Roles of leptin and resistin in metabolism, reproduction, and leptin resistance.

Increased adipose mass can cause insulin resistance and type 2 diabetes mellitus. This phenomenon is related to adipocyte-secreted signaling molecules that affect glucose balance, such as fatty acids, adiponectin, leptin, interleukin-6, tumor necrosis factor-α, and resistin. Among these hormones, leptin and resistin play important roles in regulating weight and glucose metabolism. Leptin and resistin work in both similar and opposite ways, and they interact with each other. Circulating concentrations of leptin and resistin are elevated in models of obesity and rodents fed a high-fat diet. In addition, leptin and resistin are similarly regulated by nutritional status: they are reduced by fasting and increased by feeding. This effect is mediated partially through insulin receptors and glucose transporters. Our latest data provided the first indication that in sheep, intravenous infusion of resistin increases the mean circulating concentrations of leptin and decreases luteinizing hormone in a dose-dependent manner during both the long-day (LD) and short-day seasons. Furthermore, exogenous resistin increased suppressor of cytokine signaling (SOCS)-3 mRNA expression only during the LD season, when the leptin resistance/insensitivity phenomenon was observed in the arcuate nucleus, preoptic area, and anterior pituitary. We concluded that one factor contributing to central leptin resistance is autosuppression, via which leptin and resistin stimulate the expression of SOCS-3, which inhibits leptin signaling. The increased expression of SOCS-3 in response to leptin and resistin may be a pivotal cause of leptin resistance/insensitivity, a pathological situation in obese individuals and a physiological occurrence in sheep during the LD season.

Effects of ghrelin on nocturnal melatonin secretion in sheep: An in vitro and in vivo approach.

Recent studies confirmed that pineal melatonin (MEL) secretion is regulated by ghrelin (GHRL) in seasonally reproductive sheep. The first in vivo experiment investigated whether the effect of GHRL on nocturnal secretion of MEL in sheep is mediated by type 2 serotonin receptors. Sheep ( = 16) were intravenously injected with GHRL (2.5 µg/kg of BW) and meta-Chlorophenylpiperazine (mCPP; a mixed agonist of 5-HT2B/5-HT2C receptors; 1 mg/kg BW), either combined or individually, during the short-day (SDS) and long-day (LDS) seasons. Blood samples were collected at 15-min intervals for 4 h. The second in vitro experiment examined the effect of GHRL (10 ng/mL) on MEL secretion by pineal gland (PG) explants incubated for 5 h. The expression levels and/or concentrations of tryptophan 5-hydroxylase 1 (TPH1), aralkylamine N-acetyltransferase (AA-NAT), and the phosphorylated form of AA-NAT (p31T-AA-NAT) were determined at selected time points during the SDS and LDS seasons. The experiments demonstrated that GHRL reduced MEL secretion ( < 0.01) during the SDS season. Administration of mCPP or a combination of GHRL + mCPP stimulated MEL secretion ( < 0.01) regardless of the season. Furthermore, GHRL regulated nightly MEL secretion in a TPH1-independent manner. However, during the SDS season, GHRL reduced p31T-AA-NAT expression and the AA-NAT concentration ( < 0.01) and inhibited MEL secretion ( < 0.001), whereas during the LDS season, GHRL had no effect on MEL secretion or on the expression of the examined enzymes. These findings indicate that GHRL directly and indirectly affects PG activity in sheep and that the photoperiod modulates the effects of GHRL.

Is progesterone the key regulatory factor behind ovulation rate in sheep?

Ovarian antral follicles in the ewe grow in an orderly succession, producing 3 to 4 waves per estrous cycle. In prolific sheep, some large antral follicles from the second-to-last wave of the estrous cycle are added to the ovulatory follicles emerging just before estrus to give a higher ovulation rate; it is feasible that regression of these follicles is prevented by an increase in serum concentrations of FSH or LH pulsatility at proestrus. Prolific sheep tend to have a shorter luteal phase than nonprolific ewes and there is a great deal of evidence that luteal progesterone (P4), in addition to regulating LH release, may govern the secretion of FSH heralding the emergence of follicular waves. The specific purpose of this study was to determine whether or not extending the duration of the luteal phase in prolific sheep to that typically seen in nonprolific breeds would alter the follicle wave dynamics and ovulation rate. In 2 separate experiments, exogenous P4 (7.5 mg per ewe intramuscularly) was administered on day 11 at PM and day 12 at AM (day 0 = first ovulation of the interovulatory interval studied) in moderately prolific Rideau Arcott × Polled Dorset ewes (experiment 1, n = 8) and highly prolific Olkuska ewes (experiment 2, n = 7; TRT), whereas the equinumerous groups of animals served as controls (CTR). Transrectal ovarian ultrasonography was performed daily, and jugular blood samples were drawn twice a day from day 9 until the next ovulation. Progesterone injections resulted in relatively uniform increments in serum P4 levels, but the mean duration of the interovulatory interval did not differ (P > 0.05) between TRT and CTR groups of ewes in either experiment. The mean ovulation rate post-treatment was 1.6 ± 0.2 vs 3.2 ± 0.4 (experiment 1, P < 0.001) and 3.2 ± 0.8 vs 4.0 ± 1.0 (experiment 2, P > 0.05) in TRT vs CTR, respectively. The number and percentage of ovulating follicles from the penultimate wave of the interovulatory interval studied was 0.25 ± 0.16 vs 1.75 ± 0.45 (P < 0.01) and 25.0 ± 16.4% vs 75.0 ± 16.4% (P < 0.05) in experiment 1, and 0.50 ± 0.30 vs 1.60 ± 0.40 (P < 0.05) and 13.8 ± 9.0% vs 53.4 ± 16.7% (P < 0.05) in experiment 2, for TRT vs CTR, respectively. In summary, administration of P4 at the end of diestrus decreased the incidence of ovulations from the penultimate wave of the estrous cycle in both the moderately and highly prolific strains of sheep, but it reduced the ovulation rate only in moderately prolific ewes.

Phenomenon of leptin resistance in seasonal animals: the failure of leptin action in the brain.

The core of the leptin resistance hypothesis promulgated several years ago to explain obesity as a result of environmental causes consists of 2 tenets: the extinction of leptin-induced intracellular signaling downstream of leptin binding to the long form of the neuronal receptor LTRb in the hypothalamus and the impedance to leptin entry imposed at the blood-brain barrier (BBB). A recent comprehensive investigation concluded that a central leptin insufficiency associated with obesity can be attributed to a decreased efficiency of BBB leptin transport and not to leptin insensitivity within the hypothalamus. Interestingly, anorectic leptin's effects are counteracted in some individuals by a natural resistance associated with hyperleptinemia, which is related to changes in hypothalamic sensitivity to leptin (eg, due to malnutrition, obesity, or seasonal variations due to day-length-dependent reproduction changes). In sheep, it has been observed that the hypothalamus is resistant to leptin in some periods, which is related to the adaptation of these animals to annual changes in energy supply and demand. However, a broad range of ambiguities exists regarding the implications that the intracellular signaling of signal transducer and activator of transcription-2/suppressor of cytokine signaling 3 (STAT2/SOCS3) imparts central leptin resistance. Furthermore, several plausible alternative possibilities have been proposed, such as compensatory functional and anatomic reorganizations in the appetite regulating network, rearrangements in the afferent hormonal feedback signaling involved in weight homeostasis, and modifications in leptin transport to the hypothalamus across the BBB. Taken together, these observations suggest that the contention that impaired intracellular signaling downstream of leptin entry into the appetite regulating network expedites environmentally induced obesity remains unsubstantiated and requires further evidence. Furthermore, pregnancy decreases hypothalamic sensitivity to leptin (or other unknown mechanisms), and lactation can also alter the appetite-suppressing central activity of leptin. The objective of this review was to offer an approach to understanding (1) how information regarding nutritional status is transmitted to and interpreted within the hypothalamus in animals, with special attention on seasonally breeding animals and (2) whether central leptin resistance and/or leptin insufficiency in the hypothalamus favors the development of obesity.

Influence of season and nutritional status on the direct effects of leptin, orexin-A and ghrelin on luteinizing hormone and growth hormone secretion in the ovine pituitary explant model.

The aim of this study was to examine whether leptin (anorexigenic peptide), orexin-A, and ghrelin (orexigenic peptides) could directly (ie, independently of hypothalamic influences) affect the secretion of luteinizing hormone (LH) and growth hormone (GH) by adenohypophyseal (AP) explants obtained from normally fed or fasted (48 h) ewes during the breeding and nonbreeding seasons. In addition, a specific ovine super leptin antagonist (SLAN-3) was used to assess the interactions between leptin and ghrelin and/or orexin-A. Pituitary glands from 16 ovariectomized Polish Longwool ewes that had received estradiol-releasing subcutaneous implants were collected in the breeding (November; n = 8) and nonbreeding (May; n = 8) seasons. The AP explants were incubated for 240 min in a gas-liquid interface and treated with leptin (50 ng/mL), ghrelin (100 ng/mL), orexin-A (100 ng/mL), and SLAN-3 (500 ng/mL) with orexin-A or ghrelin. Treatments with leptin and SLAN-3 + orexin-A increased (P < 0.05) LH concentrations in the cultures of AP explants from fasted animals in the breeding season. Orexin-A increased (P < 0.05) LH secretion by AP explants from both fasted and fed animals in the breeding season. Ghrelin stimulated (P < 0.05) GH secretion by AP explants collected from fasted animals in nonbreeding season and from normally fed ewes in both seasons. Leptin decreased (P < 0.05) GH secretion by AP explants collected from fasted ewes in both seasons and from nonfasted ewes in the breeding season. However, the treatment with SLAN-3 + ghrelin resulted in greater (P < 0.05) GH concentrations compared with leptin treatment of AP explants from fasted ewes in the breeding season and from normally fed ewes in nonbreeding season. In summary, leptin, orexin-A, and ghrelin exerted direct effects on AP secretory function in an ex situ model and both the reproductive season and nutritional status of the animals impinged on the direct effects of the peptides on LH and GH release. Specifically, orexin-A was more potent than leptin in directly stimulating LH secretion in cycling ewes, whereas ghrelin and leptin generally had opposing effects on the secretory function of somatotrophs in sheep.

Seasonal changes in the interactions among leptin, ghrelin, and orexin in sheep.

The adaptation of the physiology of an animal to changing conditions of light and food availability is evident at the behavioral and hormonal levels. Melatonin, leptin, ghrelin, and orexin, which exhibit rhythmic secretion profiles under ad libitum feeding conditions, are sensitive to changes in daylength, forming a tight web of interrelationships in the regulation of energy balance. The aim of this study was to determine the effects of central injections of leptin, ghrelin, and orexin on the reciprocal interactions among these hormones and the influence of photoperiod on these responses. Twenty-four ovariectomized and estradiol-implanted ewes were used in a replicated switchback design. The ewes were assigned randomly to 1 of 6 treatment groups, and the treatments were infused into their third ventricles 3 times at 0, 1, and 2 h, with 0 h being at dusk. The treatments were as follows: 1) control, Ringer-Locke buffer; 2) leptin, 0.5 µg/kg BW; 3) ghrelin, 2.5 µg/kg BW; 4) orexin B, 0.3 µg/kg BW; 5) leptin antagonist, 50 µg/kg BW, then ghrelin, 2.5 µg/kg BW; and 6) leptin antagonist, 50 µg/kg BW, then orexin B, 0.3 µg/kg BW. Blood samples (5 mL) were collected at 15-min intervals for 6 h. The administration of leptin increased (P < 0.05) plasma concentrations of melatonin during short-day (ShD) photoperiods and decreased (P < 0.05) them during long-day (LD) photoperiods, whereas ghrelin decreased (P < 0.05) melatonin concentrations during ShD photoperiod, and orexin had no effect (P > 0.1). Leptin attenuated (P < 0.05) ghrelin concentrations relative to the concentration in controls during ShD. The plasma concentrations of orexin were reduced (P < 0.05) after leptin infusions during LD and ShD photoperiods; however, ghrelin had the opposite effect (P < 0.05) on orexin concentration. Orexin increased (P < 0.05) ghrelin concentrations during LD. Ghrelin and orexin concentrations were increased (P < 0.05) after leptin antagonist infusions. Our data provide evidence that the secretion of leptin, ghrelin, and orexin are seasonally dependent, with relationships that are subject to photoperiodic regulation, and that leptin is an important factor that regulates ghrelin and orexin releases in sheep.

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells.

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10(-7)m) for 24 h. The concentrations of PGE(2) and PGF(2α) in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE(2) and PGF(2α) release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

Seasonal effects of central leptin infusion and prolactin treatment on pituitary SOCS-3 gene expression in ewes.

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 µg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

Effects of orexigenic peptides and leptin on melatonin secretion during different photoperiods in seasonal breeding ewes: an in vitro study.

The pineal gland (PG) acts as a neuroendocrine transducer of daily and seasonal time through the nocturnal release of melatonin. Here, we examined the interaction of season, orexin, ghrelin, and leptin on melatonin secretion by pineal explants in short-term culture. Glands were collected after sunset from 12 ewes during long days (LD; April and May) and from an additional 12 ewes during short days (SD; October and November). Glands were transected sagittally into strips, with each equilibrated in 2.5 mL of Dulbecco's modified Eagle's medium for 60 min, followed by a 2-h incubation in control medium or medium containing orexin B (10 and 100 ng/mL), ghrelin (10 and 100 ng/mL), or 50 ng/mL of leptin. After a 3-h incubation, some PG explants treated previously with lower doses of orexin or ghrelin were challenged with 50 ng/mL of leptin and those treated with both doses of orexin were challenged with 300 nM of the ß-agonist isoproterenol. One milliliter of medium was harvested and replaced from each well every 30 min. Treatment with the low dose of orexin during LD increased melatonin secretion about 110% (P<0.01); treatment with a high dose increased melatonin secretion about 47% (P<0.001). During the SD period, leptin stimulated (P < 0.05) melatonin secretion slightly compared with mean melatonin concentration in controls. However, together, orexin and leptin depressed (P<0.01) melatonin secretion. Both doses of ghrelin reduced (P < 0.01) melatonin concentration during the SD season compared with control culture. Addition of ghrelin and leptin to culture medium increased (P<0.01) melatonin concentration compared with ghrelin-treated culture and decreased melatonin concentration (P<0.01) compared with leptin-treated culture during SD. Isoproterenol stimulated (P<0.01) melatonin secretion compared with values observed during the pretreatment period. We conclude that orexigenic peptides (orexin B and ghrelin) and an anorectic peptide (leptin) affect PG directly. The responses of PG to those hormones depend on day length. Moreover, secretion of melatonin from the ovine PG is under an adrenergic regulation.

Seasonal effects of central leptin infusion on secretion of melatonin and prolactin and on SOCS-3 gene expression in ewes.

Recent studies have demonstrated photoperiodic changes in leptin sensitivity of seasonal mammals. Herein, we examined the interaction of season (long days (LD) versus short days (SD)) and recombinant ovine leptin (roleptin) on secretion of melatonin and prolactin (PRL) and on mRNA expression of suppressor of cytokine signaling-3 (SOCS-3) in the medial basal hypothalamus (MBH) in sheep. Twenty-four Polish Longwool ewes, surgically fitted with third ventricle (IIIV) cannulas, were utilized in a replicated switchback design involving 12 ewes per season. Within-season and replicate ewes were assigned randomly to one of three treatments (four ewes/treatment) and infused centrally three times at 0, 1 and 2 h beginning at sunset. Treatments were 1) control, Ringer-Locke buffer; 2) L1, roleptin, 0.5 microg/kg BW; and 3) L2, roleptin, 1.0 microg/kg BW. Jugular blood samples were collected at 15-min intervals beginning immediately before the start of infusions and continued for 6 h. At the end of blood sampling, a washout period of at least 3 days elapsed before ewes were re-randomized and treated with one of the treatments described above (four ewes/treatment). Ewes were then killed and brains were collected for MBH processing. Leptin treatments increased (P<0.001) circulating leptin concentrations compared with controls during both seasons in a dose-dependent manner. Overall, mean plasma concentrations of melatonin were greater (P<0.001) during LD than SD. However, leptin treatments increased melatonin concentrations during SD in a dose-dependent manner and decreased it during LD. Similarly, plasma concentrations of PRL were greater (P<0.001) during LD than SD. However, unlike changes in melatonin, circulating PRL decreased (P<0.001) in response to leptin during LD. Semi-quantitative PCR revealed that leptin increased (P<0.001) SOCS-3 expression in the MBH region during LD in a dose-dependent manner. Data provide evidence that secretion of photoperiodic hormones such as melatonin and PRL are inversely regulated by leptin during SD and LD. However, the increase in expression of SOCS-3 in the MBH during LD compared with SD fails to fully explain these effects.

Leptin as a nutritional signal regulating appetite and reproductive processes in seasonally-breeding ruminants.

Photoperiod and nutrition both exert major influences on reproduction. Thus, it seems axiomatic that seasonal rhythms in ovulation are influenced by nutrition. In this context, leptin is one of the most important hormonal signals involved in the control of energy homeostasis, feeding behavior and reproductive function in mammals. However, the number of published investigations establishing a functional interaction between leptin and photoperiodism in seasonal breeders is limited. In common with most seasonally-breeding mammals, sheep exhibit robust circannual cycles in body weight and reproduction, which are driven mainly by changes in day-length. Recently, attention has focused on the role of leptin in this process, particularly in its roles as a major peripheral signal controlling appetite, melatonin and prolactin secretion. The purpose herein is to review current concepts in the overall biology of leptin, to summarize its influence on the hypothalamic-pituitary axis, and to highlight recent developments in our understanding of its interaction with season in regulating appetite, body weight and reproduction in seasonally-breeding mammals. The latter observations may be important in delineating states of leptin resistance and obesity in humans.

The role of orexin A in the control of prolactin and growth hormone secretions in sheep--in vitro study.

Orexin A may play a special role in animals' sensitivity to the day length changes such as sheep. The localization of mRNA for prepro-orexin in the ovine hypothalamus was found to correspond to the pattern described in rodents. The results of that research also showed that the expression of the orexin gene depends on the length of a day and is higher during short days. Other study revealed that mRNA for orexin receptors (OxR)1 and OxR2 shows strong expression in the anterior, intermediate and posterior pituitary lobes of the rat. In addition, it was also found that in the anterior pituitary, OxR1 is more strongly expressed than OxR2. These observations indicate that the pituitary gland is capable of receiving the orexin signal. The aim of the study was to determine the interaction of season and orexin A on PRL and GH secretion by pituitary explants in short-term culture. Studies were carried out on pituitaries explants collected from lactating Polish Longwool sheep during the long (LD, May, n=5) and short day (SD, December, n=5). Glands were transected saggitally into halves, with each incubated in 2.5 ml of M-199 for 180-min in medium containing either 0 or 1000 ng/ml of orexin A. Treatment with orexin during LD increased significantly the secretion of PRL (P < 0.01) and GH (P < 0.05), compared to controls. In cultures from glands collected during SD, orexin significantly (P < 0.05) decreased the secretion of both hormones, compared to controls. We conclude that the secretion of PRL and GH from the ovine pituitary gland is negatively responsive to orexin A during SD; whereas orexin may stimulate PRL and GH secretion during LD.

Continuous administration of low-dose GnRH in mares II. Pituitary and ovarian responses to uninterrupted treatment beginning near the autumnal equinox and continuing throughout the anovulatory season.

We tested the hypothesis that continuous subcutaneous treatment with low-dose GnRH, administered to mares from late September/early October through March, would prevent the development of seasonal anovulation. Quarter Horse mares (n=20) were stratified by age and body condition score and assigned randomly to either a saline control (n=9) or a GnRH (n=11) treatment group. Gonadotropin-releasing hormone was delivered continuously via osmotic minipumps, with sham pumps placed in control mares. Initial pumps were inserted on Day 3 following ovulation or during the follicular phase if the next anticipated ovulation did not occur by 9 October. Delivery rate of GnRH was 2.5 microg/h (60 microg/day) for the first 60 days, followed by 5.0 microg/h (120 microg/day) thereafter. Pumps were replaced every 30 days. Eighty and 100% of all mares had become anovulatory by 1 November and 1 December, respectively, and remained anovulatory through the end of February. Neither serum concentrations of LH throughout the study nor total releasable pools of LH in March differed between groups. Although control mares that exhibited ovulatory cycles after study onset had greater (P<0.05) mean concentrations of LH during the follicular phase and metestrus compared to GnRH-treated mares, neither size of ovulatory follicles nor interovulatory intervals differed between groups. Serum concentrations of FSH were not affected by treatment, but were lowest (P<0.05) from November through January. Continuous infusion of low-dose GnRH, beginning soon after autumnal equinox and continuing until just after vernal equinox, failed to prevent the occurrence of or to hasten transition from seasonal anovulation.

Regulatory roles of leptin in reproduction and metabolism: a comparative review.

Leptin plays an important role in signaling nutritional status to the central reproductive axis of mammals and appears to be at least a permissive factor in the initiation of puberty. The expression and secretion of leptin are correlated with body fat mass and are acutely affected by changes in feed intake. Moreover, circulating leptin increases during pubertal development in rodents, human females and heifers. Effects of leptin are mediated mainly via receptor activation of the JAK-STAT pathway; however, activation of alternative pathways, such as MAP kinase, has also been reported. Although the leptin receptor (LR) has not been found on GnRH neurons, leptin stimulates the release of GnRH from rat and porcine hypothalamic explants. Moreover, leptin increases the release of LH in rats and from adenohypophyseal explants and/or cells from full-fed rats and pigs. In contrast, stimulation of the hypothalamic-gonadotropic axis by leptin in cattle and sheep is observed predominantly in animals and tissues pre-exposed to profound negative energy balance. For example, leptin prevents fasting-mediated reductions in the frequency of LH pulses in peripubertal heifers, augments the magnitude of LH and GnRH pulses in fasted cows, and enhances basal secretion of LH in vivo and from adenohypophyseal explants of fasted cows. However, leptin is incapable of accelerating the frequency of LH pulses in prepubertal heifers, regardless of nutrient status, and has no effect on the secretion of GnRH and LH in full-fed cattle or hypothalamic/hypophyseal explants derived thereof. Similar to results obtained with LH, basal secretion of GH from anterior pituitary explants of fasted, but not normal-fed cows, was potentiated acutely by low, but not high, doses of leptin. Mechanisms through which undernutrition hypersensitize the hypothalamic-gonadotropic axis to leptin may involve up-regulation of the LR. However, an increase in LR mRNA expression is not a requisite feature of heightened adenohypophyseal responses in fasted cattle. To date, leptin has not been successful for inducing puberty in ruminants. Future therapeutic uses for recombinant leptin that exploit states of nutritional hypersensitization, and identification of genetic markers for genotypic variation in leptin resistance, are currently under investigation.

Chronic administration of recombinant ovine leptin in growing beef heifers: effects on secretion of LH, metabolic hormones, and timing of puberty.

Serum concentrations of leptin increase linearly from approximately 16 wk before until the week of pubertal ovulation in beef heifers. To test the hypothesis that exogenous leptin can hasten the onset of puberty in heifers, we examined the effects of chronic administration of recombinant ovine leptin (oleptin) on timing of puberty, pulsatile and GnRH-mediated release of LH, and plasma concentrations of GH, IGF-I, and insulin. Fourteen fall-born, prepubertal heifers (Brahman x Hereford, 12 to 13 mo; 304.7+/-4.12 kg) were used. Heifers were stratified by age and BW and assigned randomly to one of two groups (seven animals per group): 1) Control; heifers received s.c. injections of saline twice daily (0700 and 1900) for 40 d; and 2) Leptin; heifers received s.c. injections of oleptin (19.2 microg/kg) twice daily at 0700 and 1900 for 40 d. Blood samples were collected at 10-min intervals for 5 h on. d 0, 5, 10, 20, 30, and 40, and twice daily, just before each treatment injection, throughout the study. On d 41, heifers received i.v. injections of GnRH at 0 (0.0011 microg/kg) and 90 min (0.22 microg/kg), with additional sampling for 5.5 h to examine releasable pools of LH. Diets promoted a gain of 0.32+/-0.09 kg/d, which did not differ between groups. Plasma concentrations of leptin increased markedly in leptin-treated heifers and were greater (P < 0.001) than controls throughout (27.8+/-0.8 vs. 4.9+/-0.12 ng/mL). None of the heifers reached puberty during the experiment, but did so within 45 d of its termination. Mean concentrations of plasma LH, GH, IGF-I, and insulin were not affected by treatment, nor was there an overall effect on the frequency of LH pulses. However, a treatment x day interaction (P = 0.02) revealed that the frequency of LH pulses (pulses/ 5 h) was greater (P = 0.03) in controls (3.6+/-0.36) than in leptin-treated heifers (1.7+/- 0.28) on d 10. Characteristics of GnRH-induced release of LH were not affected by treatment. In summary, chronically administered leptin failed to induce puberty or alter endocrine characteristics in beef heifers nearing the time of expected puberty.

Regulatory roles of leptin at the hypothalamic-hypophyseal axis before and after sexual maturation in cattle.

Studies assessed, either directly or indirectly, the role of GnRH in leptin-mediated stimulation of LH release in cattle before and after sexual maturation. In experiment 1, the objectives were to determine whether leptin could acutely accelerate the frequency of LH pulses, and putatively GnRH pulses, in prepubertal heifers at different stages of development. In experiment 2, we determined directly whether acute, leptin-mediated increases in LH secretion in the fasted, mature female are accompanied by an increase in GnRH secretion. Ten-month-old prepubertal heifers (experiment 1) fed normal- (n = 5) and restricted-growth (n = 5) diets received three injections of saline or recombinant ovine leptin (oleptin; 0.2 microg/kg body weight, i.v.) at hourly intervals during 5-h experiments conducted every 5 wk until all normal-growth heifers were pubertal. Leptin increased mean concentrations of circulating LH regardless of diet, but pulse characteristics were not altered at any age. In experiment 2, ovariectomized, estradiol-implanted cows (n = 5) were fasted twice for 72 h and treated with either saline or oleptin i.v. (as in experiment 1) on Day 3 of each fast. Leptin increased plasma concentrations of LH and third ventricle cerebrospinal fluid concentrations of GnRH, and increased the amplitude of LH and the size of GnRH pulses, respectively, on Day 3 of fasting compared to saline. Overall, results indicate that leptin is unable to accelerate the pulse generator in heifers at any developmental stage. However, leptin-mediated augmentation of LH concentrations and pulse amplitude in the nutritionally stressed, mature female are associated with modifications in GnRH secretory dynamics.

Evidence that lamprey GnRH-III does not release FSH selectively in cattle.

Experiments were performed to test the hypothesis that lamprey GnRH-III (lGnRH-III) selectively releases FSH. Primary cultures of bovine adenohypophyseal cells were treated with mammalian GnRH (mGnRH) and lGnRH-III (10(-9), 10(-8), 10(-7) and 10(-6) M) or control media in Experiment 1. All doses of mGnRH and the two highest doses of lGnRH-III stimulated (P < 0.001) a non-selective release of LH and FSH. In Experiments 2-4, Latin Square designs were utilized in vivo to examine whether physiological and hormonal milieu regulate putative selective effects of lGnRH-III. In Experiments 2 and 3, ovariectomized cows with basal levels of estradiol only (Experiment 2) or in combination with luteal phase levels of progester-one (Experiment 3) were injected with mGnRH and lGnRH-III (0.055, 0.11, 0.165 and 1.1 microg/kg body weight (BW) and saline. All doses of mGnRH released (P < 0.001) LH and FSH, but only the highest dose of lGnRH-III stimulated (P < 0.001) a non-selective release of both LH and FSH (Experiment 3). For Experiments 4A and 4B, intact, mid-luteal phase cows were injected with mGnRH and lGnRH-III (1.1 microg/kg BW; Experiment 4A), lGnRH-III (1.1 and 4.4 microg/kg BW; Experiment 4B) and saline. As before, mGnRH released (P < 0.001) both LH and FSH at all doses. In contrast, lGnRH-III at the highest dose released (P < 0.001) LH but not FSH. These findings suggest that lGnRH-III may act as a weak competitor for the mGnRH receptor and do not support the hypothesis that it selectively releases FSH in cattle.

Leptin prevents fasting-mediated reductions in pulsatile secretion of luteinizing hormone and enhances its gonadotropin-releasing hormone-mediated release in heifers.

We tested the hypothesis that leptin could prevent fasting-mediated reductions in pulsatile secretion and modify GnRH-mediated release of LH in heifers approaching puberty. Thirteen crossbred, prepubertal heifers (13.5-16 mo; 280-350 kg) exhibiting frequencies of pulses of LH between 0.67 and 1 pulse/h, were assigned randomly to two groups: 1). control (n = 6), fasted for 72 h with s.c. injections of saline at 12-h intervals, and 2). leptin (n = 7), fasted for 72 h with s.c. injections of oleptin (19.2 microg/kg) at 12-h intervals. Blood samples were collected intensively for 6 h on Days 0 and 3. This was followed on Day 3 with sequential administration of physiological (0.0011 microg/kg, i.v.) and pharmacological (0.22 microg/kg, i.v.) doses of GnRH and additional blood sampling. Leptin treatment increased (P = 0.0003) plasma concentrations of leptin 5-6-fold compared to controls. Fasting caused a marked decline (P = 0.01) between Days 0 and 3 in the frequency of LH pulses in controls; however, this effect was prevented in the leptin group, with pulse frequency increasing (P < 0.008) from Day 0 to 3. Leptin treatment increased GnRH-induced release of LH at both low (P = 0.04) and high (P = 0.02) doses. Plasma insulin and insulin-like growth factor-1 were reduced by fasting and unaffected by leptin. Leptin increased mean concentrations of growth hormone. Results indicate, for the first time, that exogenous leptin can prevent fasting-mediated reductions in the frequency of LH pulses and modify GnRH-mediated release of LH in intact, prepubertal heifers.

Effects of leptin on basal and GHRH-stimulated GH secretion from the bovine adenohypophysis are dependent upon nutritional status.

We have shown recently that leptin modulates at least two aspects of anterior pituitary LH release in ruminants: basal and GnRH-mediated release. To test the hypothesis that leptin directly affects basal and GHRH-mediated GH secretion from the adenohypophysis, we examined the effects of various doses of recombinant ovine leptin (oleptin) on perifused adenohypophyseal (AP) explants and compared responses of tIssues from control and fasted cows. Ten mature, ovariectomized and estradiol-implanted cows were assigned to one of two dietary groups: (1) normal-fed (n=5) and (2) fasted for 72 h (n=5). At the end of the fasting period, cows were euthanized and pituitaries were collected. Adenohypophyseal explants were perifused for a total of 6.5 h, including a 2-h treatment at 2.5 h with Krebs-Ringer bicarbonate buffer containing 0, 5, 10, 50, or 100 ng/ml oleptin, and a challenge with GHRH at 4.5 h. All doses of oleptin greater than 5 ng/ml decreased (P<0.01) basal GH secretion compared with controls in tIssues collected from normal-fed cows. In contrast, GH release from AP explants from fasted cows treated with the lowest dose of oleptin was 28% (P<0.002) higher than control explants, but larger doses had no effect. Leptin caused an inversely related, dose-dependent increase in GHRH-mediated GH release in tIssues from normal-fed cows. Marked increases (P<0.01-P<0.001) in GH release were observed for the 5 and 10 ng/ml oleptin, with lesser (P<0.08) and no effects observed at the 50 and 100 ng/ml doses respectively. In fasted cows, oleptin had no stimulatory effect on GHRH-induced GH release. Results show that leptin can act directly at the anterior pituitary level to modulate GH release, and this effect is dependent upon nutritional history.

Leptin acts at the bovine adenohypophysis to enhance basal and gonadotropin-releasing hormone-mediated release of luteinizing hormone: differential effects are dependent upon nutritional history.

Recombinant ovine leptin (oleptin) stimulates an acute increase in the secretion of LH in fasted, but not in normal-fed, cows through an augmentation of the magnitude of individual pulses of LH. Herein, we tested the hypothesis that this effect could be accounted for by functional changes at the adenohypophyseal (AP) level. Eleven ovariectomized, estradiol-implanted cows were assigned to one of two dietary groups: normal-fed (n = 6) and fasted (fasted for 72 h; n = 5). After the animals were killed, the adenohypophyses were collected and AP explants were perifused with Krebs-Ringer bicarbonate buffer (KRB) for a total of 6.5 h, including a 2-h treatment at 2.5 h with KRB or increasing doses of oleptin and a challenge at 4.5 h with 50 ng of GnRH. To test for effects of leptin at the hypothalamic level, explants encompassing the medial basal hypothalamus-infundibular complex (HYP) were incubated in KRB alone (control) or in KRB containing 1000 ng of oleptin. Basal release of LH from AP explants treated with leptin was greater (P < 0.02) than that from control-treated explants in fasted, but not in normal-fed, cows. To the contrary, leptin-treated explants from normal-fed, but not from fasted, cows released more (P < 0.001) LH in response to GnRH than control-treated tissues. Neither fasting nor leptin affected (P > 0.1) the secretion of GnRH from HYP explants. These observations support the hypothesis that leptin modulates the secretion of LH in mature cows, to a large extent, by its direct actions at the AP. Differential manifestations of these effects are dependent upon nutritional history.