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Orexin gene expression and regulation by photoperiod in the sheep hypothalamus
Regulatory Peptides 104 (2002) 41 – 45 www.elsevier.com/locate/regpep
Orexin gene expression and regulation by photoperiod in the sheep hypothalamus Zoe A. Archer a,*, Patricia A. Findlay a, Stewart M. Rhind b, Julian G. Mercer a, Clare L. Adam a a
Molecular Neuroendocrinology Group, Aberdeen Center for Energy Balance and Obesity, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK b Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK
Abstract Hypothalamic orexin gene expression has not been reported in the ruminant. Here, we describe the localization of preproorexin mRNA in the ovine lateral hypothalamic area (LHA) and zona incerta (ZI) using in situ hybridization. The hypothalamic localization of the orexin gene expression was similar in sheep to rodent models. Since appetite in sheep is seasonally (photoperiodically) regulated, we compared the amounts of preproorexin mRNA between long- (LD) and short-day (SD) photoperiods in both freely feeding (food intake is 20% higher in LD than SD) and food-restricted sheep (50% liveweight maintenance for 11 weeks). Gene expression was higher in SDs than in LDs but was not affected by chronic food restriction. In a second study, hypothalamic orexin gene expression in castrate sheep was not affected by a 4-day fast, irrespective of gonadal steroid (estradiol) replacement, and was not affected by the gonadal steroid per se. The results demonstrate the sensitivity of orexin gene expression to photoperiod, but up-regulation occurs in SDs when the appetite is characteristically low and no sensitivity to imposed changes in food intake. This supports the concept that orexins may not have a primary role in appetite regulation and correction of negative energy balance but since the sheep breed only in SDs, their role in seasonal reproductive activation deserves further study. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ovine; Lateral hypothalamic area; Zona incerta; Food intake
1. Introduction The orexins or hypocretins were discovered within the lateral hypothalamic area and zona incerta of rats [1,2]. Hypothalamic gene expression in the rat for the common precursor peptide preproorexin was up-regulated by fasting and by food restriction [1,2], and ICV administration of orexins A or B stimulated food intake [2]. In addition, peripheral injections of orexin B increased food intake in the pig [3], indicating that orexins play a role in food intake. Although more recent findings indicate that the primary role of orexins may be in the regulation of sleep – wakefulness and arousal [4], it is clear that orexins also impact on systems that regulate energy homeostasis. The role of orexins in domestic ruminant species, with their divergent nutritional physiology, has hitherto not been investigated. Here, we aimed to determine whether the sheep brain has a lateral hypothalamic distribution of preproorexin gene expression as in the rodent models. Since appetite in sheep is seasonally *
Corresponding author. Tel.: +44-1224-712-751; fax: +44-1224-716686. E-mail address: [email protected] (Z.A. Archer).
(photoperiodically) regulated [5,6], we compared the amount of preproorexin mRNA between long- and short-day photoperiods in both freely feeding and food-restricted sheep. Lastly, we examined the response of orexin gene expression to fasting.
2. Methods 2.1. Animals In the first study, eight adult Soay castrate male sheep (aged 1 – 2 years, 27– 38 kg liveweight) with steroid replacement (subcutaneous estradiol implants inserted 13 weeks prior to euthanasia, producing plasma concentrations of 4.0 F 0.27 pg/ml [7]) were euthanized following 11 weeks in long-day (LD, 16 h light/8 h dark) or short-day artificial photoperiod (SD, 8 h light/16 h dark) (n = 4/group). All animals received a complete diet (11 MJ metabolizable energy/kg dry matter (DM)). Two sheep in each photoperiod were fed ad libitum (AL) and two were restricted to approximately 50% of the amount required to maintain liveweight (R).
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 1 ) 0 0 3 4 7 - 0
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Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
In a second study, eight adult Suffolk Greyface male castrate sheep (aged 2 years, 60 –70 kg liveweight) were euthanized in December (57N, natural photoperiod approximately 8 h light/16 h dark, i.e. similar to artificial SD in the first study) following normal feeding for the maintenance of liveweight (complete diet containing approximately 10 MJ metabolizable energy/kg DM) (maintenance-fed, M) or 4 days without food (food-deprived, FD) (n = 4/group). Two in each group had subcutaneous estradiol implants which had been inserted 6 months previously (E+) (plasma concentrations 1.5 F 0.27 pg/ml) and two did not (E , < 0.5 pg/ml). Sheep in both studies were killed by lethal overdose of sodium pentobarbitone (Euthesate; Rhone Merieux, Harlow, Essex) between 1000 and 1600 h, approximately 4 h after the last meal for all except the FD group. The brain was rapidly removed, flash frozen in isopentane (Sigma, UK) over dry ice prior to storage at 80 C. All experimental procedures were licensed under the Animals (Scientific Procedures) Act of 1986 and received approval from the Macaulay Land Use Research Institute (first study) and Rowett Research Institute (second study) Ethical Review Committees.
sity was then computed using standard curves generated from 14C autoradiographic micro-scales (Amersham). 2.3. Statistical analysis Gene expression densitometry data for different treatments were compared by two-way analysis of variance.
3. Results The antisense preproorexin riboprobe derived from rat cDNA was hybridised to the sheep brain tissue, whereas
2.2. In situ hybridization Messenger RNA expression for preproorexin was determined by in situ hybridization in 20-mm coronal hypothalamic sections using the techniques described in detail elsewhere [8,9]. Briefly, sections were mounted on poly-Llysine slides and stored at 70 C before fixation (in 4% paraformaldehyde) and acetylation. Riboprobes were generated using a 347-bp cDNA for a partial rat preproorexin sequence cloned by RT-PCR [10]. Sections were hybridised overnight at 58 C using 35S-labelled antisense and sense riboprobes (1– 1.5 107 cpm/ml). Slides were treated with RNase A to remove the unhybridised probe, and then desalted with a final high stringency wash in 0.5 saline – sodium citrate (SSC) at 60 C for 30 min. The slides were airdried and apposed to autoradiographic film (Kodak BioMax MR; Amersham, UK) for 3 days. Slides from the first study were then coated with autoradiographic emulsion (Hypercoat LM-1; Amersham), dried and stored in the dark at 4 C for 6 days. They were then developed (D-19; Kodak), fixed (Unifix; Kodak) and counter-stained with toluidine blue. Serial sections spanning the hypothalamus caudally from the optic chiasm along the third ventricle were used to determine the orexin mRNA localization within the lateral hypothalamus. Quantification was undertaken on a uniform set of lateral hypothalamic sections (n = 3/sheep in the first study and n = 9/sheep in the second study) taken 0.5 mm caudal of the point where both sides of the optic chiasm meet beneath the third ventricle (as in Fig. 1B) using the Image-Pro Plus system (Media Cybernetics, MD, USA). This determined the intensity and area of the hybridization signal on the basis of set parameters. The integrated inten-
Fig. 1. Autoradiographic localization of preproorexin mRNA in coronal sections of ovine hypothalamus. A and B, hybridized to the antisense probe, are approximately 1 mm apart. C shows lack of hybridization to the sense probe in a section adjacent to B. LHA, lateral hypothalamic area; ZI, zona incerta; 3V, third ventricle; OC, optic chiasm; Fx, fornix; Mt, fasciculus mamillothalamicus. Scale bar = 2 mm.
Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
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Fig. 2. Photomicrographs of lateral hypothalamic area in ovine brain section hybridized with the antisense preproorexin probe. A gives 5 magnification showing part of the zona incerta (scale bar = 2 mm), and the arrows denote cells shown at 40 magnification in B and C (scale bar = 10 mm).
the sense probe showed no hybridization signal. Preproorexin gene expression was localized in the lateral hypothalamic area (LHA) and zona incerta (ZI) of the ovine hypothalamus (Fig. 1). Emulsion autoradiography confirmed the localization of preproorexin mRNA to LHA neurons (Fig. 2).
Preproorexin gene expression was significantly increased by SDs ( P < 0.05), but there was no effect of food restriction nor a photoperiod food intake interaction (Fig. 3). In the second study, there was no effect of fasting or estradiol replacement on preproorexin gene expression nor a fasting estradiol interaction (Fig. 4).
Fig. 3. Preproorexin gene expression in the lateral hypothalamic area of sheep kept in long-day (LD) or short-day (SD) photoperiod and fed ad libitum (AL) or a restricted amount (R). Values are expressed as percentage of the LD/R group value (n = 2/group).
Fig. 4. Preproorexin gene expression in the lateral hypothalamic area following normal maintenance feeding (M) or 4 days of food deprivation (FD) in castrate male sheep with (E+) and without estradiol steroid replacement (E ). Values are expressed as percentage of M/E group value (n = 2/group).
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Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
4. Discussion Orexin gene expression is reported here for the first time in the sheep hypothalamus where it is localized in the lateral hypothalamic area and zona incerta as reported in rodents (rat [2]; Siberian hamster [10]). There was no effect of chronic food restriction on orexin gene expression in the sheep LHA unlike fasting in the rat [2,11]. However, there was an effect of photoperiod with the amounts of orexin mRNA higher in SDs than LDs. The increase in orexin gene expression was unlikely to be attributable to the reduced voluntary food intake seen in SDs [6] since imposed reductions in food intake had no effect, irrespective of photoperiod. It is also counter-intuitive that this orexigenic neuropeptide should be up-regulated during the season of reduced appetite drive. Furthermore, changes in the hypothalamic orexin gene expression do not accompany the reduced appetite drive and food intake seen during SDs in seasonal Siberian hamsters [10,12]. Rather, the SD increase in orexin gene expression in sheep may relate to reproductive activation, which is confined to the SD photoperiod in this species [6,13]. A role for orexins in regulating the reproductive neuroendocrine axis has recently been proposed since ICV infusion of orexin A or B rapidly stimulates tonic LH secretion in steroid-pretreated ovariectomized rats [14]. In addition, the central administration of orexin A to estradiol-primed ovariectomized rats restored the preovulatory LH surge in fasted animals, whereas anti-orexin A antiserum abolished the LH surge in fed rats [15]. Higher levels of orexin gene expression during the breeding season are unlikely to be caused by the seasonal increase in circulating gonadal steroids since the present sheep were steroid-clamped across both photoperiods in the first study, and the presence or absence of gonadal steroid did not affect orexin mRNA levels in the second study. However, a key interaction between sex steroids and orexins in the control of tonic GnRH/LH secretion has been suggested since orexin A-given ICV inhibits pulsatile LH in ovariectomized rats that have not been primed with steroids [16,17], yet stimulates pulsatile LH secretion in steroid-primed counterparts [14]. The lack of effect of fasting on orexin gene expression in sheep agrees with the evidence from the seasonal Siberian hamster [10,12] and mouse [18] but contrasts with the data for rats [2]. It is feasible that the lack of effect of food deprivation on orexin gene expression during December may be explained by the observation that orexin mRNA is already elevated during short days. However, food restriction also had no effect in either short or long days in study 1. Fasting acutely reduces the circulating leptin in sheep [19] as well as in rodents [20,21]. Thus, an acute reduction in the leptin signal does not seem to increase orexin gene expression, suggesting that orexin probably does not play a major role in leptin-driven responses to correct inappropriate negative energy balance in the sheep. Thus, the localization of orexin gene expression in the lateral hypothalamus of sheep is similar to that in rodents.
Levels of gene expression were apparently unaffected by chronic food restriction or fasting in this seasonal ruminant species but were influenced by photoperiod. These results lend support to the concept that appetite regulation is not the primary role of orexins and provide a basis for future studies on the role of orexins in sheep, for example, in regulating the reproductive neuroendocrine axis.
Acknowledgements This work was funded by the Scottish Executive Environment and Rural Affairs Department. Dr. Z. Archer was a recipient of a Boyd Orr Research Center studentship.
References [1] Delecea L, Kilduff TS, Peyron C, Gao X, Foya PE, Danielson PE, et al. The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. PNAS 1998;95:322 – 7. [2] Sakaurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 1998;92:573 – 85. [3] Dyer CJ, Touchette KJ, Carroll JA, Allee GL, Matteri RL. Cloning of porcine prepro-orexin cDNA and effects of an intramuscular injection of synthetic porcine orexin-B on feed intake in young pigs. Domest Anim Endocrinol 1999;16:145 – 8. [4] Samson WK, Resch ZT. The hypocretin/orexin story. Trends Endocrinol Metab 2000;11:257 – 62. [5] Kay RN. Seasonal variation of appetite in ruminants. In: Haresign W, editor. Recent advances in animal nutrition. London: Butterworth; 1985. p. 199 – 210. [6] Adam CL. Nutritional and photoperiodic regulation of appetite and reproduction in seasonal domestic mammals. Reprod Domest Anim 2000;(Supplement 6):1 – 8. [7] Olster DH, Foster DL. Control of gonadotrophin secretion in the male during puberty: a decrease in response to steroid inhibitory feedback in the absence of an increase in steroid-independent drive in the sheep. Endocrinology 1986;118:2225 – 34. [8] Mercer JG, Lawrence CB, Beck B, Burlet A, Atkinson T, Barrett P. Hypothalamic NPY and prepro-NPY mRNA in Djungarian hamsters: effects of food deprivation and photoperiod. Am J Physiol 1995;38:R1099 – 106. [9] Adam CL, Findlay PA, Kyle CE, Young P, Mercer JG. Effect of chronic food restriction on pulsatile luteinizing hormone secretion and hypothalamic neuropeptide Y gene expression in castrate male sheep. J Endocrinol 1997;152:329 – 37. [10] Mercer JG, Moar KM, Ross AW, Hoggard N, Morgan PJ. Photoperiod regulates arcuate nucleus POMC, AgRp and leptin receptor mRNA in Siberian hamster hypothalamus. Am J Physiol 2000;278:R271 – 81. [11] Mondal MS, Nakazato M, Date Y, Murakami N, Yanagisawa M, Matsukura S. Widespread distribution of orexin in rat brain and its regulation upon fasting. Biochem Biophys Res Commun 1999;256:495 – 9. [12] Reddy AB, Cronin AS, Ford H, Ebling FJ. Seasonal regulation of food intake and body weight in the male Siberian hamster: studies of hypothalamic orexin (hypocretin), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). Eur J Neurosci 1999;11:3255 – 64. [13] Lincoln GA, Short RV. Seasonal breeding: nature’s contraceptive. Rec Prog Hormone Res 1980;36:1 – 51. [14] Pu S, Jain M, Kalra P, Karla S. Orexins, a novel family of hypothalamic neuropeptides, modulate pituitary luteinizing hormone secretion in an ovarian steroid-dependent manner. Regul Pept 1998;78:133 – 6.
Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45 [15] Kohsaka A, Watanobe H, Kakizaki Y, Suda T, Schioth HB. A significant participation of orexin-A, a potent orexigenic peptide, in the preovulatory luteinizing hormone and prolactin surges in the rat. Brain Res 2001;898:166 – 70. [16] Tamura T, Irahara M, Tezuka M, Kiyokawa M, Aono T. Orexins, orexigenic hypothalamic neuropeptides, suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats. Biochem Biophys Res Commun 1999;264:759 – 62. [17] Irahara M, Tamura T, Matuzaki T, Saito S, Yasui T, Yamano S, et al. Orexin-A suppresses the pulsatile secretion of luteinizing hormone via beta-endorphin. Biochem Biophys Res Commun 2001;281:2323 – 6.
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[18] Tritos NA, Mastaitis JW, Kokkotou E, Maratos-Flier E. Characterization of melanin-concentrating hormone and prepro-orexin expression in the murine hypothalamus. Brain Res 2001;895:160 – 6. [19] Marie M, Findlay PA, Thomas L, Adam CL. Daily patterns of plasma leptin in sheep: effects of photoperiod and food intake. J Endocrinol 2001;170:277 – 86. [20] Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, MaratosFlier E, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250 – 2. [21] Ahima RS, Flier JS. Leptin. Annu Rev Physiol 2000;62:413 – 37.
Orexin gene expression and regulation by photoperiod in the sheep hypothalamus Zoe A. Archer a,*, Patricia A. Findlay a, Stewart M. Rhind b, Julian G. Mercer a, Clare L. Adam a a
Molecular Neuroendocrinology Group, Aberdeen Center for Energy Balance and Obesity, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK b Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK
Abstract Hypothalamic orexin gene expression has not been reported in the ruminant. Here, we describe the localization of preproorexin mRNA in the ovine lateral hypothalamic area (LHA) and zona incerta (ZI) using in situ hybridization. The hypothalamic localization of the orexin gene expression was similar in sheep to rodent models. Since appetite in sheep is seasonally (photoperiodically) regulated, we compared the amounts of preproorexin mRNA between long- (LD) and short-day (SD) photoperiods in both freely feeding (food intake is 20% higher in LD than SD) and food-restricted sheep (50% liveweight maintenance for 11 weeks). Gene expression was higher in SDs than in LDs but was not affected by chronic food restriction. In a second study, hypothalamic orexin gene expression in castrate sheep was not affected by a 4-day fast, irrespective of gonadal steroid (estradiol) replacement, and was not affected by the gonadal steroid per se. The results demonstrate the sensitivity of orexin gene expression to photoperiod, but up-regulation occurs in SDs when the appetite is characteristically low and no sensitivity to imposed changes in food intake. This supports the concept that orexins may not have a primary role in appetite regulation and correction of negative energy balance but since the sheep breed only in SDs, their role in seasonal reproductive activation deserves further study. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Ovine; Lateral hypothalamic area; Zona incerta; Food intake
1. Introduction The orexins or hypocretins were discovered within the lateral hypothalamic area and zona incerta of rats [1,2]. Hypothalamic gene expression in the rat for the common precursor peptide preproorexin was up-regulated by fasting and by food restriction [1,2], and ICV administration of orexins A or B stimulated food intake [2]. In addition, peripheral injections of orexin B increased food intake in the pig [3], indicating that orexins play a role in food intake. Although more recent findings indicate that the primary role of orexins may be in the regulation of sleep – wakefulness and arousal [4], it is clear that orexins also impact on systems that regulate energy homeostasis. The role of orexins in domestic ruminant species, with their divergent nutritional physiology, has hitherto not been investigated. Here, we aimed to determine whether the sheep brain has a lateral hypothalamic distribution of preproorexin gene expression as in the rodent models. Since appetite in sheep is seasonally *
Corresponding author. Tel.: +44-1224-712-751; fax: +44-1224-716686. E-mail address: [email protected] (Z.A. Archer).
(photoperiodically) regulated [5,6], we compared the amount of preproorexin mRNA between long- and short-day photoperiods in both freely feeding and food-restricted sheep. Lastly, we examined the response of orexin gene expression to fasting.
2. Methods 2.1. Animals In the first study, eight adult Soay castrate male sheep (aged 1 – 2 years, 27– 38 kg liveweight) with steroid replacement (subcutaneous estradiol implants inserted 13 weeks prior to euthanasia, producing plasma concentrations of 4.0 F 0.27 pg/ml [7]) were euthanized following 11 weeks in long-day (LD, 16 h light/8 h dark) or short-day artificial photoperiod (SD, 8 h light/16 h dark) (n = 4/group). All animals received a complete diet (11 MJ metabolizable energy/kg dry matter (DM)). Two sheep in each photoperiod were fed ad libitum (AL) and two were restricted to approximately 50% of the amount required to maintain liveweight (R).
0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 1 ) 0 0 3 4 7 - 0
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Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
In a second study, eight adult Suffolk Greyface male castrate sheep (aged 2 years, 60 –70 kg liveweight) were euthanized in December (57N, natural photoperiod approximately 8 h light/16 h dark, i.e. similar to artificial SD in the first study) following normal feeding for the maintenance of liveweight (complete diet containing approximately 10 MJ metabolizable energy/kg DM) (maintenance-fed, M) or 4 days without food (food-deprived, FD) (n = 4/group). Two in each group had subcutaneous estradiol implants which had been inserted 6 months previously (E+) (plasma concentrations 1.5 F 0.27 pg/ml) and two did not (E , < 0.5 pg/ml). Sheep in both studies were killed by lethal overdose of sodium pentobarbitone (Euthesate; Rhone Merieux, Harlow, Essex) between 1000 and 1600 h, approximately 4 h after the last meal for all except the FD group. The brain was rapidly removed, flash frozen in isopentane (Sigma, UK) over dry ice prior to storage at 80 C. All experimental procedures were licensed under the Animals (Scientific Procedures) Act of 1986 and received approval from the Macaulay Land Use Research Institute (first study) and Rowett Research Institute (second study) Ethical Review Committees.
sity was then computed using standard curves generated from 14C autoradiographic micro-scales (Amersham). 2.3. Statistical analysis Gene expression densitometry data for different treatments were compared by two-way analysis of variance.
3. Results The antisense preproorexin riboprobe derived from rat cDNA was hybridised to the sheep brain tissue, whereas
2.2. In situ hybridization Messenger RNA expression for preproorexin was determined by in situ hybridization in 20-mm coronal hypothalamic sections using the techniques described in detail elsewhere [8,9]. Briefly, sections were mounted on poly-Llysine slides and stored at 70 C before fixation (in 4% paraformaldehyde) and acetylation. Riboprobes were generated using a 347-bp cDNA for a partial rat preproorexin sequence cloned by RT-PCR [10]. Sections were hybridised overnight at 58 C using 35S-labelled antisense and sense riboprobes (1– 1.5 107 cpm/ml). Slides were treated with RNase A to remove the unhybridised probe, and then desalted with a final high stringency wash in 0.5 saline – sodium citrate (SSC) at 60 C for 30 min. The slides were airdried and apposed to autoradiographic film (Kodak BioMax MR; Amersham, UK) for 3 days. Slides from the first study were then coated with autoradiographic emulsion (Hypercoat LM-1; Amersham), dried and stored in the dark at 4 C for 6 days. They were then developed (D-19; Kodak), fixed (Unifix; Kodak) and counter-stained with toluidine blue. Serial sections spanning the hypothalamus caudally from the optic chiasm along the third ventricle were used to determine the orexin mRNA localization within the lateral hypothalamus. Quantification was undertaken on a uniform set of lateral hypothalamic sections (n = 3/sheep in the first study and n = 9/sheep in the second study) taken 0.5 mm caudal of the point where both sides of the optic chiasm meet beneath the third ventricle (as in Fig. 1B) using the Image-Pro Plus system (Media Cybernetics, MD, USA). This determined the intensity and area of the hybridization signal on the basis of set parameters. The integrated inten-
Fig. 1. Autoradiographic localization of preproorexin mRNA in coronal sections of ovine hypothalamus. A and B, hybridized to the antisense probe, are approximately 1 mm apart. C shows lack of hybridization to the sense probe in a section adjacent to B. LHA, lateral hypothalamic area; ZI, zona incerta; 3V, third ventricle; OC, optic chiasm; Fx, fornix; Mt, fasciculus mamillothalamicus. Scale bar = 2 mm.
Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
43
Fig. 2. Photomicrographs of lateral hypothalamic area in ovine brain section hybridized with the antisense preproorexin probe. A gives 5 magnification showing part of the zona incerta (scale bar = 2 mm), and the arrows denote cells shown at 40 magnification in B and C (scale bar = 10 mm).
the sense probe showed no hybridization signal. Preproorexin gene expression was localized in the lateral hypothalamic area (LHA) and zona incerta (ZI) of the ovine hypothalamus (Fig. 1). Emulsion autoradiography confirmed the localization of preproorexin mRNA to LHA neurons (Fig. 2).
Preproorexin gene expression was significantly increased by SDs ( P < 0.05), but there was no effect of food restriction nor a photoperiod food intake interaction (Fig. 3). In the second study, there was no effect of fasting or estradiol replacement on preproorexin gene expression nor a fasting estradiol interaction (Fig. 4).
Fig. 3. Preproorexin gene expression in the lateral hypothalamic area of sheep kept in long-day (LD) or short-day (SD) photoperiod and fed ad libitum (AL) or a restricted amount (R). Values are expressed as percentage of the LD/R group value (n = 2/group).
Fig. 4. Preproorexin gene expression in the lateral hypothalamic area following normal maintenance feeding (M) or 4 days of food deprivation (FD) in castrate male sheep with (E+) and without estradiol steroid replacement (E ). Values are expressed as percentage of M/E group value (n = 2/group).
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Z.A. Archer et al. / Regulatory Peptides 104 (2002) 41–45
4. Discussion Orexin gene expression is reported here for the first time in the sheep hypothalamus where it is localized in the lateral hypothalamic area and zona incerta as reported in rodents (rat [2]; Siberian hamster [10]). There was no effect of chronic food restriction on orexin gene expression in the sheep LHA unlike fasting in the rat [2,11]. However, there was an effect of photoperiod with the amounts of orexin mRNA higher in SDs than LDs. The increase in orexin gene expression was unlikely to be attributable to the reduced voluntary food intake seen in SDs [6] since imposed reductions in food intake had no effect, irrespective of photoperiod. It is also counter-intuitive that this orexigenic neuropeptide should be up-regulated during the season of reduced appetite drive. Furthermore, changes in the hypothalamic orexin gene expression do not accompany the reduced appetite drive and food intake seen during SDs in seasonal Siberian hamsters [10,12]. Rather, the SD increase in orexin gene expression in sheep may relate to reproductive activation, which is confined to the SD photoperiod in this species [6,13]. A role for orexins in regulating the reproductive neuroendocrine axis has recently been proposed since ICV infusion of orexin A or B rapidly stimulates tonic LH secretion in steroid-pretreated ovariectomized rats [14]. In addition, the central administration of orexin A to estradiol-primed ovariectomized rats restored the preovulatory LH surge in fasted animals, whereas anti-orexin A antiserum abolished the LH surge in fed rats [15]. Higher levels of orexin gene expression during the breeding season are unlikely to be caused by the seasonal increase in circulating gonadal steroids since the present sheep were steroid-clamped across both photoperiods in the first study, and the presence or absence of gonadal steroid did not affect orexin mRNA levels in the second study. However, a key interaction between sex steroids and orexins in the control of tonic GnRH/LH secretion has been suggested since orexin A-given ICV inhibits pulsatile LH in ovariectomized rats that have not been primed with steroids [16,17], yet stimulates pulsatile LH secretion in steroid-primed counterparts [14]. The lack of effect of fasting on orexin gene expression in sheep agrees with the evidence from the seasonal Siberian hamster [10,12] and mouse [18] but contrasts with the data for rats [2]. It is feasible that the lack of effect of food deprivation on orexin gene expression during December may be explained by the observation that orexin mRNA is already elevated during short days. However, food restriction also had no effect in either short or long days in study 1. Fasting acutely reduces the circulating leptin in sheep [19] as well as in rodents [20,21]. Thus, an acute reduction in the leptin signal does not seem to increase orexin gene expression, suggesting that orexin probably does not play a major role in leptin-driven responses to correct inappropriate negative energy balance in the sheep. Thus, the localization of orexin gene expression in the lateral hypothalamus of sheep is similar to that in rodents.
Levels of gene expression were apparently unaffected by chronic food restriction or fasting in this seasonal ruminant species but were influenced by photoperiod. These results lend support to the concept that appetite regulation is not the primary role of orexins and provide a basis for future studies on the role of orexins in sheep, for example, in regulating the reproductive neuroendocrine axis.
Acknowledgements This work was funded by the Scottish Executive Environment and Rural Affairs Department. Dr. Z. Archer was a recipient of a Boyd Orr Research Center studentship.
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