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Regular Article |
a Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, Beeville, Texas 78102
b Department of Animal Science
c Center for Animal Biotechnology and Genomics, Texas A&M University, College Station, Texas 77843
d Department of Animal and Wildlife Sciences, Texas A&M University—Kingsville, Kingsville, Texas 78363
e Department of Animal Science, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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anterior pituitary, insulin, leptin, luteinizing hormone, neuroendocrinology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In ruminants, it is generally recognized that the link between the central reproductive axis and acute changes in nutrient intake is less distinct than that in monogastrics, and short-term feed restriction, as a general rule, does not measurably reduce pulsatile LH release [10, 11]. However, this belief has been tempered by recent observations in our laboratory [12] and the laboratories of others [13] involving heifers and castrated rams. In prepubertal heifers, circulating leptin, leptin mRNA in adipose tissue, and LH pulsatility are reduced by short-term fasting [12]. Moreover, 72-h feed restriction reduces pulsatile LH release in estradiol-implanted, castrated ram lambs [13], and subcutaneous administration of human leptin (hleptin) prevents these changes. Collectively, these and other observations indicate that the interactions between estradiol negative feedback, body energy reserves, and perhaps maturity of the central reproductive axis are important determinants of the response to acute diet restriction in ruminants. Importantly, these responses may be linked to circulating leptin, to changes in responsiveness to leptin, or to interactions between leptin and other metabolic hormones. Thus, leptin could potentially provide information on the availability of energy stores that can be used during periods of nutrient restriction. However, no investigations have examined these relationships in mature cattle.
The current experiments were designed to 1) examine the effects of short-term fasting on leptin gene activity and circulating leptin in mature, ovariectomized beef cows in the presence of physiologic levels of estradiol and 2) determine responsiveness of the hypothalamic-pituitary axis of fasted and nonfasted cows to recombinant ovine leptin (oleptin). Because nutritionally mediated changes in circulating leptin and LH are also tightly linked to insulin secretion [10, 12, 14], and because leptin has been shown to influence pancreatic endocrine function via changes in sympathetic tone [15], a secondary interest was to examine insulin secretion after centrally administered leptin in fasted and nonfasted cows.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal Model and Intracerebroventricular Delivery of Recombinant oLeptin
The animal model employed in these studies was the ovariectomized, mature beef cow bearing an estradiol implant. These implants are designed to produce basal circulating concentrations of estradiol-17ß of 2–4 pg/ml as reported previously [16]. Ovariectomized, steroid-treated females and castrated, steroid-treated males provide excellent models for studying the effects of nutrition on neuroendocrine control of gonadotropin secretion in ruminants [2, 13, 17, 18]. With this approach, hormonal implants can provide a constant level of steroid (estradiol) negative feedback without the complications associated with ovarian cyclicity. Cows used in the current experiments had mean (± SEM) concentrations of estradiol of 3.3 ± 0.4 pg/ml.
Availability of oleptin preparations for use in biologic studies is limited. In the current experiment, the recombinant oleptin [19] used previously [12] in a ruminant-specific RIA [20] was used for leptin infusion. To minimize the amounts necessary to conduct the experiments, and because leptin effects are to a large degree effected at the hypothalamic level [7–9], we chose to infuse leptin directly into the cerebroventricles using an intracerebroventricular (ICV) cannulation model similar to that established and reported previously from this laboratory [16].
Procedures
Experiment 1A tested the hypotheses that 1) short-term fasting would reduce circulating concentrations of leptin and insulin in mature, ovariectomized, estradiol-implanted cows; 2) ICV infusions of recombinant oleptin would attenuate fasting-mediated reductions in insulin; and 3) central infusion of recombinant oleptin would stimulate LH secretion, particularly in fasted cows. Thirteen mature, ovariectomized cross-bred beef cows (Texas Agricultural Experiment Station herd, Beeville, TX), each bearing a subcutaneous estradiol implant, were used in this study. Twelve of 13 cows were surgically fitted with ICV cannulas (control, n = 6; fasted, n = 6) as described previously [16], except that cannulas were inserted into the lateral rather than the third ventricle. The location and function of cannulas were verified by radiography and continuous flow of cerebrospinal fluid. A period of at least 3 wk was allowed for cows to recover from ICV surgery. Cows were fed once daily at 0700 h a diet formulated to provide 100% of the National Research Council (NRC) [21] requirements for maintenance before the start of the experiment, and their average body condition score was 6 ± 0.12 on a scale of 1 to 9 (1, emaciated; 9, obese).
Each cow was assigned to one of two dietary groups: 1) control: cows were fed 100% of the NRC requirements [21] for maintenance and had free access to water (n = 6) and 2) fasted: cows were fasted for 60 h with free access to water (n = 7). On the day before the start of dietary treatments (Day -1), cows were fitted with jugular catheters (polyethylene tubing, 1.4 mm inside diameter, 1.9 mm outside diameter; Becton Dickinson, Parsippany, NJ) for intensive blood sampling. At the same time, patency of ICV cannulas was verified, and ICV cows were treated prophylactically with antibiotics (oxytetracycline HCl [9.9 mg/kg daily] and oral sulfadimethoxine [137.6 mg/kg once]). Cows were placed in stanchions after control cows had been fed and were allowed to stand without further restraint during periods of intensive blood sampling, which started at 0900 h on Days 0 and 2. Blood was collected semiremotely at 10-min intervals for 6 h on Day 0 and for 12 h on Day 2 of the experiment via an extension connected to the jugular catheter. Blood samples were dispensed into tubes containing 150 µl of a solution containing heparin (1000 IU/ml) and 5% EDTA and placed immediately on ice. Plasma was harvested by centrifugation and stored at -20°C until hormone analysis. At the beginning of the intensive sampling periods on Days 0 (6 h) and 2 (12 h), sterile saline (200 µl) was infused as a control into the ICV cannula at 0, 1, and 2 h. Similarly, on Day 2, cows in each group received ICV infusions of 200 µg recombinant oleptin [19] in 100 µl saline, followed by a 100-µl injection of saline to flush the cannula at 6, 7, and 8 h (Fig. 1). The dose of leptin used in this experiment was determined on the basis of extrapolation from previously reported experiments in pigs [22] and sheep [23] in combination with preliminary experiments using hypothalamic explants and ICV-cannulated cows in this laboratory (unpublished results). The heads of the cows were briefly restrained during each ICV infusion. The fasted cow without an ICV cannula was not treated with saline or leptin, and data from this animal contributed only to the study of fasting effects.
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Because of confounding effects of leptin infusion, leptin gene activity could not be determined in the initial group of cows. Therefore, to test whether the leptin gene was responsive to short-term fasting in mature cows, an additional 12 ovariectomized, estradiol-implanted cows were used in a complementary experiment (experiment 1B). This experiment also allowed us to determine whether patterns of leptin in the circulation are affected by period of the day, which, if observed, could complicate our interpretation of the effects of centrally infused leptin in experiment 1A. Before the onset of the experiment, cattle were fed for maintenance according to NRC recommendations [21]. The average body condition score was 6.1 ± 0.2, as described previously. Each cow was assigned to one of two dietary groups: 1) control: cows were fed Coastal Bermuda grass hay ad libitum and had free access to water (n = 6) and 2) fasted: cows were fasted for 60 h with free access to water (n = 6). Subcutaneous fat samples were collected lateral to the tail head by aseptic biopsy using epidural anesthesia (2% lidocaine HCl) at the beginning (Day 0) and end (Day 2) of the dietary treatment. Fat samples were snap frozen in liquid nitrogen and stored at -80°C until Northern blot analysis for leptin mRNA. Blood samples were collected by caudal venipuncture every hour on Day 2 of the experiment and processed as described previously.
RIA and Colorimetric Assays
Circulating concentrations of leptin were determined using a highly specific oleptin RIA validated for use in bovine serum [20]. Use of this assay for determining plasma concentrations of leptin in the bovine has been validated and reported previously by our laboratory [12]. Determinations for circulating leptin levels were performed in samples collected every 3 h for 6 h on Days 0 (experiment 1A) and 2 (experiments 1A and 1B) and every hour for samples collected on the final 6 h on Day 2 (experiments 1A and 1B). Plasma concentrations of insulin were determined as validated previously [24] in samples collected for leptin determinations. Circulating concentrations of LH were determined with a validated assay [25] for samples collected at 10-min intervals for 6 h on Day 0 and for 12 h on Day 2 (experiment 1A). Serum estradiol was assayed in extracted samples as reported previously [26]. Intraassay and interassay coefficients of variation for the preceding assays averaged 7% and 15%, respectively. Plasma glucose was determined in samples collected at 0 and 6 h on Day 0 and at 0, 6, and 12 h on Day 2 using the Sigma colorimetric assay (510A; Sigma, St. Louis, MO) according the to manufacturer's instructions.
Northern Blot Analysis
Total cellular RNA was isolated from 0.7 g of subcutaneous adipose tissue as previously described [12]. Fifteen micrograms of RNA were loaded on 1% agarose gels, separated by electrophoresis, and transferred onto nylon membranes. UV transillumination of ethidium bromide-stained RNA was used to quantify 18S rRNA bands using a Fluor-S MultiImager System (Bio-Rad Laboratories, Hercules, CA). Blots were hybridized with a 32P-labeled RNA probe generated from a 350-base pair oleptin cDNA [27] (GenBank accession No. U62123). Hybridization signals were quantitated with an Instant Imager (Packard Instrument Co., Downers Grove, IL) and normalized with 18S rRNA.
Statistical Analysis
Hormone data were analyzed by ANOVA for repeated measures using the PROC General Linear Models (GLM) procedure of Statistical Analysis Systems (SAS 8.1) (SAS Institute Inc., Cary, NC). The frequency and amplitude of LH pulses were determined using a pulse-detection algorithm, Pulsefit 1.2 [28]. Sources of variation were diet, day, cow(diet), and appropriate interactions. The least significant means procedure was used to compare means when significant differences were detected. Because of random differences in LH concentrations between groups on Day 0, analysis of covariance (ANCOVA) was used to compare treatment means on Day 2. Mean concentrations of LH for each cow on Day 0 were used as the covariate to test effects of dietary treatment on LH secretion. Similarly, mean amplitude of LH pulses on Day 0 was used as a covariate in an ANCOVA procedure to test effects of dietary treatment on mean amplitude of LH pulses. To test the temporal effects of leptin administration on plasma LH and insulin concentrations, the 12-h intensive sampling period on Day 2 was subdivided in four periods (I–IV). Hormone data were analyzed using ANOVA for repeated measures (PROC GLM procedure of SAS). The sources of variation were diet, period, cow(diet), and appropriate interactions. When a significant difference was detected, the least significant means procedure was used to compare means. Leptin mRNA data were transformed to percentage values relative to Time 0 (Day 0) and analyzed by the t-test procedure of SAS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Short-Term Fasting on Leptin Gene Expression in Adipose Tissue; on Circulating Levels of Insulin, Leptin, and Glucose; and on Pulsatile LH Release
In experiment 1A, postprandial increases of circulating insulin in the control group resulted in overall mean concentrations of insulin that were greater (P < 0.01) in the control than in the fasted group on Day 0 (Figs. 2 and 3). In addition, fasting caused a 49% decrease (P < 0.001) in circulating insulin on Day 2 compared with Day 0, and concentrations were lower (P < 0.01) than in controls (Fig. 3). Circulating concentrations of leptin were lower in the fasted than in the control group (P < 0.04). Plasma concentrations of glucose did not differ between and within groups on any day (Fig. 3). Fasting caused a 30% reduction (P < 0.001) in leptin mRNA expression in adipose tissue on Day 2 (experiment 1B; Fig. 4).
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The mean concentration of LH and the mean amplitude of LH pulses were greater (dietxday; P < 0.01) in the fasted compared with the control group at the start of the experiment on Day 0 (6-h sampling window). Because differences in feed intake involving only a few hours do not affect LH secretion in ruminants [1], differences in LH secretion patterns on Day 0 were considered to be innate to the individual animals and not a consequence of diet. Using ANCOVA to adjust for the difference on Day 0, we observed no effect of fasting on any LH variable in this experiment.
Effects of ICV Infusions of oLeptin on Plasma Concentrations of Insulin, Leptin, and Glucose and on LH Secretion
On Day 2 of the experiment (Fig. 5), mean concentrations of insulin were lower (P < 0.01) in fasted compared with control cows during periods I and II. However, after the start of leptin infusions, plasma insulin began to increase steadily (P < 0.01) in the fasted group and reached concentrations similar to those observed before fasting and not different from those in the control group at Hours 9, 10, and 12 (P > 0.10; Fig. 5). Circulating concentrations of leptin were greater (P < 0.01) during period IV (after leptin infusion) than during previous periods in both control and fasted groups. This increase in circulating leptin was not seen in control-fed and fasted cows that were not infused centrally with leptin (experiment 1B, data not shown). Mean concentrations of glucose were not affected by leptin infusions (P > 0.1; data not shown).
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A remarkable increase (P < 0.03) in mean concentrations of LH was observed in fasted cows during period IV after ICV infusions of leptin compared with concentrations in the previous three periods and with concentrations in control cows during period IV. Although mean concentrations of LH in the control group during period IV were lower (P < 0.05) than those for periods I–III, this observation did not account for the positive effect of leptin in the fasted group. However, the mean size of LH pulses, determined by the area under each pulse, was greater (P < 0.04) in the fasted than in the control group. Increased pulse size appeared to occur as a result of an extended duration of individual pulses in four of five cows (P < 0.01) after ICV infusion of leptin, as neither amplitude nor frequency of pulses per se differed between groups. Figure 6 shows individual patterns of LH secretion in two representative fasted cows on Day 2 of the experiment. An increase in baseline concentrations of LH and area under each pulse after leptin treatment in the two representative fasted cows can be readily observed. For the two representative control-fed cows, concentrations of LH and amplitude of pulses appear to decline after leptin treatment, which is consistent with the reduction in mean concentrations observed for this group during period IV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Declines in leptin mRNA and circulating leptin were similar to those reported previously in prepubertal heifers [12] and in several other species [8, 9, 13, 29–31], confirming that leptin synthesis and secretion are acutely responsive to changes in nutritional status. Circulating concentrations of leptin increased in both control and fasted groups after ICV infusions of recombinant oleptin. The outcome of central infusions of leptin has been reported in the mouse. Radiolabeled leptin (125I-leptin) injected into the lateral ventricle appears in the circulation in less than 5 min, reaching peak concentrations within 20 min that were similar to those observed 20 min after i.v. infusion of a similar quantity [32]. In addition, periventricular diffusion of 125I-leptin was detected up to 600 µm from the midline into the hypothalamus within 30 min, demonstrating the potential for infusion of leptin into the ventricular system to act within hypothalamic neurons. In humans, leptin secretion has been reported to follow a circadian rhythm [33], with a nadir early in the morning (0800–0900 h), an increase during the day, and a peak between 2400 and 0200 h. A circadian rhythm for leptin secretion has not been reported in ruminants. However, in experiment 1B, we collected blood samples for 12 h to determine if circulating concentrations of leptin would change between 0900 and 2100 h. Had such changes been observed, their occurrence would have complicated our interpretation of the increased concentrations of leptin observed after ICV infusions in experiment 1A. No evidence of increased circulating leptin during the evening hours was observed in cows not infused with leptin (experiment 1B). Therefore, the increases observed in circulating leptin for both groups after central infusion of oleptin (experiment 1A) probably were not confounded by a potential diurnal variability.
Lower mean concentrations of insulin on Day 0 in fasted cows were due mainly to postprandial increases in insulin in control cows fed in the morning before the beginning of blood sampling and have been reported previously in cattle [34]. Such increases in insulin can occur either as a physiologic response to increasing plasma glucose levels or as parasympathetic responses to feeding [35, 36]. In our study, circulating glucose was not affected by feeding or short-term fasting or by central administration of leptin.
Importantly, ICV infusions of leptin, which resulted in an elevation of serum leptin, increased circulating concentrations of insulin in fasted cows to those observed before fasting and similar to those in controls. Because ICV leptin enters the peripheral circulation, central and peripheral effects of leptin could not be differentiated in the present experiments. However, the stimulatory effects of leptin on serum insulin in fasted cows supports previous observations in rat pancreatic beta cells [37, 38]. In those studies, leptin stimulated basal insulin secretion from isolated rat pancreatic islets cultured in 5–7 mM glucose but did not affect glucose-stimulated (25 mM) insulin secretion. Paradoxically, others have reported either negative [39–42] or neutral [13, 14] effects of leptin on insulin release. The basis for these conflicting observations is not clear but has arisen from experiments involving a variety of experimental conditions and animal models. For example, hleptin injected subcutaneously over a period of 76 h to produce concentrations of leptin above 30 ng/ml does not affect insulin secretion during fasting in the castrated, estradiol-treated male sheep [13]. This is in contrast to the present study in fasted cows in which centrally infused leptin elevated plasma concentrations of leptin to prefasting concentrations (7 ng/ml) and increased circulating concentrations of insulin. Pancreatic beta cells express leptin receptors [39, 40], and both Janus kinase (JAK), a signal transducer and activator of transcription (STAT), and mitogen-activated protein (MAP) kinase [43, 44] have been reported as intracellular mediators of leptin-receptor interaction. In addition, leptin has been shown to influence ATP-sensitive potassium channels in pancreatic beta cells [40]. Although leptin has been reported to directly influence pancreatic insulin release, an indirect effect via the central nervous system cannot be discounted. Chemical sympathectomy prevents the suppressive effects of leptin on a glucose-induced increase in secretion of insulin [15], suggesting an indirect effect of leptin on the pancreas via the autonomic nervous system. In contrast, ICV infusions of leptin resulted in normalization of circulating concentrations of insulin during fasting-induced hypoinsulinemia in cows. If this effect is mediated via the central nervous system, it is in addition to the paradoxical variety of effects seen for leptin through direct action on pancreatic beta cells. Taken together, these reports suggest the potential for a series of complex actions of leptin on the pancreas that include effects on gene expression, plasma membrane polarization potential, and sympathetic tone. However, the relevance of these pathways to ruminant physiology and metabolism remains to be determined.
As expected, short-term fasting did not suppress LH secretion in these mature, ovariectomized, estradiol-implanted cows. Yet, fasted cows responded to leptin with a remarkable increase in the mean concentrations of LH. This occurred as a result of a 46.6% increase in the size of LH pulses in four of five fasted cows after central infusion of leptin. Two of the 4 fasted cows that responded to leptin showed an increase in size of LH pulses almost immediately after ICV infusions, whereas the other 2 cows that responded to leptin showed a delayed response (note the 2 representative fasted cows in Fig. 6). This variability in temporal response to centrally infused leptin may be associated with variability among animals in diffusion of the hormone into the tissue, either directly or via the bloodstream. Nevertheless, the increased size of LH pulses was extended throughout the end of the experiment, which can be seen by the significant increase in mean concentrations of LH in the fasted group during period IV. The increase did not involve a heightened frequency of LH pulses, but this would not have been expected since pulse frequency was not suppressed in this model. Acute feed restriction causes a rapid reduction in mean concentrations of LH in rodents, humans, and monkeys [45–47], but only chronic undernutrition has been shown to suppress LH release in mature cows [48]. Nonetheless, we have reported that 48 h of total feed restriction decreases LH pulse frequency in growing prepubertal heifers [12], suggesting that the hypothalamic-pituitary axis of immature female cattle is more sensitive to acute perturbations in energy status than that of mature cows having larger adipose stores. It has been reported that undernutrition increases leptin receptor numbers in the hypothalamus of ewes [49]. Thus, it is possible that fasted cows had increased numbers of leptin receptors in the hypothalamus and became more sensitive to leptin.
Control-fed cows exhibited decreased mean concentrations of LH during period IV in the present study, which can be seen in the data for individual control cows shown in Figure 6. Whether this was also an effect of leptin is not clear. Hypothalamic explants of rats treated with low concentrations of leptin (10-12 and 10-10 M) show increased GnRH secretion compared with those of controls [50]. However, higher doses of leptin decrease secretion of GnRH into the media. Henry et al. [23] did not observe an effect of centrally infused recombinant hleptin on LH secretion in normal-fed, ovariectomized ewes without estrogen replacement; however, subcutaneous administration of hleptin prevented the decrease in mean concentrations of LH in gonadectomized, estradiol-implanted male sheep fasted for 72 h [13], indicating that not only metabolic status, but also gonadal steroids may impact the effects of leptin on the reproductive axis.
In summary, the results of the present experiments demonstrate that short-term (60-h) feed restriction diminishes synthesis of leptin in adipose tissue and levels of circulating leptin in mature cows, similar to results observed in prepubertal heifers. In contrast to the pattern found in immature cattle, secretion of LH was not affected by short-term fasting. However, fasted cows were clearly more sensitive than control-fed cows to the stimulatory effects of leptin, resulting in an augmentation of size of LH pulses and an increase in the mean circulating concentration of LH. In addition to marked effects on LH secretion, leptin also stimulated an increase in circulating insulin levels in fasted cows. This effect, similar to that observed for LH, was clearly dependent on the metabolic status of the animal. However, because of conflicting reports in the literature, the role of leptin in regulating pancreatic insulin secretion in ruminants remains unclear and requires additional study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USDA-NRI 00-35203-9132 and Project H-6881 of the Texas Agricultural Experiment Station. A preliminary report of this work was presented at the 33rd Annual Meeting of the Society for the Study of Reproduction. 
2 Correspondence: G.L. Williams, Animal Reproduction Laboratory, Texas A&M University Agricultural Research Station, 3507 Hwy. 59 E, Beeville, TX 78102-9410. FAX: 361 358 4930; glw{at}fnbnet.net
Accepted: December 19, 2001.
Received: September 26, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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