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Regular Article |
a School of Anatomy and Human Biology and The Western Australian Institute for Medical Research, University of Western Australia, Crawley, Perth, Western Australia 6009, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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hypothalamus, lactation, leptin, leptin receptor, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During pregnancy, plasma leptin concentrations increase from about midgestation [11–13], an effect that would normally be expected to decrease food consumption. On the contrary, food consumption either remains unchanged [14] or increases over this period [15], suggesting that pregnancy induces a state of leptin resistance to ensure provision of energy to meet the demands of rapid fetal growth in late pregnancy [16]. However, the mechanisms involved in this resistance remain uncertain. In the pregnant mouse, Gavrilova et al. [10] showed that leptin resistance is likely due, at least in part, to placental secretion of Ob-Re, the soluble form of the leptin receptor. Thus, plasma leptin-binding activity increases dramatically in the final third of mouse pregnancy, and this binding is thought to limit access of leptin to target tissues [10]. Although not studied in detail, the same report indicates that unlike the mouse, plasma leptin-binding activity in the rat increases only marginally in pregnancy [10], suggesting that leptin resistance in this species may involve a different mechanism. The rat placenta expresses all three major isoforms of the leptin receptor, Ob-Ra, Ob-Rb, and Ob-Re, and among these the soluble Ob-Re form is by far the most abundant (unpublished results). This finding suggests that changes in plasma leptin-binding activity in rat pregnancy may indeed be physiologically important and thus worthy of closer investigation. Alternatively or in addition, leptin resistance in pregnancy could result from a decrease in the hypothalamic expression of Ob-Rb, the only leptin receptor isoform that exhibits full signal transduction capacity. This hypothesis is supported by the observation that estrogen, which increases progressively during rat pregnancy [17], inhibits hypothalamic Ob-Rb expression in the nonpregnant rat [18]. The present study had three objectives: to confirm by measurement of plasma leptin and food consumption within individual animals that rat pregnancy is a leptin resistant state, to determine the leptin-binding activity of plasma obtained from rats at different stages of pregnancy and lactation, and to examine whether hypothalamic expression of Ob-Rb changes over the course of pregnancy and lactation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Wistar rats weighing 235 ± 17 g (mean ± SD) on the first day of pregnancy (or 228 ± 8 g for nonpregnant controls) were obtained from UWA Animal Services (Murdoch, Australia) and maintained on a 12L:12D cycle (lights-on from 0600 h to 1800 h) at an ambient temperature of 22–23°C and humidity of 35–50%. Water and food (digestible energy, 14.5 MJ/kg; 18.9% protein, 5.2% fat, 5.0% fiber, 0.77% calcium, 0.57% phosphorus, 0.41% NaCl) were provided ad libitum. Rats were mated overnight, and the morning on which a vaginal plug was present was designated as Day 1 of pregnancy (term = Day 23). All procedures involving animals were conducted with approval from the Animal Ethics Committee of the University of Western Australia.
Food Consumption and Plasma Leptin Measurements
Daily food consumption was measured in rats housed in individual metabolic cages. Pilot experiments were performed to ensure that the amount of food provided was well in excess of the expected daily intake. All remnants of food within the cage were collected and weighed to account for wastage. Seven groups of rats (five or six rats per group) were used: nonpregnant (two consecutive estrous cycles), early pregnancy (Days 3–7), midpregnancy (Days 7–12), late pregnancy (Days 13–18), prepartum (Days 18–22), peripartum (Day 19 of pregnancy to Day 1 of lactation), and lactation (Days 7–12). Immediately following the final food consumption measurement (diestrus of the cycle, Days 7, 12, 18, and 22 of pregnancy, and Days 1 and 12 of lactation), rats were anesthetised with halothane/nitrous oxide, and blood samples were obtained from the dorsal aorta into a heparinized syringe. Blood was centrifuged for 5 min at 11 000 rpm, and plasma was stored at -20°C until subsequent analyses. Plasma leptin concentrations were measured using a rat leptin RIA kit (Linco Research, St. Charles, MO); the intra-assay coefficient of variation was 6%. This RIA is assumed to measure total plasma leptin because antibody binding of leptin is not affected by the presence of Ob-Re (Linco Research).
Plasma Leptin-Binding Activity
Plasma leptin-binding activity was assayed as described previously for mouse serum [10]. Plasma samples (5 µl) were incubated with 5 µl of 125I-leptin (approximately 5 ng/ml and 20 000 cpm) overnight at 4°C. Sample buffer (20 µl) containing bromophenol blue was added, and samples were vortexed and electrophoresed in 4–12% gradient Tris/glycine gels at 120 V across the 4% gel and 160 V across the 12% gel. The gel was dried and placed against film for 48 h at -80°C. The band corresponding to specifically bound 125I-leptin was scanned, and leptin-binding activity was quantified by analysis of pixel intensity using the public domain National Institutes of Health Image program (version 1.61) as previously described [19]. Serum from a Day 17 pregnant mouse was used as a positive control.
Real-Time Reverse Transcription Polymerase Chain Reaction Assay of Hypothalamic Ob-Rb mRNA
Brains were removed from a second series of rats at diestrus of the cycle and at Days 7, 12, 18, and 22 of pregnancy and Days 1 and 12 of lactation under halothane/nitrous oxide anesthesia. The hypothalamus was dissected [20] and immediately frozen in liquid nitrogen. Total RNA was extracted using RNAzol B (Tel-Test, Friendswood, TX) and reverse transcribed using random primers (Promega, Madison, WI) and Superscript II (Invitrogen, Life Technologies, Melbourne, Australia) according to the manufacturer's instructions. Resultant cDNA was purified using an UltraClean polymerase chain reaction (PCR) kit (Mo Bio Laboratories, Solana Beach, CA), quantified by spectrophotometry, and stored at -20°C until analysis. The regular Ob-Rb PCR product (375 base pairs) was extracted (QIAquick kit protocol; Qiagen, Clifton Hill, Australia), quantified by spectrophotometry, and then used to generate a standard curve via serial dilutions in RNase-free water (1–107-fold dilutions).
Quantitative PCR and melting curve analyses were performed in 10-µl reaction volumes in capillary tubes using the LightCycler system (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Ob-Rb primers (sense 5'-ATGAAGTGGCTTAGAATCCCTTCG-3', antisense 5'-ATATCACTGATTCTGCATGCT-3'; GeneWorks, Adelaide, Australia) and MgCl2 concentrations were 0.3 µM and 3 mM, respectively. The PCR cycling conditions included an initial denaturation at 95°C for 10 min followed by 55 cycles of 95°C for 15 sec, 54°C for 5 sec, and 72°C for 16 sec. Melting curve analysis showed a single PCR product, and the presence of this product was confirmed by gel electrophoresis (data not shown). Fluorescence values were analyzed, and a standard curve was constructed using the LightCycler software. The intra- and interassay coefficients of variation were 3.0% and 3.8%, respectively.
Western Blot Analysis of Hypothalamic Ob-Rb
Hypothalami were collected from a third series of rats at the same stages of pregnancy and lactation and were homogenized in 250 µl homogenization buffer (10 mM Tris buffer containing 1.5 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF, and 100 µg/ml trypsin inhibitor). Samples were then centrifuged at 105 000 x g for 30 min, and 30 µg of supernatant protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes (0.45 µm). Membranes were blocked for 1 h in Tris-buffered saline with Tween-20 (TBS-Tween; 0.1 M Tris, 0.15 M NaCl, 0.1% Tween-20, pH 7.5) containing 5% nonfat milk powder and then exposed for 2 h to leptin receptor antibody (K-20, 1:400 dilution in TBS-Tween containing 1% nonfat milk powder; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed in three changes of TBS-Tween and incubated for 1 h with a horseradish peroxidase-conjugated donkey anti-goat secondary antibody (1:5000; Santa Cruz Biotechnology). All incubations and washes were performed at room temperature. Immunoreactive bands were visualized using a chemiluminescence detection kit (SuperSignal Substrate; Pierce Chemical, Rockford, IL) with membranes placed against film for 1 min, and resultant images were quantified by densitometry using Scion Image analysis software (release beta 3b) as previously described [19].
Statistical Analysis
Day-to-day changes in food consumption within each group were assessed by a repeated measures ANOVA. Variation among groups in food consumption, plasma leptin concentrations, and Ob-Rb mRNA were assessed by a one-way ANOVA, and variation in plasma leptin-binding activity and hypothalamic Ob-Rb protein were assessed by a two-way ANOVA (with individual blots/gels containing a single sample from each group serving as replicates). When ANOVAs showed significant variation among groups, specific group comparisons were made by least significant difference (LSD) tests [21]. For all analyses, normal distribution of data and variance consistency among groups were confirmed using statistical software (GraphPad Software, San Diego, CA).
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RESULTS |
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Food consumption differed significantly across the estrous cycle (P < 0.01) and was lower at proestrus than at all other stages. In contrast, there were no day-to-day changes in food consumption observed within each of the early pregnancy, midpregnancy, or late pregnancy groups (Fig. 1a). In both the pre- and peripartum groups, however, food consumption declined on Day 22 of pregnancy, and in the peripartum group food consumption fell further on Day 23 and remained low on Day 1 of lactation. In the lactating group, food consumption increased (P < 0.01) from Day 7 to Day 9 of lactation and remained stable thereafter.
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To assess changes across all of pregnancy and during lactation, average food consumption was calculated for each rat in those groups in which no change was observed over the 5–6 days of measurement (i.e., early pregnancy, midpregnancy, and late pregnancy). Because substantial day-to-day changes were observed in the other pregnant and lactating groups, the values for these groups were calculated only for the days before (prepartum: Days 18–21) or after (peripartum: Day 23 of pregnancy to Day 1 of lactation; lactation: Days 9–12) the onset of this change. This analysis showed that food consumption increased progressively from proestrus to the prepartum period (P < 0.001), declined markedly (P < 0.001) in the peripartum group, and then increased (P < 0.01) again to maximum levels during lactation (Fig. 1b).
Plasma Leptin Concentrations
Plasma leptin concentrations differed significantly across pregnancy and lactation (P < 0.01, one-way ANOVA, Fig. 2), rising from 2.2 ± 0.4 ng/ml in diestrous rats to maximum levels on Day 12 (4.2 ± 0.8 ng/ml) and Day 18 (4.0 ± 0.4 ng/ml, P < 0.05) of pregnancy. Plasma leptin had begun to fall by Day 22 of pregnancy, and at Day 12 of lactation levels were similar to those measured before pregnancy.
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Plasma Leptin-Binding Activity
A major band of leptin-binding activity in rat plasma migrated very closely with that previously identified in late-pregnant mouse serum (included as positive control). This band was clearly evident in plasma samples from all rats, but its intensity increased (P < 0.01) markedly from a minimum of 12.8 ± 6.0 (arbitrary density units) at diestrous to a maximum of 69.3 ± 3.6 at Day 22 of pregnancy (Fig. 3). Binding activity then fell (P < 0.05) to 51.5 ± 11.1 at 1 day postpartum (Day 1 of lactation) and had declined further by Day 12 of lactation. Additional leptin-binding activity corresponding to albumin [10] was observed in all plasma samples but did not differ among groups (data not shown).
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Hypothalamic Expression of Ob-Rb
Expression of hypothalamic Ob-Rb mRNA increased by >50% (P < 0.05) between diestrous of the cycle and Day 7 of pregnancy but then returned to prepregnancy levels by Day 12 and remained relatively stable thereafter (Fig. 4). Western blot analysis demonstrated a clear immunoreactive leptin receptor signal at the expected size for Ob-Rb (120 kDa [22]), but this signal did not change across pregnancy (Fig. 5). A weaker immunoreactive signal was apparent at around 50 kDa and also remained unchanged among groups (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The rise in plasma leptin-binding activity in pregnancy is similar to previous observations in the mouse, although in the mouse the magnitude of the increase was far greater (around 40-fold) and did not occur until much closer to term [10]. The increase in 125I-leptin binding is assumed to reflect greater plasma leptin-binding capacity in pregnancy; exchange of endogenous leptin and 125I-leptin would be expected to occur during the extended (approximately 16 h) incubation prior to nondenaturing electrophoresis. The identity of the rat leptin-binding activity is almost certainly the same as that in mouse; both exhibited similar although not identical migration in nondenaturing electrophoresis. Leptin-binding activity in the pregnant mouse has been identified as a soluble form of the leptin receptor, most likely Ob-Re of placental origin [10]. Our recent observation that this isoform is by far the most prevalent expressed by the rat placenta (unpublished results) suggests that placental Ob-Re is also the major source of leptin-binding activity in rat pregnancy. The placenta does not appear to be the only source of plasma leptin-binding activity in the rat. Unlike the mouse, it was clearly present before and after pregnancy. Further studies are required to establish whether this activity is due to Ob-Re secretion by other tissues [23].
Elevated plasma leptin-binding activity in pregnancy has important physiological implications for both leptin action in target cells and leptin clearance from plasma. Thus, as observed for several other peptides such as corticotropin-releasing hormone [24] and the insulin-like growth factors [25], plasma binding proteins are thought to restrict exit of leptin into extravascular tissues, thus reducing the metabolic clearance rate of leptin [26] and its access to target cells [27]. Therefore, the progressive and marked increase in leptin-binding activity to a peak at Day 18 of pregnancy probably accounts for the rise in plasma leptin during pregnancy in the rat. However, plasma leptin then fell before parturition, as previously reported [12], despite a further increase in leptin-binding activity to Day 22. This finding strongly suggests that the production of leptin falls just prior to parturition, consistent with a reduction in fat mass at this time [28]. Alternatively or in addition, leptin clearance from maternal plasma may increase near term, possibly as a result of greater transplacental passage of leptin to the fetus. Kawai et al. [12] observed a marked rise in fetal leptin levels after Day 17 of gestation, and we recently observed that placental expression of Ob-Ra, the short form of the leptin receptor linked to leptin transport, increases in the rat placenta near term (unpublished results). This marked rise in Ob-Ra was specific to the placental labyrinth zone, the site of maternal-fetal exchange, consistent with increased transport of maternal leptin to the fetus at this late stage of pregnancy. This transport of leptin may be important for late fetal development. Leptin has recently been proposed as a key component of the complex hormonal regulation of fetal and placental growth [29–32], yet neither the fetus [33] nor the placenta [12, 34] appear to be significant sources of leptin in the rat. It is not clear whether the prepartum decline in maternal leptin, which has also been observed by others [12], is characteristic of species other than the rat. Thus, in humans, plasma leptin peaks around midgestation and thereafter declines only slightly if at all [16, 35]; in the baboon [36] and the mouse [10], maternal leptin increases progressively to late gestation, with no apparent prepartum fall.
Despite the prepartum decline in plasma leptin, food consumption did not increase; food consumption in this period was the lowest observed at any stage before, during, or after pregnancy, suggesting that pregnancy-induced leptin resistance disappears near term. This low level of food consumption may reflect the dynamic hormonal milieu of late pregnancy, which includes marked and rapid increases in pituitary secretion of prolactin and LH [37] and decreased ovarian secretion of progesterone [38]. Progesterone may be particularly important; it stimulates food consumption in nonpregnant rats [39], and its prepartum fall may contribute directly to reduced food consumption. The hormonal milieu during the prepartum period is very similar to that of proestrus, at least in terms of pituitary gonadotropin secretion, with postpartum ovulation occurring immediately after birth. Thus, similar hormonal signals may account for the decline in food consumption observed at proestrus. As previously reported [40], plasma leptin levels remained low after birth, although they did not appear to fall below those of prepregnancy values. Therefore, the hyperphagia of lactation almost certainly involves more than changes in leptin signalling, particularly because hypothalamic Ob-Rb expression remains unchanged. Among a large number of potential stimuli, direct activation of orexigenic signals, such as neuropeptide Y, by the suckling stimulus [41, 42] is likely to be of particular importance.
Hypothalamic Ob-Rb mRNA and protein expression was very stable before, during, and after pregnancy, with the notable exception of an approximate 2-fold increase in Ob-Rb mRNA early in pregnancy. The stimulus for the rise in Ob-Rb mRNA expression is not known, but the subsequent decrease may reflect the rising level of estrogen by midgestation [17]. Estrogen suppresses Ob-Rb expression in the nonpregnant rat [18] but stimulates adipose tissue expression of leptin mRNA [43]. Regardless of the precise stimulus for the transient peak in Ob-Rb expression, it is clear that when leptin resistance became most apparent after midgestation, hypothalamic Ob-Rb expression was stable. Garcia et al. [44] previously reported that hypothalamic Ob-Rb expression fell slightly from before pregnancy to Day 18, but we did not observe any such change. Although the reason for this discrepancy is not clear, the more complete gestational profile of hypothalamic Ob-Rb expression measured by both real-time quantitative reverse transcription PCR and Western blot analyses in the present study indicates that changes in absolute levels of Ob-Rb expression do not account for leptin resistance during pregnancy. However, the increase in hypothalamic Ob-Rb in early pregnancy could potentially reset the sensitivity of the hypothalamus to leptin, such that the subsequent decline in Ob-Rb expression could contribute to leptin resistance. Moreover, changes downstream of leptin/Ob-Rb interaction or a decrease in leptin transport across the blood brain barrier could potentially occur during pregnancy, and a contribution of central mechanisms to pregnancy-induced leptin resistance cannot be excluded. During lactation, Ob-Rb expression remained relatively stable and was similar to that observed in cycling rats and those in the late stages of pregnancy. This finding is consistent with a previous report that Ob-Rb mRNA expression measured by in situ hybridization was similar between cycling and lactating rats in most areas of the hypothalamus, including the arcuate nucleus [45]. These observations further support the suggestion that the hyperphagia of lactation is driven primarily by signals other than altered leptin signalling.
The results of this study support the hypothesis that rat pregnancy is a state of leptin resistance, as determined from relative changes in food consumption and plasma leptin levels. This appearance of leptin resistance is mediated, at least in part, by increased plasma leptin-binding activity rather than changes in hypothalamic Ob-Rb expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the National Health and Medical Research Council of Australia (project grant 139104). J.T.S. was supported by an Australian Postgraduate Research Award. 
2 Correspondence: Brendan J. Waddell, School of Anatomy and Human Biology, University of Western Australia, 35 Stirling Hwy, Crawley, Perth, Western Australia 6009, Australia. FAX: 61 8 9380 1051; bwaddell{at}anhb.uwa.edu.au
Accepted: January 10, 2002.
Received: December 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxypregn-4-en-3-one on Day 16 and the morning and afternoon of Day 22 of pregnancy in the rat. Biol Reprod 1989; 41:990-996[Abstract]This article has been cited by other articles:
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  R. A. Augustine, S. R. Ladyman, and D. R. Grattan From feeding one to feeding many: hormone-induced changes in bodyweight homeostasis during pregnancy J. Physiol., January 15, 2008; 586(2): 387 - 397. [Abstract] [Full Text] [PDF]
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T. Josephs, H. Waugh, I. Kokay, D. Grattan, and M. Thompson Fasting-induced adipose factor identified as a key adipokine that is up-regulated in white adipose tissue during pregnancy and lactation in the rat J. Endocrinol., August 1, 2007; 194(2): 305 - 312. [Abstract] [Full Text] [PDF]
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S. R. Ladyman and D. R. Grattan Suppression of Leptin Receptor Messenger Ribonucleic Acid and Leptin Responsiveness in the Ventromedial Nucleus of the Hypothalamus during Pregnancy in the Rat Endocrinology, September 1, 2005; 146(9): 3868 - 3874. [Abstract] [Full Text] [PDF]
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J. T. Smith and B. J. Waddell Leptin Distribution and Metabolism in the Pregnant Rat: Transplacental Leptin Passage Increases in Late Gestation but Is Reduced by Excess Glucocorticoids Endocrinology, July 1, 2003; 144(7): 3024 - 3030. [Abstract] [Full Text] [PDF]
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