A Role for Agouti-Related Protein in Appetite Regulation in a Species with Continuous Nutrient Delivery

Abstract

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Selected hormonal and neurotransmitter mechanisms regulating feed intake in sheep

“…Upregulation of AgRP mRNA was observed in chronically underfed ewes Archer et al, 2004 andRelling et al, 2010) or castrate male sheep fasted for 4 days . In addition, dual label immunohistochemistry (for AgRP and c-Fos) indicated that fasting for 3 days increased the number of AgRPpositive neurons and the activation of AgRP neurons in the ARC (percentage of AgRP neurons also expressing c-Fos; Wagner et al, 2004). Gene expression for AgRP was also increased in sheep with a low body condition score compared to sheep with a high body condition score (Archer et al, 2004).…”

“…Agouti-related protein AgRP mRNA Adam et al, 2002;Archer et al, 2002a) and AgRP protein (Wagner et al, 2004) were localized to the ARC nucleus in sheep. Cloning of a partial sequence of AgRP mRNA indicated a high homology with bovine AgRP but the comparison of the human message indicated only about 56% homology for the portion cloned (Wagner et al, 2004).…”

“…Cloning of a partial sequence of AgRP mRNA indicated a high homology with bovine AgRP but the comparison of the human message indicated only about 56% homology for the portion cloned (Wagner et al, 2004). Upregulation of AgRP mRNA was observed in chronically underfed ewes Archer et al, 2004 andRelling et al, 2010) , 2005).…”

“…Indeed, intracerebroventricular (ICV) injection of NPY in sheep resulted in a pronounced increase in feed intake (Miner et al, 1989;Sartin et al, 2001;Wagner et al, 2004;Whitlock et al, 2005). An interesting study of the NPY receptor types mediating feed intake indicated that the major receptor mediating the increased intake due to NPY was the NPY-Y1 receptor, and to a lesser degree, the Y2 receptor.…”

“…More importantly, feed intake remained at normal levels even after the removal of NPY. Therefore, NPY has been characterized as a neurotransmitter of the appetite system that regulated increased feed intake and is in turn regulated by nutritional as well as photoperiod manipulations in sheep.Agouti-related protein AgRP mRNA Adam et al, 2002;Archer et al, 2002a) and AgRP protein (Wagner et al, 2004) were localized to the ARC nucleus in sheep. Cloning of a partial sequence of AgRP mRNA indicated a high homology with bovine AgRP but the comparison of the human message indicated only about 56% homology for the portion cloned (Wagner et al, 2004).…”