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Endocrinology Vol. 148, No. 12 5977-5983
Copyright © 2007 by The Endocrine Society

Prolactin/Leptin Interactions in the Control of Food Intake in Rats

Lindsay Naef and Barbara Woodside

Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, Canada H4B 1R6

Address all correspondence and requests for reprints to: Barbara Woodside, Center for Studies in Behavioral Neurobiology, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, Canada H4B 1R6. E-mail: barbara.woodside{at}concordia.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
 References
 
Recent evidence suggests that the peptide hormone prolactin (PRL) modulates energy balance through a number of mechanisms, including acting in the brain to increase food intake. In the current studies, we first demonstrated that chronic infusions of PRL into the lateral ventricles increased food intake in cycling rats without disrupting estrous cyclicity. In subsequent experiments the hypothesis that at least part of PRL’s ability to increase food intake resulted from PRL-induced leptin resistance was tested. Female rats given chronic infusions of PRL (5 µg/h) into the cerebral ventricles for 10 d did not show a reduction in food intake or body weight after a central injection of 4 µg murine leptin, whereas the expected reduction in both of these parameters was seen in vehicle-infused rats. Leptin injections were without effect on these parameters, whether they were administered to free feeding PRL-infused rats or after 24-h food deprivation. This lack of a behavioral response to leptin was accompanied by an attenuation in Fos induction and phosphorylation of signal transducer and activator of transcription 3 after leptin administration in PRL-infused rats in both the ventromedial hypothalamus and paraventricular hypothalamic nucleus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
 References
 
THE PEPTIDE hormone prolactin (PRL) is found throughout vertebrate species, and has been implicated in a wide range of physiological systems, including osmoregulation (1) and immune function (2). In mammals, the actions of PRL have been studied most intensively in the context of reproduction, in which it has been shown to play critical roles in both pregnancy (3) and lactation (4). In rodents, PRL acts centrally to stimulate the onset of maternal behavior (5), and a central action of PRL in the modulation of the stress axis has been proposed (6).

The presence of PRL receptors (PRLRs) in both white and brown adipose tissue, liver (7), and pancreas (8), as well as in brain areas associated with the regulation of energy balance such as the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and paraventricular hypothalamic nucleus (PVN) (9, 10, 11, 12), raises the possibility that PRL is also involved in energy balance (13). However, evidence for a role for PRL in energy balance from studies of mice with targeted deletion of either the PRL or PRLR gene is inconsistent. A reduction in adiposity in PRLR-knockout mice (14) has been reported, although no metabolic phenotype was observed in PRL-knockout mice (15). However, PRLR activation has been implicated in the development of islet cells (16), the increase in islet cell mass observed during pregnancy (8), and the stimulation of proinsulin gene expression (17). In addition, PRL decreases insulin-stimulated leptin release from brown adipose tissue (18) as well as adiponectin secretion from white adipose tissue (19).

PRL administration increases food intake in both birds (20) and mammals (21), although the effects in female rats differ as a function of route of administration. Systemic administration of PRL disrupts estrus cyclicity, as well as increasing food intake and weight gain (22, 23). However, PRL infusions into the lateral ventricle increase food intake in the absence of any significant effect on body weight and without disrupting estrous cyclicity (23, 24), suggesting that the orexigenic effects of PRL are not accomplished simply by removing the anorectic effects of estrogen. In support of this conclusion, central PRL administration increases food intake in ovariectomized rats (24).

The results of these studies point to a central action of PRL on food intake, but the mechanisms through which this effect is produced are not clear. Recently, Ladyman and Grattan (25, 26) have reported that the ability of central leptin administration to reduce food intake was eliminated in pregnant rats on d 14 after conception, suggesting a state of leptin resistance during the second half of pregnancy. They also showed that this reduction in the behavioral effects of leptin was accompanied by a decrease in the induction of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in the VMH after leptin administration in late pregnant rats. Pituitary PRL levels are low during late pregnancy in rats, but placental lactogen levels are high (27). Like PRL itself, placental lactogens cross the blood-brain barrier and can activate PRLR (27), and it is possible that chronic activation of this receptor is associated with leptin resistance. The long form of the PRLR, like that of the leptin receptor, is coupled to the Janus kinase/STAT signaling pathway (28). Activation of both receptors leads to the phosphorylation of STAT proteins (29, 30) and protein transcription, including of the suppressors of cytokine signaling (SOCS) proteins that in turn act to inhibit further phosphorylation of STAT proteins (31, 32). Suppression of STAT phosphorylation after PRLR activation is one route through which PRL might suppress leptin receptor signaling.

Together, these data raise the possibilities first, that PRLR activation plays a role in the leptin resistance of late pregnancy and, second, that the ability of PRLR activation to suppress the effects of leptin might contribute to the hyperphagic effects of PRL. In the following experiments, we tested the second of these hypotheses. In experiment 1 we investigated the effects of chronic infusions of PRL into the lateral ventricles on food intake, weight gain, and estrus cyclicity in female rats. In the second experiment, the sensitivity of PRL-infused rats to the anorectic effects of leptin was investigated in both satiated and food-deprived states. Finally, to begin an investigation of where PRL/leptin interactions might be occurring, the effect of chronic PRL infusion on the ability of leptin injections to induce the intracellular signaling molecules, i.e. Fos and pSTAT3 in the arcuate and ventromedial, supraoptic, and paraventricular nuclei of the hypothalamus, was examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
 References
 
Subjects
Virgin female Wistar rats weighing between 220 and 240 g at the start of the experiments were the subjects. Rats were individually housed in polypropylene cages (45 x 25 x 20 cm) with ß-chip bedding, were maintained on a 12-h light/dark cycle (lights on at 0800 h, lights off at 2000 h), and had free access to food (powdered rat chow; Agway Ltd., Syracuse, NY) and water throughout the experiment. The room was maintained at a temperature of 20 ± 2 C. All procedures used in this experiment were approved by the Concordia University Animal Research Ethics Committee under the Guidelines of the Canadian Council on Animal Care.

Hormones
Ovine PRL (Sigma-Aldrich Canada Ltd., Ontario, Canada) was dissolved in NaHCO3, and buffered with HCl to a pH of approximately 7 to achieve final dilutions of 2, 5, and 10 µg/µl. Leptin (PeproTech, Inc., Rocky Hill, NJ) was dissolved in dH2O and buffered with a 1 M Tris solution to achieve a final dilution of 1 µg/µl.

Surgeries
Rats were anesthetized using ketamine-xylazine (5.7 mg ketamine and 0.86 mg xylazine/100 g body weight). A 22-gauge T-shaped cannula was implanted in the lateral ventricle using the following coordinates: anteroposterior, 0:00; lateral, 0:16; and ventral, 0:50 (33). The cannula was anchored to the skull with four stainless steel screws and dental acrylic. In experiments 2 and 3, a T-shape cannula with an injection port was used so that leptin could be injected through the same cannula as the PRL was infused. The cannula was connected via polyethylene tubing to an osmotic minipump (Alzet model no. 2001 for experiment 1 and model no. 2002 for experiments 2 and 3; Alzet) filled with either saline or PRL, which was then inserted sc in the neck region.

Procedure
Experiment 1.
Food intake, body weight, and vaginal cytology were recorded daily throughout the experiment. Only rats that showed regular 4-d estrous cycles before surgery were included in the experiment. Surgery took place after at least 10 d of baseline data collection, when rats were in the metestrus stage of the estrous cycle. Rats were assigned to one of the four treatment groups (2 µg PRL/1 µl·h, 5 µg PRL/1 µl·h, 10 µg PRL/1 µl·h, and vehicle 1 µl/h), matched for weight and food intake. Food intake, body weight, and estrous cyclicity were recorded for the ensuing 7 d.

Experiments 2a and 2b.
Baseline and surgical procedures, as well as data collection, were as for experiment 1, except that 14-d osmotic minipumps with an infusion rate of 0.5 µl/h and one dose of PRL (5 µg of PRL/h) were used. In experiment 2a, animals received an intracerebroventricular (icv) injection of 4 µl saline, or 4 µg leptin in a volume of 4 µl, 1 h before the start of the dark cycle on d-10 infusion. In experiment 2b, animals were food deprived beginning 1 h before lights off on d-9 infusion and received an icv injection of 4 µl saline or 4 µg leptin 1 h before the start of the dark cycle on d 10. Each injection was given over a period of 4 min. After the injection was completed, the injector was left in place for a further 2 min to ensure that the drug was completely deposited into the target site. Food was returned to cages at the start of the dark cycle on the day of injections. In both experiments, rats were habituated to the injection procedure by removing the blocker from the cannula and inserting the injector for 2 min on the 2 d before the test day. Care was taken to ensure that there was no systematic variation between groups on the day of the cycle on which leptin injections were given.

Experiment 3.
Procedures were as described in experiment 2a, except that 30 min after leptin injection, half the rats in each group were perfused, and brains were collected and subsequently processed for pSTAT3 immunohistochemistry. The remaining rats were perfused 75 min after leptin injection and subsequently processed for Fos immunohistochemistry. Blood samples were obtained from another cohort of rats that received either chronic infusions of PRL or vehicle but were not injected on the final day.

Leptin assay.
Plasma leptin levels were obtained by ELISA using a kit obtained from LINCO Research, Inc. (St. Charles, MO). All samples were run in one assay with an intraassay variability of less than 10%.

Fos immunohistochemistry.
Each animal was injected with an overdose of sodium pentobarbital and perfused transcardially with 200 ml ice-cold saline, followed by 300 ml 4% paraformaldehyde. Brains were postfixed in a 30% sucrose-4% paraformaldehyde solution for 48 h. Forty-micrometer thick sections throughout the supraoptic nucleus (SON) paraventricular nucleus PVN, ventromedial nucleus of the hypothalamus (prosthetic valve thrombosis), and ARC were obtained and processed for Fos-like immunoreactivity (Fos-lir). The sections were incubated for 30 min in 3% H2O2 solution in Tris-buffered saline (TBS) to reduce nonspecific staining and then washed in TBS three times for 15 min. Tissue was then incubated for 90 min at 4 C in blocking serum (0.3% Triton X-100; Sigma-Aldrich, St. Louis, MO), 3% normal goat serum (NGS) (Vector, Ontario, Canada), and TBS, and then incubated in the primary antibody solution (Fos antibody, Ab-5, PC-38; Oncogene Research Products, Boston, MA) diluted 1:100,000. Forty-eight hours later, sections were washed three times in TBS for 15 min and then were incubated in secondary antibody (biotinylated rabbit antigoat; Vector) for 1 h, washed three times in TBS, and incubated in ABC reagent (Vector) for 2 h. Tissue staining was developed using an ABC Vector kit (diaminobenzene, nickel intensified). The sections were then mounted on gelatin slides and coverslipped (Permount; Fisher Scientific, Quebec, Canada) for analysis.

pSTAT3 immunohistochemistry.
Brains were collected as described previously, except that a 4-h postfixation in 4% paraformaldehyde followed by 36 h in 30% sucrose was used. Thirty-microgram floating sections of the hypothalamic areas of interest were incubated in 1% H2O2 and 1% NaOH in PBS for 20 min, 0.3% glycine for 10 min, and 0.03% sodium dodecyl sulfate for 10 min. After 3 x 5-min washes in PBS, sections were incubated for 1 h at room temperature in blocking solution (4% NGS in 0.25% Triton X-100 and 0.02% sodium azide in PBS) and overnight at 4 C in primary antibody directed against pSTAT3 (1:1000 dilution; Cell Signaling Technology, Inc., Beverly, MA) in blocking solution. The following day, sections were washed (3 x 5 min), incubated in 3% NGS and secondary antibody (biotinylated goat-antirabbit; Vector) in blocking solution without sodium azide for 1 h, and washed. Next, sections were incubated in ABC reagent (Vector) for 2 h and developed with a diaminobenzene Vector kit.


   Image analysis
Top
 Abstract
 Introduction
 Materials and Methods
 Image analysis
Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
 References
 
Sections were visualized using a Sony XC77 camera (Sony Corp., Kanagawaken, Japan) mounted on a light microscope (Labolux Leitz GMBH, Wetzlar, Germany). Images were captured using National Institutes of Health Image Analysis Software (1.60 b) (Bethesda, MD) installed on a Power Macintosh computer (G4; Apple Computer, Inc., Cupertino, CA). Fos-lir and pSTAT3-stained cells were counted from the images captured in the Tagged Image File Format picture files. To obtain an estimate of Fos-lir and pSTAT3, expression in SON, stained cells in sections from 0.80 to –0.92 mm from bregma (33) were counted, and an average of these sections was calculated. For the PVN, only sections in the medial area of the nucleus (–1.80 mm from bregma) were used, and an average over all sections was obtained. To obtain an estimate of the number of Fos-lir, pSTAT3 in the ARC and VMH sections from –2.56 to –2.80 mm from bregma were counted, and the mean number of stained cells per section was calculated. To identify stained cells, sections from each experimental group across each assay that appeared to be stained were selected, and the relative density of each cell was recorded. An average of the density of the cells was then calculated to establish the criterion for each brain region across all experimental groups. The observer counting cells was blind to group membership.


   Assessment of cannula placement
Top
 Abstract
 Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
Statistical analysis
 Results
 Discussion
 References
 
The correct placement of the cannula was assessed in experiments 1 and 2 by icv injection of angiotensin (50 ng in 2 µl) on the day after the end of the experiment. If the injection of angiotensin stimulated rapid drinking, then the cannula placement into the lateral ventricle was considered accurate. In experiment 3, correct cannula placement was assessed histologically. Only data from rats with accurate placement of the cannula were included in the analysis.


   Statistical analysis
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 Abstract
 Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
Results
 Discussion
 References
 
Data from experiment 1 were analyzed using a one-way ANOVA for independent groups. Where appropriate, pairwise post hoc tests were performed using Fisher’s least significant difference. Data from experiments 2 and 3 were analyzed using 2 x 2 ANOVAs for independent groups.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
Discussion
 References
 
Experiment 1: effects of chronic PRL infusion on food intake and estrous cyclicity
Average daily food intake during the treatment phase for all four groups is shown in Fig. 1Go. As this figure illustrates, PRL treatment increased food intake [main effect of group F(3, 22) = 4.975; P = 0.009]. Post hoc analyses using Fisher’s least significant difference further revealed that each of the PRL-treated groups had a higher food intake than the vehicle-treated group. There were no significant differences in food intake between PRL-treated groups. In contrast, PRL administration had no effect on weight gain [F(3, 23) = 0.828; P = 0.462]. Rats in all groups displayed normal 4-d estrous cycles throughout the experiment.


Figure 1
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  		FIG. 1. Chronic icv infusions of PRL at doses of 2, 5, and 10 µg/h increase food intake (upper panel) but do not change body weight (lower panel). *, P < 0.05 compared with vehicle (veh)-infused group (n = 6 per group).

 
Experiment 2
Experiment 2a: effect of leptin injections in PRL-infused sated rats.
As Fig. 2Go (top panel) shows, leptin administration reduced food intake in vehicle-infused but not in PRL-infused rats. Analyses of these data yielded a significant interaction between the chronic and acute treatments [F(1, 22) = 4.09; P < 0.05]. A similar pattern of results was observed for weight change after leptin injection (Fig. 2Go, bottom panel). However, in this case the ANOVA showed significant main effects of both chronic (vehicle vs. PRL) and acute (vehicle vs. leptin) treatment [F(1, 22) = 4.45, P < 0.05; F(1, 22) = 4.92, P < 0.05, respectively] and no significant interaction effect.


Figure 2
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  		FIG. 2. Chronic infusion of PRL (5 µg/h) for 10 d eliminates the effects of leptin injection (4 µg) on food intake (upper panel) and body weight change (lower panel) in satiated rats. *, P < 0.05 compared with Sal/Sal group. Sal/Sal, n = 7. Sal/Lep, n = 6. PRL/Sal, n = 6. PRL/Lep, n = 7. Sal, Saline; Lep, leptin.

 
Experiment 2b: effect of leptin injections in PRL-infused food deprived rats.
As shown in Fig. 3Go, the effects of leptin injection in saline and PRL-infused rats after 24-h food deprivation were similar to those observed in sated rats. Leptin produced the expected suppression of food intake and loss of body weight in saline-infused rats but was not effective in rats that were chronically infused with PRL. Analyses of these data revealed significant main effects of chronic PRL infusion [change in food intake, F(1, 23) = 8.19, P < 0.05; weight change, F(1, 23) = 13.02, P < 0.05] and acute leptin injection [change in food intake, F(1, 23) = 18.83, P < 0.05; weight change, F(1, 23) = 7.51, P < 0.05], as well as a significant interaction between these factors [change in food intake, F(1, 23) = 12.12, P < 0.05; weight change, F(1, 23) = 5.98, P < 0.05].


Figure 3
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  		FIG. 3. Chronic infusion of PRL (5 µg/h) for 10 d eliminates the effects of leptin injection (4 µg) on food intake (upper panel) and body weight change (lower panel) after 24-h food deprivation rats. *, P < 0.05 compared with Sal/Sal group. Sal/Sal, n = 5. Sal/Lep, n = 6. PRL/Sal, n = 7. PRL/Lep, n = 8. Sal, Saline; Lep, leptin.

 
Experiment 3
Plasma leptin levels.
Chronic PRL infusion did not affect circulating leptin levels (vehicle mean 2.89 ± 0.19 ng/ml; PRL mean 2.99 ± 0.37 ng/ml).

Fos-lir.
As shown in Fig. 4Go, leptin administration induced a robust expression of Fos-lir in both the ARC [top panel, significant main effect for leptin injection; F(1, 15) = 8.7; P < 0.05] and VMH [middle panel, significant main effect for leptin injection; F(1, 15) = 4.94; P < 0.05]. There was less Fos-lir expression in the VMH after leptin injection in PRL-infused rats, although the interaction between PRL and leptin treatments did not reach statistical significance. There was no significant difference in Fos-lir between saline- and PRL-infused rats in either the ARC or VMH.


Figure 4
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  		FIG. 4. Induction of Fos-lir by leptin injection in ARC (upper panel), VMN (middle panel), and PVN (lower panel) in rats pretreated with chronic infusions of vehicle or PRL (n = 3–5 per group) *, P < 0.05 significant difference between leptin and vehicle-treated groups. Lep, Leptin; Sal, saline.

 
In the PVN, leptin injection tended to increase Fos-lir in saline but not in PRL-infused rats (Fig. 4Go, bottom panel). However, these effects did not reach statistical significance. There were no statistically significant effects of either PRL or leptin treatments on Fos-lir expression in the SON.

pSTAT3 immunoreactivity.
Leptin injection increased pSTAT3 immunoreactivity in the ARC, VMH, and PVN (Fig. 5Go). As with Fos expression, leptin injection induced less pSTAT3 in the VMH and PVN of PRL-infused rats than in vehicle-infused rats. Analysis of these data yielded a significant main effect for leptin treatment in all three areas [ARC F(1, 38) = 8.75, P < 0.05; VMH F(1, 37) = 30.75, P < 0.05; and PVN F(1, 32) = 8.65, P < 0.05]. The ability of chronic PRL infusion to attenuate pSTAT3 induction in the VMH and PVN after acute leptin injection was reflected in interaction effects that approached significance [VMH F(1, 37) = 3.329, P = 0.08; and PVN F(1, 32) = 2.76, P = 0.1].


Figure 5
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  		FIG. 5. Counts of pSTAT3-stained cells in ARC (upper panel), VMN (middle panel), and PVN (lower panel) in rats pretreated with chronic infusions of vehicle or PRL (n = 8–11 per group). *, P < 0.05 significant difference between leptin and vehicle treated groups. #, P < 0.08 between Sal/Lep and PRL/Lep groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
References
 
The results of experiment 1 show that chronic infusion of PRL into the lateral ventricle increases food intake in cycling rats without producing any change in weight gain or estrous cyclicity. These data are consistent with previous studies demonstrating that injections of PRL increase food intake in female rats (21, 23, 24) as well as with similar data in ringdoves (20), and suggest that PRL has orexigenic effects in a wide variety of species. They also emphasize the importance of the central actions of PRL in the control of ingestive behavior.

The lack of effect of central PRL infusions on weight gain is also consistent with our previous results showing that bidaily central injections of PRL increase food intake without increasing weight gain in female rats (23, 24), and suggests that PRL may act to modulate both energy intake and expenditure. The pattern of results seen after central injections or infusions of PRL contrasts with that seen after systemic administration. The latter induces both an increase in food intake and increased weight gain (21, 22, 23), and, because this treatment also suppresses estrous cyclicity (23), it is has been suggested that some of the effects of peripheral PRL are mediated through reductions in circulating estrogen. Although the current studies do not examine potential PRL and estrogen interactions directly, the fact that there was no effect of central PRL infusions on estrous cyclicity and that we have previously shown that central PRL injections increase food intake in ovariectomized rats (24) suggests that such an interaction is unlikely to make a major contribution to the effects of PRL in the model used here.

The absence of a dose response relationship between central PRL infusion and increased food intake contrasts with previous results using bidaily central injections (24). This suggests that the doses used here were at the high end of the effective range and that in future studies the dose range should be extended to include lower doses.

The results of experiment 2 suggest that one mechanism through which PRL acts to increase food intake is by inducing leptin resistance. Rats given chronic infusions of PRL did not show the expected decrease in food intake or body weight after a central injection of leptin, whereas vehicle-infused rats did. The lack of response to leptin was seen in PRL-infused rats whether they were ad libitum fed or food deprived for 24 h before leptin injection, suggesting that endogenous levels of leptin do not influence this effect.

The ability of central leptin administration to decrease food intake has been shown to vary with reproductive state. Ladyman and Grattan (25) found that pregnant rats did not show a reduction in food intake after icv injections of leptin when tested on d 14 after conception, when placental lactogen concentrations are high and can activate PRLRs in the brain (27). Therefore, the ability of chronic PRLR activation to induce leptin resistance might be the mechanism through which leptin resistance is induced in late pregnancy. During lactation, suckling stimulation from the young stimulates pituitary PRL release, and PRL from the periphery can cross the blood-brain barrier and activate PRLRs within the brain (34, 35). Therefore, it is possible that lactation too is associated with a reduction in leptin sensitivity. Previous data have shown that lactating rats had only a transient reduction in food intake when leptin was infused from d 8–15 postpartum (36), but whether this reflects leptin resistance or increased activity in orexigenic pathways is not yet known. However, it is likely that the pattern of PRLR activation is critical in inducing leptin resistance. Ladyman and Grattan (25) have shown the twice daily surges of PRL that occur in early pregnancy are not sufficient to induce leptin resistance, although they are accompanied by an increase in food intake.

The results of experiment 3 indicate that leptin signaling, as reflected in the induction of Fos-lir and phosphorylation of STAT3, is reduced in the VMH and PVN of PRL-infused rats. Leptin injections induced Fos-lir and pSTAT3 in the ARC, VMH, and PVN, and PRL treatment attenuated staining in the VMH and PVN. The effects on pSTAT3-immunoreactivity observed in this experiment are similar to those obtained by Ladyman and Grattan (26), who found that leptin signaling was reduced in the VMH, but not the ARC, in the latter half of pregnancy at a time when central leptin injections do not reduce food intake.

Precisely how PRLR activation might suppress leptin signaling within these areas is not yet clear. The two most direct pathways would be through an effect of PRLR activation on leptin receptor density or its interference with the intracellular signaling cascade induced by leptin receptor activation. Although there is evidence that leptin receptor mRNA expression within the VMH is decreased in late pregnancy (26), thus far there is no evidence that hyperprolactinemic states are associated with a decrease in leptin receptors in the PVN. Activation of PRLR induces SOCS3 expression in the hypothalamus (37), and SOCS3 is able to suppress leptin signaling by preventing the phosphorylation of STAT3 (32). Either of these direct interactions between PRL and leptin depends on the colocalization of PRL and leptin receptor to the same neurons, and although some neurons within the VMH and PVN express PRLR (38), there is, thus far, no evidence that these neurons also express the leptin receptor.

Alternative mechanisms could involve a process in which PRLR activation results in the regulation of a mediator that in turn acts locally or more distally to modulate leptin signaling. PRL has been shown to interact with neuropeptides implicated in the control of energy balance in multiple ways. There is evidence that PRL administration can reduce corticotropin-releasing factor mRNA expression within the PVN of ovariectomized rats exposed to a stressor (39). In addition, suckling-induced PRL secretion increases neuropeptide Y (NPY) mRNA expression within the dorsomedial hypothalamus (40), and it has been suggested that these neurons then project to the PVN to stimulate food intake (41). In late pregnancy and lactation, when central PRLR activation is increased, there is also an increase in agouti-related peptide (AGRP) mRNA expression within the ARC (42, 43), and this peptide increases food intake, in part, by acting as an endogenous antagonist at the melanocortin 4 receptor (44). Interestingly, Buntin and colleagues (45, 46) have shown that PRL administration increases both AGRP mRNA and NPY mRNA expression in the ringdove. Whether such effects occur after PRL administration in the rat and whether they are secondary to the induction of leptin resistance or they represent a leptin-independent route through which PRL modulates neuropeptides involved in energy balance remains to be seen.

Pro-opiomelanocortin (POMC) and NPY neurons in the ARC are considered major sites for the action of leptin on food intake (47), and the phosphorylation of STAT3 and subsequent protein synthesis is a critical step in this pathway (48). Thus, it may seem somewhat surprising that the reduction in leptin’s ability to reduce food intake that occurs in PRL-treated rats is not accompanied by major changes in intracellular signaling in this nucleus. It is possible, of course, that PRL treatment does influence leptin signaling within the ARC, but through a pathway that involves neither Fos induction nor phosphorylation of pSTAT3.

However, despite the recent emphasis on the central role of the ARC in the control of energy balance, there is ample data to support a role for both the PVN and VMH in the control of food intake. The PVN is a projection site for NPY/AGRP neurons occurring from the ARC and NTS, as well as from POMC/CART neurons whose cell bodies are in the ARC. The PVN also has reciprocal connections with the VMH and LH, and contains melanocortin 4 receptors together with receptors for numerous other neurotransmitters and neuropeptides involved in the control of energy balance (49). Moreover, a previous study showed that bidaily PRL injections directly into the PVN, at doses ineffective when administered into the ventricles, significantly increase food intake in female rats (50).

Recent evidence has also underscored the importance of the VMH in the control of energy balance. Mice with a targeted deletion of the steroidogenic factor 1 gene have a malformation of the VMH and develop an obese phenotype (51), and mice lacking leptin receptors on steroidogenic factor 1 positive neurons within the VMH have a higher body weight and are more prone to diet-induced obesity (52). Moreover, data demonstrating an excitatory pathway between the medial VMH and POMC neurons within the ARC suggest that activity in the VMH can modulate anorectic pathways within this area (53). Interestingly, however, in our earlier study we showed that bidaily injections of PRL into the VMH did not increase food intake (50), suggesting that either the effect of PRL on leptin signaling in the VMH is not critical for PRL-induced increase in food intake or that this effect is mediated through a more distal site of PRLR activation.

In summary, PRL has multiple effects on energy balance, one of which is to act centrally to increase food intake. One correlate of PRL’s orexigenic effects is the induction of central leptin resistance and a reduction in the intracellular signals of leptin receptor activation within the VMH and PVN. Whether these are the primary sites at which PRL acts to attenuate leptin’s effects on food intake and body weight, and whether each area has a similar role in this effect remain to be clarified. However, these results do point to a novel mechanism through which PRL modulates energy balance and provide some insight into the connection between hyperprolactinemic states and metabolic disturbances.


   Footnotes
 
This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to B.W.). L.N. was the recipient of an Undergraduate Summer Research Fellowship from the Natural Sciences and Engineering Research Council of Canada.

Present address for L.N.: Douglas Hospital Research Centre, McGill University, Montreal, Canada H4H 1R3.

Disclosure Summary: The authors have nothing to declare.

First Published Online September 13, 2007

Abbreviations: AGRP, Agouti-related peptide; ARC, arcuate nucleus; Fos-lir, Fos-like immunoreactivity; icv, intracerebroventricular; NGS, normal goat serum; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; PRL, prolactin; PRLR, prolactin receptor; pSTAT3, phosphorylated signal transducer and activator of transcription 3; PVN, paraventricular hypothalamic nucleus; SOCS, suppressors of cytokine signaling; SON, supraoptic nucleus; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline; VMH, ventromedial hypothalamus.

Received April 11, 2007.

Accepted for publication September 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Image analysis
 Assessment of cannula placement
 Statistical analysis
 Results
 Discussion
 References
 

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D. R. Grattan
Fetal Programming from Maternal Obesity: Eating Too Much for Two?
Endocrinology, November 1, 2008; 149(11): 5345 - 5347.
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