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APPETITE, OBESITY AND METABOLISM
Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, Quebec H4B 1R6, Canada
Submitted 10 March 2003 ; accepted in final form 12 August 2003
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ABSTRACT |
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 TOP ABSTRACT GENERAL METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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reproduction; food intake; postpartum; energy availability
Leptin, the protein product of the ob gene, is thought to be a signal by which the state of long-term energy stores is transmitted to the central nervous system (9, 14, 20) and hence one pathway through which the energetic state of the animal could control the reproductive axis. Circulating leptin levels correlate positively with the amount of adipose tissue and decrease during periods of food deprivation or restriction (12). Leptin gains access to the brain through an active transport mechanism (13), and leptin receptors have been localized in many brain areas, including those implicated in the control of food intake and reproduction (11). In addition to its role as a signal of adiposity, there is also evidence that leptin can modulate short-term signals of energy balance through stimulation of sympathetic outflow, increases in metabolic substrates, and glucose metabolism (17, 29, 30).
Support for a permissive role of leptin in reproduction comes from studies showing that exogenous leptin administration given during an acute fast can prevent the effect of food deprivation both on estrous cyclicity (2, 24) and on lactational infertility (35). The question of whether leptin's actions are mediated primarily by direct effects on the pathway integrating energy balance cues and the reproductive axis or indirectly through leptin's ability to increase the availability of oxidative fuels, however, is still a matter of debate (23, 24). For example, it has been demonstrated that, in hamsters, the ability of leptin to reverse food deprivation-induced anestrus is blocked by concurrent treatment with inhibitors of metabolic fuel utilization (26). These data are consistent with an extensive body of research demonstrating that inhibiting metabolic fuel utilization alone is sufficient to suppress the reproductive axis (see Ref. 32 for review).
In rats, lactation is accompanied by a decrease in circulating leptin levels and a period of infertility (15, 16, 36). Lowered leptin levels during lactation are not a necessary prerequisite for lactational infertility because preventing milk delivery in suckled rats results in hyperleptinemia but does not disrupt the anovulatory state (36). Moreover, chronic leptin administration has no effect on the length of lactational anovulation in ad libitum-fed animals (36). By contrast, the effects of a 48-h fast on days 13 and 14 postpartum, which both prolongs lactational infertility and causes a further drop in leptin levels, can be eliminated by exogenous leptin administration (35). Together these findings are consistent with the contention that leptin plays a permissive role in reproductive function in lactating rats, as it does in rats in other reproductive states, but that the effect of leptin on reproductive function only becomes apparent at levels below those seen during lactation in animals fed ad libitum.
To examine this possibility further, we used another model in which restricting food availability has been shown to prolong lactational infertility: chronic food restriction for the first 2 wk postpartum. We first compared circulating leptin levels between ad libitum-fed and chronically food-restricted rats and then tested the ability of chronic intracerebroventricular leptin administration to attenuate the effects of food restriction on lactational infertility. In contrast to the effects seen previously in acutely food-deprived lactating rats, exogenous leptin administration did not attenuate the prolongation of lactational infertility induced by food restriction. In experiment 3, we used the acute food deprivation model to determine the contribution of route of leptin administration and dose to the different results obtained.
Food-restricted females continue to deliver milk as reflected in continued litter growth, whereas litters nursed by food-deprived females lose weight dramatically even on the first day of deprivation. The hypothesis that the continued energetic demand of lactation detracted from the ability of leptin replacement to eliminate the effects of chronic food restriction on reproductive function was tested in experiment 4 by administering leptin intracerebroventricularly to food-restricted females prevented from delivering milk. Given the heavy energetic demands of lactation, this experiment allowed us to examine the interaction between energy expenditure and leptin in the control of reproductive function.
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GENERAL METHODS |
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TOPABSTRACTGENERAL METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Virgin female Wistar rats (Charles River Breeding Farms, St.-Constant, Quebec) weighing between 220 and 240 g at mating were used in these experiments. The animals were maintained in a 12:12-h light-dark cycle (lights on at 0800, lights off at 2000) at a room temperature of 20 ± 2°C. Animals were mated by group-housing them with one male. Three weeks later, the pregnant females were moved to individual plastic cages (20 x 45 x 50 cm) with Beta Chip bedding. The day of parturition was designated as day 0 postpartum, and litters were culled to eight pups on day 1 postpartum.
Food Intake and Body Weight Measures
Animals assigned to food-restricted conditions were given 50% of a previously ascertained ad libitum ration for each of the first 14 days postpartum. Animals in the food-deprived condition were completely deprived of food from 0900 on the morning of day 13 postpartum until 0900 on the morning of day 15 postpartum. Food intake was recorded daily until day 17 postpartum, the day at which the pups start to eat by themselves. Dam and litter weight were recorded daily throughout the experiment. Beginning on the first day of treatment, litters of animals that received leptin were switched daily with the litters of the vehicle animals to control for any effects of leptin on milk delivery.
Vaginal Cytology
Vaginal smears were obtained daily from all animals starting on day 4 postpartum. The smears were rated for the number of cornified epithelial cells by two independent judges. When 70% of the cells present on the slide were of the cornified type, the female was considered to be in estrus. This day marked the end of lactational diestrus.
Experiment 1: Effect of Chronic Food Restriction on Leptin Levels in Lactating Rats
Twenty-six females served as subjects in this experiment. Females were assigned to either the food-restricted (FR) or ad libitum-fed (AL) condition on day 1 postpartum. Blood samples were taken on either day 15 (AL: n = 6, FR: n = 8) or day 20 (AL: n = 6, FR: n = 6) postpartum.
Cannulation. Females were equipped with a jugular catheter 2 days before blood sampling. After anesthesia with a ketamine-xylazine mixture (6 mg ketamine and 0.86 mg xylazine/100 g body wt), a 26-mm length of Silastic tubing (Dow Corning, Midland, MI, OD = 0.047 in., ID = 0.025 in.) was introduced into the right jugular vein to the atrium of the heart, secured to the vein, and exteriorized between the scapulas. The cannula was filled with heparinized sterile saline (50 U heparin/1 ml), and the wound was rapidly sutured. A topical antibiotic (Cicatrin, Wellcome, Kirkland, Quebec, Canada) was applied to prevent infection at the wound site.
Sampling. The day before sampling, dams and their litters or nonlactating females were transferred to experimental cages (similar to housing cages) and kept in a quiet experimental room. The next morning (0800-0900), the Silastic jugular cannula was connected to 
70 cm (200 µl dead volume) of PE-50 tubing (Becton Dickinson, Parsippany, NJ), which was exteriorized out of the cage, and rats were left undisturbed with their litters for 2-3 h before sampling. Sampling was initiated between 1100 and 1200 when blood (300-400 µl) was withdrawn through the extension tubing. The sampling procedure lasted 3 min and did not appear to disturb the behavior of the animals, most of which slept or nursed their litters throughout the sampling process. Blood was collected on ice and centrifuged for 3 min at 13,000 rpm, and plasma was stored at -20°C before hormonal determination.
RIA. Plasma leptin concentrations were determined using a Rat Leptin RIA kit obtained from Linco Research (St. Charles, MO). Determinations for each sample were made in duplicate within one assay. Intra-assay variability was <8%, and nonspecific binding was <5%.
Statistical analyses. Maternal weight gain was calculated by subtracting the day 1 postpartum weight from the weight recorded on the test day (day 15 or day 20 postpartum). Litter growth was determined by calculating the mean daily increase in weight of litters nursed by each female either from days 1-15 or days 1-20, depending on group. Two-way (diet condition x day postpartum) ANOVA for independent groups was used to analyze these parameters. A one-way between-groups ANOVA was used to compare maternal body weight on day 1 postpartum.
Experiment 2: Ability of Intracerebroventricular Leptin Administration To Attenuate the Effect of Chronic Food Restriction on Lactational Infertility
Experiment 2a: effects of intracerebroventricular leptin administration from days 8-15 postpartum. In this experiment, females and their litters were assigned to one of three groups on day 1 postpartum: a low leptin group (n = 8) that received continuous intracerebroventricular infusions of leptin (1 µg/day) from day 8 to day 15; a high leptin group (n = 6) that received intracerebroventricular infusions of leptin (10 µg/day) for a similar time period; and a vehicle-treated group (n = 7) that received infusions of vehicle alone. All groups were food restricted from day 1 to day 14 postpartum. Litters of females in the leptin groups were switched daily with those of females either in the vehicle group or in a group of food-restricted nonoperated controls.
INTRACEREBROVENTRICULAR CANNULA AND MINIPUMP IMPLANTATION. Twenty-two gauge L-shaped cannulas (Plastic One, Richmond, VA) were used and connected to the osmotic minipumps by plastic tubing (PE-50, ID = 0.69 mm, OD = 1.14 mm). Before implantation, osmotic minipumps filled either with vehicle or leptin were connected to the cannulas with a 4-cm length of plastic tubing and primed by being placed in a tube filled with sterile saline in a Haake incubator at 37°C for 6 h.
On day 8 postpartum, cannulas were implanted into the right lateral ventricle under ketamine-xylazine anesthesia (6 mg of ketamine and 0.86 mg of xylazine/100 g body wt). After being anesthetized, the animals were placed in the stereotaxic apparatus. The scalp was shaved slightly posterior to the eyes up to the base of the skull. A sagittal incision 2.5 cm in length was made, the skin was retracted, and the skull was exposed and cleaned. The cannulas were aimed at the lateral ventricle (coordinates: anteroposterior 0.02 cm, lateral 0.16 cm, dorsoventral 0.5 cm with incisor bars 5 mm above interaural line) and were held in place with jeweler screws inserted in the cranium and dental cement. On solidification of the dental cement, the osmotic minipumps were inserted subcutaneously in the neck region. Sutures were used to close the wound. Antibiotic powder Cicatrin (Wellcome) was applied to prevent infection and facilitate healing.
OSMOTIC MINIPUMP REMOVAL. Seven days after implantation, the osmotic minipumps were removed under Metofane (Janssen Pharmaceutical, Mississauga, Ontario, Canada) anesthesia. In those animals receiving central infusions, the plastic tubing connecting the osmotic minipump and the intracerebroventricular cannula was cut, and the free end of the plastic tubing was sealed to prevent infections. The wound was closed using Autoclip (Becton Dickinson, Sparks, MD) surgical staples. Cicatrin was applied.
HISTOLOGY. Correct placement of intracerebroventricular cannula was determined by histological examination. After completion of the experiment, females were euthanized using CO2. The animals were decapitated, and their brains were extracted and stored in a solution of 10% formalin for 4 days. The brains were sliced into 50-µm sections with the aid of a vibratome and mounted on gelatin-coated microscope slides. The sections were stained with cresyl violet, and the tips of the cannulas were located. Animals in which the cannulas were found to be misplaced were removed from all subsequent analyses.
STATISTICAL ANALYSES. Pretreatment litter growth was determined by calculating the mean daily increase in weight of litters nursed by each female from day 1 to day 7. Both this parameter and maternal weight on day 1 postpartum were analyzed using a one-way ANOVA for independent groups. A two-way (diet condition x day postpartum) ANOVA for independent groups was used to compare length of lactational infertility among groups. Maternal weight gain for the pretreatment phase was calculated by taking the difference between the day 1 postpartum weight and that recorded on day 8 postpartum for each female, the treatment phase weight change was calculated by taking the difference between the day 15 and day 8 weight, and that for the posttreatment phase was calculated by subtracting the day 15 postpartum weight from that obtained on day 22 postpartum. A two-way mixed ANOVA with groups as the between-subject variable and phase of the experiment as the within-subjects variable was used to analyze these parameters. Weight gain of litters nursed by females within each treatment condition was analyzed using a two-way mixed ANOVA with groups as the between-subject variable and days of treatment as the within-subjects variable.
Experiment 2b: effects of intracerebroventricular leptin administration from day 15 to day 22 postpartum. The design and methods used in this experiment were identical to those for experiment 2a with the exception that cannulas and minipumps were inserted on day 15 postpartum and removed on the morning of day 22 postpartum, and because this surgery occurred close to the expected date of estrus termination, a nonoperated control group was included. Each group contained eight females and litters, with the exception of the lowleptin group that contained only seven. Statistical analyses were as for experiment 2a.
Experiment 3: Comparison of Effects of Intracerebroventricular and Intraperitoneal Leptin Administration on Length of Lactational Infertility in Food-Deprived Lactating Rats
In this experiment, we compared the effect of administering leptin by intraperitoneal injection (2.5 mg·kg-1·day-1) and via intracerebroventricular infusion (10 µg/day) during a 48-h fast on days 13 and 14 postpartum on the duration of lactational infertility.
Lactating females and their litters, adjusted to eight pups in number, were assigned to one of four groups: ad libitum-fed, vehicleinfused (AL ICV-VEH, n = 5); food-deprived, vehicle-infused (FD ICV-VEH, n = 5); food-deprived, leptin-infused (FD ICV-LEP, n = 5); and food-deprived, intraperitoneal leptin (FD IP-LEP, n = 5).
Intracerebroventricular drug administration. Intracerebroventricular drug administration was as described in experiment 2a, with the exception that animals underwent surgery on day 12 postpartum and pumps were removed on day 15 postpartum.
Systemic drug administration. Murine leptin (Preprotech) was mixed in Tris-buffered saline to a dilution of 1.25 mg/ml (pH 7.35) and administered intraperitoneally at a dose of 1.25 mg/kg every 12 h during the deprivation period.
Statistical analysis. Length of lactational infertility was compared among groups using a one-way ANOVA for independent groups. Maternal weight change and litter weight gain data were analyzed using two-way mixed ANOVA with group as the between-subject variable and days as the within-subject variable. Postfast food intake (day 15 postpartum) was compared among groups using a one-way ANOVA for independent groups.
Experiment 4: Ability of Intracerebroventricular Leptin Administration To Attenuate the Effects of Chronic Food Restriction on Lactational Infertility in Galactophore-Cut Rats
Females and their litters were assigned to one of four groups: ad libitum fed, vehicle infused (AL-VEH, n = 7); sham operated, food restricted, vehicle infused (SOFR-VEH, n = 7); galactophore cut, food restricted, vehicle infused (GCFR-VEH, n = 9); and galactophore cut, food restricted, leptin infused (GCFR-LEP, n = 9). Animals in the FR groups were food restricted from day 1 to day 15 postpartum, intracerebroventricular surgery was carried out on day 8 postpartum, and leptin (10 µg/day) or vehicle was infused for the following 7 days. Drug and minipump preparation was as described for experiment 1.
Galactophore transection. To eliminate milk delivery, the tubes that carry milk from the mammary gland to the nipple (the galactophores) were severed under anesthesia with a ketamine-xylazine mixture (6 mg ketamine and 0.86 mg xylazine/100 g body wt). Galactophores were exposed by making midline incisions in the skin of the ventrum. One incision was made between the anterior three pairs of nipples and the other between the posterior three pairs of nipples. After the incisions were made, the skin was deflected, all the galactophores were cut, a topical antibiotic powder (Cicatrin, Wellcome) was applied, and the midline incisions were sutured closed. Animals in the sham-operated groups were subjected to a similar procedure, but the galactophores were left intact. Galactophore transection and sham surgery were performed 1 wk before mating. Litters were switched between galactophore-cut and foster dams every 12 h (0900 and 2100) to ensure that equal suckling stimulation was received by all dams and to prevent starvation of the litters. The efficacy of the galactophore-cut manipulation was assessed by weighing the litter before and after switching between the galactophore-cut dams and foster mothers and by inspection of the mammary glands at autopsy.
Although the galactophore-cut manipulation eliminates milk delivery, sensory stimulation associated with suckling, as reflected in the maintenance of high prolactin levels, lactational infertility, and increased food intake, is maintained (36).
Food restriction. Animals in the SOFR-VEH group were given 50% of intact ad libitum intake from day 1 to day 14 postpartum, while those in the GCFR groups were given 19% of intact ad libitum intake. The ration of the SOFR-VEH group is one that we have used in many previous studies. The ration for the GCFR group was used because pilot studies demonstrated that this ration resulted in the same depletion in fat stores in galactophore-cut females that we typically observe after food restriction in sham-operated or intact lactating females.
Statistical analysis. Length of lactational infertility and weight change during days 1-15 postpartum were compared among groups using a one-way ANOVA for independent groups.
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RESULTS |
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TOPABSTRACTGENERAL METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Figure 1 shows plasma leptin levels of ad libitum-fed and food-restricted lactating rats on days 15 and 20 postpartum. As expected, food restriction for the first 14 days postpartum reduced circulating leptin levels in food-restricted females. By day 20 postpartum, 5 days after refeeding, there was no difference in leptin levels between diet conditions. A two-way (diet condition x day postpartum) ANOVA performed on these data yielded both statistically significant effects of diet condition and diet condition x day postpartum interaction [F(df = 1,22) = 10.14; P < 0.05; F (1,22) = 12.47; P < 0.05, respectively] but no statistically significant effect of day postpartum.
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Maternal body weight did not differ significantly among the groups on day 1 postpartum (means: day 15, AL = 284.80 ± 6.14 g, FR = 289.89 ± 8.27 g; day 20, AL = 282.73 ± 4.29 g, FR = 286.25 ± 6.06 g). As expected, maternal weight change during the experiment was affected by both diet condition and day postpartum. Ad libitum-fed females had gained weight by both day 15 and day 20 postpartum (means: 29.97 ± 6.39 g, 47.52 ± 9.61 g, respectively). By contrast, food-restricted females lost weight during the first 15 days postpartum (mean = -43.09 ± 7.38 g) but gained weight rapidly on refeeding so that by day 20 they had gained weight (mean = 14.63 ± 3.95 g). Analysis of these data yielded significant main effects of both diet condition and day postpartum as well as a significant interaction of these two factors [F(1,22) = 52.56, P < 0.05; F(1,22) = 26.53, P < 0.05; F(1,22) = 7.56, P < 0.05, respectively].
Litter growth. Litters nursed by ad libitum-fed females gained more weight than those nursed by food-restricted females over both the first 15 and 20 days postpartum (means: day 15, AL = 13.37 ± 0.86 g, FR = 7.88 ± 0.43 g; day 20, AL = 14.93 ± 0.58 g, FR = 9.26 ± 0.23 g). Analysis of these data revealed significant effects of both diet condition and day postpartum [F(1,22) = 99.58, P < 0.05; F(1,22) = 6.90, P < 0.05] but no significant interaction of these parameters.
Experiment 2: Ability of Intracerebroventricular Leptin Administration To Attenuate the Effect of Chronic Food Restriction on Lactational Infertility
Experiment 2a: effects of intracerebroventricular leptin administration from day 8 to day 15 postpartum. LENGTH OF LACTATIONAL INFERTILITY. As can be seen in Fig. 2A, leptin infusion had no effect on the length of lactational infertility in chronically food-restricted rats [F(2,18) = 0.17, P > 0.05].
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BODY WEIGHT. Maternal body weight did not differ between the groups on day 1 postpartum [means: FR saline treated, 297.81 ± 9.58 g; FR 1 µg leptin, 295.46 ± 6.79 g; FR 10 µg leptin, 295.38 ± 9.01 g, F(2,18) = 0.03, P > 0.05]. As expected, all females lost weight during the food restriction period and gained weight after refeeding (see Fig. 2B), resulting in a statistically significant effect of phase [F(2,36)= 532.88, P < 0.05]. Leptin-treated animals lost slightly more weight during treatment than the vehicle-treated group, but this did not result in either a statistically significant effect of group or of a group x phase interaction [F(2,18) = 2.44, P > 0.05; F(4,36) = 0.80, P > 0.05]
LITTER GROWTH. Groups did not differ with respect to average daily litter weight gain in the 7 days before treatment [means: FR saline treated, 8.50 ± 0.25 g; FR 1 µg leptin, 8.04 ± 0.45 g; FR 10 µg leptin, 7.61 ± 0.30 g; F(2,18) = 1.36, P > 0.05]. Analysis of litter growth during the treatment phase of the experiment showed that, as expected, litters gained less weight per day as the food restriction progressed, resulting in a statistically significant effect of days [F(5,90) = 11.46, P < 0.05]. Leptin administration tended to increase litter weight gain (see Fig. 2C), and simple main effects analysis showed that there were statistically significant effects of leptin administration on litter weight gain on days 10, 11, and 14 postpartum.
Experiment 2b: effects of intracerebroventricular leptin administration from days 15-22 postpartum. LENGTH OF LACTA-TIONAL INFERTILITY. There was no effect of leptin administration on length of lactational infertility (see Fig. 3A).
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BODY WEIGHT. Groups did not differ in mean body weight on day 1 postpartum [means: FR saline treated, 290.05 ± 9.21 g; FR 1 µg leptin, 295.84 ± 9.33 g; FR 10 µg leptin, 291.93 ± 4.38 g; FR nonoperated controls, 292.88 ± 4.68 g; F(3,27) = 0.11, P > 0.05]. Maternal body weight change was calculated for each of three phases of the experiment; pretreatment (days 1-15), treatment (days 15-22), and posttreatment (days 22-25). As is shown in Fig. 3B, females in all groups lost weight during the pretreatment (food restriction) phase and gained weight during the treatment and posttreatment phases [significant main effect for phase F(2,54) = 733.64, P < 0.05]. Leptin treatment modulated this pattern of weight change [significant effects of group and group x phase interaction F(3,27) = 5.20, P < 0.05; F(6,54) = 5.52, P < 0.05]. Post hoc analyses using simple main effects showed that females in the FR 10 µg leptin group gained less weight during the treatment phase than the other groups, which did not differ significantly.
LITTER GROWTH. Groups did not differ with respect to daily litter growth for the first 2 wk of the experiment [means: FR saline treated, 7.69 ± 0.43 g; FR 1 µg leptin, 8.14 ± 0.27 g; FR 10 µg leptin, 8.19 ± 0.35 g; FR nonoperated controls, 7.48 ± 0.39 g; F(3,27) = 0.97, P > 0.05]. Because pups begin to eat independently early in the third postpartum week, the effect of treatment on litter growth was determined by examining litter weight change only for days 16-18. As can be seen in Fig. 3C, litters increased their weight gain across these 3 days, and litters nursed by females in the food-restricted, nonoperated control group gained the greatest amount of weight during this time [significant main effects for group and day, F(3,27) = 7.32, P < 0.05; F(2,54) = 5.84, P < 0.05, respectively]. There was also a tendency for litters nursed by females in the FR 10 µg leptin group to gain less weight than those in the FR saline group.
Experiment 3: Comparison of Effects of Intracerebroventricular and Intraperitoneal Leptin Administration on Length of Lactational Infertility in Food-Deprived Lactating Rats
Lactational infertility. As shown in Fig. 4A, both intracerebroventricular leptin administration at a dose of 10 µg/day and intraperitoneal leptin administration at a dose of 2.5 mg·kg-1·day-1 given during the period of food deprivation shortened the period of lactational infertility relative to vehicletreated, food-deprived females. A one-way ANOVA performed on these data yielded a significant main effect for group, and post hoc analyses using Fisher's least significant difference test showed that food-restricted rats treated with vehicle had a longer period of lactational infertility than any other group.
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Body weight. As expected, females in all the food-deprived groups lost weight during the food deprivation period and rapidly regained weight afterward [significant main effects for group, F(3,17) = 9.39, P < 0.05; days, F(4,68) = 231.95, P < 0.05; and group x days interaction, F(12,68) = 30.10, P < 0.05]. Subsequent simple main effects analyses showed that there was no effect of leptin administration on either weight loss during food deprivation or weight gain after deprivation (see Fig. 4B).
Litter growth. Food deprivation of the dams resulted in litter weight loss compared with the weight gain showed by litters nursed by ad libitum-fed females, resulting in significant main effects for group [F(3,17) = 11.88, P < 0.05] and days [F(7,119) = 58.70, P < 0.05]. The effect of food deprivation on litter growth was rapidly reversed when females were refed [statistically significant group x days interaction, F(21,119) = 11.17, P < 0.05]. Post hoc simple main effects analyses showed that leptin did not modulate the pattern of litter growth in the food-deprived groups (see Fig. 4C).
Food intake. Both intracerebroventricular and intraperitoneal leptin administration suppressed refeeding food intake on day 15 postpartum. Whereas females in both the ad libitum-fed and food-deprived groups ate similar amounts (means 65.25 + 3.06 and 64.78 + 6.47 g, respectively) on this day, food-deprived rats treated with intracerebroventricular or intraperitoneal leptin ate only 48.12 + 1.74 and 49.64 + 4.03 g, respectively.
Experiment 4: Ability of Intracerebroventricular Leptin Administration To Attenuate the Effects of Chronic Food Restriction on Lactational Infertility in Galactophore-Cut Rats
Lactational infertility. As shown in Fig. 5A, intracerebroventricular leptin infusion for days 8-15 postpartum was sufficient to attenuate the effects of food restriction on lactational infertility in galactophore-cut, suckled females. Analysis of these data showed a significant main effect for group [F(3,28) = 13.84, P < 0.05], and post hoc pairwise analyses showed that the length of lactational infertility of food-restricted, galactophore-cut rats treated with leptin was shorter than that of the other two food-restricted groups and was no different from that of the sham-operated, ad libitum-fed group.
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Body weight. As expected, females in all of the food-restricted groups lost weight during the first 2 wk postpartum, whereas females in the ad libitum-fed group gained weight (Fig. 5B). Analysis of these data yielded a significant main effect of group [F(3,28) = 147.05, P < 0.05]. Pairwise post hoc comparisons showed that sham-operated, ad libitum-fed females gained more weight than those in the food-restricted groups and that there were no differences among the three food-restricted groups.
Litter growth. As expected, litters lost weight when being nursed by galactophore-cut dams, whereas they gained weight when being nursed by females in the sham-operated groups (data not shown).
Food intake. Postrestriction food intake was lower in food-restricted, galactophore-cut rats treated with leptin (mean 28.58 ± 3.24 g) than in those treated with vehicle (mean 37.22 ± 1.33, P < 0.05).
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DISCUSSION |
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TOPABSTRACTGENERAL METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Despite the decrease in leptin levels observed in experiment 1, the results of experiment 2 show that intracerebroventricular administration of leptin is not sufficient to attenuate the effects of food restriction on reproductive function. These results contrast with the well-established ability of exogenous leptin administration to attenuate the effects of energetic challenge on ovulation in other reproductive states (1, 13, 24) and after acute food deprivation in lactating rats (35). They are similar, however, to those that we previously obtained in ad libitum-fed lactating rats (36). Furthermore, they suggest that in the latter case the failure of leptin administration to restore reproductive function may not simply have resulted from a reduction in circulating leptin levels too small to have impacted on reproductive function.
One obvious explanation for the failure of the leptin administration protocols used in experiment 2 to restore reproductive function in food-restricted lactating rats is simply because the doses used were not high enough. This seems unlikely, however; the higher dose used was found by Gruaz et al. (13) to be sufficient to oppose the effects of chronic food restriction on pubertal delay. As the present results also show, both doses used produced the expected effects on weight loss and postrestriction food intake. Moreover, when administered from day 8 to day 15 postpartum, both doses tended to increase litter growth (see Fig. 2). In addition, previous work has shown that these doses produce a decrease in food intake in ad libitum-fed lactating rats, although the effect was only transitory (36).
Another possibility is that, although central leptin administration is sufficient to eliminate the effect of food restriction and food deprivation in other reproductive states, in lactating rats, leptin is required to act both centrally and peripherally to restore reproductive function. Leptin receptors have been localized throughout the endocrine system, and leptin has been shown to have direct effects at both the pituitary and the ovary (4, 5). The results of experiment 3 show, however, that both central and peripheral leptin administration are sufficient to attenuate the effects of acute food deprivation on lactational fertility. These data demonstrate that the central action of leptin at the higher of the doses used in experiment 2 is sufficient to offset the effects of reduced energy availability on lactational infertility. These results do not rule out the possibility, however, that, when chronic food restriction is combined with lactation, the resulting reduction in leptin levels has effects on peripheral tissues.
An unexpected effect of leptin administration to food-restricted rats seen in experiment 2 was the increase in litter growth observed on some treatment days when the protein was administered from day 8 to day 15 postpartum (shown in Fig. 2). Given that leptin can increase the availability of oxidizable fuels through increasing sympathetic outflow (29), it seems reasonable to suppose that during lactation these fuels are used for milk synthesis. Such an effect might not be as apparent when leptin infusions are made later in lactation, because at that time pups begin to eat independently. Diversion of fuels to milk synthesis in food-restricted females might divert oxidizable fuels away from mechanisms that control estrous cyclicity and serve to counter the ability of leptin to stimulate the reproductive axis. By contrast, in situations where there is no energetic drain of milk delivery as in nonlactating animals or in food-deprived lactating rats, the increased availability of oxidizable fuels in sensors such as those in the area postrema, which give rise to short-term signals of energy availability, would provide a second source of permissive signals to the reproductive axis.
This analysis provides a third alternative explanation for the inability of leptin to attenuate the effects of food restriction on reproductive function in lactating rats and was explored in experiment 4. The results of this experiment demonstrated that intracerebroventricular leptin administration at the higher of the doses used in experiment 2 to food-restricted, galactophorecut rats is sufficient to offset the effects of food restriction on lactational infertility. It is unlikely that differences in relative amounts of food restriction account for the effectiveness of leptin administration in this experiment. The food restriction regimen used was chosen because it resulted in similar fat pad weights in sham-operated, food-restricted and galactophorecut, food-restricted rats. In addition, this leptin administration schedule had no effect on length of lactational infertility in ad libitum-fed rats. Because the galactophore surgery does not interfere with the ability of suckling stimulation to elevate circulating prolactin levels and maintain lactational infertility (36), it is also unlikely that the effectiveness of leptin administration in food-restricted, galactophore-cut rats reflects differences in gonadal steroid and peptide hormone levels. Rather these data tend to support the hypothesis that the ability of leptin to maintain reproductive function in food-restricted rats depends on both its direct and indirect actions as well as the presence of other energy balance cues.
If leptin is able to eliminate the effects of food deprivation and, under some circumstances, those of food restriction on the length of lactational infertility, the issue is how it does so. The fact that limiting food availability has effects that outlast the manipulation itself, e.g., food deprivation on days 13-14 postpartum affects the timing of an event that would usually occur around day 21 postpartum, suggests that limiting food availability interferes with a process leading to ovulation rather than with the mechanism of ovulation itself. Thus leptin treatment can be seen as restoring this process rather than providing a trigger for ovulation. One of the results of chronic food restriction during lactation is the prolonged suppression of luteinizing hormone (LH) levels, which would be expected to delay follicular growth and ultimately ovulation (33). This prolonged suppression of LH has been associated with high levels of NPY in the arcuate nucleus (1), and there is compelling evidence that NPY provides the link between energy balance and the reproductive system (see Ref. 27 for review). Thus one would expect that leptin treatment would reduce NPY levels, thus alleviating the suppression of gonadotropin-releasing hormone and restoring LH release and follicular growth.
Together the results of these experiments have shown that food restriction and lactation have additive effects in the reduction of leptin levels. Moreover, they demonstrate that intracerebroventricular leptin administration is sufficient to attenuate the effects of both acute food deprivation and chronic food restriction on length of lactational infertility but apparently only when the energetic drain of milk production and delivery is eliminated. We propose that, in the presence of this drain, increased metabolic fuels liberated by leptin administration are diverted to milk synthesis, and hence both decrease the permissive cues on the reproductive axis and potentially increase the strength of other signals of negative energy balance.
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ACKNOWLEDGMENTS |
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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.
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