Clare L. Adam

Increased maternal nutrition alters development of the appetite-regulating network in the brain

Individuals exposed to an increased nutrient supply before birth have a high risk of becoming obese children and adults. It has been proposed that exposure of the fetus to high maternal nutrient intake results in permanent changes within the central appetite regulatory network. No studies, however, have investigated the impact of increased maternal nutrition on the appetite regulatory network in species in which this network develops before birth, as in the human. In the present study, pregnant ewes were fed a diet which provided 100% (control, n=8) or ∼160% (well‐fed, n=8) of metabolizable energy requirements. Ewes were allowed to lamb spontaneously, and lambs were sacrificed at 30 days of postnatal age. All fat depots were dissected and weighed, and expression of the appetite‐regulating neuropeptides and the leptin receptor (OBRb) were determined by in situ hybridization. Lambs of well‐fed ewes had higher glucose (Glc) concentrations during early postnatal life (F=5.93, P<0.01) and a higher relative subcutaneous (s.c.) fat mass at 30 days of age (34.9±4.7 g/kg vs. 22.8±3.3 g/kg;P<0.05). The hypothalamic expression of proopiomelanocortin was higher in lambs of well‐fed ewes (0.48±0.09 vs. 0.28±0.04, P<0.05). In lambs of over‐nourished mothers, but not in controls, the expression of OBRb was inversely related to total relative fat mass (r2=0.50, P=0.05, n=8), and the direct relationship between the expression of the central appetite inhibitor CART and fat mass was lost. The expression of neuropeptide Y and AGRP was inversely related to total relative fat mass (NPY, r2=0.28, P<0.05;agouti‐related peptide, r2=0.39, P<0.01). These findings suggest that exposure to increased nutrition before birth alters the responses of the central appetite regulatory system to signals of increased adiposity after birth.—Muhlhausler, B. S., Adam, C. L., Findlay, P. A., Duffield, J. A., and McMillen, I. C. Increased maternal nutrition alters development of the appetite‐regulating network in the brain. FASEB J. 20, E556–E565 (2006)

Early origins of obesity: programming the appetite regulatory system

There is evidence that changes in perinatal nutrition programme the development of relative fat mass and the regulation of appetite in adult life. These studies have been primarily in the rodent utilizing maternal overnutrition or undernutrition imposed at different stages of pregnancy and beyond, mapping of neuropeptide localization and activity and appropriate null mutant models. Whilst the rodent offers significant advantages in terms of a short gestation and the availability of useful transgenic and null mutant models, there are also advantages to using an animal model more akin to the human, in which all components of the ‘fat–brain axis’ are present before birth, such as the sheep. This review summarizes recent work on the expression and localization of the ‘appetite regulatory’ peptides in the fetal rodent and sheep hypothalamus and their potential role in the early programming of postnatal appetite and obesity.

Photoperiod regulates growth, puberty and hypothalamic neuropeptide and receptor gene expression in female Siberian hamsters.

In seasonal mammals, both the growth and reproductive axes are regulated by photoperiod. Female Siberian hamsters were kept, for up to 12 weeks, in long-day (LD) or short-day (SD) photoperiod, from weaning at 3 weeks of age (Exp 1). LD hamsters had characteristically faster growth and higher asymptotic body weight, adiposity, and leptin gene expression in adipose tissue. Only LD females attained puberty. Gene expression in the hypothalamic arcuate nucleus for leptin receptor (OB-Rb), POMC, and melanocortin 3-receptor (MC3-R) was higher in LD but did not change from weaning levels in SD. In contrast, gene expression in the arcuate nucleus for cocaine and amphetamine-regulated transcript (CART) was higher in SD than LD, a difference that was apparent at 2 weeks post weaning. Transfer of SD females to LD at 15 weeks post weaning (Exp 2) increased body weight, leptin signal, and gene expression for POMC but failed to induce normal puberty onset or to increase gene expression for OB-Rb and MC3-R. Therefore, ph...

Hypothalamic gene expression in sheep for cocaine- and amphetamine-regulated transcript, pro-opiomelanocortin, neuropeptide Y, agouti-related peptide and leptin receptor and responses to negative energy balance.

Hypothalamic pathways involved in the regulation of energy balance have not been widely studied in ruminants to date. Here, we used in situ hybridisation to study the gene expression of a number of leptin-sensitive receptors and neuropeptides in the ovine hypothalamus. Gene expression was first localised for cocaine- and amphetamine-regulated transcript (CART) and agouti-related peptide (AGRP). We then examined in adult male castrated sheep the effects of acute negative energy balance induced by a 4-day fast on the amounts of these mRNAs and those for leptin receptor (OB-Rb), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC). CART mRNA was localised in the arcuate nucleus (ARC), paraventricular nucleus, median eminence and ventromedial hypothalamic nucleus, and extensive co-localisation with POMC mRNA was demonstrated in the ARC. AGRP mRNA was localised in the ARC. Fasting up-regulated gene expression for OB-Rb and for the orexigenic neuropeptides NPY and AGRP in the ARC. There was a trend towards down-regulation of gene expression for the anorexigenic neuropeptide CART and no effect on POMC in the ARC, although these results are inconclusive. The presence or absence of oestradiol-containing subcutaneous implants did not influence gene expression or the effects of fasting. The hypothalamic changes were consistent with responses to the observed reduction in circulation leptin and suggest that the peripheral feedback and central mechanisms for restoring the energy balance may be largely conserved across monogastric and ruminant species.

Expression of orexin receptors in the brain and peripheral tissues of the male sheep

Orexins exert their effects through two specific receptors (OX1R and OX2R) that have been found mainly in the brain and also in peripheral tissues of rats and humans. Here, we demonstrate expression of mRNA encoding for ovine OX1R and OX2R in central and peripheral tissues of sheep. Gene expression for orexin receptors in the hypothalamus and the preoptic area was localised by in situ hybridisation. OX1R was detected in arcuate nuclei (ARC), median eminence (ME), the lateral hypothalamic nuclei and preoptic area (POA) and it was scattered along the third ventricle from the paraventricular (PVN) to the ventromedial hypothalamic nuclei (VMH). OX2R was localised in the PVN, ARC, ME, ventral VMH and a small region of the ventral POA. Gene expression for OX1R and OX2R in central and peripheral tissues was analysed using quantitative real time RT-PCR. Both orexin receptor genes were expressed in the hypothalamus, POA, hippocampus, amygdala, olfactory bulb, pineal gland and recess and pituitary gland, whereas only OX1R mRNA was detected in the testis, kidney and adrenal gland. The expression of the genes for orexin receptors in this range of ovine tissues suggests roles for orexins in multiple physiological functions, with actions at both central and peripheral levels.

Appetite regulatory neuropeptides are expressed in the sheep hypothalamus before birth

In the adult, a hypothalamic neural network acts to maintain energy balance in response to nutritional feedback from the periphery. Although there is an immediate requirement for this system to be functional at birth, it is unknown whether the components of this central neural network are expressed in the developing brain before birth. We therefore examined in the fetal sheep hypothalamus during late gestation gene expression for leptin receptor (OB‐Rb) and neuropeptides that regulate energy balance in the adult. Brains were collected from fetal sheep at 110 days (n = 12) and 140 days of gestation (n = 5) (term = 150 days) and gene expression was detected in all hypothalami using in situ hybridization with radiolabelled riboprobes for OB‐Rb, neuropeptide Y (NPY), agouti‐related peptide, pro‐opiomelanocortin and cocaine‐ and amphetamine‐regulated transcript (CART). All mRNAs were expressed in the arcuate nucleus of fetuses at both time points. Additional sites of mRNA expression were the dorsomedial hypothalamus (DMH) for NPY, the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and lateral hypothalamic area for CART, and the DMH, PVN and VMH for OB‐Rb. We have therefore demonstrated that adult‐like localization of gene expression for OB‐Rb and key appetite regulatory neuropeptides is established in the ovine hypothalamus before birth. Thus, the fetus possesses a central appetite regulatory neural network with the potential to respond to changes in nutrient supply, which could impact on energy balance regulation both before and after birth.

Impact of glucose infusion on the structural and functional characteristics of adipose tissue and on hypothalamic gene expression for appetite regulatory neuropeptides in the sheep fetus during late gestation

In the present study, our aim was to determine whether intrafetal glucose infusion increases fetal adiposity, synthesis and secretion of leptin and regulates gene expression of the ‘appetite regulatory’ neuropeptides neuropepetide Y (NPY), agouti‐related peptide (AGRP), pro‐opiomelanocortin (POMC) and cocaine‐ and amphetamine‐regulated transcript (CART) and receptors (leptin receptor (OB‐Rb) and melancortin 3 receptor (MC3R)) within the fetal hypothalamus. Glucose (50% dextrose in saline) or saline was infused (7.5 ml h−1) into fetal sheep between 130 and 140 days gestation (term = 150 ± 3 days gestation). Glucose infusion increased circulating glucose and insulin concentrations, mean lipid locule size (532.8 ± 3.3 μm2versus 456.7 ± 14.8 μm2) and total unilocular fat mass (11.7 ± 0.6 g versus 8.9 ± 0.6 g) of the perirenal fat depot. The expression of OB‐Rb mRNA was higher in the ventromedial nucleus compared to the arcuate nucleus of the hypothalamus in both glucose and saline infused fetuses (F= 8.04; P < 0.01) and there was a positive correlation between expression of OB‐Rb and MC3R mRNA in the arcuate nucleus (r= 0.81; P < 0.005). Glucose infusion increased mRNA expression for POMC, but not for the anorectic neuropeptide CART, or the orexigenic neuropeptides NPY and AGRP, in the arcuate nucleus of the fetal hypothalamus. These findings demonstrate that increased circulating glucose and insulin regulate gene expression of the neuropeptides within the fetal hypothalamus that are part of the neural network regulating energy balance in adult life.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep

BackgroundThe gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA).MethodsAntibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fishers least significant difference.ResultsIn adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01).ConclusionEvidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.

Photoperiod influences the central effects of ghrelin on food intake, GH and LH secretion in sheep

Ghrelin is a circulating peptide, primarily secreted by the gut, that has reported actions within the hypothalamo-pituitary axis to stimulate food intake, inhibit GnRH/LH secretion and stimulate GH secretion in monogastric species. Here, we examine responses to centrally administered ghrelin in a seasonal ruminant. Estradiol-implanted castrated male sheep with indwelling intracerebroventricular (i.c.v.) cannulae were kept with unrestricted food for 16 weeks in long day photoperiod (LD, 16 h light/day) then 16 weeks in short days (SD, 8 h light/day). In week 16 of each photoperiod they were given a control (saline) i.c.v. injection on day 1 and ghrelin i.c.v. injection on day 2. Mean circulating endogenous plasma ghrelin concentrations showed no diurnal pattern and were similar between the photoperiods. Central ghrelin injection increased voluntary food intake 2-fold in the first hour after administration in LD but not in SD, decreased LH pulse frequency and amplitude in SD but not in LD, and stimulated GH release in both photoperiods, although there was a 1.5-fold larger response in LD. Therefore, central injection of ghrelin to sheep acutely stimulated food intake in LD, suppressed reproductive neuroendocrine output in SD, and stimulated GH secretion irrespective of photoperiod, although more pronounced in LD. These data indicate that photoperiod can influence hypothalamic appetite and reproductive neuroendocrine responses to ghrelin in seasonal species.

Appetite regulation and seasonality: implications for obesity.

High circulating concentrations of leptin in obesity are associated with an apparent loss of its characteristic anorexic action within the hypothalamic region of the brain. Central insensitivity to leptin may therefore contribute to the aetiology of this disease, and an increased understanding of the underlying mechanisms will identify potential means of prevention and/or therapeutic targets. Seasonal animals such as sheep and Siberian hamsters (Phodopus sungorus) exhibit annual photoperiod-driven cycles of appetite and body weight. Increased food intake and weight gain in long days (summer) are associated with high circulating leptin, and decreased intake and weight loss in short days (winter) with low leptin. Critically, these cycles are associated with reversible changes in sensitivity to leptin. High sensitivity is seen in short days and relative insensitivity in long days, demonstrated both in sheep given leptin centrally via intracerebroventricular cannulas and in hamsters given leptin peripherally. In addition, primary hypothalamic appetite-regulating targets for leptin (i.e. neuropeptide Y, melanocortin and cocaine- and amphetamine-regulated transcript pathways) respond differently in these species to changes in circulating leptin and nutritional status induced by photoperiod as opposed to such changes induced by food restriction. Studies of seasonal animals will help to resolve causes of altered sensitivity to leptin and whether these changes reflect altered transport into the brain and/or altered signalling at the receptor or post-receptor level. Increased knowledge of the mechanism(s) and time-course for development and reversal of reduced central leptin sensitivity will provide new insights into the development and control of obesity.