 2, 4, 6, 8, 10 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 5, 7, 9, 11, 13 7, 9, 11, 13, 15, 17, 19, 21 12, 13, 14, 15 10, 12, 14, 16, 18, 20 14, 16, 18, 20 15, 17, 19, 21
Duration, % of time No. squares entered crawling vs. walking Open? Success? Distance, no. falls, latency Latency
1
All tests had a maximum duration of 3 minutes. Normal ages are based on Altman and Sudarshan (Altman and Sudarshan, 1975). 3 Age at which 50% of normal subjects perform. 4 Age at the which 50% of subjects obtain asymptotic level. 5 Age first observed in nearly all animals. 2
opposite to one another, and two arms enclosed by vertical walls. The maze was placed in a room having a low level of illumination. Rats were individually placed on the maze in the center position and allowed to explore for 5 minutes. Total arm entries, proportion of entries to open arms, and proportion of time spent in open arms was determined for each subject. 2.5. Repeated spatial reversal. One male and one female rat from each experimental and control litter were tested on repeated spatial reversal, a task requiring behavioral flexibility, for five days beginning at ages 54 and 89 days. Each rat, 12 hours food deprived, was placed in the startbox of an elevated open T-maze that was positioned in a normally lighted room. For the first trial, food (sugar coated cereal) was placed in each goalbox. Whichever arm the animal selected then became the reinforced arm. The rat was reinforced in that arm until it attained the criteria of entering that arm on four consecutive trials. Upon attaining criterion, the arm containing reinforcement was switched. The rat was then reinforced for entering the new arm until it again attained criterion. At that time, the arms was again switched. Each rat was given 10 trials per day for 5 days. Number of correct choices and criteria attained was recorded. 2.6. Maze activity and response to novelty. The same pups tested in the elevated plus maze and for repeated spatial reversal were tested for maze activity and response to a novel alley at age 112 days. The test involved two massed trials in an enclosed Y-maze. During the first trial entrance to one arm was blocked by a barrier. On each trial, the rats were placed individually into the starting arm and permitted to explore for 5 minutes. Number of arms entered on each trial and first arm entered on the second trial were recorded. Also, for the second trial the proportion of entries in to the novel arm and proportion of time spent in the novel arm were determined.
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2.7. Tissue isolation. At various time points (2, 7, 28, 42 and 112 days), individual pups from each acutely treatment group were killed (using chilling and decapitation or CO2 asphyxiation) and the brains isolated and freed from associated membranes. The brains were weighed to the nearest milligram and divided along the medial sagittal fissure. The right hemispheres were taken for glucose uptake studies and the left hemispheres were quick frozen (liquid nitrogen) for transporter protein determination. Because of the relatively small brain size, three brains from two day old animals were pooled to provide sufficient tissue for analysis. 2.8. Glucose transporter protein assay. Glucose transporter proteins (Glut 1, 45kD and 55kD isoforms) were assayed as described by Postic, et al. [24] via semi-quantitative Western blotting using a primary polyclonal antibody against Glut 1 that was raised in New Zealand white rabbits by Dr. G. Lynis Dohm (Department of Biochemistry, East Carolina University). Transporter protein levels from brain tissue preparations of individual animals were separated by SDS-PAGE (10% gel, two hours at 30 milliamps/gel, 4 – 6° C). Following Western blotting to Immobilon (one hour at 400 milliamps/blot, 4 – 6° C) using Towbin’s buffer solution, the transporter proteins were compared using a horseradish peroxidaselabeled second antibody with quantitation by Imagequant software following scanning of the resulting films. Both the 45kD and 55kD isoforms of Glut 1 from each brain were determined on the same blot. To allow comparisons between blots, a standard adult rat brain tissue preparation was run on each gel/blot. 2.9. Brain 2-deoxyglucose uptake. 2-deoxyglucose uptake by brain tissue was measured as previously described [25]. 2.10. IV glucose tolerance tests. In order to compare the previously reported [26] effects of acute malnutrition to the effects of chronic in utero malnutrition on the offspring’s subsequent response to a glucose challenge, pups from chronically malnourished pregnant animals were examined at 60 days of age to determine their fasting plasma insulin and glucose levels and for their response to an intravenous glucose challenge. For the I.V. glucose challenge, the pups were fasted for six hour with water available, ad lib. and a “0” time blood sample (500 microliters) was taken from an indwelling carotid catheter placed 48 hours previous to the glucose challenge. An I.V. dose of glucose was administered (50% glucose in sterile water) to give a peak blood glucose level of 9 –10 mM). Blood samples (500 microliters) were taken from the carotid catheter at 1, 3, 5, 10, 30 and 60 minutes after the IV dose of glucose. This procedure and all others procedures involving animals were approved by the East Carolina University Institutional Animal Care and Use Committee. 2.11. Statistical analyses. For all behavioral observations and biochemical assays, group means were tested for significant differences using the appropriate analysis of variance (ANOVA) and Least Mean Square (LMS) post-hoc tests using SAS for Windows. For all tests, p⬍0.05 was used to conclude a statistically significance difference was present.
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Fig. 1. The effect of chronic (A) or acute (B) maternal malnutrition on maternal weight gain during pregnancy. In both frames the “n” for each time point is ⱖ 6. The data points marked by an “*” are significantly different (p ⬍ 0.05) from the ad lib data point on the same day of pregnancy. Note that following parturition, the acutely malnourished mothers were still significantly lighter than were the ad lib controls, indicating that the acutely malnourished mothers shared in the weight loss along with their fetuses. For this figure and all others data points represent group means ⫾ S.E.M.
3. Results Maternal Weight. Chronic malnutrition or seventy-two hours of acute food deprivation on days 17 through 19 of pregnancy resulted in a significant inhibition of maternal weight gain (Figure 1). As previously reported [26], the loss of mass during acute starvation was shared by both mothers and fetuses because, following parturition, the weights of the malnourished mothers were significantly less than the weights of the chow-fed control mothers (Figure 1B) and the fetal/neonatal weights of the malnourished pups were also significantly lower (Figure 2A). Conversely, chronic maternal malnutrition resulted in only the offspring being small compared to controls as the control and malnourished maternal weights were identical following parturition (Figure 1A). Fetal and Neonatal Body Weights. Weight differences between control and malnourished pups were similar for both the acute and chronic models, although the chronically malnourished pups may have faired somewhat better in terms of overall growth following parturition
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Fig. 2. The effect of acute (A) and (C) maternal malnourish and chronic maternal malnourish (B) on pup growth during the neonatal period (A) and (B) and during adulthood (C). In all frames the “n” for each time point is ⱖ 3. The data points marked by an “*” are significantly different (p ⬍ 0.05) from the data point for ad lib pups on the same day.
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Fig. 3. Effect of acute (A and B) and chronic (C) maternal malnutrition on brain growth in the offspring. In each panel, “*” indicates a value that is significantly different form the ad lib value (p⬍0.05).
(compare Figure 2A and 2B) The weight differences between control and malnourished pups were maintained throughout the time period that the offspring were studied (Figure 2C). Fetal and Neonatal Organ Weights. The brain weights of pups from acutely food-deprived and control litters were essentially identical (Figure 3A and 3B), suggesting that the brain, at least in terms of weight, was spared in the acute model. In the chronic model of malnutrition, offspring brain weights were lower than controls but the heart and liver of the chronically malnourished animals were not significantly different (Figure 3C).
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Fig. 4. Brain glucose uptake by female (A) and male (B) pups from ad lib and acutely malnourished litters. The data points marked by an “*” are significantly different (p⬍0.05). The values for males at 2 days were identical while the female values were 20 –25% higher in the malnourished females. The difference in uptake between males on day 7 is significant but not robust.
Brain Glucose Uptake. In female pups from litters acutely malnourished, glucose uptake by brain showed a small but statistically significant change on day 2 and small changes on days 42 and 112 compared to pups from control litters (Figure 4A). Male pups from acutely malnourished litters showed only a small decrease in transport at 7 days of age (Figure 4B) but their uptake was identical to that of the control animals at all other ages examined. These small changes were judged not to be of sufficient magnitude to alter overall glucose uptake. Chronically malnourished pups had a significant increase in basal glucose uptake compared to controls but the significance of this difference was lost in the presence of exogenous insulin (Figure 5). Response to a Glucose Challenge. As previously reported for the acute model [26], chronic in utero malnutrition resulted in the offspring being hyperglycemic in response to an
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Fig. 5. Effect of malnutrition on brain basal and insulin-stimulated glucose uptake by 18 day fetuses. The increase in basal uptake by the brains of malnourished fetuses was significant (p ⬍ 0.05). Notable variance in the insulin-stimulated data resulted in the differences not being significant.
intravenous glucose challenge (Figure 6). At 60 days of age, both male and female malnourished progeny were hyperglycemic in response to a glucose challenge but the female animals were more notably altered (Figures 6A and 6B). Brain Glucose Transporters. Maternal malnutrition resulted in small, but statistically significant, changes in brain Glut1 glucose transporter protein levels (55kD isoform). The small increases in the 55kD isoforms at two days of age in both sexes were paralleled by a modest but significant increased in glucose uptake in females at this age but there was no difference in male glucose uptake. Further, the inversion between control and malnourished 55kD isoform levels seen at day 42 in the female was not paralleled by a change in the uptake of glucose at this time point (compare Figure 4B and Figure 7D) 2.12. Behavioral milestones. The only developmental milestone on which the malnourished and control pups were found to differ significantly was latency to descend the wire mesh platform (Figure 8). The malnourished rats took longer to descend than did the control animals (155.1⫾5.4 s vs. 126.2⫾9.8 seconds, p⬍0.05). 2.13. Maze behaviors. No significant differences between pups from acutely malnourished or control litters were observed on any of the elevated plus maze measures, T-maze repeated reversal, or Y-maze activity or response to a novel alley. However, regardless of fetal diet, females were found to be more active in mazes than were males. Females entered more arms than did males in the plus maze when tested at 61 days of age (29.9⫾ vs. 19.6⫾), and entered more arms in the Y-maze on both the first (16.2⫾ vs. 12.2⫾) and second (18.9⫾ vs. 13.0⫾) day of testing at ages 112–113 days. However, the males and females did not differ on open field activity when tested at ages 7, 9, 11, 13, 15, 17, 19 and 21 days or on total arms entered in the plus maze at 30 days of age.
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Fig. 6. Response to an IV glucose challenge by 60 day old female (A) and male (B) offspring from litters fed ad lib and chronically malnourished. In all panels, data point for which the error bars do not overlap are significantly different (p⬍0.05).
4. Discussion In the current study, individual animals from control and acutely malnourished litters were compared on several of the developmental milestones established by Altman and Sudarshan [27]. In addition, the animals were compared on measures of emotionality and cognitive behaviors. These developmental milestone behaviors provided an excellent method to detect delayed maturation. Since rats reach each of the behavioral milestones on different days, testing of several behaviors, over time, permits the detection of not only developmental delay, but also any developmental recovery that may occur as the animals mature. Behavior on the elevated plus maze has been shown to be sensitive to chronic fetal malnutrition and is considered an excellent measure of exploration and anxiety [28]. The results reveal that
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Fig. 7. Brain Glut 1 45 kD isoform (A&C) and Glut 1 55 kD isoform (B&D) at various ages. Those data points marked by an “*” are significantly different from the comparable ad lib fed pup values. Error bars have been omitted for clarity at certain data points.
only coordination and balance as evidenced by the slower descent of the ramp were significantly impaired by the fetal malnutrition. The offspring of acutely or chronically malnourished dams weighed less than control offspring at birth and the difference in body weights was maintained throughout the study
Fig. 8. Time to descend a wire mesh ramp elevated at a 45° angle. The malnourished and control pups were found to differ significantly in latency to descend the wire mesh platform. The malnourished rats took 155.1 ⫾ 5.4 seconds versus 126.2 ⫾ 9.8 seconds for the offspring from control litters (p⬍0.05)
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period. However, the brain weight of the acutely malnourished offspring was lower only at one time point. This outcome suggests that mechanisms exist to protect brain growth from short-term malnutrition. Consistent with the rapid brain weight recovery, the acutely malnourished offspring were impaired on only a single developmental behavioral paradigm. In comparison, the brain weights of pups from chronically malnourished litters were significantly less than control pup brain weights. Previous studies [7,8,9,11,13,14] have demonstrated the behavioral deficits resulting from chronic protein/calorie deprivation throughout gestation. These data suggest that whereas three days of maternal fasting has a profound effect on maternal weight and has a sustained effect on offspring body weight, brain development and behavioral maturation are spared unless malnutrition is prolonged. As has also been observed in earlier research [29], female offspring were more active than males regardless of the dietary treatment of the mothers. At the molecular level, the small changes in female brain glucose uptake seen on days 2 and 42 were paralleled by small but significant changes in the Glut1 55kD transporter. Because the 55kD isoform is associated with the microvasculature [23] and is involved in transport out of the microvasculature, the data suggest that the 55kD isoform may be the rate limiting factor. This relationship is speculative given the limited data available, but fits with a previous report of such changes in 55kD mediated-transport in a growth suppressed embryonic chick brain model [30,31]. Overall, Glut1 transporter protein isoforms levels and glucose uptake by the brains of both male and female acutely malnourished pups show minimal differences relative to the control brains; again suggesting that the malnourished brains were “protected” during the fasting period. Furthermore, the slight differences in brain transporter proteins isoform levels were paralleled by minimal behavioral differences between these two groups. Thus, the probability of observing maternal malnutrition-induced biochemical and behavioral changes in the progeny appears to be a function not only of the intensity of the of malnutrition but also of the duration of the treatment. Although not studied here, the timing of the maternal malnourished period relative to the developmental stage of the fetus/embryo may also play a significant role in determining the growth and behavioral outcome for the offspring.
5. Conclusions 1. Acute maternal malnutrition during late pregnancy (via a 72 hour maternal fast) resulted in a significant weight deficiency in both the mothers and their offspring. 2. Chronic malnutrition (90% of control calories from day 1 to day 20 of the pregnancy) resulted in a fetal/neonatal weight deficit that was not shared by the mothers 3. Acute maternal malnutrition had minimal effects on the offspring’s brain weight, Glut 1 transporter isoform levels and glucose uptake was increased only circa parturition 4. Acute maternal malnutrition had little affect on the offspring’s maturation rates or learning behaviors 5. In parallel with other reports, chronic maternal malnutrition inhibited fetal brain growth and stimulated fetal glucose uptake 6. Acute maternal malnutrition produced minimal biochemical and behavioral changes in
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the progeny as compared with prolonged malnutrition. Thus, the reliability of observing fetal malnutrition-induced biochemical and maturation changes appeared to be a function of the duration as well as of the intensity of the of maternal malnutrition.
Acknowledgments This work was supported in part by grants from the University of North Carolina Nutrition Institute to LWM and SNP and from the Children’s Miracle Network and Grant # AA10681 from NIAAA to SNP.
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The effect of maternal malnutrition during pregnancy in the rat on the offspring’s weight, glucose uptake, glucose transporter protein levels and behaviors Sam N. Penningtonb,*, J. Sue Penningtonc, Lisa D. Ellingtona, F. Melinda Carverb, Ivan A. Shibley Jrd, Neil Jeansonneb, Steven A. Lynchb, Lori A. Robersona, Debbie S. Milesb, Elaine P. Wormingtona, Larry W. Meansa a
Department of Psychology, College of Arts and Sciences, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA b Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA c Department of Comparative Medicine, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA d Department of Chemistry, Penn State Berks Campus, Box 7009 Reading, PA 19610, USA Received 17 January 2000; received in revised form; 30 July 2000; accepted 31 July 2000
Abstract Intrauterine growth retardation (IUGR) is associated with multiple molecular and physiological changes in the offspring. In the current study, offspring of dams acutely fasted on days 17–19 were compared to the offspring of non-food deprived dams with respect to postnatal growth, glucose uptake, brain glucose transporter Glut 1 protein levels and age-specific behaviors. Further, in a chronic study, control mothers were given ad lib. access to a nutritionally balanced liquid diet from day 0 until day 18 or day 20 of pregnancy. These offspring were compared to the offspring of mothers who consumed 90% of the ad lib mothers’ calories. Overall, the malnourished offspring had: 1) IUGR, 2) small changes in brain glucose uptake and in Glut 1 transport protein content and, 3) minor alterations in their behavior. In both models, pups whose dams were malnourished weighed less than control offspring at birth and the growth retardation was sustained through six months of age. In the acute model, behavioral comparisons revealed that rats from acutely malnourished litters were impaired only on incline descent, requiring more time to descend than did the non-malnourished control pups. Brain glucose uptake showed only modest differences. Western blotting of glucose transporter
* Corresponding author. Tel.: ⫹1-252-816-2076; fax: ⫹1-252-816-3260. E-mail address: [email protected] (S.N. Pennington). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 7 0 - 6
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proteins indicated that there was little correlation between brain glucose uptake and Glut 1 protein levels in pups from either malnourished or control litters. At 60 days, malnourished offspring were glucose intolerant relative to offspring from control litters. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Malnutrition; Glucose uptake; Glucose transporters; Brain; Behavior; Growth suppression
1. Introduction Poor maternal nutrition during gestation leads to intrauterine fetal growth retardation (IUGR) of the developing organism and is associated with a number of unfavorable outcomes for the offspring. Both human and animal studies have shown that intrauterine growth retardation is associated with behavioral, metabolic and physiological abnormalities in the progeny that may not fully appear until adult life [1– 4]. Furthermore, studies examining the effects of pre- and postnatal malnutrition on brain anatomy and physiology have shown that gestation is a highly vulnerable period [5,6]. Malnutrition throughout gestation in rats has many documented behavioral consequences in the offspring. For example, chronic protein/ calorie under nutrition throughout pregnancy increases exploratory activity [7,8], open arm entries in a plus maze [8,9], and produces deficits in the differential reinforcement of low rates of responding at 18 second (DRL 18r) acquisition [10], brightness discrimination [11,12] and reversal [12], water maze reversal [13] and repeated reversal learning [12]. In contrast, prenatal malnutrition has not been found to significantly impair inhibitory avoidance [8,11,12] or performance on working memory tasks [7,8,11,12,14]. In the current studies, behavioral comparisons were made of one male pup and one female pup from each acutely malnourished and control litter on righting reflex, elevation of shoulders, elevation of head, geotactic reaction, open field activity, wire mounting, narrow bridge crossing, and incline descent at ages 1 through 21 days, response in an elevated plus maze at ages 29 and 61 days, repeated spatial reversal at ages 54 –58 and 89 –93 days, and response to a novel alley at age 113 days. Retrospective studies in humans [15–17] as well as prospective studies in animal models [18] have shown that significant correlation also exists between IUGR and molecular alterations in glucose uptake/metabolism. Cell division and fetal growth require a complex series of biochemical events including a concurrent increase in energy metabolism. One of the earliest consequences of entry into the cell cycle is an increase in the uptake of glucose. The focal role of glucose uptake in growth and metabolism has led to a substantial literature describing the glucose transporter (Glut) proteins [19]. Glut 1 is expressed in most tissues, in most species and is the major transporter in brain and in the developing fetus [20 –24]. In the brain, the Glut 1 55 kilodalton (kD) isoform is found at the blood brain barrier while the 45kD isoform is associated primarily with neurons and glial cells [19]. In the present studies, brain tissue glucose uptake was determined and brain Glut 1 protein levels (45kD and 55kD isoforms) were assayed in the total membrane fraction of brains isolated from young and adult rat offspring from control and acutely malnourished pregnancies.
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2. Materials and methods 2.1. Animal model (acute). Twelve timed-pregnant rats (Harlan Sprague-Dawley, primigravida) were purchased from Harlan (Indianapolis, IN). The animals were individually housed and on day 17 of their pregnancy, randomly divided into two groups. Group one (controls) continued to have access to chow ad lib. while group two (IUGR) continued to have access to water but was fasted for 72 hours (from 08:00 hours of day 17 through 08:00 hours of day 20). On the morning of day 20, the starved animals were returned to ad lib. chow feeding and all pregnancies (6 controls and 6 IUGR animals) were allowed to go to term. The offspring from these litters were behaviorally tested as described below. In addition, one male and one female offspring from each of these litters were killed at 2, 7, 28, 42 and 112 days and their brains assayed for glucose uptake and glucose transporter protein content, also described below. 2.2. Animal model (chronic). Virgin Sprague Dawley female rats (200 –225 gm, Harlan) were given access to rat chow and a nutritionally balanced liquid diet (BioServe) for one week prior to mating. The diet is designed for pregnant mothers and contains maltose dextrin. Each female was placed with a fertile male during the dark portion of the light cycle. At the start of pregnancy (vaginal plug ⫽ day 0), each female was randomly assigned to one of two dietary groups. Group 1 (Controls) were given free access only to the maltose dextrincontaining liquid diet through out their pregnancy. Group 2 (Experimental) received a reduced amount of the liquid diet (based on kcal/kg) calculated to provide 90% of the calories consumed by the paired Control female on the previous day. On day 20 of their pregnancies, all mothers was returned to lab chow until the birth of their pups. Within 48 hours of parturition, the neonates were culled to 4 males and 4 females per litter (whenever possible) and each litter of pups was surrogate fostered to a nursing female who had received free access to only lab chow through out her pregnancy. All pups were weaned at 21 days of age and received free access to lab chow. 2.3. Maturation: behavioral milestones. During the first 21 postnatal days, one male and one female pup from each litter were tested for developmental level using several behaviors (see Table 1). Pups were tested individually for righting on a surface (time to turn from back on to all four paws), geotaxic reaction (time to turn head 1800 to upward orientation when placed facing down on an incline), elevation of shoulders (duration that shoulders were raised above the supporting surface), elevation of head (duration that the head was held above the supporting surface), hind limb support (ability to grasp a 2-mm diameter wire with one or both hind paws after being suspended from the wire by front paws), traversing a 1.2-mm wide 60-cm long wooden platform, descending a 450 wire mesh surface, and number of squares entered in an open field. The pups were also observed for the first appearance of walking in the open field and examined for day of eye opening. For all behavioral measurements, the operator was blinded as to the animals in utero nutritional treatment. 2.4. Elevated plus maze. One male and one female pup from each control and experimental litter were tested at both 29 and 61 days of age. The maze had two open arms, located
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Table 1 Behavioral tests given to acutely malnourished and control pups during first 21 postnatal days Behavior1
Normal age2
Ages of testing (days)
Dependent variables
Righting on a surface Geotaxic reaction Elevation of shoulders
13 2⫹3 64
Latency Latency Duration, % of time
Elevation of head Open field activity, walking
114 12–135
Eye opening Hind limb support Traversing 1.2 cm path Descending on wire mesh
14–155 153 173 183
1–9 2, 4, 6, 8, 10 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 5, 7, 9, 11, 13 7, 9, 11, 13, 15, 17, 19, 21 12, 13, 14, 15 10, 12, 14, 16, 18, 20 14, 16, 18, 20 15, 17, 19, 21
Duration, % of time No. squares entered crawling vs. walking Open? Success? Distance, no. falls, latency Latency
1
All tests had a maximum duration of 3 minutes. Normal ages are based on Altman and Sudarshan (Altman and Sudarshan, 1975). 3 Age at which 50% of normal subjects perform. 4 Age at the which 50% of subjects obtain asymptotic level. 5 Age first observed in nearly all animals. 2
opposite to one another, and two arms enclosed by vertical walls. The maze was placed in a room having a low level of illumination. Rats were individually placed on the maze in the center position and allowed to explore for 5 minutes. Total arm entries, proportion of entries to open arms, and proportion of time spent in open arms was determined for each subject. 2.5. Repeated spatial reversal. One male and one female rat from each experimental and control litter were tested on repeated spatial reversal, a task requiring behavioral flexibility, for five days beginning at ages 54 and 89 days. Each rat, 12 hours food deprived, was placed in the startbox of an elevated open T-maze that was positioned in a normally lighted room. For the first trial, food (sugar coated cereal) was placed in each goalbox. Whichever arm the animal selected then became the reinforced arm. The rat was reinforced in that arm until it attained the criteria of entering that arm on four consecutive trials. Upon attaining criterion, the arm containing reinforcement was switched. The rat was then reinforced for entering the new arm until it again attained criterion. At that time, the arms was again switched. Each rat was given 10 trials per day for 5 days. Number of correct choices and criteria attained was recorded. 2.6. Maze activity and response to novelty. The same pups tested in the elevated plus maze and for repeated spatial reversal were tested for maze activity and response to a novel alley at age 112 days. The test involved two massed trials in an enclosed Y-maze. During the first trial entrance to one arm was blocked by a barrier. On each trial, the rats were placed individually into the starting arm and permitted to explore for 5 minutes. Number of arms entered on each trial and first arm entered on the second trial were recorded. Also, for the second trial the proportion of entries in to the novel arm and proportion of time spent in the novel arm were determined.
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2.7. Tissue isolation. At various time points (2, 7, 28, 42 and 112 days), individual pups from each acutely treatment group were killed (using chilling and decapitation or CO2 asphyxiation) and the brains isolated and freed from associated membranes. The brains were weighed to the nearest milligram and divided along the medial sagittal fissure. The right hemispheres were taken for glucose uptake studies and the left hemispheres were quick frozen (liquid nitrogen) for transporter protein determination. Because of the relatively small brain size, three brains from two day old animals were pooled to provide sufficient tissue for analysis. 2.8. Glucose transporter protein assay. Glucose transporter proteins (Glut 1, 45kD and 55kD isoforms) were assayed as described by Postic, et al. [24] via semi-quantitative Western blotting using a primary polyclonal antibody against Glut 1 that was raised in New Zealand white rabbits by Dr. G. Lynis Dohm (Department of Biochemistry, East Carolina University). Transporter protein levels from brain tissue preparations of individual animals were separated by SDS-PAGE (10% gel, two hours at 30 milliamps/gel, 4 – 6° C). Following Western blotting to Immobilon (one hour at 400 milliamps/blot, 4 – 6° C) using Towbin’s buffer solution, the transporter proteins were compared using a horseradish peroxidaselabeled second antibody with quantitation by Imagequant software following scanning of the resulting films. Both the 45kD and 55kD isoforms of Glut 1 from each brain were determined on the same blot. To allow comparisons between blots, a standard adult rat brain tissue preparation was run on each gel/blot. 2.9. Brain 2-deoxyglucose uptake. 2-deoxyglucose uptake by brain tissue was measured as previously described [25]. 2.10. IV glucose tolerance tests. In order to compare the previously reported [26] effects of acute malnutrition to the effects of chronic in utero malnutrition on the offspring’s subsequent response to a glucose challenge, pups from chronically malnourished pregnant animals were examined at 60 days of age to determine their fasting plasma insulin and glucose levels and for their response to an intravenous glucose challenge. For the I.V. glucose challenge, the pups were fasted for six hour with water available, ad lib. and a “0” time blood sample (500 microliters) was taken from an indwelling carotid catheter placed 48 hours previous to the glucose challenge. An I.V. dose of glucose was administered (50% glucose in sterile water) to give a peak blood glucose level of 9 –10 mM). Blood samples (500 microliters) were taken from the carotid catheter at 1, 3, 5, 10, 30 and 60 minutes after the IV dose of glucose. This procedure and all others procedures involving animals were approved by the East Carolina University Institutional Animal Care and Use Committee. 2.11. Statistical analyses. For all behavioral observations and biochemical assays, group means were tested for significant differences using the appropriate analysis of variance (ANOVA) and Least Mean Square (LMS) post-hoc tests using SAS for Windows. For all tests, p⬍0.05 was used to conclude a statistically significance difference was present.
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Fig. 1. The effect of chronic (A) or acute (B) maternal malnutrition on maternal weight gain during pregnancy. In both frames the “n” for each time point is ⱖ 6. The data points marked by an “*” are significantly different (p ⬍ 0.05) from the ad lib data point on the same day of pregnancy. Note that following parturition, the acutely malnourished mothers were still significantly lighter than were the ad lib controls, indicating that the acutely malnourished mothers shared in the weight loss along with their fetuses. For this figure and all others data points represent group means ⫾ S.E.M.
3. Results Maternal Weight. Chronic malnutrition or seventy-two hours of acute food deprivation on days 17 through 19 of pregnancy resulted in a significant inhibition of maternal weight gain (Figure 1). As previously reported [26], the loss of mass during acute starvation was shared by both mothers and fetuses because, following parturition, the weights of the malnourished mothers were significantly less than the weights of the chow-fed control mothers (Figure 1B) and the fetal/neonatal weights of the malnourished pups were also significantly lower (Figure 2A). Conversely, chronic maternal malnutrition resulted in only the offspring being small compared to controls as the control and malnourished maternal weights were identical following parturition (Figure 1A). Fetal and Neonatal Body Weights. Weight differences between control and malnourished pups were similar for both the acute and chronic models, although the chronically malnourished pups may have faired somewhat better in terms of overall growth following parturition
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Fig. 2. The effect of acute (A) and (C) maternal malnourish and chronic maternal malnourish (B) on pup growth during the neonatal period (A) and (B) and during adulthood (C). In all frames the “n” for each time point is ⱖ 3. The data points marked by an “*” are significantly different (p ⬍ 0.05) from the data point for ad lib pups on the same day.
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Fig. 3. Effect of acute (A and B) and chronic (C) maternal malnutrition on brain growth in the offspring. In each panel, “*” indicates a value that is significantly different form the ad lib value (p⬍0.05).
(compare Figure 2A and 2B) The weight differences between control and malnourished pups were maintained throughout the time period that the offspring were studied (Figure 2C). Fetal and Neonatal Organ Weights. The brain weights of pups from acutely food-deprived and control litters were essentially identical (Figure 3A and 3B), suggesting that the brain, at least in terms of weight, was spared in the acute model. In the chronic model of malnutrition, offspring brain weights were lower than controls but the heart and liver of the chronically malnourished animals were not significantly different (Figure 3C).
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Fig. 4. Brain glucose uptake by female (A) and male (B) pups from ad lib and acutely malnourished litters. The data points marked by an “*” are significantly different (p⬍0.05). The values for males at 2 days were identical while the female values were 20 –25% higher in the malnourished females. The difference in uptake between males on day 7 is significant but not robust.
Brain Glucose Uptake. In female pups from litters acutely malnourished, glucose uptake by brain showed a small but statistically significant change on day 2 and small changes on days 42 and 112 compared to pups from control litters (Figure 4A). Male pups from acutely malnourished litters showed only a small decrease in transport at 7 days of age (Figure 4B) but their uptake was identical to that of the control animals at all other ages examined. These small changes were judged not to be of sufficient magnitude to alter overall glucose uptake. Chronically malnourished pups had a significant increase in basal glucose uptake compared to controls but the significance of this difference was lost in the presence of exogenous insulin (Figure 5). Response to a Glucose Challenge. As previously reported for the acute model [26], chronic in utero malnutrition resulted in the offspring being hyperglycemic in response to an
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Fig. 5. Effect of malnutrition on brain basal and insulin-stimulated glucose uptake by 18 day fetuses. The increase in basal uptake by the brains of malnourished fetuses was significant (p ⬍ 0.05). Notable variance in the insulin-stimulated data resulted in the differences not being significant.
intravenous glucose challenge (Figure 6). At 60 days of age, both male and female malnourished progeny were hyperglycemic in response to a glucose challenge but the female animals were more notably altered (Figures 6A and 6B). Brain Glucose Transporters. Maternal malnutrition resulted in small, but statistically significant, changes in brain Glut1 glucose transporter protein levels (55kD isoform). The small increases in the 55kD isoforms at two days of age in both sexes were paralleled by a modest but significant increased in glucose uptake in females at this age but there was no difference in male glucose uptake. Further, the inversion between control and malnourished 55kD isoform levels seen at day 42 in the female was not paralleled by a change in the uptake of glucose at this time point (compare Figure 4B and Figure 7D) 2.12. Behavioral milestones. The only developmental milestone on which the malnourished and control pups were found to differ significantly was latency to descend the wire mesh platform (Figure 8). The malnourished rats took longer to descend than did the control animals (155.1⫾5.4 s vs. 126.2⫾9.8 seconds, p⬍0.05). 2.13. Maze behaviors. No significant differences between pups from acutely malnourished or control litters were observed on any of the elevated plus maze measures, T-maze repeated reversal, or Y-maze activity or response to a novel alley. However, regardless of fetal diet, females were found to be more active in mazes than were males. Females entered more arms than did males in the plus maze when tested at 61 days of age (29.9⫾ vs. 19.6⫾), and entered more arms in the Y-maze on both the first (16.2⫾ vs. 12.2⫾) and second (18.9⫾ vs. 13.0⫾) day of testing at ages 112–113 days. However, the males and females did not differ on open field activity when tested at ages 7, 9, 11, 13, 15, 17, 19 and 21 days or on total arms entered in the plus maze at 30 days of age.
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Fig. 6. Response to an IV glucose challenge by 60 day old female (A) and male (B) offspring from litters fed ad lib and chronically malnourished. In all panels, data point for which the error bars do not overlap are significantly different (p⬍0.05).
4. Discussion In the current study, individual animals from control and acutely malnourished litters were compared on several of the developmental milestones established by Altman and Sudarshan [27]. In addition, the animals were compared on measures of emotionality and cognitive behaviors. These developmental milestone behaviors provided an excellent method to detect delayed maturation. Since rats reach each of the behavioral milestones on different days, testing of several behaviors, over time, permits the detection of not only developmental delay, but also any developmental recovery that may occur as the animals mature. Behavior on the elevated plus maze has been shown to be sensitive to chronic fetal malnutrition and is considered an excellent measure of exploration and anxiety [28]. The results reveal that
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Fig. 7. Brain Glut 1 45 kD isoform (A&C) and Glut 1 55 kD isoform (B&D) at various ages. Those data points marked by an “*” are significantly different from the comparable ad lib fed pup values. Error bars have been omitted for clarity at certain data points.
only coordination and balance as evidenced by the slower descent of the ramp were significantly impaired by the fetal malnutrition. The offspring of acutely or chronically malnourished dams weighed less than control offspring at birth and the difference in body weights was maintained throughout the study
Fig. 8. Time to descend a wire mesh ramp elevated at a 45° angle. The malnourished and control pups were found to differ significantly in latency to descend the wire mesh platform. The malnourished rats took 155.1 ⫾ 5.4 seconds versus 126.2 ⫾ 9.8 seconds for the offspring from control litters (p⬍0.05)
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period. However, the brain weight of the acutely malnourished offspring was lower only at one time point. This outcome suggests that mechanisms exist to protect brain growth from short-term malnutrition. Consistent with the rapid brain weight recovery, the acutely malnourished offspring were impaired on only a single developmental behavioral paradigm. In comparison, the brain weights of pups from chronically malnourished litters were significantly less than control pup brain weights. Previous studies [7,8,9,11,13,14] have demonstrated the behavioral deficits resulting from chronic protein/calorie deprivation throughout gestation. These data suggest that whereas three days of maternal fasting has a profound effect on maternal weight and has a sustained effect on offspring body weight, brain development and behavioral maturation are spared unless malnutrition is prolonged. As has also been observed in earlier research [29], female offspring were more active than males regardless of the dietary treatment of the mothers. At the molecular level, the small changes in female brain glucose uptake seen on days 2 and 42 were paralleled by small but significant changes in the Glut1 55kD transporter. Because the 55kD isoform is associated with the microvasculature [23] and is involved in transport out of the microvasculature, the data suggest that the 55kD isoform may be the rate limiting factor. This relationship is speculative given the limited data available, but fits with a previous report of such changes in 55kD mediated-transport in a growth suppressed embryonic chick brain model [30,31]. Overall, Glut1 transporter protein isoforms levels and glucose uptake by the brains of both male and female acutely malnourished pups show minimal differences relative to the control brains; again suggesting that the malnourished brains were “protected” during the fasting period. Furthermore, the slight differences in brain transporter proteins isoform levels were paralleled by minimal behavioral differences between these two groups. Thus, the probability of observing maternal malnutrition-induced biochemical and behavioral changes in the progeny appears to be a function not only of the intensity of the of malnutrition but also of the duration of the treatment. Although not studied here, the timing of the maternal malnourished period relative to the developmental stage of the fetus/embryo may also play a significant role in determining the growth and behavioral outcome for the offspring.
5. Conclusions 1. Acute maternal malnutrition during late pregnancy (via a 72 hour maternal fast) resulted in a significant weight deficiency in both the mothers and their offspring. 2. Chronic malnutrition (90% of control calories from day 1 to day 20 of the pregnancy) resulted in a fetal/neonatal weight deficit that was not shared by the mothers 3. Acute maternal malnutrition had minimal effects on the offspring’s brain weight, Glut 1 transporter isoform levels and glucose uptake was increased only circa parturition 4. Acute maternal malnutrition had little affect on the offspring’s maturation rates or learning behaviors 5. In parallel with other reports, chronic maternal malnutrition inhibited fetal brain growth and stimulated fetal glucose uptake 6. Acute maternal malnutrition produced minimal biochemical and behavioral changes in
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the progeny as compared with prolonged malnutrition. Thus, the reliability of observing fetal malnutrition-induced biochemical and maturation changes appeared to be a function of the duration as well as of the intensity of the of maternal malnutrition.
Acknowledgments This work was supported in part by grants from the University of North Carolina Nutrition Institute to LWM and SNP and from the Children’s Miracle Network and Grant # AA10681 from NIAAA to SNP.
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