Early onset of ghrelin production in a marsupial

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Abstract

Ghrelin regulates appetite in mammals and can stimulate growth hormone (GH) release from the pituitary. In rats and humans, ghrelin cells appear in the stomach during late fetal life. Nevertheless, the role of ghrelin in early mammalian development is not well understood. Marsupials deliver highly altricial young that weigh less than 1 g so they must feed and digest milk at a comparatively immature stage of development. Since they complete their growth and differentiation while in the pouch, they are accessible models in which to determine the time course of ghrelin production during development. We examined the distribution of gastric ghrelin cells, plasma ghrelin concentrations and pituitary expression of the ghrelin receptor (ghsr-1α) and GH in the tammar wallaby, Macropus eugenii. There were ghrelin immunopositive cells in the developing mesenchyme of the stomach from day 10 post partum (pp) to day 150 pp. Subsequently ghrelin protein in the fore-stomach declined and was absent by day 250 pp but remained in the gastric cells of the hind-stomach. Ghrelin was detected in the developing pancreas from day 10 pp but was absent by day 150 pp and in the adult. Pituitary ghsr-1α expression and plasma concentrations of ghrelin increased significantly up to day 70–120 pp while GH expression was also elevated, declining with GH to reach adult levels by day 180 pp. These results demonstrate an early onset of gastric ghrelin expression in the tammar in concert with a functional stomach at a relatively earlier stage than that of developmentally more mature eutherian young.

Introduction

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor 1α, GHSR-1α (Kojima et al., 1999, van der Lely et al., 2004). Ghrelin acts via a different mechanism from that of hypothalamic growth hormone-releasing hormone (GHRH) to stimulate GH release (Howard et al., 1996). The main focus in ghrelin physiology since its discovery in 1999 has been to determine the function of ghrelin, but relatively few studies have investigated the ontogeny of ghrelin secretion during development. This may be due to the fact that knockout studies in mice did not produce any measurable growth deficits (Sun et al., 2003). However, ghrelin may provide an important link in understanding how the growth axis is regulated during development.

The primary site of ghrelin synthesis and secretion in post-natal life is the stomach, specifically, the endocrine cells of the gastric epithelium that contain four major cell types, namely enterochromaffin cells that produce serotonin, enterochromaffin-like cells that produce histamine and uroguanylin, D cells that synthesize somatostatin and X/A-like cells that produce ghrelin (Date et al., 2000, Rindi et al., 2002). Ghrelin cells are scattered throughout the gastric mucosa but are more prevalent in the fundic rather than pyloric regions (Tomasetto et al., 2000, Yabuki et al., 2004). Ghrelin is also expressed at high levels in the α-cells of the pancreatic islets where it is thought to act locally on islet β-cells in the control of insulin secretion (Date et al., 2002, Wierup et al., 2002, Chanoine and Wong, 2004). However, pancreatic ghrelin expression appears to be more prevalent during fetal life in humans and rats (Rindi et al., 2004, Kojima and Kangawa, 2005).

There is an inverse relationship in the developmental regulation of gastric and pancreatic ghrelin. In the fetal rat stomach there are few ghrelin cells. These are first detected at day 18–21 of the 22-day pregnancy in the rat (Hayashida et al., 2002, Wierup and Sundler, 2005). After birth, the number of ghrelin cells rapidly increases, presumably in association with its fundamental role in appetite stimulation (Sakata et al., 2002). In the human fetal pancreas, ghrelin cells comprise 10% of islet cells from mid- to late-gestation (Wierup and Sundler, 2005). In the late fetal rat, plasma ghrelin concentrations are approximately double that of adults, while pancreatic ghrelin expression and protein levels are approximately five times that of the stomach (Chanoine and Wong, 2004). Ghrelin expression in the pancreas is down regulated after birth and the stomach becomes the main source of ghrelin (Chanoine and Wong, 2004, Wierup and Sundler, 2005). Thus, there appear to be different mechanisms regulating ghrelin physiology before and after birth in eutherian mammals.

The elevated plasma concentrations of ghrelin in the late fetal rat may stimulate GH secretion from the fetal pituitary since plasma GH is also elevated in the rat and sheep fetus at this time (Gluckman et al., 1979, Glasscock and Nicoll, 1981). Injection of ghrelin into 1- and 3-week-old rats increases plasma GH concentrations (Hayashida et al., 2002). This confirms the presence of pituitary GHSR-1α in the rat during early post-natal life, but there is no information concerning the ontogeny or expression characteristics of GHSR-1α during fetal life in any species other than human where the receptor is present in the pituitary at 18 and 31 weeks of fetal life and is sensitive to the GH releasing effects of ghrelin in culture (Shimon et al., 1998).

Marsupials are useful comparative models in which to profile gastric ghrelin because they have a very short gestation and deliver young with a mix of altricial and precocial development of their physiological and biochemical systems (Tyndale-Biscoe and Janssens, 1988). Marsupial young must consume milk for a lengthy period post-natally and from a relatively earlier developmental stage than most eutherian young (Tyndale-Biscoe and Renfree, 1987). Despite this immaturity, the pituitary is fully functional at birth in the tammar, and receives neural connections from the hypothalamus by approximately day 10–20 pp with the majority of hypothalamic nuclei fully differentiated by day 25 pp (Leatherland and Renfree, 1982, Leatherland and Renfree, 1983, Renfree, 1994, Cheng et al., 2002). Gastric ghrelin secretion may therefore be required at a relatively earlier developmental time point in marsupials to stimulate the young to suck milk. The pituitary may be receptive to the growth hormone secreting actions of ghrelin at this time, inducing the elevated plasma GH levels that are characteristic of early fetal life in eutherian mammals but occur during post-natal life in marsupials such as the possum (Trichosurus vulpecula) and bandicoot (Isoodon macrourus) (Bassett et al., 1970, Glasscock et al., 1990, Fisher, 1998, Saunders et al., 2000, Saunders et al., 2002).

The tammar wallaby has a ruminant physiology with a complex fore-stomach and hind-stomach that changes size and cellular composition dramatically throughout the 9–10 months of pouch life while it is dependent on milk. At birth, the stomach appears as a smooth, half coiled structure that develops folds in the fore-stomach region known as haustra by day 30 pp (Waite et al., 2005). This is followed by the development of a spiraling structure similar to that of the adult stomach by day 60 pp. However, there is no difference in the stomach epithelium between hind- and fore-stomach regions up to day 130 pp. After this period, and certainly by day 170 pp, the hind-stomach develops mature gastric glands typical of the fundic regions of adult marsupials with all of the endocrine cell types described previously (Langer et al., 1980, Waite et al., 2005). In contrast, the fore-stomach develops into a distinct cardia region which consists of a stratified columnar epithelium lacking parietal cells and with shallower glands relative to the hind-stomach, which retains parietal cells (Waite et al., 2005). After day 170 pp, the morphology of the tammar stomach is essentially identical to the adult. From about 200 days pp the young begins to consume grass and is weaned between approximately 300 and 350 days pp (Green and Merchant, 1988, Janssens and Messer, 1988, Menzies et al., 2007).

At the same time as these morphological changes are taking place in the stomach, the sucking pattern of the pouch young as well as the quantity and composition of the milk changes dramatically. From 0 to 120 days pp the young is permanently attached to the teat but from day 120 pp onwards the young is intermittently attached and as lactation proceeds individual sucking bouts become less frequent but more vigorous (Green et al., 1988). During the period of permanent attachment, the milk is low in fats and relatively high in carbohydrate (Green and Merchant, 1988). However, after this phase, the relative proportion of lipid increases and carbohydrate declines (Green and Merchant, 1988). More importantly, the specific protein and amino acid composition of marsupial milk changes dynamically throughout the whole of lactation in tune with the changing developmental demands of the maturing young (Nicholas, 1988).

The ability of the marsupial young to suck and digest milk from a very early stage of development provides a unique physiological system in which to profile the expression of ghrelin, which regulates appetite in adult mammals. This study describes the cloning and characterization of the GH, ghrelin and ghrelin receptor genes in a marsupial mammal, the tammar wallaby and investigates the onset of ghrelin secretion in the developing pouch young.

Section snippets

Animals

Tammar wallabies of Kangaroo Island origin were housed in outdoor grassy yards in our breeding colony. Animals were maintained on a diet of pasture supplemented with lucerne cubes and vegetables. Water was provided ad libitum. Ages of pouch young were determined by head length measurement and reference to published growth curves (Poole et al., 1991). All experiments were approved by the University of Melbourne Animal Ethics and Experimentation Committees and conformed to the Australian National

Cloning and sequence analysis of tammar GH, ghrelin and ghrelin receptor (GHSR-1α) genes

The wallaby pituitary GH mRNA was identical in length (831 bp) and structure to that described for T. vulpecula (Saunders et al., 1998). This included the 5′-untranslated region (60 bp), signal peptide (75 bp), mature protein (575 bp, including stop codon), and 3′-untranslated region (121 bp). The sequence predicts a translated mature protein of 190 amino acids similar to other vertebrate species with a high degree of sequence conservation (Table 1). The complete sequence contained structural

Discussion

Gastric and pancreatic ghrelin were present in the developing tammar from shortly after birth and at a developmental stage much earlier than that observed in eutherian mammals, suggesting that ghrelin may be an important factor in survival after birth to stimulate appetite in the sucking pouch young. During the period when pituitary GH expression was high, plasma ghrelin concentrations and pituitary expression of the ghrelin receptor GHSR-1α also reached a peak suggesting that ghrelin may be

Acknowledgements

We thank Dr. Andrew Pask and Ms. Helen Gehring for advice and assistance with molecular aspects of the study and to Dr. Danielle Hickford for assistance with the immunohistochemistry. This work was supported by a Loftus-Hills Memorial grant to BRM and a Federation Fellowship to MBR.

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