Corticotropin-releasing factor receptor subtypes mediating nutritional suppression of estrous behavior in Syrian hamsters

Caloric deprivation inhibits reproduction, including copulatory behaviors, in female mammals. Decreases in metabolic fuel availability are detected in the hindbrain, and this information is relayed to the forebrain circuits controlling estrous behavior by neuropeptide Y (NPY) projections. In the forebrain, the nutritional inhibition of estrous behavior appears to be mediated by corticotropin-releasing factor (CRF) or urocortin-signaling systems. Intracerebroventricular (ICV) infusion of the CRF antagonist, astressin, prevents the suppression of lordosis by food deprivation and by NPY treatment in Syrian hamsters. These experiments sought to determine which CRF receptor type(s) is involved. ICV infusion of the CRF receptor subtype CRFR2-selective agonists urocortin 2 and 3 (UCN2, UCN3) inhibited sexual receptivity in hormone-primed, ovariectomized hamsters. Furthermore, the CRFR2-selective antagonist, astressin 2B, prevented the inhibition of estrous behavior by UCN2 and by NPY, consistent with a role for CRFR2. On the other hand, astressin 2B did not prevent the inhibition of behavior induced by 48-h food deprivation or ICV administration of CRF, a mixed CRFR1 and CRFR2 agonist, suggesting that activation of CRFR1 signaling is sufficient to inhibit sexual receptivity in hamsters. Although administration of CRFR1-selective antagonists (NBI-27914 and CP-154,526) failed to reverse the inhibition of receptivity by CRF treatment, we could not confirm their biological effectiveness in hamsters. The most parsimonious interpretation of these findings is that, although NPY inhibits estrous behavior via downstream CRFR2 signaling, food deprivation may exert its inhibition via both CRFR1 and CRFR2 and that redundant neuropeptide systems may be involved.     

Disinhibition of female sexual behavior by a CRH receptor antagonist in Syrian hamsters

Several conditions that inhibit female sexual behavior are thought to be associated with altered corticotropin-releasing hormone (CRH) activity in the brain. The present experiments examined the hypothesis that endogenous CRH receptor signaling mediates the inhibition of estrous behavior by undernutrition and in other instances of sexual dysfunction. Intracerebroventricular (ICV) infusion of CRH or urocortin inhibited estrous behavior in ovariectomized steroid-primed hamsters. Conversely, ICV infusion of the CRH receptor antagonist astressin prevented the suppression of estrous behavior by food deprivation or by ICV administration of neuropeptide Y. Astressin treatment also induced sexual receptivity in nonresponders, animals that do not normally come into heat when treated with hormones, and this effect persisted in subsequent weekly tests in the absence of any further astressin treatment. Activation of the hypothalamo-pituitary-adrenocortical axis was neither necessary nor sufficient to inhibit estrous behavior, indicating that this phenomenon is due to other central actions of CRH receptor agonists. This is the first direct evidence that CRH receptor signaling may be a final common pathway by which undernutrition and other conditions inhibit female sexual behavior.     

Metabolic control of food intake and estrous cycles in syrian hamsters. I. Plasma insulin and leptin

The "adipostat hypothesis" refers to the idea that circulating hormone concentrations reflect levels of body adiposity and act as signals to control food intake and reproduction. Implicit in the adipostatic hypothesis are the following two assumptions: 1) plasma levels of adipostatic hormones accurately reflect body fat content and 2) decreased plasma concentrations of adipostatic hormones necessarily result in increased food intake and inhibited reproductive processes. The present experiments are designed to test these assumptions. Fat and lean Syrian hamsters were either fasted for 12, 24, 36, or 48 h or allowed ad libitum access to food. Contrary to the first assumption, plasma leptin and insulin levels in fat hamsters dropped dramatically by 12 h after the start of a fast, with no significant change in body fat content and no postfast hyperphagia. Lean hamsters showed anestrus after a 48-h fast but not after a 24-h fast. Contrary to the second assumption of the lipostatic hypothesis, lean hamsters fasted for 24 h and then refed for the next 24 h had leptin levels that were not significantly elevated compared with those of 48-h-fasted hamsters. Thus, in adult female Syrian hamsters, plasma leptin concentrations do not accurately reflect body fat content under all conditions; normal estrous cyclicity does not necessarily require plasma leptin concentrations higher than those of fasted hamsters; and decreased plasma leptin levels do not result in increased food intake.     

Food deprivation inhibits estrous behavior in hormone-treated Syrian hamsters despite elevated estradiol levels

Estradiol and progesterone (P) induce female mammalian reproductive behaviors, which are, in turn, sensitive to food availability. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. While this suppression of estrous behavior may be due to alterations in neural steroid receptor levels, it is also possible that decreased levels of circulating estradiol could be involved in mediating this suppression. Ovariectomized Syrian hamsters given varying doses of estradiol benzoate (EB) and P were tested to determine whether increasing doses of sex steroids would overcome the suppressive effects of food deprivation on estrous behavior. As expected, lordosis duration decreased in food-deprived animals. Increasing the levels of EB, but not P, increased lordosis duration in the food-deprived animals so that animals who were given 20 microg of EB had lordosis durations significantly longer than food-deprived hamsters that received 1.5 microg and 2.5 microg EB. Following an injection of 2.5 microg of EB, food-deprived hamsters actually had higher circulating levels of estradiol than ad libitum-fed animals. Therefore, increasing circulating levels of estradiol can increase lordosis durations in fasted animals; however, the suppression of estrous behavior occurs despite increased circulating estradiol levels in ovariectomized, steroid-treated animals. The most parsimonious explanation for this phenomenon is a deprivation-induced reduction in neural responsiveness to estradiol.     

Leptin Indirectly Affects Estrous Cycles by Increasing Metabolic Fuel Oxidation

In previous experiments, lean Syrian hamsters fasted on days 1 and 2 of the estrous cycle failed to show sex behavior and ovulation normally expected to occur on the evening of day 4. The first goal of the present experiment was to determine whether systemic treatment with theob(obese) protein leptin could reverse the effects of fasting on estrous cyclicity, social behaviors, and ovulation rate. Fasting-induced anestrus was reversed and normal sex and social behavior and ovulation rate were restored in hamsters injected intraperitoneally with 5 mg/kg leptin every 12 h during fasting on days 1 and 2 of the estrous cycle. A second goal was to test whether the effects of leptin could be prevented by treatment with pharmacological agents that block the oxidation of metabolic fuels. Glucose oxidation was blocked by treatment with 2-deoxy- -glucose (2DG) and fatty acid oxidation was blocked by treatment with methyl palmoxirate (MP). 2DG (1000 mg/kg) or MP (20 mg/kg) was administered at doses that did not induce anestrus in hamsters fedad libitum.As in the first experiment, fasting-induced anestrus was reversed by leptin treatment. However, when each injection of leptin was preceded by an injection of 2DG or MP, leptin treatment did not reverse fasting-induced anestrus. In summary, estrous cyclicity was not restored when oxidation of metabolic fuels was blocked, despite high endogenous levels of leptin. These results are consistent with the hypothesis that leptin acts indirectly on the reproductive system by increasing fuel oxidation.     

Aggression in the female golden hamster: effects of reproductive state and social isolation

To gain information on possible hormonal correlates, the aggressive behavior of intact female hamsters towards males was observed at various times during the estrous cycle, pseudopregnancy, pregnancy, and lactation. For methodological information, estrous cycle females also were tested after varying periods of social isolation. It was found that pregnant and especially lactating hamsters were more aggressive than pseudopregnant or estrous cycling females. Comparisons of days within each reproductive condition showed that aggression tended to be higher on certain days: the day preceding behavioral estrus of the estrous cycle, Day 10 of pregnancy, and the first 5 days of lactation. Except for pseudopregnancy, sexual behavior unaccompanied by aggression occurred at some time during all reproductive conditions, and both sexual behavior and aggression were found to occur together on Day 10 of pregnancy and Day 1 of lactation. The changes in aggressive behavior associated with reproductive states were attributed to increased male interest, inhibition by ovarian hormones, and facilitation by prolactin. Increasing periods of social isolation also were found to be associated with increased aggression. It was suggested that this effect, too. might have been due to increased prolactin levels.     

Mating stimulation required for mating-induced estrous abbreviation in female rats: effects of repeated testing

Mating stimulation, particularly vaginal-cervical stimulation, causes estrous abbreviation in female rats. In most previous studies, female rats were repeatedly tested for sexual behavior until estrous termination occurred. Thus, it was not clear whether sensory stimulation (e.g., flank stimulation, olfactory cues) received during the repeated testing procedure contributed to estrous abbreviation. In Experiment 1, we determined the effect of premating to two or four ejaculations on the rate of estrous termination when a repeated testing procedure was used. We compared ovariectomized, hormone-primed, female rats receiving (1) four ejaculations, (2) two ejaculations, or (3) no premating. Females premated to either two or four ejaculations showed significantly lower levels of sexual receptivity 12 h later than did nonpremated females. These results confirm that premating induces estrous abbreviation when a repeated testing procedure is used. In Experiment 2, we determined whether the repeated testing procedure was necessary for estrous abbreviation. Ovariectomized, hormone-primed female rats were premated to two ejaculations or not premated. The rats were then tested for sexual behavior repeatedly or only once. Females that were premated and repeatedly tested for sexual behavior showed a statistically significant decrease in sexual receptivity compared to females that were not premated; however, the level of sexual receptivity in premated females did not differ from that in non-premated females when they were tested only once. The results suggest that heat duration is the result of a complex interplay between those factors that promote the expression of sexual receptivity and those that inhibit it.     

Hormonal changes and energy substrate availability during the hibernation cycle of Syrian hamsters

Animals have to adapt to seasonal variations in food resources and temperature. Hibernation is one of the most efficient means used by animals to cope with harsh winter conditions, wherein survival is achieved through a significant decrease in energy expenditure. The hibernation period is constituted by a succession of torpor bouts (hypometabolism and decrease in body temperature) and periodic arousals (eumetabolism and euthermia). Some species feed during these periodic arousals, and thus show different metabolic adaptations to fat-storing species that fast throughout the hibernation period. Our study aims to define these metabolic adaptations, including hormone (insulin, glucagon, leptin, adiponectin, GLP-1, GiP) and metabolite (glucose, free fatty acids, triglycerides, urea) profiles together with body composition adjustments. Syrian hamsters were exposed to varied photoperiod and temperature conditions mimicking different phases of the hibernation cycle: a long photoperiod at 20 °C (LP20 group), a short photoperiod at 20 °C (SP20 group), and a short photoperiod at 8 °C (SP8). SP8 animals were sampled either at the beginning of a torpor bout (Torpor group) or at the beginning of a periodic arousal (Arousal group). We show that fat store mobilization in hamsters during torpor bouts is associated with decreased circulating levels of glucagon, insulin, leptin, and an increase in adiponectin. Refeeding during periodic arousals results in a decreased free fatty acid plasma concentration and an increase in glycemia and plasma incretin concentrations. Reduced incretin and increased adiponectin levels are therefore in accordance with the changes in nutrient availability and feeding behavior observed during the hibernation cycle of Syrian hamsters.     

Initiation of hyperinsulinemia and hyperleptinemia is diet dependent in C57BL/6 mice.

C57BL/6 female mice were fed high fat diets containing different types of carbohydrate (sucrose or corn starch) and contents of cholesterol (0.03 % or 1 %) to identify early metabolic changes leading to increases in leptin levels and eventual insulin resistance. Under identical dietary fat conditions, type of carbohydrate and cholesterol content contributed to the timing of leptin increases. Mice fed a high-fat, high-sucrose diet showed early (4 weeks) and robust increases in circulating insulin and leptin levels (2-fold and 5-fold, respectively). In contrast, mice fed this diet with added cholesterol or with sucrose substituted by corn starch led to marked delays (8-10 weeks) in the elevations of insulin and leptin, although body weight gains were nearly identical among test diet groups. Thus, sucrose in combination with saturated fat played a specific role in initiating early metabolic changes associated with elevated leptin and insulin levels. Because leptin levels were most reflective of changes in insulin, our data support a role for insulin in determining plasma leptin levels in mice.     

Blockage of neonatal leptin signaling induces changes in the hypothalamus associated with delayed pubertal onset and modifications in neuropeptide expression during adulthood in male rats

The neonatal leptin surge, occurring from postnatal day (PND) 5 to 13 and peaking at PND9 in rodents, is important for the development of neuroendocrine circuits involved in metabolic control and reproductive function. We previously demonstrated that treatment with a leptin antagonist from PND 5 to 9, coincident with peak leptin levels in the neonatal surge, modified trophic factors and markers of cell turnover and neuronal maturation in the hypothalamus of peri-pubertal rats. The kisspeptin system and metabolic neuropeptide and hormone levels were also modified. Here our aim was to investigate if the timing of pubertal onset is altered by neonatal leptin antagonism and if the previously observed peripubertal modifications in hormones and neuropeptides persist into adulthood and affect male sexual behavior. To this end, male Wistar rats were treated with a pegylated super leptin antagonist (5 mg/kg, s.c.) from PND 5 to 9 and killed at PND102–103. The appearance of external signs of pubertal onset was delayed. Hypothalamic kiss1 mRNA levels were decreased in adult animals, but sexual behavior was not significantly modified. Although there was no effect on body weight or food intake, circulating leptin, insulin and triglyceride levels were increased, while hypothalamic leptin receptor, POMC and AgRP mRNA levels were decreased. In conclusion, alteration of the neonatal leptin surge can modify the timing of pubertal onset and have long-term effects on hypothalamic expression of reproductive and metabolic neuropeptides.     

Effects of naltrexone and CCK on estrous behavior and food intake in Syrian hamsters

Food deprivation inhibits estrous behavior in several species of rodents, but little is known about the neurotransmitter systems mediating this phenomenon. We determined whether partial blockade of opioid receptors by continuous infusion of naltrexone and/or acute peripheral injection of cholecystokinin (CCK) administration would overcome the suppressive effects of food deprivation on estrous behavior in Syrian hamsters. Contrary to expectation, naltrexone produced a slight suppression of estrous behavior, and systemic CCK administration had no effect. This dose of naltrexone was sufficient to reduce in vivo binding of [(3)H]naloxone in the brain, and both compounds affected other parameters such as food intake and body weight gain. Thus, the doses of CCK and naltrexone that were used were physiologically effective. These findings suggest that neither peripheral CCK nor opioid systems are likely to play a major role in the suppression of hamster estrous behavior by food deprivation.     

Differential effect of long-term food restriction on fatty acid synthase and leptin gene expression in rat white adipose tissue.

Long-term food restriction (85%, 70% and 50% of ad libitum energy intake for one month) induced a substantial fall in serum leptin concentration and leptin mRNA levels in epididymal white adipose tissue in rats. Surprisingly, this suppression was not reversed by refeeding ad libitum for 48 h. The reduction in serum leptin concentration and leptin mRNA level did not strictly correlate with reduction in fat or body mass. Unlike serum leptin concentration and epididymal adipose tissue leptin mRNA levels, fatty acid synthase activity, fatty acid synthase protein abundance and fatty acid synthase mRNA levels increased significantly in white adipose tissue after refeeding rats subjected to food restriction. The increase in serum insulin concentration was observed in all groups on different degrees of food restriction and refed ad libitum for 48 h compared to controls. A decrease in serum insulin concentration was found in the rats not refed before sacrifice. Long-term food restriction did not significantly affect serum glucose concentrations in either refed or non-refed rats. The data reported in this paper indicate that there is no rapid rebound in serum leptin concentration or leptin gene expression in contrast to the increase in serum insulin concentration and fatty acid gene expression in white adipose tissue of rats refed ad libitum after one month's food restriction.     

Energy Budget,Behavior and Leptin in Striped Hamsters Subjected to Food Restriction and Refeeding

Food restriction induces a loss of body mass that is often followed by rapid regaining of the lost weight when the restriction ends, consequently increasing a risk of development of obesity. To determine the physiological and behavioral mechanisms underlining the regaining, striped hamsters were restricted to 85% of initial food intake for 4 weeks and refed ad libitum for another 4 weeks. Changes in body mass, energy budget, activity, body composition and serum leptin level were measured. Body mass, body fat mass and serum leptin level significantly decreased in food-restricted hamsters, and increased when the restriction ended, showing a short “compensatory growth” rather than over-weight or obesity compared with ad libitum controls. During restriction, the time spent on activity increased significantly, which was opposite to the changes in serum leptin level. Food intake increased shortly during refeeding, which perhaps contributed to the rapid regaining of body mass. No correlation was observed between serum leptin and energy intake, while negative correlations were found in hamsters that were refed for 7 and 28 days. Exogenous leptin significantly decreased the time spent on activity during food restriction and attenuated the increase in food intake during refeeding. This suggests that low leptin in restricted animals may function as a starvation signal to induce an increase in activity behavior, and high leptin likely serves as a satiety signal to prevent activity during refeeding. Leptin may play a crucial role in controlling food intake when the restriction ends, and consequently preventing overweight.     

Refeeding-Induced Brown Adipose Tissue Glycogen Hyper-Accumulation in Mice Is Mediated by Insulin and Catecholamines

Brown adipose tissue (BAT) generates heat during adaptive thermogenesis through a combination of oxidative metabolism and uncoupling protein 1-mediated electron transport chain uncoupling, using both free-fatty acids and glucose as substrate. Previous rat-based work in 1942 showed that prolonged partial fasting followed by refeeding led to a dramatic, transient increase in glycogen stores in multiple fat depots. In the present study, the protocol was replicated in male CD1 mice, resulting in a 2000-fold increase in interscapular BAT (IBAT) glycogen levels within 4–12 hours (hr) of refeeding, with IBAT glycogen stores reaching levels comparable to fed liver glycogen. Lesser effects occurred in white adipose tissues (WAT). Over the next 36 hr, glycogen levels dissipated and histological analysis revealed an over-accumulation of lipid droplets, suggesting a potential metabolic connection between glycogenolysis and lipid synthesis. 24 hr of total starvation followed by refeeding induced a robust and consistent glycogen over-accumulation similar in magnitude and time course to the prolonged partial fast. Experimentation demonstrated that hyperglycemia was not sufficient to drive glycogen accumulation in IBAT, but that elevated circulating insulin was sufficient. Additionally, pharmacological inhibition of catecholamine production reduced refeeding-induced IBAT glycogen storage, providing evidence of a contribution from the central nervous system. These findings highlight IBAT as a tissue that integrates both canonically-anabolic and catabolic stimulation for the promotion of glycogen storage during recovery from caloric deficit. The preservation of this robust response through many generations of animals not subjected to food deprivation suggests that the over-accumulation phenomenon plays a critical role in IBAT physiology.     

Nonphotic phase shifting in female Syrian hamsters: interactions with the estrous cycle

Nonphotic phase shifting of circadian rhythms was examined in female Syrian hamsters. Animals were stimulated at zeitgeber time 4.5 by either placing them in a novel running wheel or by transferring them to a clean home cage. Placement in a clean home cage was more effective than novel wheel treatment in stimulating large (> 1.5 h) phase shifts. Peak phase shifts (ca. 3.5 h) and the percentage of females showing large phase shifts were comparable to those found in male hamsters stimulated with novel wheels. The amount of activity induced by nonphotic stimulation and the amount of phase shifting varied slightly with respect to the 4-day estrous cycle. Animals tended to run less and shift less on the day of estrus. Nonphotic stimulation on proestrus often resulted in a 1-day delay of the estrous cycle reflected in animals' postovulatory vaginal discharge and the expression of sexual receptivity (lordosis). This delay of the estrous cycle was associated with large phase advances and high activity. These results extend the generality of nonphotic phase shifting to females for the first time and raise the possibility that resetting of circadian rhythms can induce changes in the estrous cycle.     

Sexual receptivity in hamsters: brain nuclear estrogen and cytosolic progestin receptors after single and multiple steroid treatments and during the estrous cycle

This series of experiments investigated the relationship between various treatments consisting of estradiol benzoate (EB) and progesterone (P) on sexual receptivity and on concentrations of nuclear estrogen receptors (NER) and cytosolic progestin receptors (CPR) in the hypothalamus-preoptic area in female hamsters. The injection of 1 microgram EB at 0 and 24 hr resulted in higher levels of receptivity (after 0.25 or 0.5 mg P), NER and CPR compared to those obtained after a single injection of 2 micrograms EB. Animals treated with 5 micrograms EB at 0 and 24 hr displayed greater levels of receptivity (after 0.5 mg P) and had higher NER concentrations than animals given a single injection of 10 micrograms EB. Groups treated with either 1 microgram EB at 0 hr or 0.5 microgram EB at 0 and 24 hr did not differ and showed low levels of receptivity, NER and CPR, NER and CPR were also measured on each day of the estrous cycle. NER levels rose between Days 1 and 2, again between Days 2 and 3, and remained elevated on Day 4. CPR levels increased between Days 2 and 3, and there was no difference between Days 3 and 4. Taken together, these data suggest that receptivity in hamsters after estrogen exposure is correlated with the accumulation and maintenance of relatively high NER levels and on the induction of CPR. This can be accomplished by a single large injection of EB or by smaller split doses.     

Anabolic-Androgenic Steroid Effects on Sexual Receptivity in Ovariectomized Rats

Anabolic-androgenic steroid (AAS) compounds are synthetic androgens taken by athletes to increase physical strength and endurance. Recent studies in our laboratory have demonstrated that AAS administration disrupts the estrous cycle of Long–Evans rats. The present experiments examined the effects of six commonly abused AAS compounds on sexual receptivity in ovariectomized rats. Adult female Long–Evans rats received estradiol benzoate (EB; 2.0 μg/day sc) for 6 consecutive days followed by 15 days of EB concurrent with daily sc injections of 7.5 mg/kg of one of the following AAS compounds: 17α-methyltestosterone, methandrostenolone, nandrolone decanoate, stanozolol, oxymetholone, testosterone cypionate, or the oil vehicle. On Day 15, all female rats received progesterone (1.0 mg/rat) 4 h before testing. Tests for sexual receptivity were conducted on Days 3, 6, 14, and 15 of AAS treatment. Although the time course of AAS effects on sexual receptivity varied, some overall effects were clear. For example, 17α-methyltestosterone, methandrostenolone, nandrolone decanoate, and stanozolol interfered with the display of sexual receptivity on Day 14, whereas oxymetholone and testosterone cypionate had no effect. Rats in all groups displayed high levels of sexual receptivity after receiving progesterone on Day 15. Our results show that AAS compounds vary in their degree of inhibition of female sexual behavior in ovariectomized rats.     

Acute insulin-induced elevations of circulating leptin and feeding inhibition in lean but not obese rats

Insulin has been shown to stimulate leptin mRNA expression acutely in rat adipose tissue, but its short-term effects on circulating leptin levels, and subsequent feeding behavior, have not been well described. We used 11-mo-old female selectively bred obesity-resistant (OR) and obesity-prone (OP) Sprague-Dawley rats maintained on laboratory chow to investigate this question. At testing, body weights and basal leptin levels of the OP rats were significantly elevated compared with the OR rats. In the 3-h fasted state, injection of 2.0 U insulin/kg ip resulted in significant elevations of plasma leptin at 4 h postinjection in both OP and OR groups (hour 4, +2.50 and +5.98 ng/ml, respectively). In separate feeding tests with the same groups, intake of laboratory chow pellets was significantly inhibited during hours 2-4 after 2.0 U/kg of insulin in the OR (-80.1%, P < 0.05), but not in the OP group, compared with intake after saline injections. In feeding tests with palatable moderately high-fat pellets after 2.0 and 3.0 U insulin/kg ip, significant decreases between hours 2 and 4 in intake were seen in the OR group only (-41.0 and -68.3%, respectively). Thus feeding inhibition coincides with insulin-induced elevations of plasma leptin in lean but not obese Sprague-Dawley rats. Our data suggest that elevations of leptin within the physiological range may contribute to short-term inhibition of food intake in rats and that this process may be stimulated by feeding-related insulin release.     

Leptin concentrations in plasma and follicular fluid from prepubertal gilts as influenced by fasting, refeeding and insulin.

This study's aim was to examine whether fasting and refeeding would influence leptin levels in both plasma and follicular fluid from prepubertal gilts, and whether insulin affects leptin levels in fasting gilts. In experiment 1, four gilts were fasted for 72 h and then refed. Blood samples were withdrawn during normoalimentation, at the end of fasting, and for 4 h after refeeding. All samples were assayed for leptin; alternate samples were assayed for insulin, glucose and non-esterified fatty acids (NEFA). Fasting caused a decrease in leptin, glucose and insulin levels in plasma, while NEFA concentrations increased. In experiment 2, four gilts were given insulin as a bolus (0.2 IU/kg body weight) after 68 h of fasting. Blood samples were collected every 15 min around insulin administration and were assayed for leptin, insulin and glucose. This experiment shows that insulin administration increases leptin levels during fasting. In experiment 3, gilts were ovariectomized during normal alimentation (n=4), after 48 h of fasting (n=4), and after 48 h of realimentation following 48 h of fasting (n=4). Leptin levels in both plasma and follicular fluid collected after 48 h of fasting were significantly lower than those observed during normoalimentation or refeeding. In conclusion, a transient increase in insulin during fasting is effective in restoring leptin concentrations; in addition, leptin levels in follicular fluid parallel those in plasma.