Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.
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CL Adam, TS Gadd, PA Findlay, and DC Wathes
M Marie, PA Findlay, L Thomas, and CL Adam
Circulating concentrations of leptin in sheep correlate with body fatness and are affected by level of food intake and photoperiod. The present objective was to elucidate the short-term dynamics of leptin secretion. Frequent blood samples were taken over 48 h from 12 Soay rams after 16 weeks in short-day photoperiod (SD, 16 h darkness:8 h light) with freely available food, and then after 16 weeks in long days (16 h light:8 h darkness) with food freely available (LD) or restricted to 90% maintenance (LDR) (n=6/group). During the second 24 h of sampling, half were food deprived (n=6, SD and LD) and half had their meal times shifted (n=6, SD and LDR). A homologous RIA was developed, using antibodies raised in chicken against recombinant ovine leptin, to measure plasma concentrations. Simultaneous 24 h profiles of plasma insulin, glucose and non-esterified fatty acids (NEFA) were measured. Plasma leptin was higher in LD than SD, and in LD than LDR, associated with higher food intake, liveweight and body condition score (adiposity), but tended to be lower in LDR than SD, associated with lower food intake, liveweight and body condition score. There was no evidence for a circadian rhythm of plasma leptin, but clear evidence for post-prandial peaks of low amplitude (15-36%) 2-8 h after meals given at normal and shifted times. Complete food deprivation caused a dramatic fall in plasma leptin to basal levels within 24 h. There was a positive association of plasma leptin with plasma insulin, and negative association with NEFA, both between meals and during fasting. Thus, plasma leptin concentrations in sheep are sensitive to short-term changes in energy balance, as well as to long-term photoperiod-driven changes in food intake and adiposity.
ZA Archer, PA Findlay, SR McMillen, SM Rhind, and CL Adam
Sheep exhibit photoperiod-driven seasonal changes in appetite and body weight so that nutritional status increases in long days (LD) and decreases in short days (SD); additionally, they are reproductively active in SD and inactive in LD. We addressed the hypothesis that appetite-regulatory genes in the hypothalamus respond differently to changes in nutritional feedback induced by photoperiod as opposed to food restriction, and that responses would be influenced by gonadal steroid status. Castrated oestradiol-implanted male sheep were kept in SD (8 h light/day) or LD (16 h light/day) for 11 weeks, with ad libitum or restricted food (experiment 1; n=8/group). Rams were kept in SD or LD for 12 weeks with ad libitum or restricted food (experiment 2; n=6/group). Gene expression (by in situ hybridisation) in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), neuropeptide Y (NPY), pro-opiomelanocortin (POMC) and agouti-related peptide (AGRP) was unaffected by photoperiod treatment, but food restriction increased NPY and AGRP mRNAs, in experiment 1. In experiment 2, mRNAs for POMC and cocaine- and amphetamine-regulated transcript (CART) were up-regulated and AGRP down-regulated in SD, while food restriction increased OB-Rb mRNA, increased NPY and AGRP mRNAs only in LD and decreased POMC mRNA only in SD. Thus, gene expression responded differently to photoperiod and food restriction, and the melanocortin pathway was up-regulated in SD in reproductively activated rams but not in oestradiol-implanted castrates. These data support the hypothesis that hypothalamic appetite-regulatory pathways respond differently to changes in nutritional feedback induced by photoperiod as opposed to food restriction, with gonadal steroid feedback additionally influencing the responses.
DW Miller, PA Findlay, MA Morrison, N Raver, and CL Adam
The role of leptin in neuroendocrine appetite and reproductive regulation remains to be fully resolved. A series of three experiments was conducted using adequately nourished oestradiol-implanted castrated male sheep. In a cross-over design (n=6), responses to a single i.c.v. (third ventricle) injection of leptin (0.5, 1.0 and 1.5 mg ovine leptin (oLEP) and 1.0 mg murine leptin (mLEP)), N-methyl-D-aspartate (NMDA, 20 micro g) or 0.9% saline (control) were measured in terms of LH secretion (4 h post-injection compared with 4 h pre-injection) and appetite (during 2 h post-injection) in autumn (Experiment 1). NMDA and 1.0 mg oLEP treatments were repeated in the same sheep in the following spring (Experiment 2). With an additional 12 sheep (n=18 in cross-over design), responses to low-dose 'physiological' i.c.v. infusion of leptin (8 ng/h for 12 h daily for 4 days), insulin (0.7 ng/h) and artificial cerebrospinal fluid were measured in the next spring (Experiment 3). LH was studied over 8 h and appetite over 1 h on days 1 and 4 of infusion. In Experiment 1 (autumn), oLEP overall increased LH pulse frequency by up to 110% (P<0.05), decreased LH pulse amplitude (P<0.05) and decreased appetite (P<0.05). mLEP reduced LH pulse amplitude (P<0.05) without significant effect on appetite, while NMDA reduced appetite (P<0.05) but had no effect on LH. In Experiment 2 (spring), LH responses were 'surge-like' with highly significant increases in the moving average LH concentration after 1.0 mg oLEP (P<0.001) and after NMDA (P<0.001). Compared with similar analysis of experiment 1 results, the LH response in spring was greater than that in autumn for both 1.0 mg oLEP (P<0.05) and NMDA (P<0.005). Conversely, unlike in autumn (Experiment 1), there was no effect of 1.0 mg oLEP or NMDA on appetite in the spring (Experiment 2). In Experiment 3 (spring), 'physiological' i.c.v. infusion of oLEP or insulin increased LH pulse frequency by up to 100% (P<0.001) compared with the control infusion on both days 1 and 4, but there were no effects on appetite. These results indicate that intracerebral leptin both stimulates reproductive neuroendocrine output and decreases appetite in adequately nourished sheep. However, the responses of these two axes were dose-dependent and differentially affected by the time of year, suggesting dissociation of the neural pathways involved.
ZA Archer, SM Rhind, PA Findlay, CE Kyle, L Thomas, M Marie, and CL Adam
Body reserves (long-term) and food intake (short-term) both contribute nutritional feedback to the hypothalamus. Reproductive neuroendocrine output (GnRH/LH) is stimulated by increased food intake and not by high adiposity in sheep, but it is unknown whether appetite-regulating hypothalamic neurons show this differential response. Castrated male sheep (Scottish Blackface) with oestradiol implants were studied in two 4 week experiments. In Experiment 1, sheep were fed to maintain the initial body condition (BC) score of 2.0+/-0.00 (lower BC (LBC), n=7) or 2.9+/-0.09 (higher BC (HBC), n=9), and liveweight of 43+/-1.1 and 59+/-1.6 kg respectively. LBC and HBC sheep had similar mean plasma LH concentration, pulse frequency and amplitude, but HBC animals had higher mean plasma concentrations of insulin (P<0.01), leptin (P<0.01) and glucose (P<0.01). Gene expression (measured by in situ hybridisation) in the hypothalamic arcuate nucleus (ARC) was higher in LBC than HBC sheep for neuropeptide Y (NPY; 486% of HBC, P<0.01), agouti-related peptide (AGRP; 467%, P<0.05) and leptin receptor (OB-Rb; 141%, P<0.05), but lower for cocaine- and amphetamine-regulated transcript (CART; 92%, P<0.05) and similar between groups for pro-opiomelanocortin (POMC). In Experiment 2, sheep with initial mean BC score 2.4+/-0.03 and liveweight 55+/-0.8 kg were fed a liveweight-maintenance ration (low intake, LI, n=7) while sheep with initial mean BC score 2.0+/-0.03 and liveweight 43+/-1.4 kg were fed freely so that BC score increased to 2.5+/-0.00 and liveweight increased to 54+/-1.4 kg (high intake, HI, n=9). Compared with LI, HI sheep had higher mean plasma LH (P<0.05), baseline LH (P<0.01) and pulse amplitude (P<0.01) and showed a trend towards higher pulse frequency. Although there were no differences in final mean plasma concentrations, there were significant increases over time in mean concentrations of insulin (P<0.001), leptin (P<0.05) and glucose (P<0.001) in HI sheep. Gene expression for AGRP in the ARC was higher in HI than LI animals (453% of LI; P<0.05), but expression levels were similar for NPY, OB-Rb, CART and POMC. Thus, the hypothalamus shows differential responses to steady-state adiposity as opposed to an increase in food intake, in terms of both reproductive neuroendocrine activity and hypothalamic appetite-regulating pathways. Differences in hypothalamic gene expression were largely consistent with contemporary levels of systemic leptin and insulin feedback; however, increased nutritional feedback was stimulatory to GnRH/LH whereas constant high feedback was not. The hypothalamus therefore has the ability to retain a nutritional memory that can influence subsequent responses.