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Centre for Neuroendocrinology and Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin 9001, New Zealand
Address all correspondence and requests for reprints to: Dr. David Grattan, Department of Anatomy and Structural Biology, University of Otago, P.O. Box 913, Dunedin 9001, New Zealand. E-mail: dave.grattan{at}anatomy.otago.ac.nz.
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Abstract |
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Introduction |
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Like other members of the class 1 cytokine receptor family, one of the major intracellular signaling mechanisms of the leptin receptor is a JAK/STAT pathway (JAK, Janus kinase; STAT, signal transducers and activators of transcription). In response to leptin, Ob-Rb rapidly becomes phosphorylated, a process which is mediated by JAK proteins that are associated with the leptin receptor (9, 10, 11). Cytoplasmic STAT3 proteins bind to the activated Ob-Rb, undergo tyrosine-phosphorylation induced by JAK2, then dimerize and translocate to the nucleus, where they bind to specific promoter sequences of target genes to regulate gene transcription. Leptin-induced translocation of STAT3 to the nucleus has been observed at high levels in neurons within the arcuate nucleus, the dorsomedial nucleus, and the ventromedial nucleus of the hypothalamus (12).
During pregnancy, the maternal body must support the growing conceptus as well as prepare for the subsequent demands of lactation. This is achieved by the development of a state of positive energy balance, mainly due to increased food intake. In the rat, food intake is increased by the second week of pregnancy and remains high throughout pregnancy until the day before parturition (13, 14, 15). Fat stores are also increased during pregnancy, in preparation for the increased energy demands of lactation (14). In rats, mice, and humans, leptin concentrations increase during pregnancy (16, 17, 18, 19, 20, 21). Despite elevated leptin concentrations, hyperphagia persists during pregnancy, suggesting a state of leptin resistance. As well as being obese, ob/ob mice are also infertile, and daily treatment of leptin can restore fertility in these mice (22). In pregnant ob/ob mice, food intake increases from midgestation despite daily leptin treatment, also consistent with the hypothesis that a state of leptin resistance develops during pregnancy (23).
The aim of this study was to directly test whether pregnant rats are resistant to the acute satiety action of leptin by measuring food intake after a single intracerebroventricular (i.c.v.) dose of leptin. Furthermore, to determine the mechanisms responsible for the hyperphagia observed during pregnancy in the presence of increased leptin concentrations, we examined whether hypothalamic STAT3 activation in response to leptin was altered during pregnancy.
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Materials and Methods |
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Food intake, fat mass, and serum leptin concentrations during pregnancy
Daily food intake, water intake, and body weight were measured in 11 individually housed rats for the duration of three estrous cycles and the subsequent 22 d of gestation. Food and water intake were not measured on the day of mating. To measure fat mass and leptin concentrations, groups of rats were killed by decapitation between 0800 and 1000 h on diestrus; d 7, d 14, and d 21 of pregnancy; and d 7 of lactation. Trunk blood was collected and allowed to clot at 4 C overnight, then serum was stored at –20 C until leptin concentrations were determined by RIA (Linco Research, Inc., St. Charles, MO). Total abdominal adipose tissue was dissected out and weighed.
Pattern of plasma leptin concentration during fasting and refeeding
Changes in plasma leptin concentrations during a period of fasting and refeeding were determined in both nonpregnant rats and midpregnant (d 12–14) rats to ensure that leptin concentrations decreased in response to fasting. Animals were anesthetized with halothane on d 11 of pregnancy or proestrus for the nonpregnant rats, and an indwelling atrial cannula of SILASTIC brand silicon tubing (Dow Corning Corp., Midland, MI; internal diameter 0.025 in., external diameter 0.047 in., Delgania Silicone) was placed in the right atrium via the right jugular vein. The end of the cannula was exteriorized through the skin between the scapulae so that it could be readily accessed for blood sampling. Blood sampling began at 1800 h on the day after surgery, and 300-µl samples were collected every 4 h for 72 h. At 1800 h on the second day of sampling, food was removed from the cages. Food was returned for the final 24 h of the sampling protocol. After collection of each sample, blood was centrifuged, and red cells were resuspended in an equivalent volume of sterile saline and replaced into the rat after the subsequent sample collection. Plasma samples were stored at –20 C until leptin concentrations were determined by RIA. All samples were measured in one assay, and the intraassay coefficient of variation was 11.8%.
Feeding response to i.c.v. leptin administration
The feeding response to i.c.v. leptin administration was determined in nonpregnant (diestrus), early pregnant (d 7), and midpregnant (d 14) rats. The i.c.v. cannulae (Plastics One, Roanoke, VA) were surgically implanted into diestrous and pregnant rats. Surgery took place on d 1 for the early pregnancy group and d 7 for the midpregnancy group. Animals were anesthetized with ip injections of 2% xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (80 mg/kg) and placed in a stereotaxic frame. A 23-gauge stainless steel guide cannula was implanted 1.3 mm lateral to the midline at bregma (0.0 anterior/posterior position) and 3 mm below the level of the skull. Cannulae were fixed to the skull with stainless steel screws and dental cement. The open end of the cannula was sealed with a plastic cap until the time of injection. After surgery, rats were housed individually; and food intake, water intake, and weight gain were monitored daily.
On d 6 or 13 of pregnancy, and on metestrus for the nonpregnant rats, food was removed from the cages, 1 h before the start of the dark phase. Rats were fasted to ensure that at the time of injection both pregnant and nonpregnant rats had similar low endogenous leptin concentrations. Twenty-four hours later, rats received an injection of either 4 µg leptin (recombinant mouse leptin obtained from Dr. A. F. Parlow, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) diluted in artificial cerebrospinal fluid (2 µg/µl) or vehicle into the left lateral ventricle using a 2-µl Hamilton syringe connected to a stainless steel injection cannula. The injection cannula was designed to protrude 2 mm beyond the tip of the guide cannula. The injection was carried out over a 30-sec period, and the injection cannula was left in position for an additional 30 sec. After the injection cannula was removed, the guide cannula was sealed to prevent leakage. One hour post injection, directly before the start of the dark phase, pre-weighed food pellets were returned to the rats. The amount of food consumed 3 h and 24 h after the injection was measured.
At the conclusion of the experiment, the placement of the cannula was confirmed. Rats received an injection of 2 µl of 0.1% methylene blue to the left lateral ventricle approximately 5 min before being killed by decapitation. The presence of dye in the third ventricle confirmed correct cannula placement.
STAT3 phosphorylation in response to i.c.v. leptin administration
As described above, i.c.v. cannulae were surgically implanted into diestrous and d-7 pregnant rats. On d 13 of pregnancy, and on metestrus for the nonpregnant rats, food was removed from the cages, 1 h before the start of the dark phase. Sixteen hours later, rats received either an injection of leptin or vehicle as described above. Thirty minutes after injection, rats were killed by decapitation. The brains were removed and immediately frozen on dry ice and stored at –80 C until the microdissection of hypothalamic nuclei using a micropunch technique (24). Frozen coronal brain sections (300 µm) were cut in a cryostat at –9 C, then thaw-mounted onto glass slides and refrozen. Five hypothalamic areas were punched from these sections using a 500-µm diameter micropunch needle (Table 1
). Tissue was placed in 45 µl of 62.5 mM Tris-HCl (pH 6.8) containing 1% sodium dodecyl sulfate (SDS) and Complete protease inhibitor (Roche Diagnostics, Mannheim, Germany). Additional protease inhibitors, phenylmethylsulfonyl fluoride (1 mM), and sodium orthovanadate (1 mM) were also present. Samples were briefly sonicated, then stored at –80 C. Protein concentration was determined by a modification of the Lowry method using a protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA).
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Statistical analysis
Statistical data were analyzed by one-way ANOVA except for the sequential plasma samples, where leptin concentration data were assessed by repeated-measures ANOVA. Fisher’s post hoc tests were applied where appropriate. The significance level for all statistics was set at P < 0.05. All data are presented as the mean ± SEM.
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Results |
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). On d 7 and 14 of pregnancy, serum leptin concentrations were significantly greater than that observed in nonpregnant rats (Fig 1B). By d 21 of pregnancy, the day before parturition, serum leptin concentrations had decreased to levels similar to the nonpregnant values. During lactation, leptin concentrations further decreased, to become significantly lower than nonpregnant levels. Using total weight of abdominal fat as a measure, fat mass was increased during pregnancy, being significantly higher than nonpregnant levels on d 14 and 21 of pregnancy (Fig 1C). Total abdominal fat mass on d 21 of pregnancy (11.8 ± 2.3 g) was almost double the amount in nonpregnant rats (6.9 ± 1 g). The amount of abdominal fat present on d 7 of lactation was not significantly different from nonpregnant values.
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). Fasting led to the suppression of the nocturnal increase in leptin, and plasma leptin concentrations were significantly decreased in all rats compared with prefasting levels (Fig. 2). Once food was returned, leptin concentrations increased, but the nocturnal surge did not occur in the following 24-h period of refeeding.
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). Injections were timed such that they were administrated 1 h before the end of fasting, at a time when endogenous leptin concentrations were still very low (see Fig 2
). Consistent with the data above, during the 24 h before fasting, midpregnant rats had significantly higher food intake levels compared with the nonpregnant rats. However, in contrast to the above results, early pregnant rats in this experiment did not have increased food intake, compared with nonpregnant rats. In nonpregnant rats, leptin treatment resulted in a significant reduction in food intake compared with vehicle-treated nonpregnant rats after 3 and 24 h. Similarly, in early pregnant rats, leptin treatment also resulted in significantly decreased food intake compared with the vehicle-treated d-7 pregnant rats. In the midpregnant rats, there was no difference in food intake between the leptin-treated and vehicle-treated rats at any timepoint. There was a significant increase between pre- and postfasting food intake levels in the vehicle-treated nonpregnant rats. However, in the vehicle-treated early pregnant rats, there was no significant increase between pre- and postfasting food intake levels. Also, on d 14 of pregnancy, the leptin- and vehicle-treated rats consumed similar amounts of food during the days before and after the 24-h period of fasting. Hence, the pregnant rats did not show a fasting-induced hyperphagic response.
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). Although not significantly different, vehicle-treated pregnant rats tended to have a higher level of basal STAT3 activation compared with vehicle-treated nonpregnant rats. In the VMH, leptin treatment led to a robust increase in STAT3 phosphorylation in nonpregnant rats, with a significantly smaller increase in the percentage of STAT3 that became phosphorylated in pregnant rats (Fig 5B). In contrast with the arcuate nucleus and the VMH, leptin treatment in the dorsomedial hypothalamus and lateral hypothalamus induced a similar degree of STAT3 activation in the pregnant and nonpregnant groups (Fig. 5, C and D). In the supraoptic nucleus, there was no difference in the levels of STAT3 in nonpregnant and pregnant rats, and leptin treatment did not induce STAT3 phosphorylation in this area in either group (results not shown).
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Discussion |
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In agreement with our results, it has been shown that during late pregnancy (d 17 onwards), daily i.c.v. infusion of leptin for 6 d does not decrease food intake until the day after parturition (27). Pregnancy d 14 was used in the present study because at this time, food intake, fat mass, and leptin concentrations are all significantly increased compared with nonpregnant values. Pregnancy d 7 was also used because our initial results suggested that food intake and leptin concentrations were significantly increased at this time compared with nonpregnant values. When early pregnant rats were used in a separate experiment to measure the feeding response after i.c.v. leptin administration, however, food intake was not significantly increased compared with nonpregnant controls. This discrepancy is also reflected in the literature, where previous studies report a wide range of timepoints during pregnancy at which food intake becomes significantly increased from nonpregnant levels (13, 14, 15, 26, 28, 29). These data, together with our observations, suggest that pregnancy-induced hyperphagia develops gradually and that there is a degree of variation as to when food intake becomes reliably increased compared with nonpregnant levels. The early pregnant rats showed a hypophagic response to leptin similar to that of the nonpregnant rats, indicating that the pregnancy-induced leptin resistance we observed on d 14 of pregnancy develops between d 7 and 14 of pregnancy. Based on our earlier observations, this state of leptin resistance appears to develop after food intake increases, suggesting that leptin resistance is not the primary cause of pregnancy-induced hyperphagia. However, the suppression of the satiety action of leptin during pregnancy would facilitate the increase in food intake during pregnancy.
In contrast to results obtained in rats, i.c.v. leptin administration to mice decreases food intake during all stages of pregnancy (30). Mice have increased levels of leptin-binding proteins in the blood during pregnancy, suggesting that leptin resistance in mice may be due to increased interactions with binding proteins rather than a central insensitivity to leptin (16). Although no increase in binding proteins has been seen in the rat (16), an increase in plasma leptin binding activity has been reported from midgestation (26). In other models of leptin resistance, such as diet-induced obese mice and age-related obese rats, defects in both transport of leptin into the brain and leptin signal transduction in the hypothalamus appear to contribute to leptin resistance (31, 32, 33). The leptin dose used in this study was clearly effective in nonpregnant rats and indeed is a maximal dose to reduce food intake and induce STAT3 phosphorylation (31, 34). Due to the delivery method, our results demonstrate a central resistance to leptin but do not rule out the possibility that peripheral sites may also contribute to the state of leptin resistance during midpregnancy. Although the early pregnant rats did not display a central resistance to leptin, it is possible that, at this time, there is a state of peripheral leptin resistance. The absence of any difference in food intake between leptin- and vehicle-treated rats by d 14 of pregnancy in the present study, however, indicates that this insensitivity to leptin at the central level is more than adequate to suppress the satiety actions of leptin during pregnancy. Redundancy in this system would not be unexpected, however, because maternal malnutrition during pregnancy has a number of negative consequences for the offspring, such as increased susceptibility to obesity, type 2 diabetes, and hypertension (35, 36).
Similar to nonpregnant rats, leptin concentrations during pregnancy displayed a noctural increase. Both light- and dark-phase leptin concentrations in pregnancy were increased compared with nonpregnant rats. Previously it has been reported that during the dark phase, there was no difference between leptin concentrations in pregnant and nonpregnant rats (27). The reasons for this inconsistency between results is unknown. In the present study, leptin concentrations were increased before a detectable increase in fat mass. This is consistent with previous results where leptin concentrations do not correlate with the amount of adipose tissue in pregnant rats, mice, and humans (16, 19, 20, 21, 37). The source of the increase in leptin concentrations, above that which can be accounted for due to increased fat mass, remains unknown. Although it has been reported that the placenta produces increasingly high levels of leptin during gestation (17, 18), a number of studies in both rats and mice have detected only minimal levels of leptin mRNA in the placenta (16, 19, 37). Furthermore, in vitro, the mouse placenta has not demonstrated the ability to secrete any detectable levels of leptin (38). In the present study, fasting induced significant decreases in plasma leptin concentrations in the pregnant rats as well as in the nonpregnant rats. This suggests that any placental secretion of leptin must be regulated in the same manner as that from adipose tissue. Alternatively, this observation is consistent with the hypothesis that the placenta does not contribute any significant amount of leptin to circulating concentrations in rats.
Both the early and midpregnant rats did not show a compensatory hyperphagic response when food was returned after 24 h of food deprivation. Little is known about the exact causes of the fasting-induced hyperphagic response that was observed in the vehicle-treated nonpregnant animals. The low levels of leptin during fasting (39) have been implicated in this increased hyperphagia, because the administration of leptin inhibits this response in fasted animals (40). It would appear that low leptin concentrations do not result in increased food intake in midpregnant rats, further supporting the idea that during pregnancy there is a state of insensitivity to changes in leptin concentrations.
To elucidate the mechanisms behind the observed pregnancy-induced central leptin resistance, we examined STAT3 phosphorylation after i.c.v. administration of leptin in d 14 pregnant rats. One of the major intracellular signaling pathways activated by leptin in the hypothalamus is a JAK/STAT pathway, and the phosphorylation of STAT3 is a vital step in this pathway (41). In the present study, we observed impaired leptin signal transduction through the JAK/STAT3 pathway in the hypothalamus on d 14 of pregnancy. This impaired signal transduction was limited to the VMH and arcuate nucleus. In the arcuate nucleus, leptin treatment increased pSTAT3 levels in the nonpregnant rats but not in the pregnant rats. There was less STAT3 protein in the VMH of the pregnant rats compared with the nonpregnant rats, and the percentage of STAT3 that became phosphorylated in the pregnant rats was less than that in the nonpregnant rats. In combination, these data demonstrated markedly less leptin-induced activation STAT3 in the VMH.
Our methods do not allow us to identify whether these changes occur in specific cell types; but previously, it has been shown that leptin-induced activation of the JAK/STAT3 pathway in the hypothalamus is limited to neurons and not glial cells (12). There are two main leptin-responsive neuronal populations in the arcuate nucleus involved in appetite regulation, the proopiomelanocortin (POMC) neurons and the neuropeptide Y/agouti-related protein (AgRP) neurons. The regulation of POMC and AgRP mRNA by leptin is mediated through the JAK/STAT3 intracellular signaling pathway (42). The suppressed STAT3 activation in the present study would suggest that this regulation of POMC and AgRP by leptin is impaired during pregnancy, but there is currently no direct evidence for this. However, AgRP has previously been implicated in pregnancy-induced hyperphagia, because mRNA for this neuropeptide is increased during pregnancy (29). Leptin normally has an inhibitory effect on AgRP mRNA expression (43), so the suppression of leptin-induced activation of STAT3 observed in the arcuate nucleus may help explain the increase in AgRP mRNA during pregnancy despite high leptin concentrations. A functional JAK/STAT pathway is critical to the regulation of POMC mRNA by leptin (42); therefore, our results of suppressed activation of STAT3 suggest that the satiety effect of leptin through the melanocortin system is likely to be reduced during pregnancy. In support of this is the decrease in 
-MSH, the anorexigenic peptide product of the POMC gene, that has been observed during pregnancy (44).
The VMH has long been recognized as one of the primary regions of the brain involved in suppression of appetite. Administration of leptin directly into the VMH leads to decreased food intake, even at very low doses that are unable to decrease food intake when administrated to the lateral ventricle (45). The decrease in the activation of the JAK/STAT3 pathway in the VMH during pregnancy potentially results in a decrease of this inhibitory effect of the VMH on appetite, consistent with the increased food intake seen during pregnancy.
The mechanisms suppressing leptin-induced STAT3 phosphorylation in both the VMH and arcuate nucleus during pregnancy are unknown. One possibility is that this decrease in activation may reflect a decrease in the receptor number in the hypothalamus, because both leptin receptor mRNA and protein levels have been shown to be down-regulated in obese, leptin-resistant rats (31, 46). However, there is conflicting evidence as to whether this occurs during pregnancy. Garica et al., 2000 (17), found a decrease in mRNA for the long form of the receptor in the hypothalamus during pregnancy, whereas others have demonstrated that mRNA levels and protein levels are not down-regulated (26, 29). Further work is required to clarify the levels of the leptin receptor in discrete hypothalamic nuclei during pregnancy, specifically in the VMH and the arcuate nucleus.
Pregnancy is associated with numerous changes in hormone secretion that are involved in the adaptation of the maternal body to the gestational conditions. It is likely that the changes in leptin sensitivity during pregnancy are induced by these hormonal changes that accompany pregnancy. Progesterone concentrations increase during pregnancy (47), and progesterone is a known stimulator of appetite and food intake (48, 49). Furthermore, the orexigenic effects of progesterone occur without changes to plasma leptin concentrations, suggesting that one mechanism by which progesterone may stimulate food intake is by modulating the sensitivity of the hypothalamus to leptin (49). Although the role of progesterone in the insensitivity to leptin observed during pregnancy in the present study is unknown, there is potential for an interaction between these signaling pathways, because progesterone receptors are found in both the VMH and arcuate nucleus (50, 51), the areas we found impaired leptin activation of the JAK/STAT pathway during pregnancy.
In conclusion, our results indicate that by d 14 of pregnancy, i.c.v. leptin administration is unable to suppress food intake as it does in nonpregnant rats, thus supporting the hypothesis that a state of leptin resistance develops during pregnancy. In addition, we have demonstrated that this state of leptin resistance is associated with a decrease in leptin-induced phosphorylation of STAT3 in target areas of the hypothalamus. In the arcuate nucleus, leptin treatment did not increase pSTAT3 levels in pregnant rats. In the ventromedial nucleus, there were lower levels of STAT3 in pregnant rats compared with nonpregnant rats, and leptin treatment lead to a smaller percentage increase in STAT3 phosphorylation in the pregnant rats. Although the mechanisms for the impairment of the JAK/STAT signaling pathway in the hypothalamus during pregnancy are yet to be determined, they appear to be site specific.
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Footnotes |
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Abbreviations: AgRP, Agouti-related protein; i.c.v., intracerebroventricular; JAK, Janus kinase; pSTAT, phosphorylated STAT; POMC, proopiomelanocortin; SDS, sodium dodecyl sulfate; STAT, signal transducer and activator of transcription; VMH, ventromedial hypothalamus.
Received March 16, 2004.
Accepted for publication May 3, 2004.
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-melanocyte-stimulating hormone during the gestational and postpartum periods in the rat. Proc Soc Exp Biol Med 177:318–326[Abstract]
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, significant with respect to prefasting leptin concentrations for each physiological group (P < 0.05).
