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Endocrinology, doi:10.1210/en.2007-0534
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Endocrinology Vol. 148, No. 11 5313-5322
Copyright © 2007 by The Endocrine Society

Nutritional Influences on Reproductive Neuroendocrine Output: Insulin, Leptin, and Orexigenic Neuropeptide Signaling in the Ovine Hypothalamus

David W. Miller, Joanne L. Harrison, Ellen J. Bennett, Patricia A. Findlay and Clare L. Adam

Obesity and Metabolic Health Division (J.L.H., E.J.B., P.A.F., C.L.A.), Rowett Research Institute, Aberdeen Centre for Energy Regulation and Obesity, Aberdeen AB21 9SB, United Kingdom; Sustainable Livestock Systems Group (D.W.M., E.J.B.), Scottish Agricultural College, Aberdeen AB21 9YA, United Kingdom; and Integrative Physiology Group, School of Biological Sciences (J.L.H.), University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom

Address all correspondence and requests for reprints to: Dr. Clare Adam, Obesity and Metabolic Health Division, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom. E-mail: cla{at}rowett.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study investigated how changing nutritional status may alter reproductive neuroendocrine (LH) output via circulating leptin and insulin signaling through orexigenic hypothalamic pathways. Thin sheep were given an increasing nutritional plane (INP), sheep with intermediate adiposity a static nutritional plane (SNP), and fat sheep a decreasing nutritional plane (DNP) for 6 wk. Mean group adiposities converged by wk 6, LH output increased in INP, remained unchanged in SNP, and decreased in DNP sheep. Plasma and cerebrospinal fluid (CSF) insulin and plasma leptin concentrations increased in INP but did not change in the SNP and DNP groups. In INP sheep, LH output correlated positively with adiposity and plasma and CSF insulin concentrations and negatively with orexigenic neuropeptide Y gene expression in the hypothalamic arcuate nucleus (ARC). In DNP sheep, LH output correlated positively with adiposity, CSF leptin concentrations, and ARC proopiomelanocortin gene expression and negatively with leptin receptor (OB-Rb) and agouti-related peptide gene expression in the ARC. These data are consistent with the feedback response to an increasing nutritional plane being mediated by increasing circulating insulin entering the brain and stimulating LH via inhibition of hypothalamic neuropeptide Y and the response to a decreasing nutritional plane being mediated by altered hypothalamic leptin signaling brought about by increased OB-Rb expression and decreased melanocortin signaling. Because end point adiposity was similar yet LH output was different, the hypothalamus apparently retains a nutritional memory, based on changes in orexigenic neuropeptide expression, that influences contemporary neuroendocrine responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REPRODUCTIVE STATUS IS regulated by nutritional feedback to the hypothalamus that controls GnRH and hence LH pulsatile output (reviewed in Ref. 1). Improved nutritional status is stimulatory and decreased nutritional status is inhibitory (2, 3). However, nutritional feedback comprises information about both current food intake and the body reserves, or adiposity, of the animal, and we previously reported differential effects of these components on reproductive neuroendocrine output in sheep (4). In that study increasing food intake stimulated GnRH/LH output, whereas constantly high adiposity did not, indicating that nutritional history may be important in determining responses. It remains to be determined how a dynamic nutritional status is conveyed to and processed within the hypothalamus to then influence reproductive neuroendocrine output and ultimately gonadal function. In this study we investigate the role of key peripheral metabolic hormones, their entry into the brain, and influence on candidate hypothalamic pathways that may be involved in mediating the effects of increasing or decreasing nutritional status on GnRH/LH output.

Both leptin and insulin have been shown to be involved in the long- and short-term control of reproductive neuroendocrine function in several species, including sheep (5, 6, 7, 8). Insulin stimulates rodent hypothalamic GnRH neurons in culture (9) and insulin administered centrally to sheep via intracerebroventricular (icv) cannulae stimulates LH secretion (5). Similarly, leptin stimulates GnRH from rat hypothalamic explants (10) and can increase LH secretion after icv injection in sheep (11). The presence of insulin and leptin receptors on GnRH neurons is equivocal (12, 13), and it is likely that other neuropeptides, especially those associated with energy balance regulation, act as intermediaries (7, 14, 15). Indeed, it is well established that leptin receptors (OB-Rb) and insulin receptors (Ins-R) in the hypothalamus colocalize with the orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AgRP), and anorexigenic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) (14, 16, 17, 18). Thus, we hypothesize that GnRH/LH stimulation by increasing nutritional status is mediated by increased amounts of circulating leptin and insulin entering the brain, down-regulating hypothalamic expression of NPY and AgRP, and up-regulating expression of POMC and CART, whereas GnRH/LH inhibition by decreasing nutritional status is mediated by decreased amounts of circulating leptin and insulin entering the brain, up-regulating hypothalamic NPY and AgRP expression, and down-regulating POMC and CART expression. However, we additionally hypothesize that different elements of these reciprocal regulatory pathways will predominate according to the direction of nutritional change.

Our group previously gathered evidence that sheep on a dynamic as opposed to a static plane of nutrition have differential associations between LH secretion and key metabolic hormones and/or hypothalamic expression of NPY, AgRP, and OB-Rb, dependent on the nutritional history of the animal (4). The differential correlation of putative feedback pathways with the LH response indicates that the pathways are likely to be interactive and dynamic, and this complexity may contribute to the creation of a hypothalamic nutritional memory. In other words, the hypothalamus may dissociate components of nutritional feedback by its capacity to sense the magnitude, direction, and duration of nutritional change. Although our studies focus on reproductive neuroendocrine output in sheep, the putative existence of hypothalamic nutritional memory clearly has wider implications for neuroendocrine and body weight regulation across mammalian species, including man.

Therefore, the present study examined the hypothesis that nutritional modulation of central reproductive control pathways involves circulating leptin and/or insulin signaling through hypothalamic pathways shared with the regulation of energy balance to modulate GnRH/LH secretion and that the responses depend on whether an animal is on an increasing, static, or decreasing plane of nutrition. Our approach was to prepare sheep with contrasting adiposity, which was held stable for a period before the start of the experiments and then to impose increasing, static, or decreasing nutritional regimens that caused adiposity to converge to similar levels in all three groups at the end. During these experiments we measured longitudinally the peripheral and central concentrations of insulin and leptin (preprandially to reflect net metabolic state rather than short-term responses to food intake), pulsatile GnRH/LH secretion, and the underlying changes in hypothalamic gene expression for OB-Rb, Ins-R, NPY, AgRP, POMC, and CART.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experimental procedures involving animals were conducted under the authority of the U.K. Animals (Scientific Procedures) Act of 1986 and received prior approval from the local Ethical Review Committee.

Animals
All sheep were Suffolk x Greyface adult male castrates (~1.5 yr old), and exogenous gonadal steroid was administered at a constant level (steroid clamp) via two sc estradiol-containing implants made from SILASTIC tubing (19). Implants were inserted approximately 4 wk before the start of the experiment and raised plasma estradiol concentrations to an average physiological level of 3.05 ± 0.09 pg/ml (measured at three time points during each experiment by RIA) (20). The sheep were housed in individual pens in natural photoperiod (in Aberdeen, UK, 57°N) and given a complete diet (comprising 50% chopped hay, 30% rolled barley, and 9% soybean meal, with molasses, minerals, vitamins, and trace elements) twice daily at approximately 0800 and 1600 h. Water was provided ad libitum. The main experiments were conducted in the spring (March–April) of two consecutive years.

Experiment 1
Preparatory treatments.
The 54 sheep had initial mean body weight 44 ± 0.7 kg and body condition score (BCS) 2.0 ± 0.03. BCS provides an adiposity score from assessment by palpation of the prominence and degree of cover of the spinous and transverse processes of the anterior lumbar vertebrae, scale 0 (emaciated) to 5 (obese), according to Russel et al. (21). Eighteen sheep with average BCS 2.0 (~20% body fat) were given sufficient food to maintain body weight and BCS (maintenance, 800 g/d). The remaining animals continued to receive ad libitum food until reaching BCS of about 2.75 (~27% body fat) after about 4 wk, at which time 18 animals were transferred to maintenance feeding (1100 g/d). Four weeks later, the final 18 sheep on ad libitum intake had reached BCS of about 3.25 (~34% body fat) and they were transferred to maintenance feeding (1400 g/d). All three groups were held on maintenance intakes for a further 2 wk before the start of the main experiment (Fig. 1AGo).


Figure 1
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  		FIG. 1. Plan of dietary treatments (feeding levels) used in the preparatory period and main experiment for sheep on increasing (INP), static (SNP), or decreasing (DNP) planes of nutrition in experiment 1 (A) and experiment 2 (B). Maintenance food intake for sheep in INP, SNP, and DNP groups was 800, 1100, and 1400 g/d, respectively. Sheep in experiment 2 were surgically prepared with indwelling icv cannulae.

 
Experimental treatments.
Initial BCS for the three groups (n = 18/group) were 2.06 ± 0.03 (low), 2.81 ± 0.05 (intermediate), and 3.19 ± 0.06 (high). Maintenance feeding continued for the first week of the main experiment (wk 0). For the next 1–6 wk of the experiment, sheep in the low BCS group were transferred to ad libitum feeding [increasing nutritional plane (INP) group], sheep with the high BCS were transferred to a feeding regimen calculated to provide 50% of that required to maintain current body weight and adiposity [decreasing nutritional plane (DNP) group], and sheep at the intermediate BCS were kept on a maintenance feeding regimen [static nutritional plane (SNP) group] (Fig. 1AGo).

Measurements and sample collection.
Body weight and BCS were measured once a week, and voluntary food intake by the ad libitum-fed sheep was measured daily. Blood samples were collected three times a week at 0800 h (before the morning feed) for insulin and leptin analyses. During wk 0, 1, 3, and 6, blood samples were collected via temporary jugular catheters every 15 min for 8 h midweek, starting at 0800 h, to determine pulsatile LH secretion. All blood samples were immediately centrifuged and plasma stored at –20 C until analysis. At the end of wk 1, 3 and 6, six sheep from each nutritional treatment group were killed by a lethal iv dose of sodium pentobarbitone (Euthesate; Willows Francis Veterinary, Crawley, Sussex, UK), and the whole brains were immediately excised, snap frozen in isopentane over dry ice, and stored at –80 C.

Hypothalamic gene expression.
Coronal cryostat sections (20 µm) of hypothalamic tissue were thaw mounted onto slides double coated with gelatin and poly-L-lysine and stored at –80 C. Gene expression for OB-Rb, Ins-R, NPY, AgRP, POMC, and CART was measured by in situ hybridization, using techniques described in detail elsewhere (22, 23). A riboprobe complementary to fragments of the intracellular domain of OB-Rb was generated from a cloned sheep cDNA as described previously (24). The riboprobe for Ins-R was generated from a partial ovine cDNA (25), the NPY probe from a rat cDNA (23), the CART probe from a cloned sheep cDNA (26), and AgRP and POMC probes from cloned Siberian hamster cDNAs (27). All probes have been validated on sheep brain tissue (23, 25, 28). Briefly, sections were fixed, acetylated, and hybridized overnight at 58 C using 35S-labeled cRNA probes (1–1.5 x 107 cpm/ml). They were then treated with RNase A, desalted with a final high stringency wash (30 min) in 0.5x saline sodium citrate at 60 C, dried, and apposed to Hyperfilm β-max (Amersham Pharmacia Biotech U.K. Ltd., Little Chalfont, Buckinghamshire, UK). Intensity and total area of hybridization were quantified for the hypothalamic arcuate nucleus (ARC) on each autoradiographic image, using the Image-Pro Plus system (Media Cybernetics, Silver Spring, MD). The integrated intensity of the hybridization signal was then computed using standard curves generated from 14C autoradiographic microscales (Amersham Pharmacia Biotech UK). For each probe, three comparable sections from the medial hypothalamus (~1.5–1.75 mm rostral from the opening of third ventricle) were analyzed for each brain and the results averaged to give a single value for the ARC of each animal. All sections for a single probe were processed together and sections from the same time point for all groups were placed against the same sheet of autoradiographic film.

Experiment 2
Preparatory period.
Eighteen sheep with initial mean body weight 43 ± 0.9 kg and BCS 2.0 ± 0.06 were nutritionally managed to achieve three groups (n = 6) with similar starting BCS to experiment 1. They were transferred to maintenance feeding and surgically prepared with indwelling icv cannulae directed toward the two lateral ventricles, using the method previously described by Miller et al. (6), and then held on maintenance feeding for at least 4 wk before the main experiment (Fig. 1BGo).

Experimental treatments.
Mean initial BCS were 2.29 ± 0.11 (low), 2.86 ± 0.05 (intermediate), and 3.50 ± 0.10 (high). As in experiment 1, for 6 wk, sheep with low BCS were transferred to ad libitum feeding (INP), sheep with high BCS were transferred to a feeding regimen calculated to provide 50% of that required to maintain current body weight and adiposity (DNP), and sheep at the intermediate BCS were kept on maintenance feeding (SNP) (Fig. 1BGo).

Measurements and sample collection.
As in experiment 1, body weight and BCS were measured once a week, and voluntary food intake by the ad libitum-fed sheep was measured daily. At 0800 h (before the morning feed), blood samples were collected three times a week for insulin and leptin analyses; at the same time, cerebrospinal fluid (CSF) samples (0.5–1.0 ml) were taken via the lateral cerebral ventricular cannulae using the method previously described (6). During wk 0, 1, 2, 4, and 6, blood samples were collected via temporary jugular catheters every 15 min for 8 h midweek, starting at 0800 h, to determine pulsatile LH secretion. Plasma and CSF samples were stored at –20 C until analysis.

Plasma and CSF analyses
Plasma concentrations of LH were measured by RIA (29) using reagents provided by the National Institute of Diabetes and Digestive and Kidney Diseases (Rockville, MD) and expressed in terms of the reference standard NIDDK-oLH-1–2. The assay sensitivity was 0.2 ng/ml, and the intra- and interassay coefficients of variation (CVs) values were 4.4 and 4.5%, respectively. Leptin concentrations in plasma and CSF were determined by homologous RIA (30) in a single assay with sensitivity less than 0.2 ng/ml and intraassay CV 10.0%. Insulin concentrations in plasma and CSF were measured by RIA (31) in a single assay run showing sensitivity of 0.2 µIU/ml and intraassay CV 2.7%. Plasma glucose concentrations (for experiment 1 only) were measured by an automated analyzer (KONElab; Labmedics Ltd., Salford Quays, Manchester, UK; method based on glucose hexokinase) with detection limit 0.3 mmol/liter.

Statistical analyses
LH pulse characteristics were analyzed using the Munro pulse analysis program (Zaristow Software, Haddington, East Lothian, UK). The baseline was calculated as a moving average over a 90-min window (45 min each side of the sample being tested). The individual peaks were tested against a threshold, in which a pulse was accepted if the concentration at the peak exceeded the concentration at the previous nadir by a prescribed number of SDs (see below), and the interval to the previous pulse was more than one sampling interval (i.e. > 15 min). Baxter parameters, describing the parabolic relationship between the concentrations of a hormone in the sample and the SD, were derived from the precision in the quality controls for each assay by regressing the mean concentrations of the quality controls against their SD. The G parameters (the number of SDs by which a peak must exceed the baseline to be accepted), G1 to G5, were set at 3.0, 2.5, 1.9, 1.2, and 0.9 SDs for pulses containing one to five samples above baseline concentration, respectively. LH pulse frequency data were tested for homogeneity of variance and were found to be normally distributed.

Factorial ANOVA was used to compare treatment and time effects on BCS, body weight, mean LH concentrations, LH pulse frequency, plasma and CSF insulin, plasma and CSF leptin, and hypothalamic gene expression. Post hoc Fisher’s protected least significant difference analysis was used to test for specific differences between treatments at each time point. Because a major objective was to compare the effects of dynamically increasing or decreasing nutritional status, correlation analysis was used to explore further the relationships between LH secretory variables, hypothalamic gene expression, insulin, and leptin within INP and DNP groups over time. Given that hypothalamic gene expression data in experiment 1 were available for only the euthanized subgroups (n = 6 at time points 1, 3, and 6 wk), correlation analyses included data only from these animals. Variables showing significant correlations with LH secretion were subject to additional covariate analysis whereby the residuals from these regressions were used as dependent variables in a stepwise regression approach, and then the variables themselves were included as independent variables in the stepwise regression. All statistical tests were carried out using Statview for Windows (version 4.57; Abacus Concepts Inc., Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1
The number of animals per group decreased from 18 at wk 0 and wk 1, to 12 at wk 3, and 6 at wk 6. Comparison of data from the euthanized subgroups at wk 1, 3, and 6 (n = 6) with data from remaining animals indicated that these subgroups were representative of their groups. Therefore, the statistical analyses presented here used food intake, BCS, LH, insulin, and leptin data only from these subgroups at wk 1, 3, and 6 to correspond with the hypothalamic gene expression data.

Food intake, BCS, and body weight.
Over the 6 wk of the experiment, food intake increased (P < 0.001) in the INP group (ad libitum) but remained constant in SNP and DNP groups (restricted) so that, at all time points after week 0, intake was higher in INP than SNP and higher in SNP than DNP groups (P < 0.001) (Fig. 2AGo). BCS increased over time in the INP group (P < 0.001), decreased in the DNP group (P < 0.001), and remained constant in the SNP group (Fig. 2BGo). There was a significant difference in mean BCS between groups (P < 0.001–0.05) at all time periods except wk 6, at which time the values in the three groups had converged to similar levels. Final mean body weights for INP, SNP, and DNP groups were 65 ± 1.1, 59 ± 1.7, and 60 ± 1.6 kg, respectively.


Figure 2
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  		FIG. 2. Food intake (A), BCS (adiposity) (B), mean LH concentration over 8 h (C), LH pulse frequency (D), plasma insulin concentrations (E), and plasma leptin concentrations for sheep on INP (black bars), SNP (gray bars), or DNP (white bars) plane of nutrition (F) in experiment 1. The data presented for wk 0 includes all animals (n = 18 per treatment), whereas the data for wk 1, 3, and 6 include only the representative subgroups euthanized for hypothalamic gene expression analysis at each of these time points (n = 6 per treatment). Significant differences between groups at each time point are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant changes within groups over time are annotated above each panel.

 
LH secretion.
In wk 0, when all sheep were on maintenance food intake, mean LH concentration (P < 0.001) and LH pulse frequency (P < 0.05) were lower in animals with relatively low BCS (INP group) than in animals with relatively high BCS (DNP group), with intermediate values in the animals with intermediate BCS (SNP group) (Fig. 2Go, C and D). Mean LH increased over time in the INP group (P < 0.001), decreased in the DNP group (P < 0.05) and did not change in the SNP group (Fig. 2CGo). Mean LH was higher in DNP than SNP (P < 0.05) and INP (P < 0.001) groups at wk 1 but lower in the DNP than the INP group at wk 6 (P < 0.001). LH pulse frequency decreased over time in the DNP group (P < 0.001) but did not change in the INP and SNP groups (Fig. 2DGo). LH pulse frequency was higher (P < 0.05) in the DNP than INP group at wk 1 but lower (P < 0.05) at wk 6. There were no differences between groups for mean LH or LH pulse frequency at wk 3.

Insulin and leptin.
At wk 0, preprandial plasma insulin concentrations were lower in the relatively low BCS group (INP) than in the high BCS group (DNP) (P < 0.05), with intermediate levels in the intermediate BCS group (SNP) (Fig. 2EGo). Preprandial plasma leptin concentrations were lower (P < 0.05) in the low BCS animals (INP) than the intermediate BCS animals (SNP) but not different from the high BCS (DNP) animals (Fig. 2FGo). Plasma insulin and leptin concentrations increased over time in the INP group (both P < 0.001) but did not change in DNP or SNP groups. Plasma insulin in the INP group was lower than in the DNP group (P < 0.001) at wk 1, higher than in the SNP group at wk 3 (P < 0.05), and higher than both SNP and DNP groups at wk 6 (P < 0.001); it was also higher in SNP than the DNP group at wk 6 (P < 0.05). Plasma leptin was higher in INP than SNP (P < 0.05) and DNP (P < 0.001) groups at wk 6 and higher in SNP than the DNP group at wk 6 (P < 0.05), with no differences between groups at wk 1 and 3.

Glucose.
Plasma glucose was slightly lower (4.1 ± 0.05 mmol/liter) in the thin INP sheep at wk 0 than in the fatter SNP and DNP sheep (4.3 ± 0.06 and 4.4 ± 0.06 mmol/liter, respectively; P < 0.05). There were no significant changes over time in DNP and SNP groups, but plasma glucose increased in the INP group to reach significantly higher concentrations at wk 6 (5.1 ± 0.06 mmol/liter), compared with the other groups (P < 0.001).

Hypothalamic gene expression.
All probes hybridized to the ARC. NPY gene expression decreased over time in the INP group (P < 0.001) but did not alter significantly in DNP and SNP groups (Fig. 3AGo). AgRP and OB-Rb gene expression increased over time in the DNP group (P < 0.01 and P < 0.05, respectively) but did not change in the INP and SNP groups (Fig. 3Go, B and E). There was no effect of time on CART, POMC, and Ins-R gene expression (Fig. 3Go, C, D, and F). At wk 1, NPY and OB-Rb gene expression was higher in the INP group (with relatively low BCS) than the DNP group (with relatively high BCS) (both P < 0.05), and CART gene expression was lower in DNP and INP groups than the SNP group (both P < 0.05). At wk 3, AgRP gene expression was lower in the INP than the SNP group (P < 0.05), and CART gene expression was lower in the INP than the DNP group (P < 0.05). At wk 6, NPY and AgRP gene expression was lower in the INP than the DNP (both P < 0.01) and SNP (both P < 0.05) groups, CART and POMC gene expression was higher in the INP than the DNP group (both P < 0.05) (Figs. 3Go and 4Go), and Ins-R gene expression was higher in DNP than SNP and INP groups (both P < 0.05).


Figure 3
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  		FIG. 3. Hypothalamic gene expression in the ARC for NPY (A), AgRP (B), CART (C), POMC (D), OB-Rb (E), and Ins-R (F) for sheep on INP (black bars), SNP (gray bars), or DNP (white bars) plane of nutrition in experiment 1 (n = 6 per group at each time point). Significant differences between groups at each time point are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant changes within groups over time are annotated where appropriate above each panel.

 

Figure 4
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  		FIG. 4. Representative autoradiographs showing differential riboprobe hybridization to the hypothalamic ARC for NPY (A), AgRP (B), and POMC (C) in sheep with similar adiposity (BCS 2.6) but that had been on an INP or DNP for 6 wk in experiment 1. 3V, Third ventricle. Bar, 2.5 mm.

 
Correlation analyses.
To explore further the associations apparent from the group mean data, correlation analysis was carried out between the measured variables in the two groups with dynamically changing nutritional status (Table 1Go).


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  		TABLE 1. Correlation among LH secretion, adiposity, plasma metabolic hormones, and hypothalamic gene expression in sheep on INP or DNP plane of nutrition (experiment 1)

 
For the INP group, there were significant positive relationships between mean LH and BCS (P < 0.05) and plasma insulin (P < 0.01) and between AgRP and OB-Rb gene expression (P < 0.01) and negative relationships between NPY gene expression and mean LH (P < 0.05) and BCS (P < 0.001). For the DNP group, there were significant positive relationships between LH pulse frequency and mean LH (P < 0.05), BCS (P < 0.01) and POMC gene expression (P < 0.001), between BCS and POMC gene expression (P < 0.05), between plasma insulin and leptin (P < 0.001), between gene expression for OB-Rb and NPY (P < 0.05) and AgRP (P < 0.05), and between NPY and AgRP (P < 0.05) and negative relationships between LH pulse frequency and gene expression for AgRP (P < 0.01) and OB-Rb (P < 0.05), between BCS and AgRP gene expression (P < 0.01), between insulin and Ins-R gene expression (P < 0.05), between POMC and NPY (P < 0.05) and AgRP (P < 0.05), and between OB-Rb gene expression and mean LH (P < 0.05) and BCS (P < 0.01). CART gene expression was not significantly related to any of the other variables measured in either the INP or DNP groups. Additional covariate analysis used the residuals of the regressions of BCS, insulin, leptin, Ob-Rb, Ins-R, NPY, AgRP, or POMC on LH secretion as dependent variables in stepwise regressions for both INP and DNP results; and BCS, insulin, leptin, Ob-Rb, NPY, and AgRP were then included as independent variables in the stepwise regressions. No significant covariates were identified (data not shown).

Experiment 2
Food intake, BCS, and body weight.
Food intake increased over time (P < 0.001) in the INP group (ad libitum) but remained constant in the SNP and DNP groups (restricted); at all time points after wk 0, intake was higher in the INP than SNP and higher in the SNP than DNP groups (P < 0.001) (Fig. 5AGo). BCS increased over time in the INP group (P < 0.01), decreased in the DNP group (P < 0.01), and did not change in the SNP group (Fig. 5BGo). There was a significant difference (P < 0.001) in mean BCS between groups at all time periods except wk 4 and 6, at which time values for BCS in the three groups had converged to similar levels. Final mean body weights for the INP, SNP, and DNP groups were 73 ± 1.3, 72 ± 2.0, and 70 ± 1.3 kg, respectively.


Figure 5
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  		FIG. 5. Food intake (A), BCS (adiposity) (B), mean LH concentration over 8 h (C), and LH pulse frequency (D) for sheep on INP (black bars), SNP (gray bars), or DNP (white bars) plane of nutrition in experiment 2 (n = 6 per treatment). Significant differences between groups at each time point are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant changes within groups over time are annotated above each panel.

 
LH secretion.
At wk 0, when all sheep were on maintenance food intake, mean LH was not different among the three groups (Fig. 5CGo), but LH pulse frequency was lower (P < 0.05) in animals with relatively low BCS (INP group) than in animals with relatively high BCS (DNP group) (Fig. 5DGo). Mean LH increased over time in the INP group (P < 0.05), decreased in the DNP group (P < 0.05), and did not change in the SNP group (Fig. 5CGo). Mean LH was lower (P < 0.05) in the DNP than SNP and INP groups at wk 4 and 6. LH pulse frequency increased over time in the INP group (P < 0.05), decreased in the DNP group (P < 0.01), but did not change in the SNP group (Fig. 4DGo). LH pulse frequency was lower in the DNP than the SNP and INP groups at wk 2 (P < 0.05), 4 (P < 0.01), and 6 (P < 0.01 and P < 0.001, respectively).

Insulin and leptin.
At wk 0, preprandial plasma and CSF concentrations of insulin were not different among the three groups (Fig. 6Go, A and B). Plasma leptin was higher (P < 0.05) in the DNP group (with relatively high BCS at this time) than the INP group (with relatively low BCS) (Fig. 6CGo), and CSF leptin concentrations were higher in the DNP (P < 0.01) and INP (P < 0.05) groups than in the SNP group (Fig. 6DGo). Plasma insulin (P < 0.05), plasma leptin (P < 0.01), and CSF insulin (P < 0.05) increased over time in the INP group but did not change in the DNP or SNP groups. Plasma insulin in the INP group was higher than in the DNP and SNP groups at wk 1, 2, 4, and 6 (P < 0.001–0.01). CSF insulin in the INP group was higher than in the DNP group at wk 2 (P < 0.05) and higher than in the DNP and SNP groups at wk 4 and 6 (P < 0.001). Plasma leptin in the INP group was higher than in the DNP and SNP groups at wk 4 and 6 (P < 0.001). CSF leptin was higher in both the DNP (P < 0.01) and INP groups (P < 0.05) than in the SNP group at wk 1 (P < 0.01) and higher in the INP group than in the DNP (P < 0.01) and SNP groups (P < 0.01) at wk 4 but not at wk 6.


Figure 6
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  		FIG. 6. Insulin concentrations in plasma (A) and CSF (B) and leptin concentrations in plasma (C) and CSF (D) for sheep on INP (black bars), SNP (gray bars), or DNP (white bars) plane of nutrition in experiment 2 (n = 6 per treatment). Significant differences between groups at each time point are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significant changes within groups over time are annotated where appropriate.

 
Correlation analyses.
To explore further the associations between central and peripheral changes in insulin and leptin and associations between central insulin/leptin and LH secretion, correlation analysis was carried out between these variables within the INP and DNP groups (Table 2Go).


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  		TABLE 2. Correlation among mean LH secretion, plasma, and CSF insulin and leptin concentrations for sheep on INP or DNP (experiment 2)

 
For the INP group, there were significant positive relationships between plasma leptin and CSF leptin (P < 0.05) and CSF insulin (P < 0.001), between plasma insulin and CSF insulin (P < 0.05) and LH pulse frequency (P < 0.01), and between and LH pulse frequency and CSF insulin (P < 0.05). For the DNP group, there were significant positive relationships between plasma leptin and CSF leptin (P < 0.01) and CSF insulin (P < 0.01), between CSF leptin and CSF insulin (P < 0.05), between LH pulse frequency and CSF leptin (P < 0.01) and CSF insulin (P < 0.001), and between mean LH and CSF insulin (P < 0.01). Additional covariate analysis used the residuals of the regressions of plasma insulin, CSF insulin, plasma leptin, or CSF leptin on LH secretion as dependent variables in stepwise regressions for both INP and DNP results; plasma insulin, CSF insulin, plasma leptin, and CSF leptin were then included as independent variables in the stepwise regressions. No significant covariates were found (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study supports the hypothesis that nutritional modulation of reproduction involves insulin and leptin signaling through hypothalamic orexigenic pathways to modulate GnRH/LH secretion. Moreover, mediation of the effects of changing nutritional status appeared to differ according to the direction of nutritional change. The data were consistent with the response to increasing nutritional plane being mediated by increasing circulating insulin, which was able to enter the CSF and stimulate GnRH/LH output, whereas the response to decreasing nutritional feedback appeared to be mediated by changes in leptin signaling brought about by altered OB-Rb expression rather than by decreasing circulating concentrations. Additionally, because end point adiposity was similar yet GnRH/LH output was different between the groups in both experiments, the hypothalamus seemed to retain a nutritional memory based on changes in orexigenic neuropeptide gene expression that influenced contemporary neuroendocrine responses. Others have also suggested the existence of a metabolic memory to explain the complex interactions between reproductive function and nutritional status in male sheep (32).

The positive association between adiposity (BCS) and LH output (and by inference GnRH output) is well established in sheep and other mammals (6, 33, 34, 35, 36). However, the failure of previous studies to elucidate fully the underlying mechanism may be attributable to the overriding influence of a dynamically changing nutritional status, as highlighted in this present study. Thus, different associations are seen in these sheep at the start of experiment, when they had different levels of adiposity but a similar steady-state nutritional plane, compared with the end of the experiment, when they had similar levels of adiposity but were on an increasing or decreasing nutritional plane. At the start (time 0 wk), LH output was higher in relatively fat vs. relatively thin sheep, which was associated with elevated plasma insulin and to a lesser extent plasma leptin in experiment 1 but with higher plasma leptin and no difference in plasma insulin or in CSF leptin or insulin in experiment 2. In other words, no clear endocrine mediator of adiposity effects on GnRH/LH emerged. The somewhat equivocal findings at the start of the experiments may have been attributable to their slightly differing nutritional histories during the preparatory period (i.e. different lengths of the static, maintenance nutritional holding period) given the dominant influence of changing nutritional status revealed in the main experiments.

The present data indicate a possible causal relationship between insulin, adiposity and LH secretion in the sheep that gained body fat over time. Their nutritional history was reflected in coordinated changing levels of adiposity, plasma, and CSF insulin and hypothalamic NPY gene expression in the ARC, without coordinated changes in plasma and CSF leptin or the other hypothalamic neuropeptides examined. Insulin, and not leptin, therefore provided the most likely signal of increasing nutritional status that led to increased GnRH/LH output. Centrally administered insulin inhibits hypothalamic NPY gene expression (37), increased hypothalamic NPY gene expression is associated with reduced pulsatile LH secretion (23), and centrally administered NPY inhibits GnRH/LH output (38). Therefore, the present data are consistent with the increased concentrations of circulating insulin in fattening animals causing the reduction in hypothalamic NPY gene expression and thereby increasing GnRH/LH output. There was some evidence of peripheral insulin resistance developing in these fattening animals because their preprandial plasma glucose concentrations increased significantly along with the increase in insulin concentrations during experiment 1.

By contrast, the present sheep that lost body fat over time did not have their nutritional history reflected in changing circulating plasma concentrations of either insulin or leptin but rather in increasing hypothalamic ARC expression of OB-Rb and AgRP over the 6 wk, and hypothalamic expression of POMC was decreased at the end. Because the decreased GnRH/LH output in these sheep correlated with decreased CSF concentrations of both insulin (39) and leptin (28), it is tempting to speculate that either of these CSF changes might have brought about the increase in OB-Rb expression and. Increased OB-Rb expression during negative energy balance increases hypothalamic sensitivity to hypoleptinemia (16), and it is plausible that the decreasing CSF leptin, acting through the increasing OB-Rb, may have been responsible for the increasing AgRP gene expression during the experiment and for the decreased POMC gene expression seen at the end (40). This decreased activity of the melanocortin pathway may have led to the reduced GnRH output because central infusion of AgRP, an endogenous melanocortin antagonist, suppresses pulsatile LH in rhesus monkeys (41). Moreover, melanocortins (products of POMC) generally stimulate reproduction and are involved in mediating actions of leptin on GnRH (42). The present data are consistent with decreased intrahypothalamic leptin signaling causing decreased melanocortin signaling and leading to decreased GnRH/LH output in slimming animals.

The critical importance of (regulated) brain uptake of peripheral circulating molecules, such as insulin and leptin, to perform a central signaling role is fully recognized (reviewed in Ref. 43). These peptides enter the CSF from circulating blood via transporter mechanisms that may become saturated at high concentrations. We recently reported reduced efficiency of blood-brain leptin transport with increased leptinemia in sheep (44); however, this phenomenon was not evident in the present sheep, probably because plasma leptinemia remained lower and the duration of nutritional treatments was shorter than in our earlier trial. Plasma and CSF concentrations of leptin were positively correlated in both fattening (INP) and slimming (DNP) sheep, indicating that peripheral and central changes in concentration were matched in direction if not in magnitude. However, although reduced CSF leptin signaling may have contributed to GnRH/LH inhibition in DNP sheep, there was no evidence to link CSF leptin changes with changes in GnRH in INP sheep. On the other hand, plasma and CSF insulin concentrations were only correlated in fattening (INP) sheep and not in slimming (DNP) sheep, indicating that peripheral and central increases in concentration were matched in the former and provided a credible link between increased adiposity and increased GnRH output. However, reduced peripheral insulin in slimming animals was not matched centrally and therefore did not provide a credible link between decreased adiposity and decreased GnRH output. The mechanism whereby blood-brain insulin transport is apparently regulated differentially according to the direction of nutritional change deserves further study, and it is interesting that insulin receptor gene expression was increased in slimming as opposed to fattening sheep at the same terminal adiposity. Altogether it is pertinent to note that peripheral measurements of insulin and leptin may be misleading in assessing the magnitude of signals received and the expression of their receptors within the hypothalamus.

Group differences in hypothalamic neuropeptide gene expression at specific time points in this trial further revealed the confounding effects of adiposity and nutritional plane, as reported earlier by Archer et al. (4). For example, at wk 1 relatively thin sheep with higher food intake had higher NPY gene expression than fat sheep with lower food intake, but at wk 6 sheep with lower food intake had higher NPY than sheep with higher intake but equal adiposity. This may be interpreted as a dominant influence of current food intake on NPY expression, surpassing the effect of adiposity, and emphasizes the importance of recent nutritional history in evaluating contemporary hypothalamic responses. Similar arguments may be advanced for the other neuropeptides measured, AgRP, CART, and POMC, which demonstrated greatest divergence in gene expression between the INP and DNP groups at wk 6 when adiposity had converged. In other words, snapshot hypothalamic neuropeptide profiles of individuals in a given nutritional state may be misleading without an appreciation of their preceding nutritional history (exemplified in Fig. 4Go). The hypothalamic orexigenic/anorexigenic pathways appear more sensitive to a changing nutritional status than to absolute nutritional status per se and there are clearly downstream consequences for reproductive neuroendocrine output.

In conclusion, this study has demonstrated that hypothalamic reproductive neuroendocrine pathways respond differentially to changing as opposed to constant levels of nutritional feedback and that different directions of nutritional feedback (increasing vs. decreasing) enact different signaling pathways. The GnRH/LH response to an increasing plane of nutrition appears to be mediated by changes in circulating insulin, which enters the hypothalamic CSF and stimulates reproductive neuroendocrine output by inhibiting NPY expression. The GnRH/LH response to a decreasing plane of nutrition appears to be mediated by changes in leptin signaling via increased leptin receptor expression, which inhibits reproductive neuroendocrine output by inhibiting melanocortin activity. These findings have important implications for understanding the variation in reported responses to nutritional feedback, not only in terms of reproductive outcome but also in terms of appetite and body weight regulation across the species, including humans. The hypothalamus apparently retains a nutritional memory that influences its contemporary responses.


   Acknowledgments
 
We are grateful to Duthie Farm staff and the Rowett Bioresources Group for assistance with the experimental animals.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council Grant D17281 and the Scottish Executive Environment and Rural Affairs Department.

Current address for D.W.M. and J.L.H.: School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch 6150, Western Australia, Australia.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 16, 2007

Abbreviations: AgRP, Agouti-related peptide; ARC, arcuate nucleus; BCS, body condition score; CART, cocaine- and amphetamine-regulated transcript; CSF, cerebrospinal fluid; CV, coefficient of variation; DNP, decreasing nutritional plane; icv, intracerebroventricular; INP, increasing nutritional plane; Ins-R, insulin receptor; NPY, neuropeptide Y; OB-Rb, leptin receptor; POMC, proopiomelanocortin; SNP, static nutritional plane.

Received April 24, 2007.

Accepted for publication August 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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