| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Neuroendocrinology |
Animal Reproduction Laboratory,3 Texas A&M University Agricultural Research Station, Beeville, Texas 78102
Federal University of Santa Maria–CAPES,4 Santa Maria, RS, Brazil
Department of Animal Science,5 Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas 77843
Department of Animal Science,6 University of Missouri, Columbia, Missouri 65211
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gonadotropin-releasing hormone, leptin, luteinizing hormone, neuroendocrinology, pituitary hormones
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Recently, significant progress has been made in understanding how changes in nutrition are communicated to the hypothalamic-pituitary axis, thereby controlling gonadotropin secretion. At least part of this control involves the adipocyte-derived hormone leptin. In rodents, leptin normalizes body weight and restores fertility in leptin-deficient (ob/ob) mice [8], hastens the onset of puberty in normal mice [9], and stimulates hypothalamic GnRH and pituitary LH secretion in vitro [10, 11]. However, in cattle and sheep, the effects of leptin on gonadotropin secretion are observed mainly during states of acute or chronic feed restriction [12, 13]. Fasting hypersensitizes mature cows to leptin, with leptin treatment increasing mean circulating concentrations of LH and augmenting the size of individual pulses of LH [13], effects that appear to be mediated largely at the adenohypophyseal level [14]. Importantly, in castrated rams bearing estradiol implants, leptin prevented a fasting-mediated decline in the frequency of LH pulses [7]. To date, this effect has not been demonstrated in the intact, ruminant female. However, it suggests that in ruminant models in which fasting is capable of modulating frequency of LH pulses, leptin is acting at hypothalamic sites to influence the frequency of GnRH discharge [2, 5]. The present study examined this hypothesis in intact heifers approaching their first pubertal ovulation, an animal model known to respond to fasting with a decrease in the frequency of LH pulses [6]. Objectives were to determine if exogenous leptin could prevent a fasting-induced reduction in pulsatile secretion of LH, with corollary examinations of the effects of leptin on GnRH-mediated LH release and on growth hormone (GH) secretion.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals and Procedures
Thirteen fall-born, prepubertal beef heifers (13.5–16 mo of age, 319.1 ± 9.12 kg) were used for this study. Before the beginning of the experiment, heifers were maintained in pens (25 x 9 m) where serum concentrations of progesterone were determined in twice-weekly blood samples collected by caudal venipuncture to monitor pubertal status. During this period, all heifers received diets formulated to promote a daily gain of approximately 0.45 kg/day [15].
Based on previous experience with heifers of this age, body weight, and breed type [6], we expected heifers to exhibit a frequency of LH pulses ranging from 0.50 to 1 pulse/h and to have their first pubertal ovulation within 30–90 days. Twenty-four hours before the start of the experiment, each heifer was fitted with a jugular catheter (Silastic tubing, 1.4 mm inside diameter, 1.9 mm outside diameter; Dow Corning Corporation, Midland, MI) for intensive and daily blood sampling. Heifers were assigned randomly to two groups: 1) control (n = 6) heifers were fasted for 72 h, with free access to water, and received s.c. injections of saline and 2) leptin (n = 7) heifers were fasted for 72 h (84.5 h including sampling period on Day 3), with free access to water, and received s.c. injections of recombinant ovine leptin (oleptin) in saline at a dose rate of 19.2 µg/kg per injection. Heifers were injected once on Day - 1 (1900 h); twice at 0700 and 1900 h on Days 0, 1, and 2; and once on Day 3 (0700 h) for a total of eight injections. The volume of each injection was approximately 2 ml, with volume varying slightly depending on individual body weight. Highly purified recombinant oleptin was provided by Dr. Arieh Gertler as reported previously [16]. The dose of leptin and treatment intervals were derived from both preliminary and published experiments [17] conducted in this laboratory with heifers (Fig. 1).
|
Heifers were placed in indoor stanchions, and blood samples were collected at 10-min intervals for 6 h (0800–1400 h) on Days 0 and 3. At the end of the sixth hour on Day 3, all heifers received an i.v. injection of GnRH (0.001 µg/kg) intended to produce a physiological pulse of LH [18], with 10-min blood samples collected for an additional 90 min (1400–1530 h). At this time, a second GnRH injection (0.22 µg/kg) intended to stimulate release of all releasable pools [19] of LH was administered, and intensive bleeding continued for 4 h. During this period, samples were collected at 15-min intervals during the first hour (1530–1630 h) and at 1-h intervals during the last 3 h (1630–1930 h). Blood samples were also collected twice daily throughout the study, just before each saline or leptin injection. Blood samples (10 ml) were dispensed into tubes containing a solution of 150 µl heparin (10 000 IU/ml) and 5% EDTA and placed immediately on ice. During intensive blood sampling, the volume of blood collected (10 ml) was replaced with an equal volume of saline or heparanized (300 IU/ml) saline during catheter flushing. Serum (nonintensive, caudal blood samples) or plasma was obtained by centrifugation and stored at -20°C until hormone assays were conducted. After each intensive and daily blood sampling, heifers were returned to outside pens. A time line depicting the temporal sequence of animal procedures is shown in Figure 1.
Hormone and Biochemical Assays
Serum concentrations of progesterone were assayed in twice-weekly samples using the Coat-A-Count assay kit (Diagnostics Product Corporation, Los Angeles, CA) as reported previously from this laboratory [20]. Plasma concentrations of leptin were determined in samples collected during the week before the start of the experiment and in 12-h samples collected on Days 0, 1, 2, and 3 using a highly specific oleptin RIA validated for use in bovine serum or plasma [21]. Plasma concentrations of LH [22] and GH [23] were determined in samples collected at 10-min intervals for 6 h on Days 0 and 3, with all samples collected after GnRH injections on Day 3 assayed for LH.
To further assess metabolic status, circulating concentrations of insulin were determined in samples collected at 2-h intervals during the intensive sampling period on Day 0 and in samples collected at 10-min (0 and 10 min) and 30-min (30–360 min) intervals during intensive sampling on Day 3 using an assay reported previously [24]. Plasma concentrations of insulin-like growth factor-1 (IGF-1) were determined in samples collected every hour during intensive sampling on Days 0 and 3 [23]. Plasma concentrations of nonesterified fatty acids (NEFA) were analyzed using an enzymatic colorimetric assay kit (Wako Chemicals, Richmond, VA) in the first sample collected on Day 0 (0700 h) and in the last sample collected on Day 3 (1930 h). Intra- and interassay coefficients of variation ranged from 5% to 11% and 11% to 22%, respectively, for all assays reported.
Statistical Analysis
The frequency and amplitude of LH pulses were determined with the aid of a pulse-detection algorithm (Pulsefit 1.2) [25]. Pulse frequency data were analyzed using general linear mixed models for repeated measures using the mixed procedure (PROC MIXED) of the Statistical Analysis System (SAS) [26]. Because small but significant differences in LH pulse frequency existed between groups on Day 0, covariate analyses (ANCOVA) were used to test main effects on Day 3. Circulating concentration of LH, GH, IGF-1, insulin, NEFA, and leptin were analyzed as a repeated measure experiment using the PROC MIXED procedure of SAS. Sources of variation were treatment, day, and their interaction. Day was used as the repeated variable, and heifer (treatment) was used as the subject. When differences in circulating concentrations of hormones were detected between groups on Day 0, analysis of covariance (ANCOVA) was performed to compare treatment means on Day 3. The least squares means procedure was utilized to compare means.
To analyze GnRH-induced release of LH, samples collected before and after both the low and the high doses of GnRH were grouped into four periods (Fig. 1): period I, samples -10 to 0 (time of low-dose injection); period II, samples 1 to 4 after the low dose of GnRH; period III, samples 5 to 9 (sample 9 = time of high dose of GnRH) after the low dose of GnRH; and period IV, samples 10 to 16 after the high dose of GnRH. Because of differences noted in concentrations of LH among groups during period I, analyses of covariance were used to test main effects during periods II, III, and IV. When a significant difference was detected, the least squares procedure of SAS was used to compare means.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mean plasma concentrations of leptin decreased in five and increased in one heifer in the fasted control group between Days 0 and 3. Because of this variability, analysis of variance indicated that mean concentrations of leptin in this group did not differ over the 3-day experiment. However if data from the single heifer that increased were deleted from the analysis, a 12% reduction in mean concentrations of leptin was observed between Day 0 and Day 3 (5.0 vs. 4.4 ng/ml; P < 0.02) in this group. Recombinant oleptin markedly increased plasma concentrations of leptin in the fasted, leptin-treated group, with mean concentrations greater (P = 0.0003; Fig. 2) than in controls at all times after treatment onset.
|
Effects of Leptin on Pulsatile Secretion of LH and GnRH-Mediated Release of LH
A marked decline (P = 0.01) in the frequency of LH pulses was observed in fasted, control heifers between Day 0 (4.01 ± 0.3 pulses/6 h) and Day 3 (2.18 ± 0.3 pulses/6 h) (Figs. 3 and 4). To the contrary, the frequency of LH pulses increased (4.98 vs. 3.69 ± 0.2 pulses/6 h; P = 0.008) in leptin-treated, fasted heifers during the same period and was greater (P < 0.005) than in controls on Day 3 (Figs. 3 and 5). Coincident with the decrease in frequency of LH pulses in saline-treated controls, the amplitude of LH pulses was greater (P = 0.04) in the control than in the leptin group on Day 3 (0.86 ± 0.12 vs. 0.63 ± 0.04 ng/ml).
|
|
|
After adjusting for differences in individual animal baselines during period I, GnRH-mediated release of LH during both periods II and III (low dose of GnRH) and IV (high dose of GnRH) were greater (P = 0.04 and P = 0.02, respectively) in leptin-treated than in control-treated heifers (Fig. 6A). Mean concentrations of LH peaked between 20 and 30 min after both the low and high doses of GnRH, and mean maximal peaks did not differ between groups after either dose. Instead, the increase in LH release between leptin and control groups occurred as a result of enhanced duration of release after GnRH (Fig. 6B).
|
Metabolic Hormones and NEFA
Mean plasma concentrations of GH did not change throughout the fasting period in the control group, and neither the frequency nor the amplitude of GH pulses differed between groups. However, one heifer in each group had concentrations of GH that were greater than two standard deviations above the mean of all other heifers. With these heifers included in the analysis, leptin had no effect on GH concentrations. However, if data from these two animals were not included, mean concentrations of GH increased (P = 0.009) in the leptin group during the fasting period and were greater than in controls on Day 3 (P = 0.001; Fig. 7). Plasma concentrations of insulin and IGF-1 (P = 0.0001) decreased during the fasting period in both groups and did not differ because of treatment on any day (Fig. 8). In contrast, mean concentrations of NEFA increased markedly (P < 0.0001) between Days 0 and 3 in both groups but also were unaffected by treatment (Fig. 8). Serum concentrations of progesterone remained below 0.3 ng/ml throughout the study, indicative of a continuance of the prepubertal state.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Importantly, short-term fasting also caused a marked decline in the frequency of LH pulses in fasted, control heifers, similar to that observed in our previous reports [6], and this effect was prevented in the leptin group, with pulse frequency actually increasing in the latter. The noted increase in pulse amplitude that occurred coincident with a decrease in pulse frequency in controls is a commonly observed, reciprocal relationship for these two pulse parameters [27]. The ability of leptin to prevent fasting-mediated reductions in LH pulse frequency, demonstrated here for the first time in intact heifers, is in agreement with previous studies in rodents [28], monkeys [29], and estradiol-implanted wethers [7]. Consequently, our findings suggest that leptin was able to prevent a reduction in the frequency of GnRH pulses. In fact, leptin treatment appeared to increase GnRH pulse frequency based on the increase in the number of pulses of LH on Day 3 compared to Day 0. Both the expression of mRNA for the long form of the leptin receptor and leptin binding are increased in the arcuate nucleus during fasting in rats [30, 31]. Furthermore, undernutrition is associated with an increase in the number of leptin receptors in the ventromedial hypothalamus of ewes [32]. Therefore, although the decline in circulating leptin in the current study was not dramatic, the physiologic consequences of fasting that hypersensitized the hypothalamo-hypophyseal axis to leptin may have included an up-regulation of the leptin receptor. Currently, mechanisms through which leptin may stimulate GnRH secretion remain unclear. Leptin receptor signaling pathways most often involve successive activation of Janus tyrosine kinases and signal transducers and activators of transcription [33], but other direct or indirect pathways may also be operable [34]. For example, dietary energy restriction prolongs the period of inhibition of LH secretion by estradiol negative feedback in prepubertal heifers [35] and reduces LH secretion and up-regulates the number of estrogen receptors in the hypothalamus in female lambs [36]. Moreover, short-term fasting suppresses LH secretion in gonadectomized males receiving physiological doses of estradiol but not in gonadectomized females without estradiol replacement [7]. These observations accentuate the important contribution of estradiol in regulating GnRH neuronal activity and, ultimately, LH secretion [37]. Thus, it is possible that the ability of exogenous oleptin to prevent fasting-mediated reductions in LH pulse frequency in the current experiment also involves interactions among the leptin receptor, estradiol, and nutritional status.
In the current study, GnRH-mediated release of LH was greater in leptin-treated heifers compared with saline-treated controls, and this is in partial agreement with in vitro studies reported recently from this laboratory [14]. In those experiments, leptin-treated adenohypophyseal explants from fasted cows exhibited a marked increase in basal secretion of LH before GnRH treatments were applied. Therefore, only leptin-treated explants from normal-fed, mature cows (ovariectomized with an estradiol implant) exhibited an increased response to GnRH, as releasable pools of LH in tissues from fasted cows appeared to have been reduced because of enhanced secretion rates during the basal incubation period. The direct action of leptin at the pituitary level in other species is supported by the presence of leptin receptors on gonadotropes of ewes [38] and studies demonstrating increased release of LH from pituitary explants in rats [10]. One of the potential signaling pathways through which leptin could interact with GnRH in the exocytosis of secretory granules of LH is that involving inositol triphosphate (IP3). The latter, a second messenger for GnRH action at the level of the gonadotroph, activates IP3 via JAK2 [39].
As expected, mean concentrations of GH in saline-treated control heifers did not change throughout the study, confirming that short-term fasting does not affect the secretion of this hormone in prepubertal heifers [6]. However, our results (in the absence of data from two outlier heifers) provided evidence that leptin can stimulate GH secretion in heifers and supports a role for leptin in the regulation of the somatotropic axis in ruminants. Results are also consistent with data showing a stimulation of GH by leptin in fasted sheep [7] and in anterior pituitary explants from cows in the fasted state [40]. With one reported exception [12], leptin does not appear to consistently influence the secretion of GH in well-fed sheep [41, 42] or anterior pituitaries from well-fed cows [40]. Expression of leptin receptor mRNA is increased in the pituitary and hypothalamus of fasted rats [43]. Thus, it is possible that the increase in GH secretion observed in leptin-treated heifers was a result of leptin receptor interaction in somatotropes.
Fasting-mediated decreases in circulating concentrations of IGF-1 and insulin and increases in plasma NEFA observed in the current study are consistent with previous reports from this laboratory and others in sheep and cattle [6, 7, 13, 44]. Recent studies from this laboratory also demonstrated that intracerebroventricular or peripheral infusions of oleptin normalize fasting-mediated declines in circulating insulin and these effects were dependent on the metabolic status and the dose of leptin [13, 45]. In one of these studies, doses of 0.2 and 20 µg/kg of oleptin increased circulating insulin briefly, while the intermediate dose of oleptin (2.0 µg/kg) elevated insulin concentrations for at least 3 h [45].
In the current study, high doses of oleptin over a 3-day period of fasting were unable to prevent a reduction in circulating insulin, and these results are in agreement with others described previously in sheep [7]. The presence of leptin receptors in pancreatic islets [46] indicates that leptin can regulate directly the secretion of insulin. However, it has been reported that tissues exposed to relatively large concentrations of leptin accumulate excessive amounts of suppressors of leptin signaling, which can create a state of leptin resistance [33]. Thus, our failure to observe an effect of leptin on fasting-mediated decreases in circulating insulin may be a result of a state of leptin resistance created by the prolonged administration of high doses of leptin during the 3-day experiment. Increases in mean concentrations of NEFA are representative of the lipolytic state that animals typically undergo during fasting. Leptin treatment had no measurable effect on this process.
In summary, results of the current study demonstrate, for the first time in prepubertal, intact heifers, the ability of exogenous leptin to prevent fasting-mediated reductions in LH pulse frequency, to enhance responsiveness of the anterior pituitary to GnRH, and to increase basal GH secretion.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: G.L. Williams, Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, 3507 Highway 59E, Beeville, TX 78102-9410. FAX: 361 358 4930; glw{at}fnbnet.net
Received: 18 July 2003.
First decision: 18 August 2003.
Accepted: 12 September 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Crane, N. Akhter, B. W. Johnson, M. Iruthayanathan, F. Syed, A. Kudo, Y.-H. Zhou, and G. V. Childs Fasting and Glucose Effects on Pituitary Leptin Expression: Is Leptin a Local Signal for Nutrient Status? J. Histochem. Cytochem., October 1, 2007; 55(10): 1059 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Gasser, G. A. Bridges, M. L. Mussard, D. E. Grum, J. E. Kinder, and M. L. Day Induction of precocious puberty in heifers III: Hastened reduction of estradiol negative feedback on secretion of luteinizing hormone J Anim Sci, August 1, 2006; 84(8): 2050 - 2056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Maciel, D. A. Zieba, M. Amstalden, D. H. Keisler, J. P. Neves, and G. L. Williams Chronic administration of recombinant ovine leptin in growing beef heifers: Effects on secretion of LH, metabolic hormones, and timing of puberty J Anim Sci, October 1, 2004; 82(10): 2930 - 2936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.A. Zieba, M. Amstalden, S. Morton, M.N. Maciel, D.H. Keisler, and G.L. Williams Regulatory Roles of Leptin at the Hypothalamic-Hypophyseal Axis Before and after Sexual Maturation in Cattle Biol Reprod, September 1, 2004; 71(3): 804 - 812. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
