Rearing animals in small litters induces a permanent increase in body weight and body fat. To determine whether changes in sympathoadrenal activity contribute to this effect, litter size was adjusted the day after birth and maintained until weaning at 21 days. Sympathetic nervous system (SNS) activity was measured in adult animals using [3H]norepinephrine ([3H]NE) turnover in peripheral tissues. Although litter size was without effect on [3H]NE turnover in chow-fed animals, acceleration of [3H]NE turnover by dietary sucrose was completely abolished in heart and attenuated in interscapular brown adipose tissue and kidney of rats reared in small litters. Body and epididymal fat-pad weights were heavier in rats reared in small litters; however, weight gain in response to dietary enrichment with sucrose did not differ as a function of litter size. Thus litter size alters dietary activation of the SNS, and this effect presumably reflects changes in central nervous system regulation.
- interscapular brown adipose tissue
- body fat
- nervous system activity
- sympathoadrenal function
postnatal experience affects the development of numerous components of the mammalian nervous system. Visual and auditory pathways (11, 15, 26) as well as somatosensory and olfactory systems (2, 29) are dependent on sensory input for normal maturation. The sympathoadrenal system is also susceptible to environmental influences during development. For example, early exposure to a hot environment affects the development of sudomotor function (7), whereas neonatal exposure to cool temperatures induces hyperplasia in sympathetic efferent pathways to brown adipose tissue (16). Moreover, neonatal handling (brief, daily separation of mothers and pups between birth and weaning) leads to diminished sympathetic nervous system (SNS) activity in spleen and heart and to exaggerated adrenal medullary responsiveness to fasting in adult male rats (30).
The size of the litter in which an animal is reared represents another neonatal manipulation with potential implications for SNS development. Rats reared in large litters accumulate less [3H]norepinephrine ([3H]NE) in the heart after subcutaneous injection of tracer than animals reared in smaller litters (12). Similarly, cardiac NE concentrations are inversely related to litter size at 15 days of age, although not at weaning (10). Finally, cardiac and renal NE levels are lower at 40 days of age in rats reared in large litters than in those reared in smaller litters (24). However, the consequences of litter size for sympathoadrenal function (in contrast to tissue NE levels) in adult animals has not been examined.
Rearing animals in small litters has long been known to increase body size, an effect that has been attributed to neonatal “overnutrition” (14, 20, 28). Because rearing in small litters has been employed in several experimental models of obesity or hypertension (1, 3, 9, 13, 19), the possibility arose that developmentally acquired alterations in sympathoadrenal function might contribute to the increased susceptibility to obesity or hypertension in these animals. Because neonatal overnutrition is commonly followed by overfeeding after weaning, the relative contributions of litter size and the postweaning diet are not always clear. Consequently, these studies were undertaken to examine specifically the impact of litter size alone on the regulation of SNS activity.
Male or female CD rats (1-day old) with multiparous foster mothers were obtained from Charles River Breeding Laboratories (Wilmington, MA). On the day of arrival, male rats were redistributed to produce litters of 6, 12, or 18 pups each, whereas in separate experiments, female rats were reared in litters of 4, 10, or 16 pups. Rats were weaned at 21 days; all pups from a single litter-sized group were pooled and redistributed to 3 pups per cage for subsequent housing except during the weight-gain study, when animals were housed 2 pups per cage. The room in which the animals were housed for the duration of the studies was maintained at 21 ± 2°C on a 14:10-h light-dark cycle. Animals used in this study were kept in accordance with the guidelines and approval of the Animal Care and Use Committee of Northwestern University Medical School.
Unless otherwise specified, animals were provided free access to water and standard laboratory chow (NIH-07 Open Formula Mouse/Rat Diet, Harlan Teklad, Madison, WI). In the sucrose-feeding studies, animals received either lab chow or lab chow supplemented with 10% sucrose (wt/vol) to drink. Sucrose feeding commenced 7 days before the start of the turnover study and continued throughout the turnover measurement. Bottles containing sucrose were replaced daily.
[3H]NE Turnover Procedure
l-[ring-2,5,6-3H]NE (sp act 40–60 Ci/mmol; DuPont NEN Research Products, Boston, MA) was diluted with 0.9% NaCl and injected intravenously into the tail veins of unanesthetized animals in a total volume of 1.0 ml. The dose of [3H]NE used in the current studies was 30–45 μCi/kg of body wt (∼0.12–0.18 μg of NE/kg of body wt). The rats were killed at preselected times via CO2 inhalation. For each time point in the NE turnover studies, 4–6 animals were killed from each experimental group. The tissues were rapidly removed, frozen on dry ice, and stored at −20°C for later processing (usually within 2 wk).
Extraction and Analysis of Tissue Catecholamines
For NE analysis, the organs were weighed and homogenized in iced 0.2 N perchloric acid in a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) to extract the catecholamines. After addition of the internal standard, 3,4-dihydroxybenzylamine (DHBA, Sigma, St. Louis, MO), catecholamines were isolated from the perchloric acid extract by adsorption onto alumina (Woelm neutral, ICN Nutritional Biochemicals) in the presence of 2 M Tris buffer (pH 8.7; Sigma) containing 2% EDTA. Catecholamines were eluted from the alumina with 0.2 N perchloric acid. Aliquots of the alumina eluate were injected onto a liquid chromatographic system for catecholamine analysis following the method of Eriksson and Persson (8) with slight modification. Unless otherwise specified, all chemicals were obtained from Fisher Scientific (Fair Lawn, NJ). Aliquots of the alumina eluates were counted for [3H]NE by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation analyzer (Packard Instrument, Meriden, CT). Efficiency for 3H is ≥58% in this system.
Data are displayed as means ± SE unless otherwise noted. Statistical ANOVA and analyses of covariance (ANCOVA) were performed using Data Desk 6.1 statistical software (Data Description, Ithaca, NY) (27). Post hoc pairwise comparisons after ANOVA utilized Scheffé's test. Analyses employed P < 0.05 as the criterion for statistical significance; P values between 0.05 and 0.1 are provided in text and tables, whereas Pvalues ≥0.1 are indicated as not significant (NS).
In studies of NE turnover, the data were plotted semilogarithmically. The method of least squares was used to calculate the slope of decline in NE specific activity over time after tracer injection (17). In all measurements of [3H]NE turnover, no significant variation in endogenous NE was observed over the 24 h of the experiment. The statistical significance of each computed regression line was assessed by ANOVA, and ANCOVA was used in comparison of fractional turnover rates. In the studies comparing sucrose-induced changes in [3H]NE turnover between animals reared at different litter sizes, indicator variables were used for litter size and diet, and the slopes of the regression lines were analyzed in a 2 × 2 ANCOVA model (18). Goodness of fit for each regression line was evaluated by examination of externally studentized residuals. NE turnover rates were calculated as the product of the fractional turnover rate and the endogenous NE concentration.
Effect of Litter Size on Tissue NE Levels in Male and Female Rats
Because rearing male and female rats in large litters was previously shown to diminish NE levels in heart and kidney (24), our initial studies examined the impact of litter size on organ weight and NE content in tissues from male and female rats. The results are presented in Tables1 and2, respectively. In males (Table 1), although body and organ weights and tissue NE levels were inversely related to litter size at both 25 and 53 days of age, NE levels in relation to tissue weights did not differ significantly among rearing groups. In a subsequent experiment (Table3), tissue NE concentrations were reduced 9% (P < 0.03) in heart but not in kidney or spleen of 65-day-old male rats reared in large litters. In female rats (see Table2), although body weights were less affected by litter size than in male animals, tissue NE levels were uniformly reduced in rats reared in large litters. NE concentrations in heart and kidney were also lower in rats reared in large litters (P < 0.02 andP < 0.025, respectively). By contrast, in a subsequent experiment (Table 4), tissue NE concentrations did not differ between 73-day-old female rats reared in large or small litters. Thus, although tissue NE levels in several peripheral tissues were lower in rats reared in large litters, the reduction in tissue NE was usually commensurate with the reduction in organ weight.
Effects of Litter Size on Sucrose-Induced Stimulation of Tissue [3H]NE Turnover in Male and Female Rats
Because NE levels alone provide little information regarding the activity of peripheral sympathetic nerves, studies were undertaken to assess the dynamic nature of tissue NE stores using the [3H]NE turnover technique. In an initial study, no differences in [3H]NE turnover rates were noted among groups of male rats reared in litters of 6, 12, and 18 pups that were fed lab chow ad libitum (data not shown). Subsequent experiments were therefore performed comparing [3H]NE turnover in rats reared in small or large litters that were fed either lab chow or lab chow supplemented with sucrose (10% solution to drink) for 7 days before and during the turnover measurement. These experiments provided simultaneous comparisons of the effects of litter size and dietary sucrose. The results for male rats studied at 65 days of age are presented in Fig. 1 and Table 3; results for female rats at 73 days of age are shown in Fig.2 and Table 4. Because sucrose supplementation of a lab-chow diet is known to accelerate [3H]NE turnover in heart and other tissues (31-33), these experiments were designed to test the impact of litter size on dietary stimulation of sympathetic activity in several peripheral tissues simultaneously.
In male rats (see Fig. 1 and Table 3), the impact of sucrose feeding on [3H]NE turnover in peripheral tissues differed markedly between rats reared in large and small litters. In heart, the expected rise in [3H]NE turnover with sucrose feeding was observed in rats reared in large but not small litters (litter size × diet interaction; P = 0.0003). Fractional NE turnover increased from 6.3 ± 0.6 to 9.0 ± 0.9%/h (P < 0.02) in rats reared in large litters but not in rats reared in small litters (from 6.4 ± 0.6 to 6.7 ± 0.5%/h; P = NS), and although total NE turnover was 30% greater in sucrose-fed rats from large litters, it was 6% lower in sucrose-fed animals from small litters. In interscapular brown adipose tissue (IBAT), fractional NE turnover was lower overall in rats reared in small litters (P < 0.05) and increased only 47% with sucrose feeding in these animals compared with the 77% increment observed in rats reared in large litters. Likewise in kidney, fractional NE turnover increased from 7.4 ± 0.8 to 11.3 ± 1.0%/h (P < 0.004) in rats reared in large litters but not at all in animals reared in small litters, and total NE turnover increased 40% in rats from large litters but fell 8% in animals from small litters. In spleen, effects of litter size on [3H]NE turnover were not statistically significant. Thus the size of the litter in which a male rat was reared markedly influenced the SNS response to dietary sucrose as assessed by measurements of [3H]NE turnover in heart, IBAT, and kidney.
In female rats (see Fig. 2 and Table 4), similar findings were noted. Cardiac fractional NE turnover increased from 7.3 ± 0.5 to 9.6 ± 0.8%/h (P < 0.025) in rats reared in large litters but not in animals reared in small litters (from 7.8 ± 0.3 to 7.6 ± 0.6%/h, P = NS; litter size × diet interaction, P = 0.0007), and although total NE turnover increased 23% with sucrose feeding in rats from large litters, it fell 7% in sucrose-fed animals from small litters. In IBAT, fractional NE turnover increased from 7.6 ± 1.1 to 10.7 ± 1.0%/h (P < 0.05) in rats reared in large litters but fell in animals reared in small litters (from 8.6 ± 1.0 to 8.1 ± 1.2%/h, P = NS; litter size × diet interaction, P < 0.06), and total NE turnover increased 47% in rats from large litters but fell 9% in animals from small litters. Similar results were obtained in kidney. Renal NE turnover rose 8% with sucrose feeding in rats from large litters but fell 16% in animals from small litters. In spleen, differences in [3H]NE turnover were not apparent in chow-fed animals, although among sucrose-supplemented animals, fractional [3H]NE turnover was less in rats reared in small litters than in those from large litters (P < 0.05). Thus, in female as well as in male rats, sympathetic activation in heart, IBAT, and kidney by dietary sucrose was generally less in animals reared in small than in large litters.
Effect of Litter Size on Weight Gain in Chow- and Sucrose-Fed Male Rats
To determine whether litter size also affected an animal's propensity to gain weight when exposed to a sucrose-enriched diet, 59-day-old male rats reared in litters of 6 or 18 were divided into two weight-matched groups. One group received lab chow and the other received lab chow plus 10% sucrose to drink. Food intake and body weight were measured for 8 wk, and the results are presented in Table5. Animals reared in small litters were heavier both at the start and at the end of the feeding period (P < 0.0001). Although energy intake and weight gain were slightly greater over the course of the 8-wk study in sucrose-fed than in chow-fed rats and in rats reared in small than in large litters, these differences were not statistically significant. Thus rearing in small litters does not increase a male rat's propensity to gain weight when presented with a sucrose-supplemented diet, in contrast to the impact of rearing male rats in a cool environment, which does (34).
Effect of Litter Size on Epididymal Fat-Pad Weight in Male Rats
Epididymal fat-pad weights were obtained at the end of the 8-wk feeding study as a measure of abdominal fat deposition. The results are presented in Table 5 and Fig.3. Epididymal fat pads were heavier in rats reared in small litters and in those fed the sucrose-supplemented diet both in raw weights and in relation to body size (Table 5). ANCOVA of epididymal fat-pad weights in relation to body weights also revealed that after taking body weight into account (P < 0.0001), there was a significant interaction between litter size and body weight. This is illustrated in Fig. 3. Regression lines relating epididymal fat-pad weight to body weight are significantly steeper in rats reared in small than in large litters, and the effect of sucrose supplementation is to increase the height (P = 0.002) but not the slope of these regression lines (interaction between body weight and diet; P = NS). For every 100-g increase in body weight, epididymal fat-pad weight rises by 2.0 ± 0.8 g in rats reared in large litters and by 5.9 ± 0.8 g in those reared in small litters (P = 0.0003). Thus rearing rats in a small litter promotes fat accretion in epididymal fat depots, an effect to which a sucrose-enriched diet makes an additive contribution.
The findings presented here demonstrate that although tissue weight and NE content were inversely related to litter size, these effects were usually of similar proportion. Consequently, the impact of litter size on NE concentration (ng of NE/g of tissue) was minor and inconsistent across experiments in the tissues examined. In contrast, litter size induced marked and reproducible effects on the dynamic behavior of NE stores as measured by [3H]NE turnover in peripheral tissues. Although rates of [3H]NE turnover did not differ as a function of litter size in chow-fed animals, rats reared in small litters consistently exhibited less of an increment in [3H]NE turnover in heart, brown fat, and kidney with addition of sucrose to the diet than did animals reared in large litters. Because under steady-state conditions [3H]NE turnover provides a generally accepted index of SNS activity in peripheral tissues of unanesthetized animals, these findings imply that the magnitude of dietary-induced changes in SNS activity is determined in part by the number of littermates with which an animal shares the postnatal environment.
Potential mechanisms that are responsible for diminished sucrose-induced activation of sympathetic nerves in rats reared in small litters were not addressed in the current study. Because SNS activity is under the direct control of centers in the hypothalamus and brain stem, it is likely that litter size affects the functional development of these regions as well. In support of this conjecture, Seidler and colleagues (24) showed that NE turnover within the cerebral cortex and cerebellum of the rat was affected by litter size in developing animals. More recently, Plagemann and colleagues (5, 6, 21, 22) have demonstrated effects of litter size on the structure, neurochemistry, and responsiveness to leptin of neurons in the paraventricular, ventromedial, and arcuate nuclei of the hypothalamus. Whether any of these differences in central nervous system function is responsible for or contributes to the current findings is unknown but warrants further investigation.
Animals reared in small litters are consistently fatter than their companions reared in large litters, a finding that has been noted repeatedly (1, 9, 19) and was also identified in the current studies (see Table 5 and Fig. 4). Although diminished SNS activation in thermogenic tissues such as brown fat may contribute to this propensity for fat accumulation, no difference was noted between rearing groups in weight gain during an 8-wk exposure to a sucrose-enriched diet (Table 5). This finding was somewhat surprising given the lesser activation of SNS by dietary sucrose in rats reared in small litters and given previous reports that weight gain is exaggerated in rats reared in small litters when the animals are fed a high-fat diet (9, 19). Although current studies did not address this, it is conceivable that ingestion of a high-fat diet in adult animals exerts additive effects to those due to litter size per se. One possibility for such an interaction may relate to expression of β3-adrenergic receptors, which is the subtype principally associated with thermogenesis and lipolysis in rodents. Gene expression for the β3-adrenergic receptor is reduced in adipose tissue of mice fed a high-fat diet (4), whereas expression of this gene in IBAT is enhanced when rats are fed a sucrose-enriched diet similar to that employed in the current study (J. B. Young, unpublished observation). Thus obesity in animals may arise as a result of interactions between environmental exposures during neonatal and adult life. Diet or other factors in the adult environment that diminish end-organ responsiveness to adrenergic stimulation may amplify the modest reduction in SNS activation associated with small litter size noted here. Whether such environment-to-environment interactions affect adrenergic regulation of energy metabolism is unknown but is worthy of further study.
Because the great majority of pregnancies result in singleton births, variation in litter size per se is unlikely to contribute importantly to SNS development in humans. On the other hand, the physiological mechanisms involved in programming SNS function by manipulation of litter size may well participate in human development under other circumstances. For example, insulin levels in neonatal rats are inversely related to litter size (22, 23). Because administration of exogenous insulin to neonatal rats appears to impair sympathetic responses to carbohydrate in adulthood (J. B. Young, unpublished observation), the possibility exists that circulating levels of insulin during neonatal life may induce selective and permanent changes in sympathetic responsiveness. Infants born to diabetic mothers represent a human cohort known to exhibit hyperinsulinemia during perinatal life and a propensity to develop obesity later in childhood (25). Thus studies of otherwise normal rats reared in small litters may provide insight into factors contributing to acquisition of obesity-susceptibility traits in humans.
The skillful technical assistance of Yiu-Kuen Chow and Martin Fetzer is acknowledged.
These studies were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-20378 and DK-54904.
Address for reprint requests and other correspondence: J. B. Young, Northwestern Univ.-Chicago, Ward 4-161, 303 East Chicago Ave., Chicago, IL 60611-3008 (E-mail:).
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.
- Copyright © 2002 the American Physiological Society