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BOR - Papers in Press, published online ahead of print June 13, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060434
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BIOLOGY OF REPRODUCTION 77, 492–503 (2007)
DOI: 10.1095/biolreprod.107.060434
© 2007 by the Society for the Study of Reproduction, Inc.

Central Insulin-Like Growth Factor 1 Receptors Play Distinct Roles in the Control of Reproduction, Food Intake, and Body Weight in Female Rats1

Brigitte J Todd 2 3, Gregory S Fraley 7, Alison C Peck 4, Gary J Schwartz 3 5, and Anne M Etgen 3 6

Departments of Neuroscience,3 Obstetrics & Gynecology,4 Medicine,5 and Psychiatry & Behavioral Sciences,6 Albert Einstein College of Medicine, Bronx, New York 10461 Biology Department,7 Hope College, Holland, Michigan 49422

ABSTRACT

Estradiol and progesterone induction of the LH surge in ovariectomized female rats requires concurrent activation of brain insulin-like growth factor 1 (IGF1) receptors. The present study determined whether brain IGF1 receptor signaling is required for estrous cyclicity in gonadally intact female rats. A selective IGF1 receptor antagonist (JB-1) or vehicle was continuously administered into the third ventricle by osmotic minipumps. Following surgical placement of the minipumps, all rats temporarily reduced food intake, lost weight, and suspended estrous cycles. Control rats resumed cycles within a few days and exhibited compensatory hyperphagia until they returned to presurgical body weight. Animals receiving JB-1 had severely delayed or absent estrous cycles, failed to show rebound feeding, and regained body weight more slowly. Vehicle-infused animals pair fed to JB-1-treated rats had even lower body weights but resumed estrous cycles sooner than those given drug alone. Chronic infusion of IGF1 alone had no effect on any of these parameters, but coinfusion of IGF1 with the antagonist completely reversed JB-1 effects on food intake and estrous cyclicity and partially reversed the effects on body weight. There were no significant differences in the expression of galanin-like peptide (Galp) or Kiss1 mRNA in the arcuate or periventricular hypothalamic area of control and JB-1-treated animals at a time point when food intake and estrous cycles were different between controls and JB-1-treated rats. These data suggest that brain IGF1 signaling is necessary for normal estrous cycles as well as compensatory hyperphagia and that IGF1 modulation of the reproductive axis is not secondary to reduced food intake.

brain, estrous cyclicity, feeding, hypothalamus, insulin-like growth factor receptor, luteinizing hormone, neuroendocrinology

INTRODUCTION

Insulin-like growth factor 1 (IGF1) signaling in the brain participates in ovarian steroid hormone control of female reproductive physiology and behavior. Blockade of brain IGF1 signaling abolishes the estradiol (E2)- and progesterone (P)-induced LH surge in ovariectomized (OVX) rats and partially blocks reproductive behavior [1, 2]. Moreover, IGF1 signaling is necessary for E2-induced synaptic remodeling in the arcuate nucleus of the hypothalamus, which is thought to be necessary for induction of the preovulatory LH surge [3, 4]. IGF1 also regulates other neuroendocrine processes, such as growth hormone release [5] and pubertal timing [6, 7]. IGF1 receptors are present in GnRH neurons [8, 9] and throughout the hypothalamus [10], where they colocalize with estrogen receptors [11].

In female mammals, reproductive activity and energy balance are linked such that fertility is suppressed during times of energy shortage. This ensures that pregnancy and lactation, which are energetically costly, occur when nutritional demands can be met. For example, food restriction suppresses LH release and reproductive behaviors in a number of species, including rodents and primates [12–16]. Similarly, reducing the availability of oxidizable metabolic fuels inhibits estrous cycles and reproductive behaviors in hamsters [17, 18]. These deficiencies in reproductive parameters in fasted animals can be restored by refeeding [13, 19, 20]. Because circulating levels of IGF1 are influenced by nutritional status [21, 22], it is conceivable that circulating IGF1 may signal energy balance information to the brain [23, 24], and this action may be important for its regulation of the female reproductive system.

Various circulating factors involved in metabolism and food intake have been proposed to communicate nutritional status to the brain regions controlling reproduction. These include insulin, glucose, leptin, and ghrelin [25–29], all of which play essential roles in metabolism and control of food intake. While our previous research implicates IGF1 in neuroendocrine regulation of reproduction, whether IGF1 acts in the brain to control metabolic activity or feeding is more equivocal. IGF1 binds to the insulin receptor with approximately 100-fold lower affinity than insulin [30], and synthesis and secretion of both IGF1 and insulin are regulated by changes in food intake [21, 22]. Insulin infusion into the brain reduces food intake and body weight of normal [31] and diabetic [32] rats and inhibits the fasting and diabetes-induced increase of neuropeptide Y (Npy) gene expression in the hypothalamus [32, 33]. By contrast, IGF1 infusion into the brain significantly reduces food intake after 2 days in diabetic male rats but not in controls [34]. In castrated male sheep, intracerebroventricular (icv) infusion of IGF1 has no effect on food intake or body weight [35].

The mediobasal hypothalamus is a major site at which peripheral metabolic signals are integrated to inform the reproductive system of nutritional status. Among the hypothalamic neurons participating in this signaling function are those expressing Npy, agouti-related protein, proopiomelanocortin, and galanin [36–40]. Galanin-like peptide (GALP) has recently been implicated in the hypothalamic integration of nutrition and reproduction [41, 42]. GALP is a small peptide expressed predominantly in the arcuate nucleus that has been proposed to act as a link between metabolism and reproduction [42, 43] that is regulated by insulin [44] and leptin [45]. Treatment with exogenous GALP rescues reproductive function in rats with experimentally induced diabetes [46]. The product of the Kiss1 gene, KISS1, is also present in the arcuate nucleus and anteroventral periventricular area (AVPV) of the hypothalamus, and it stimulates LH secretion when administered peripherally or centrally [47–49]. Because KISS1 neurons are regulated by leptin [50], they may also regulate metabolism and food intake.

The purpose of the present experiments was to test the hypothesis that brain IGF1 signaling is necessary for the maintenance of estrous cyclicity in gonadally intact female rats. We used a selective antagonist of IGF1 receptors administered into the third ventricle to chronically block brain IGF1 signaling in female rats. We assessed the impact of this treatment on function of the hypothalamic-pituitary-gonadal axis. We also investigated the effects that this manipulation has on food intake and body weight, as there is evidence that IGF1, like insulin, might be a signal of positive energy balance from the periphery to the brain [23, 24]. If this is the case, one might predict that chronic antagonism of brain IGF1 receptors would increase food intake. Finally, we performed in situ hybridization for Galp and Kiss1 mRNAs to determine whether chronic blockade of IGF1 receptors affects expression of genes known to play important roles in reproduction and energy balance. Our findings demonstrate that blockade of brain IGF1 receptors interferes with estrous cycles and that this effect is not attributable simply to reduced food intake or body weight. We also discovered that brain IGF1 signaling is required for the expression of compensatory hyperphagia following a period of reduced food intake.

MATERIALS AND METHODS

Materials

Guide cannulae and injection cannulae were purchased from Plastics One (Roanoake, VA). Alzet osmotic minipumps were purchased from Durect Corp. (Cupertino, CA). JB-1 and human IGF1 were purchased from Bachem (San Carlos, CA). The vehicle used to dissolve JB-1 and infused into all control animals was artificial cerebrospinal fluid (aCSF; 140 mM NaCl, 3 mM KCl, 1.2 mM Na2HPO4, 1.0 mM MgCl2, 0.27 mM NaH2PO4, 1.2 mM CaCl2, and 7.2 mM dextrose, pH 7.4). Plasmids containing Kiss1 cDNA and a partial cDNA for rat Galp were the generous gift of Dr. Robert Steiner, University of Washington. SP6 RNA polymerase was purchased from New England Biologicals (Beverly, MA). 33P-UTP was purchased from Perkin-Elmer (Indianapolis, IN). T7 RNA polymerase and RNase were purchased from Roche Biochemicals (Indianapolis, IN). Citrasol was purchased from VWR (Seattle, WA). DPX was purchased from Sigma Biologicals (St. Louis, MO).

Animals

Adult female Sprague-Dawley rats (170–190 g) were purchased from Taconic Farms (Germantown, NY). They were housed individually on wire-bottomed cages and maintained on a 14L:10D cycle (lights-off at 2000 h) with standard laboratory chow and tap water freely available. At the end of experiments, animals were given an overdose of ketamine and xylazine and decapitated. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the Albert Einstein College of Medicine.

Assessment of Food Intake, Body Weight, and Estrous Cyclicity

Food intake and estrous cyclicity were monitored daily, both before and after cannula and minipump placement. Twenty-four-hour food intake and body weight were measured daily at 1600 h. Vaginal smears were taken daily with an inoculation loop, and cytology was monitored to follow estrous cycles. Animals with irregular estrous cycles before cannula placement were excluded. Food intake varied with estrous cycle phase, with lowest food intake occurring on the day of estrus (Fig. 1). There were no significant differences among any groups in baseline food intake or body weight. After placement of the osmotic minipumps (designated day 0), daily food intake, body weight, and vaginal cytology continued to be monitored daily for the remainder of the experiment. In some experiments, aCSF-infused animals were pair fed to the level of another ad libitum-fed group. The amount of food given to the pair-fed rats was determined by averaging the previous day's 24-h food intake by the ad libitum-fed group and feeding all members of the pair-fed group that amount.


Figure 01
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  		FIG. 1 Food intake varies with estrous cyclicity. A and B) Representative examples of food intake (g/g BW*100) throughout the estrous cycle in two rats. A) Rat with a 4-day estrous cycle. B) Rat with a 5-day estrous cycle (approximately 20% of our rats had 5-day cycles). The asterisk denotes the day of estrus. C) Mean food intake on estrus, proestrus, and diestrus I and II (collapsed) in the 12 rats of the first experiment (see Fig. 2) during the presurgical period. All groups are significantly different from each other (P < 0.05) as denoted by *.

Third Ventricle Cannula and Osmotic Minipump Placement

Animals were anesthetized with a ketamine/xylazine mixture (40 and 7 mg/kg, respectively, i.m.) and placed into a Kopf stereotaxic apparatus with the nosebar set at +5.0 mm. A 22-gauge guide cannula was placed into the third ventricle (A/P, +0.2 mm; M/L, +0.0 mm; D/V, –9.8 mm with respect to Bregma). Immediately following cannula implantation, an osmotic minipump (7- or 14-day delivery; Alzet model 2002 or 1007D; flow rate, 0.5 µl/h) was placed subcutaneously between the scapulae and connected with polyethylene tubing to a 26-gauge internal cannula that extended 1 mm below the guide cannula. Model 2002 (14 day) was used in all experiments, except for the second pair-feeding experiment, in which model 1007D (7 day) was used. Osmotic minipump placement was performed on the same day as cannula placement to avoid two exposures to anesthesia and surgery, which cause body weight reduction. Cannula placement was verified at the end of the experiments by dye injection into the third ventricle or, for the in situ hybridization experiments, by tracing the cannula track in the brain sections.

Drug Treatment

Osmotic minipumps were used to provide constant icv infusion of drugs or vehicle into the third ventricle. IGF1 receptors were blocked by infusing JB-1, a synthetic peptide analog of IGF1 that is a highly selective, competitive antagonist of IGF1 binding to the IGF1 receptor [51]. In vitro, JB-1 blocks IGF1-induced cellular proliferation and IGF1-dependent autophosphorylation of IGF1 receptors and has no activity at IGF2 or insulin receptors [51]. The dose of JB-1 (20 µg/ml) used was identical to what blocks E2-dependent neuroprotection in the hippocampus [52]. In one experiment, human IGF1 was also administered by osmotic minipump, alone or in combination with JB-1. The dose of IGF1 (2 µg/ml) used was based on the concentration of IGF1 in vitro that can successfully overcome the antagonistic effects of JB-1 to induce cellular proliferation [51].

In Situ Hybridization for Kiss1 and Galp mRNA

Animals were implanted with icv cannulae that were connected to osmotic minipumps containing either aCSF or JB-1 as described above. At approximately 1600 h on the fifth day following implantation, animals were decapitated, and the brain was rapidly removed and frozen on dry ice. Five complete sets of 20-µm sections were cut through the hypothalamus from the preoptic area to the level of the mammillary bodies. Sections were mounted on slides and stored in a –80°C freezer until in situ hybridization.

Probe Preparation. Antisense and sense mouse Kiss1 probes were generated as previously described [47]. The Kiss1-specific sequence spanned bases 76–486 of the mouse cDNA sequence (GenBank accession number AF472576). We predicted that a mouse riboprobe would hybridize to rat Kiss1 mRNA, because there is 90% homology between mouse and rat Kiss1 cDNA in the cloned region (shown by Smith et al. [53]).

Plasmids containing a partial cDNA for rat Galp were cloned as previously described [45]. Linearized Galp cDNA was transcribed for antisense riboprobes with SP6 RNA polymerase in the presence of 33P-UTP. Sense transcriptions were carried out with T7 RNA polymerase. Appropriate riboprobe length was determined with formaldehyde gel electrophoresis. Riboprobes were quantified in a scintillation counter following purification.

In Situ Hybridization Protocol. One complete set of hypothalamic sections was used for each probe. Sections were removed from the freezer, pretreated, and allowed to air dry prior to the hybridization procedure. The final volume of probe mix plus hybridization buffer was 100 µl/slide. The volume of probe was calculated (0.2 pmol/slide) and allowed to thaw on ice. The probe was combined with 1:20 volume yeast tRNA (Roche) in TE (0.1 M Tris-0.01 M EDTA, pH = 8.0) to produce the probe mix. Probe mix was heat denatured by placing into boiling water for 3 min and then returned to ice for 5 min. The denatured probe mix was added to prewarmed hybridization buffer at a ratio of 1:4. After adding the hybridization mix to the slides, the sections were covered with glass coverslips. Slides were placed in humidity chambers, which were then put into ovens at 55°C for 16 h. Following hybridization, humidity chambers were removed from the ovens and allowed to return to room temperature. Coverslips were removed, and the slides were returned to slide racks and then washed twice in 4x sodium chloride-sodium citrate (SSC) for 15 min each at room temperature. Slides were then placed into 37 mg/ml RNase in 0.15 M NaCl, 10 mM Tris, and 1 mM EDTA (pH 8.0) for 30 min at 37°C and then in RNase buffer without RNase at 37°C for another 30 min. After a 3-min wash in 2x SSC at room temperature, slides were washed twice with agitation in 0.1x SSC at 62°C for 30 min each. After a 3-min room temperature wash in 0.1x SSC, sections were dehydrated in graded ethanols (3 min each) and allowed to air dry. The slides were dipped in Kodak NTB emulsion, air dried, and stored at 4°C for approximately 1 wk. They were then removed under safe light conditions, developed, dehydrated in graded ethanols, and cleared in Citrasol, and coverslips were applied with DPX.

In Situ Hybridization Quantification and Analysis. Slides from all of the animals were assigned a random three-letter code, alphabetized, and read under dark-field illumination with custom-designed software designed to count the number of silver grains (corresponding to radiolabeled mRNA) over each cell [54]. Cells were counted as mRNA positive when the number of silver grains in a cluster exceeded that of background. The image processing system consisted of a Leica DM460 5 Mpix digital camera mounted on a Leica DB5000b microscope (both from Nuhsbaum). The camera was attached to a Scion VG-5 Frame Grabber (Scion Corp., Frederick, MD) mounted on a Macintosh G5 computer (Apple Computer, Cupertino, CA) running UW Grains Software [54].

Radioimmunoassays

Serum was collected for analysis of LH, FSH, triiodothyronine (T3), insulin, leptin, and glucose. Serum for analysis of LH, FSH, and T3 was collected from the animals used for the in situ hybridization studies (JB-1-treated and aCSF-treated groups) at the time of decapitation. For analysis of insulin, leptin, and glucose, animals were implanted with icv cannulae that were connected to osmotic minipumps containing either aCSF or JB-1 as described above. Half of the aCSF-treated group was pair fed to the level of the JB-1-treated rats, as described in the pair-feeding experiment. Animals were decapitated, and serum was collected at 1600 h on the fifth day following cannula implantation.

Serum LH, FSH, and T3 were measured at Northwestern University Radioimmunoassay Core (Evanston, IL). Serum LH was measured with reagents from the National Institutes of Health. For LH, the antiserum was anti-rLH-S11, the standard was rLH-RP3, the assay sensitivity was 0.1 ng/ml, and the intraassay coefficient of variation was 2.2%. The T3 assay was performed with a solid-phase RIA kit (MP Biomedicals, Orangeburg, NY) with an intraassay variance of 13.0%. For FSH, the limit of sensitivity was 1.0 ng/ml, and the interassay coefficient of variation was 9.2%. Serum insulin, leptin, and glucose were sent to Linco Diagnostics (St. Charles, MO) for determination of concentrations by commercial RIA or chemistry kits.

Statistical Analysis

Most data are expressed as mean ± SEM. In some cases, the percentage of rats exhibiting 4- to 5-day estrous cycles is shown. Food intake is expressed as grams ingested over a 24-h period per gram of body weight x 100. Surgical placement of cannulae (day of surgery = Day 0 in all cases) resulted in a temporary disruption of estrous cycles (a prolonged diestrus). The day of resumption of estrous cycles was defined by the first appearance of a proestrous or estrous smear that was followed by resumption of normal estrous cyclicity. Thus, if a proestrous or estrous smear occurred in isolation (without subsequent return to normal cyclicity), it was not deemed to be a resumption of estrous cyclicity. If an animal failed to resume estrous cycling for the duration of the monitoring period, a value equivalent to the day when the experiment ended was assigned. Postsurgical body weight was expressed as a percentage of presurgical body weight. Body weight and food intake were analyzed by mixed-design ANOVA (treatment as a between-subjects factor, postsurgical day as a within-subjects factor) with the Fisher protected least-squares difference (PLSD) post hoc test when significant differences were found. Estrous cycle data were analyzed by group factorial ANOVA or the Student t-test when appropriate. Serum hormone and glucose data were analyzed by the Student t-test or group factorial ANOVA with the Fisher PLSD test where applicable. In situ hybridization data were analyzed by the Student t-test.

RESULTS

Chronic Blockade of Brain IGF1 Receptors Interferes with Compensatory Hyperphagia and Estrous Cycles

We initially investigated the effects of 14-day blockade of brain IGF1 receptors on food intake, body weight, and estrous cyclicity by chronic infusion of the selective IGF1 receptor antagonist JB-1 or aCSF (controls) into the third ventricle (Fig. 2). For all animals, surgical placement of the cannula and osmotic minipump resulted in a 10%–15% loss of presurgical body weight and a temporary disruption of estrous cycles. Food intake was also significantly reduced in all animals on the day of surgery and the day following surgery. Anesthetics are known to delay the LH surge in rodents [55], so that surgery on the day of proestrus can potentially affect the generation of the LH surge, and thus estrous cyclicity. In these studies, at least one animal per group was in proestrus on the day of surgical cannula placement (see individual figure legends), but there was no correlation between return to estrous cyclicity and estrous cycle phase on the day of surgery.


Figure 02
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  		FIG. 2 Effects of continuous icv infusions of JB-1, a selective IGF1 receptor antagonist (20 µg/ml; infusion rate, 0.5 µl/h) or aCSF (controls) on 24-h food intake (A), body weight expressed as percentage of baseline (B), and percentage of rats initiating normal estrous cyclicity on the days following surgery (C) in female rats. Day 0 denotes the day of surgery. In B, line at 100% indicates baseline body weight on the day of surgery. Data represent mean ± SEM (n = 6 rats per group). A total of 2/6 aCSF rats and 1/6 JB-1-treated rats were in proestrus on the day of surgery. In A, * indicates significantly different from JB-1. In B, * represents significantly different from baseline. P < 0.05 in all cases.

There were significant differences in postsurgical food intake between the two groups (F = 13.8; P < 0.005). On Days 3–8 following surgery, control animals exhibited compensatory hyperphagia, eating significantly more food than in the baseline monitoring period. JB-1-treated animals did not exhibit this compensatory hyperphagia and thus had significantly lower 24-h food intake than control animals on Days 3–8 (P < 0.05; Fig. 2A). However, JB-1-infused animals resumed presurgical food intake by Day 3 following surgery. JB-1-treated animals returned to presurgical body weight more slowly than controls (Fig. 2B). By Day 8 postsurgery, control animals regained their baseline body weight, whereas the JB-1-treated animals failed to regain baseline body weight for the duration of the treatment. Finally, estrous cyclicity was also disrupted by JB-1 (Fig. 2C). Three days following surgery, 70% of control animals had initiated a normal 4- or 5-day cycle. By contrast, the majority of JB-1-treated animals failed to resume estrous cyclicity by Day 14, instead remaining in diestrus. On average, control animals resumed normal cycles after 4.5 ± 1.5 days, compared with 8.6 ± 0.9 days in JB-1-treated animals (t-test, P < 0.05).

Pair Feeding

Most animals infused chronically with the IGF1 receptor antagonist failed to resume estrous cycles, despite a return to baseline food intake (Fig. 2), suggesting that brain IGF1 receptors regulate estrous cyclicity independent of their effects on food intake and body weight. To determine whether the delayed return of estrous cycles in JB-1-treated animals was due to reduced food intake, we performed pair-feeding experiments. Cannulae were placed in the third ventricle of gonadally intact female rats and connected to 14-day osmotic minipumps containing either JB-1 or aCSF vehicle. The aCSF-treated rats were pair fed to the level of the JB-1-treated rats, which were ad libitum fed. Again, JB-1-treated rats did not exhibit compensatory hyperphagia (Fig. 3A). Interestingly, even though food intake was identical in both groups, JB-1-treated rats regained body weight significantly more rapidly than aCSF-treated, pair-fed animals (Fig. 3B; F = 6.2; P < 0.05). This suggests that blockade of brain IGF1 receptors affects energy storage or expenditure in addition to preventing compensatory hyperphagia. Although body weight was significantly lower in pair-fed animals, return to estrous cyclicity occurred significantly sooner in this group than in JB-1-treated animals (mean time to cycle resumption = 11.9 ± 0.7 days in JB-1-treated animals, 5.2 ± 1.7 days in pair-fed animals; t-test, P < 0.05; Fig. 3C). In all, only two of the seven JB-1-treated animals resumed cycles, but five of the six pair-fed animals resumed cycles.


Figure 03
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  		FIG. 3 Effects of pair feeding aCSF-treated rats to JB-1-treated rats on 24-h food intake (A), body weight expressed as percentage of baseline (B), and percentage of rats initiating normal estrous cyclicity on the days following surgery (C) in female rats. In B, line at 100% indicates baseline body weight on the day of surgery. Data represent mean ± SEM (n = 7 rats in JB-1 ad libitum-fed group, 6 in aCSF pair-fed group). A total of 2/7 JB-1 and 2/6 aCSF-treated, pair-fed rats were in proestrus on the day of surgery. In B, * indicates significantly different from baseline (P < 0.05).

We also investigated the effects of terminating pair feeding or JB-1 treatment on food intake, body weight, and estrous cycle resumption (Fig. 4). Cannulae were placed in the third ventricle of gonadally intact female rats and connected to 7-day osmotic minipumps containing either JB-1 or aCSF vehicle. Half of the aCSF-treated rats were ad libitum fed (controls); the other half were pair fed to the level of the JB-1-treated rats, which were ad libitum fed. At the end of 7 days, minipumps were disconnected, and the pair-fed group was permitted to resume ad libitum feeding. All animals were followed for another 7 days. As before, control animals exhibited compensatory hyperphagia in the days following the surgery, whereas JB-1-treated animals did not. Following minipump disconnection, pair-fed aCSF-treated animals immediately exhibited a compensatory hyperphagia, whereas the JB-1-treated animals did not, despite termination of drug administration (Fig. 4A).


Figure 04
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  		FIG. 4 Effects of pair feeding and release from pair feeding/JB-1 treatment on 24-h food intake (A), body weight expressed as percentage of baseline (B), and percentage of rats initiating normal estrous cyclicity on the days following surgery (C) in female rats. Day 0 denotes the day of surgery. In B, line at 100% indicates baseline body weight on the day of surgery. Data represent mean ± SEM (n = 5 rats in aCSF ad libitum-fed, 6 in JB-1 ad libitum-fed, 7 in aCSF pair-fed groups). A total of 3/5 aCSF ad libitum-fed, 1/6 JB-1 ad libitum-fed, and 2/7 aCSF pair-fed rats were in proestrus on the day of surgery. In A, * indicates significantly different from JB-1 and pair fed; **, significantly different from control and JB-1. In B, * indicates all groups significantly lower than baseline; **, control group significantly different from other groups; ***, JB-1 group significantly different from baseline; #, all three groups significantly different from each other. P < 0.05 in all cases.

Figure 4B shows that control ad libitum-fed animals regained body weight more rapidly than the other two groups (P < 0.05); however, these animals and the aCSF-treated, pair-fed group both returned to presurgical body weight on Day 9 following surgery. In contrast, body weight of JB-1-treated animals did not return to baseline until Day 11. In agreement with the first pair-feeding experiment, on Days 6 and 7 following surgery, body weight in the aCSF-treated, pair-fed group was significantly lower than in the JB-1-treated rats (P < 0.05).

Estrous cyclicity resumed quickly in control ad libitum-fed animals following surgery and in aCSF-treated, pair-fed animals upon resumption of ad libitum feeding (mean day of cycle resumption, 5.7 ± 0.7 and 10.9 ± 0.6 days postsurgery, respectively). JB-1-treated animals were slower than both other groups to resume cycles (13.2 ± 0.6 days following surgery; P < 0.05), even though they weighed significantly more than pair-fed animals when treatments and pair feeding ended on day 7. However, all JB-1-treated rats exhibited estrous cycles by 15 days following surgery and 8 days following disconnection of osmotic minipumps (Fig. 4C).

Serum Levels of Insulin, Glucose, and Leptin in Pair-Fed Rats

We next determined whether JB-1 infusion produced differences in serum levels of nutrients or feeding-associated hormones that are known to affect reproductive physiology. We prepared additional aCSF ad libitum-fed; JB-1-treated, ad libitum-fed; and aCSF pair-fed animals and killed them at 1600 h on Day 6 following surgery, a time when body weight differed significantly among all three groups (aCSF ad libitum-fed animals: 95.3% ± 0.3%; JB-1 ad libitum: 90.0% ± 0.4%, aCSF pair fed: 86.9% ± 0.5% of baseline; F = 6.57, P < 0.05). The ad libitum-fed control animals were also hyperphagic at this time (data not shown). Figure 5 shows serum levels of insulin, glucose, and leptin in these rats. Despite significant differences in ingestive behavior and body weight, there were no significant differences among the three groups in serum insulin or glucose. Serum leptin concentrations were significantly higher in JB-1-treated, ad libitum-fed animals than in aCSF-treated, pair-fed animals (P < 0.05). The normal levels of leptin seen in the JB-1-treated animals may contribute to their failure to exhibit rebound feeding.


Figure 05
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  		FIG. 5 Effects of continuous icv infusions of JB-1 and pair feeding on serum insulin (A), glucose (B), and leptin (C) concentrations. Animals were killed 6 days following surgery, and serum was collected for glucose determination and hormone RIA. Data represent mean ± SEM (n = 6 rats per group). *P < 0.05.

Coinfusion of IGF1 with IGF1 Receptor Antagonist

We next determined whether the observed effects of JB-1 were attributable to interactions with brain IGF1 receptors by coinfusing a low dose of IGF1 with the antagonist. At the dose used (2 µg/ml), it is unlikely that IGF1 would interact with insulin receptors. Four groups of females were included: aCSF (controls), JB-1, IGF1 alone, and both JB-1 and IGF1. Figure 6A shows that all animals, except for those treated with JB-1, exhibited compensatory hyperphagia for several days after surgery. The overall difference in 24-h food intake among the four groups (F = 16.2; P < 0.0001) was entirely attributable to lower food intake in the rats treated only with the IGF1 receptor antagonist. Importantly, IGF1 alone at the dose used did not alter rebound feeding or the return to baseline food intake. However, concurrent administration of IGF1 with JB-1 completely blocked the effects of the IGF1 antagonist on compensatory hyperphagia.


Figure 06
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  		FIG. 6 Effects of continuous icv infusions of aCSF, IGF1, JB-1, or both IGF1 and JB-1 on food intake (A), body weight expressed as percentage of baseline (B), and percentage of rats initiating normal estrous cyclicity on the days following surgery (C) in female rats. Day 0 denotes the day of surgery. Data represent mean ± SEM (n = 6 for all groups, except for JB-1, where n = 7). A total of 1/6 aCSF-treated, 2/6 IGF1-treated, 2/6 JB-1-treated, and 1/6 IGF1+JB-1-treated rats were in proestrus on the day of surgery. In A, * indicates JB-1 significantly different from all other groups. In B, * indicates all groups significantly lower than baseline; **, JB-1 significantly different from all other groups; ***, JB-1 and JB-1+IGF1 significantly different from aCSF and IGF1 alone and from each other; #, JB-1 and JB-1+IGF1 significantly different from other groups but not from each other. P < 0.05 in all cases.

Return to baseline body weight was also significantly different among groups (F = 8.6; P < 0.005; Fig. 6B). Interestingly, animals receiving concurrent administration of IGF1 and its antagonist did not return to baseline body weight at the same rate as control animals or animals receiving IGF1 alone (these two groups did not differ). Instead, these animals regained weight at a rate intermediate between controls and JB-1-treated animals (Fig. 6B). Estrous cycle resumption was also significantly different among groups (F = 32.2; P < 0.0001; Fig. 6C). Rats infused with JB-1 failed to resume estrous cycles during the 14-day treatment period, whereas rats infused with IGF1 plus the antagonist resumed estrous cyclicity at the same time as controls and animals given IGF1 alone.

In Situ Hybridization for Kiss1 and Galp mRNA

We hypothesized that blockade of brain IGF1 receptors by JB-1 would alter regulation of neuropeptides known to be involved in food intake and reproduction. The gene product of Kiss1, KISS1, is a potent positive regulator of gonadotropin secretion [47, 56, 57]. GALP has recently been implicated in the regulation of both food intake and reproduction [43, 46, 58]. We used in situ hybridization to quantify expression levels of Galp and Kiss1 mRNA in the hypothalamus of control and JB-1-treated animals killed at 1600 h on Day 6 following cannula implantation, when differences in food intake are maximal (controls: 9.11 ± 0.2; JB-1: 6.28 ± 0.4 g/100 g of body weight for the 24 h prior to sacrifice). As before, JB-1-infused rats did not exhibit compensatory hyperphagia (Fig. 7E). There were no significant differences between controls and JB-1-treated animals in number of cells positive for Galp or Kiss1 mRNA or in mean number of silver grains present in cells in the arcuate nucleus (Figs. 7 and 8). We also measured Kiss1 mRNA in the AVPV and found no significant differences between groups in either number of cells labeled or intensity of labeling (see Fig. 8). We also measured serum concentrations of LH, FSH, and T3 in these animals and saw no significant differences between groups in any of these hormones (Table 1, although note that all but one animal showed a diestrus smear at the time of killing).


Figure 07
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  		FIG. 7 Expression of Galp mRNA in the arcuate nucleus of rats receiving continuous icv infusions of JB-1 or aCSF (controls). A and B) Representative examples of Galp mRNA in control (A) or JB-1-treated (B) rats. C) Mean number of Galp-positive cells per arcuate nucleus of rats. D) Mean number of silver grains per cell. E) Food intake before and after surgical placement of cannula (Day 0). F) Body weight in animals as a percentage of presurgical body weight. Animals were killed on Day 6 after surgery, when control animals exhibit compensatory hyperphagia but JB-1-treated animals do not. Data represent mean ± SEM. In E, *P < 0.05. Bar = 50 µm (A, B).


Figure 08
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  		FIG. 8 Expression of Kiss1 mRNA in the arcuate nucleus and AVPV of rats receiving continuous icv infusions of JB-1 or aCSF (controls). A–D) Data from the arcuate nucleus. E–H) Data from the AVPV. A, E, B, F) Representative examples of Kiss1 mRNA in control (A, E) or JB-1-treated (B, F) rats. C, G) Mean number of Kiss1-positive cells per arcuate nucleus of rats. D, H) Mean number of silver grains per cell. Sections are taken from the same animals as in Figure 7. Data represent mean ± SEM. Original magnification A, B, E, F x200.


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  		TABLE 1 Serum hormone levels of control and JB-1-treated rats.a

DISCUSSION

We report the novel finding that ongoing IGF1 signaling in the brain is necessary for normal function of the hypothalamic-pituitary-gonadal axis in female rats. Of 26 rats receiving chronic infusions of JB-1 into the third ventricle, only 4 showed evidence of estrous cycles during the treatment period, and the resumption of estrous cycles in these four rats was delayed when compared with vehicle-infused females (see Figs. 2, 3, and 5). This observation is consistent with our previous research on OVX rats showing that brain IGF1 signaling is necessary for the positive feedback effects of E2 and P that lead to the LH surge [1]. Interestingly, the suppressive effects of blocking brain IGF1 receptors on estrous cyclicity continued for a minimum of 4 days, even after the antagonist treatment was terminated by removal of the minipump. By contrast, when control animals were pair fed to JB-1-treated females, resumption of ad libitum feeding initiated estrous cycles in all but one rat within 2–3 days (see Fig. 4). One explanation for the delay in resumption of estrous cyclicity after withdrawal of JB-1 may be that chronic blockade of IGF1 receptors modifies the neural circuitry underlying GnRH secretion, and the delay in resumption of estrous cycles reflects restoration of synaptic connectivity. This would be consistent with the studies of Fernandez-Galaz et al. [3] and Cardona-Gomez et al. [4], who demonstrated that brain IGF1 receptor activity is required for E2-dependent synaptic remodeling in the arcuate nucleus, an important regulator of GnRH release.

Actions of IGF1 on the Reproductive Axis Are Not a Consequence of Reduced Nutrient Availability

Taken together, the present findings suggest that the suppression of the reproductive axis produced by blocking brain IGF1 signaling is not a consequence of reduced food intake or inadequate nutrient availability. Body weights of control and JB-1-infused rats were not different by Day 5 following surgery, yet in all of the experiments, only the control animals had initiated estrous cycles at this time. In addition, food intake in animals receiving chronic antagonist infusions returned to presurgical baseline levels within 2–3 days after surgery, even though estrous cycles often failed to resume for the entire 14-day treatment period. Thus, it is unlikely that JB-1-treated animals had a deficient supply of oxidizable metabolic fuels, a treatment known to suppress estrous cycles [17, 59]. In addition, when IGF1 and JB-1 were coinfused, body weight was not fully restored to control levels, whereas the inhibition of compensatory hyperphagia and estrous cycles was completely reversed. Furthermore, control animals that were pair fed to JB-1 rats regained weight significantly more slowly but resumed estrous cycles more rapidly than JB-1 rats (although more slowly than aCSF controls). Therefore, the suppression of estrous cycles by the IGF1 receptor antagonist is likely to be via a direct action on the hypothalamic-pituitary-gonadal axis, perhaps on the GnRH neurons, which express both IGF1 and IGF1 receptors [8, 9] and/or their afferent inputs. This interpretation receives further support from experiments in peripubertal animals, in which central administration of IGF1 increases LH secretion, an effect that can be blocked with prior immunoneutralization of GnRH [6]. Central administration of IGF1 can also advance puberty in female rats [6] (although see Zeinoaldini et al. [60]).

Circulating IGF1 concentrations are related to nutritional state [21, 22], and plasma levels of free IGF1 have been proposed to be a key factor signaling nutritional status to the reproductive axis [23, 24]. Our observations, however, indicate that brain IGF1 receptors do not simply signal nutrient status to the brain centers controlling reproductive function. Blockade of brain IGF1 receptors inhibited reproductive capacity, evidenced by the prolonged cessation of estrous cycles. Importantly, estrous cycles in JB-1-treated rats were inhibited even when food intake and body weight were not significantly different from control animals. Moreover, chronic JB-1 infusion did not increase food intake under any circumstances, the response that would be predicted if blockade of brain IGF1 receptors were interpreted as an indicator of negative energy balance. In fact, blockade of brain IGF1 receptors prevented the compensatory hyperphagia seen in vehicle-infused animals, an effect similar to the administration of exogenous leptin following a period of food restriction [61]. Our data thus show the novel and unexpected finding that brain IGF1 signaling is required for the rebound feeding that normally occurs following a period of reduced food intake [61, 62]. Ongoing brain IGF1 receptor signaling may thus be required to detect the nutritional deficit that triggers rebound feeding.

Actions of IGF1 on Metabolic Parameters and Rebound Feeding

Serum levels of glucose, insulin, and leptin did not differ between control and JB-1-treated animals at 5 days following cannula implantation, a time point when their 24-h food intake differed significantly. However, this was the only time at which serum glucose, insulin, and leptin concentrations were measured, and body weights were not significantly different at this time. It is possible that at other time points, circulating levels of these nutrient signals differ between control and JB-1-treated animals. It is not unexpected that serum leptin concentrations were significantly different between JB-1-treated animals and animals that were pair fed to them. The reduced leptin in pair-fed animals correlates with the reduced body weight seen in this group compared with JB-1-treated or control rats. It is possible that this difference contributes to the rebound feeding that pair-fed rats exhibit upon termination of the pair feeding. JB-1-treated animals do not exhibit rebound feeding upon withdrawal of the drug, possibly because their circulating leptin concentrations are not appreciably reduced.

The effects of JB-1 on body weight could be related to the ability of IGF1 to increase fat oxidation and energy expenditure in rats [63, 64]. The absence of brain IGF1 receptor signaling in JB-1-infused rats may signal reduced availability of fuels for oxidation, leading animals to curtail energy expenditure. It is conceivable that energy expenditure is reduced to the extent that the animals actually gain weight, even without compensatory hyperphagia. Indeed, the weight gain in JB-1 animals is greater than in pair-fed animals (see Figs. 3 and 4). Thus, pair-fed animals with restricted food intake, but normal brain IGF1 receptor signaling, may utilize more energy than JB-1 animals, and hence, they are unable to overcome their energy deficit. The concept that blockade of brain IGF1 receptors may increase energy storage is also supported by the fact that JB-1 animals have significantly higher serum leptin concentrations than pair-fed animals. The reduced serum leptin concentration in pair-fed animals is consistent with the fact that free fatty acids downregulate leptin secretion [65]. Although we did not measure free fatty acids, the release of free fatty acids from lipid stores into the circulation is a well-known response to negative energy balance.

Even though anesthesia and surgery had significant effects on body weight and estrous cyclicity, estrous cycles returned quickly in control animals, and the rebound feeding that occurred in control animals postsurgically is consistent in amount and duration with that occurring in postfood deprivation [62]. Thus, it is possible that the effects of JB-1 on rebound feeding in a specific food-deprivation model would show the same requirement for IGF1 signaling. As noted above, the current data suggest that the JB-1-treated animals fail to perceive some signal of compromised nutritional status. This may be due to reduced expression or activity of some molecule regulated by IGF1 that normally drives animals to eat.

Activation of Brain IGF1 Receptors, Not Insulin Receptors, Is Permissive for Estrous Cyclicity and Compensatory Hyperphagia

Our findings strongly suggest that JB-1 disrupts estrous cycles by blocking IGF1 receptors rather than insulin receptors. It is important to consider the role of insulin in the regulation of energy balance and reproductive homeostasis. Insulin is a low-affinity ligand for the IGF1 receptor, and IGF1 can bind to insulin receptors [66]. Supraphysiological doses of insulin affect E2-dependent neurite outgrowth, most likely through activation of IGF1 receptors [67]. Icv administration of insulin also reduces food intake and decreases body weight [31]. Moreover, insulin deficiency, as in diabetes, produces hyperphagia [32]. Hence, if JB-1 blocked brain insulin signaling, this should result in hyperphagia, as observed in mice with a neuron-specific insulin receptor knockout [68]. Instead, we observed an inhibition of rebound feeding in animals chronically infused with the IGF1 receptor antagonist. To ensure that JB-1 specifically blocked IGF1 receptor signaling, we combined JB-1 with a 10-fold lower dose of IGF1. Coinfusion of this low level of IGF1, which by itself had no effect on food intake, body weight, or estrous cycles, completely reversed the effects of the antagonist on feeding and estrous cyclicity. Therefore, it is highly unlikely that the actions of JB-1 on food intake or the reproductive axis can be attributed to insulin receptors, a conclusion consistent with the in vitro research showing that this peptide binds selectively to IGF1 receptors and has a very low affinity for insulin receptors [51].

It is interesting that animals receiving a combination of JB-1 and IGF1 returned to baseline body weight more rapidly than JB-1-treated animals but more slowly than controls or animals given IGF1 alone. Therefore, although exogenous IGF1 completely rescued estrous cyclicity and food intake in JB-1-treated females, it did not completely reverse JB-1 effects on body weight. As discussed above, this may indicate a role for brain IGF1 receptors in energy storage or expenditure that is independent of their actions on food intake and the reproductive axis. Because we were unable to monitor metabolic rate or activity levels in our animals, we cannot distinguish between these possibilities.

Chronic Blockade of IGF1 Receptors Does Not Alter Galp or Kiss1 Expression

It is possible that IGF1 exerts its effects on the reproductive axis via afferent systems that innervate GnRH neurons or their axon terminals. We investigated two potential mediators of IGF1 inhibition of reproduction and rebound feeding, Galp and Kiss1 mRNA, in the hypothalamus of control and JB-1-treated animals. GALP, which is expressed only in the arcuate nucleus of the hypothalamus, is a potent regulator of food intake and metabolism [43, 69]. GALP neurons are regulated by fasting, leptin, insulin, and glucose [27, 44, 45]. Administration of GALP stimulates LH secretion and sexual behavior in male rodents and primates [43, 69, 70] and rescues reproductive function in diabetic rats [46]. These observations suggest that GALP is an integrator of metabolism and reproduction [42]. KISS1 neurons are present in the arcuate nucleus and AVPV, and KISS1 directly activates GnRH neurons, stimulating LH release [47, 56, 57]. Moreover, Kiss1 neurons are targets for leptin [50], placing them in a position to respond to nutritional status.

We therefore hypothesized that blockade of IGF1 receptor signaling in the brain would chronically alter expression of these genes and that this might account for the observed changes in reproductive function and food intake. However, there were no differences in the number of Galp- or Kiss1-positive cells, or in the number of grains per labeled cell, between control and JB-1-treated rats killed at a time when estrous cyclicity and food intake differed. These data are consistent with our findings that serum leptin, insulin, and glucose concentrations did not differ between JB-1-treated rats and controls. Nonetheless, our data do not preclude a role for GALP or KISS1 at earlier times after drug infusion. We killed animals at a time when estrous cycles were reinstated in controls, but not in JB-1 animals, and when food intake was significantly different between these two groups. However, mRNA expression of these neuropeptide systems may have differed at earlier time points, or mRNA expression may not be regulated at all. Protein expression and peptide release are other likely points of regulation. Thus, we cannot rule out a role for GALP or KISS1 in the suppression of estrous cycles by JB-1. Moreover, it may be important to direct future efforts toward examination of other neuropeptides and neuromodulators (e.g., NPY, proopiomelanocortin) known to regulate both food intake and reproduction.

At the time of brain collection for mRNA measurement, we also took blood samples from the rats and measured serum LH, FSH, and T3. There were no significant differences between control and JB-1-treated rats in any of these hormones; however, again, these are single data points, and further investigation into the pulsatile secretion of these hormones may reveal differences between groups. The LH finding is consistent with the fact that there were no differences in Kiss1 mRNA expression in the brain. Because estrous cyclicity is typically suppressed in JB-1-treated animals but not in control females 6 days after surgery, one might have expected that gonadotropin levels would be higher in controls. However, six of seven of the control animals were killed on the day of diestrus, when serum LH and FSH are normally low. If we collected serum from aCSF-treated, gonadally intact animals on the day of proestrus, we would expect to observe significantly higher serum LH, and possibly FSH, concentrations in controls.

In summary, the present findings demonstrate a requirement for ongoing brain IGF1 receptor activity to maintain normal hypothalamic-pituitary-gonadal function in female rats. Brain IGF1 receptor regulation of the reproductive axis is not attributable to its effects on food intake, nutrient availability, or body weight. Our observations also reveal a novel permissive role for brain IGF1 receptors in compensatory hyperphagia induced by anesthesia and surgical manipulation, a role that is distinct from that of reproductive regulation.

ACKNOWLEDGMENTS

The authors thank Ms. Brigitte Mann at Northwestern University for performing the LH, FSH, and T3 assays. We also thank Dr. Alice Shu for technical assistance.

FOOTNOTES

1Supported by NIH grants T32 DK07513 (B.J.T.), R01 HD29856 (A.M.E.), DK066238 (G.S.F.), R01DK066618 (G.J.S.), and the Skirball Foundation (A.M.E. and G.J.S.). Back

Correspondence: 2Brigitte J. Todd, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., F113, Bronx, NY 10461. FAX: 718 430 8654; e-mail: btodd{at}aecom.yu.edu

Received: 29 January 2007.

First decision: 6 March 2007.

Accepted: 11 June 2007.

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