Detection of ghrelin, obestatin and nesfatin-1 in sheep milk: Three-way evaluation in ewe plasma, milk and lamb plasma

1. Introduction

Pregnancy and lactation involve neuroendocrine and hormonal alterations as mothers naturally adapt to these metabolically challenging conditions. Lactation not only modifies the serum hormone profile but also alters the ability of specific tissues to respond to hormones (Vernon, 1989). Energy intake and consumption management during the lactation phase are crucial for neonatal metabolism in ruminants. The initiation and maintenance of lactation in sheep, particularly with increasing day length, require the presence of many hormones (Toner et al., 2008). The balance between energy and food intake is regulated by the ghrelin, obestatin, and nesfatin-1 hormones (Stengel et al., 2010). Ghrelin is a gastric hormone that controls food consumption and energy balance (Date et al., 2002). Ghrelin has been identified in the stomachs of humans, rats, cows, pigs, dogs, and horses (Hayashida et al., 2001; Tomasetto et al., 2001). Obestatin is produced by the epsilon cells of the pancreas and plays a critical role in metabolic control. It has been proposed to function as a ghrelin antagonist (Lacquaniti et al., 2011). Obestatin levels are negatively correlated with the homeostasis model evaluation of body mass index (BMI), glucose, insulin, leptin, and insulin resistance, while exhibits a positive relationship with the insulin-sensitive index. These findings suggest a specific role for obestatin in regulating energy homeostasis. The significant correlation between plasma obestatin and ghrelin levels suggests a simultaneous release of these two hormones from a common origin (proghrelin/C-ghrelin) (Zhang et al., 2005). Nesfatin-1 has been proven to reduce food consumption in the rat hypothalamus and third ventricle following injection. Nesfatin is named after the NUCB2 (Nucleobindin-2) protein, which regulates satiety and adiposity (Shimizu et al., 2009). Multiple studies indicate that nesfatin-1 is secreted by peripheral tissues, including adipose tissue, gastric mucosa, endocrine pancreatic β-cells, testes, and the central nervous system (García-Galiano et al., 2010a; Price et al., 2007; Stengel et al., 2010). Nesfatin-1 has been associated with the regulation of body weight, glucose homeostasis, gastrointestinal processes, and the onset of puberty in animals (Atsuchi et al., 2010; Garcia-Galiano et al., 2010b; Su et al., 2010). Recent investigations have demonstrated the presence of ghrelin in humans, notably in colostrum (Aydin et al., 2006) and in regular milk (Aydin et al., 2007). Another study (Aydin, 2013) reported the presence of ghrelin and nesfatin-1 in the milk of dairy cattle. The statistics on dairy cattle may also be relevant to sheep’s milk.

This study was aimed at identifying the presence of the peptide hormones ghrelin, obestatin, and nesfatin-1 in sheep’s milk. Serum levels were analyzed to evaluate the compatibility of these peptide hormones in nursing ewes. Finally, a three-way hormone analysis was conducted in sheep, sheep’s milk, and lambs by monitoring the plasma levels of ghrelin, obestatin, and nesfatin-1 peptides in the lambs of the studied ewe. The presence of ghrelin, obestatin, and nesfatin-1 in sheep’s milk has not yet been reported. If the presence of these hormones in sheep’s milk is confirmed, it would indicate that the consumption of this milk by lambs post-birth may support appetite regulation and energy metabolism.

2. Materials and Methods

3. Results

3.1 Changes of hormone levels in postpartum sheep over 5 weeks

Plasma ghrelin levels in postpartum lambs significantly increased from the 3^rd to the 5^th week (p < 0.001; Table 2, Fig. 1). Plasma obestatin concentrations exhibited a significant increase from 3^rd to 5^th week in postpartum ewes, as did ghrelin levels (p < 0.001, Table 2, Fig. 2). At weeks three, four, and five, plasma nesfatin-1 levels steadily decreased in contrast to ghrelin and obestatin levels (p < 0.001; Table 2, Fig. 3).

Table 2. Mean plasma levels of hormones analyzed during the weekly early lactation period in sheep.

Plasma hormone concentrations in postpartum sheep by week
Analyzed hormones (n=30)	Week 3 postpartum (mean ± SEM)	Week 4 postpartum (mean ± SEM)	Week 5 postpartum (mean ± SEM)	p value	CV % (intra- assay)
Ghrelin (pg/ml)	50.94 ± 5.37	64.98 ± 6.94	103.08 ± 9.69	<0.001	3.4
Obestatin (pg/ml)	41.03 ± 4.37	59.15 ± 6.16	89.51 ± 7.72	<0.001	3.2
Nesfatin-1 (ng/ml)	4.73 ± 0.31	3.54 ± 0.30	2.56 ± 0.26	<0.001	2.5

Close

Fig. 1.

Mean plasma levels of Ghrelin analyzed during the weekly early lactation period in sheep’s plasma, milk and lambs. *denotes significant differences among treatments.

Export to PPT

Close

Fig. 2.

Mean plasma levels Ghrelin and Obestatin analyzed during the weekly early lactation period in sheep.

Export to PPT

Close

Fig. 3.

Mean plasma levels of Obestatin analyzed during the weekly early lactation period in sheep’s plasma, milk and lambs. *denotes significant differences among treatments.

Export to PPT

3.2 Changes in ghrelin, obestatin, and nesfatin-1 levels in sheep’s milk at weeks 3, 4, and 5 of lactation

Sheep’s milk demonstrated weekly rises in ghrelin and obestatin levels (p<0.001; Table 3, Figs. 1 and 3) during weeks 3, 4, and 5 of the early lactation phases. Moreover, the detection of obestatin and nesfatin-1 hormones in sheep’s milk has been documented for the first time in this study. The results indicated that obestatin and ghrelin concentrations in milk were higher in week 5 compared to weeks 3 and 4 (p < 0.001; Table 3, Fig. 2). Nonetheless, the concentrations of nesfatin-1 in the milk exhibited a statistically significant weekly reduction, in contrast to the levels of ghrelin and obestatin (p < 0.001; Table 3, Fig. 4).

Table 3. Mean plasma levels of hormones analyzed during the weekly early lactation period in lambs.

Plasma hormone concentrations in lambs by week
Analyzed hormones (n=30)	Week 3 postpartum (mean ± SEM)	Week 4 postpartum (mean ± SEM)	Week 5 postpartum (mean ± SEM)	p value	CV % (intra- assay)
Ghrelin (ng/ml)	2.51 ± 0.45	4.89 ± 0.89	5.01 ± 0.72	<0.001	1.7
Obestatin (pg/ml)	21.44 ± 0.26	29.58 ± 0.47	31.68 ± 0.56	<0.001	3.6
Nesfatin-1 (ng/ml)	1.25 ± 0.22	1.03 ± 0.18	0.89 ± 0.11	<0.001	1.2

Close

Fig. 4.

Mean plasma levels of Nesfatin-1 analyzed during the weekly early lactation period in sheep’s plasma, milk and lambs. *denotes significant differences among treatments.

Export to PPT

4. Discussion

We measured the plasma concentrations of ghrelin, obestatin, and nesfatin-1 hormones involved in energy metabolism to examine these physiological changes during weeks 3, 4, and 5 after parturition. Our results showed a statistically significant increase in plasma ghrelin and obestatin levels from week 3 onwards (p<0.001). Contrastingly, nesfatin-1, leptin, and insulin levels decreased statistically over the same period (p<0.001) in sheep. Ghrelin has two primary functions: it secretes growth hormone and stimulates the neuropeptide Y/Agouti-related peptide (NPY/AgRP) system to stimulate appetite (Kojima et al., 1999). Results indicated that the increase of ghrelin during lactation in sheep could be a stimulation of the negative energy balance caused by the excess energy expended by the mother due to increased milk yield.

Energy expenditure in terms of breastfeeding and milk production supports nutrient intake during lactation (Woodside, 2007). Moreover, NPY: Neuropeptide Y AgRP: Agouti-related peptide mRNA: Messenger ribonucleic acid expression increases, leading to hunger (Pickavance et al., 1996). Ghrelin is reported to stimulate negative energy balance in dairy cows (Bradford and Allen, 2008) and mice (Sun et al., 2008). It has been demonstrated that increased ghrelin peptide promotes negative energy balance by facilitating stomach emptying (Asakawa, 2005). It is essential to acknowledge that metabolic effects of ghrelin are influenced by age and genetic predisposition. It has been demonstrated that the ghrelin/GHS-R signaling pathway serves as a modulator of energy balance rather than a primary regulator (Sun et al., 2008). The rise of plasma ghrelin in nursing sheep observed in the present study is likely related to ghrelin’s function as a modulator in response to the energy deficit.

Obestatin was identified as an anorexigenic peptide, unlike ghrelin (Lagaud et al., 2007), although further research does not corroborate these results (Gourcerol et al., 2006; Green et al., 2007). In our previous study on exogenous acute and chronic administration (by intraperitoneal administration) of ghrelin and obestatin to mice, we found that ghrelin and obestatin worked together, and in the food-restricted mice group, ghrelin and obestatin concentrations increased in parallel (Udum et al., 2016). In this study, it was investigated whether plasma obestatin in lactating sheep is in harmony with ghrelin, and consistent results with our previous study were observed. Plasma obestatin levels of lactating ewes increased statistically significantly in parallel with ghrelin, again supporting the theory that ghrelin and obestatin work together to regulate metabolism and energy balance.

The function of nesfatin-1 in ruminant metabolism is still unclear. A recent study indicated that nesfatin-1 levels decreased in lambs who received dietary supplements and had superior body condition ratings. Nesfatin-1 concentrations were decreased in this group of animals due to heightened hunger and increased energy requirements (Barbato et al., 2021). Therefore, low levels of nesfatin-1 promote the body’s recovery of reserves, underscoring its significance as a peptide in energy metabolism. In our study, nesfatin-1 concentrations in both plasma and milk of sheep were inversely related to ghrelin and obestatin levels. As the weeks progressed, ghrelin and obestatin levels increased, while nesfatin-1 values decreased. In this case, nesfatin-1 levels decreased because the ewes needed more energy as their milk yield increased, and ghrelin and obestatin levels increased in response to the increased energy needs. We support the earlier study (Barbato et al., 2021) with our blood and milk data and showed a statistically significant decrease in nesfatin-1 values in sheep’s plasma with increasing numbers of weeks.

This is the first study to our knowledge showing the presence and levels of ghrelin, obestatin, and nesfatin-1 in sheep’s milk. Previous studies (Aydin et al., 2008, 2006) have reported the presence of obestatin and ghrelin in human milk and noted the presence of nesfatin-1 in dairy cattle (Aydin, 2013). In our investigation, concentrations of ghrelin and obestatin in sheep’s milk were consistently increased in accordance with plasma levels in sheep. Aydın et al. (2008) reported that ghrelin and obestatin levels in human milk were higher than those in plasma. However, ghrelin and obestatin concentrations in milk were lower than sheep’s plasma levels in our study. Nesfatin-1 values in sheep’s milk decreased over the weeks, like levels in sheep’s plasma, but remained lower than plasma nesfatin-1 levels. We hypothesize that ghrelin, obestatin, and nesfatin-1 peptides in sheep’s milk transfer from plasma into milk; however, we need evidence of mammary gland production of these hormones.

Aydin et al. (2008) suggest that the mammary gland might secrete these hormones, as they found higher ghrelin and obestatin levels in milk. However, in our investigation, we are unable to substantiate this assertion, as the hormone concentrations in milk were lower than those in plasma. Another study (Ilcol and Hizli, 2007)found that ghrelin levels in human milk were lower than those in maternal plasma, which is consistent with our data. Furthermore, our study focused exclusively on weeks 3, 4, and 5, during which the ewes exhibited elevated milk supply. Determining the levels of these peptides involved in energy metabolism during periods of high milk production may be significant. Nevertheless, further studies are required to determine whether cells in the mammary gland can produce these peptide hormones. Ghrelin, obestatin, and nesfatin-1 levels were measured in blood samples taken from lambs at weeks 3, 4, and 5 after birth, and we observed increased ghrelin and obestatin plasma levels in lambs while nesfatin-1 levels decreased (p<0.001). Although these results were consistent with the hormone and milk values of ewes at weeks 3, 4, and 5, the levels were lower. The plasma levels of ghrelin, obestatin, and nesfatin-1 in lambs might be transmitted from the mother’s milk to the offspring or could vary based on the energy needs of the lambs. Given the growing energy requirements of lambs, it is more logical that ghrelin and obestatin levels increase and that nesfatin-1 levels decrease. This is supported by its ability to stimulate growth hormone, considering the function that gave ghrelin its name (growth hormone releasing), and the increase in ghrelin levels by weeks is directly proportional to the growth of lambs. The discovery of ghrelin and obestatin in maternal milk confirms studies suggesting that these compounds may have a lasting impact on the control of eating behavior, metabolism, and energy balance (Savino et al., 2012).

5. Conclusions

We detected the presence of ghrelin, obestatin, and nesfatin-1 in sheep’s milk during early lactation. Concentrations of these hormones in milk were lower than those in sheep’s plasma. Nonetheless, we demonstrated that plasma ghrelin and obestatin levels in sheep rose at a rate comparable to that observed in milk. In contrast, nesfatin-1 levels decreased in parallel in both sheep’s plasma and milk. Although nesfatin-1 levels diminished concurrently in both sheep plasma and milk. Additionally, we noted that plasma levels of ghrelin, obestatin, and nesfatin-1 in lambs had similar rises and declines relative to the plasma and milk levels of their maternal sheep. Our triadic analysis study on the hormone levels of sheep’s plasma, sheep’s milk, and lamb’s plasma showed that these peptide hormones were detectable in all samples. We report that these peptides that regulate energy metabolism in ruminants collaborate, and the presence of these peptides in the mother’s milk could be transmitted to their offspring. Alternatively, the production of these peptide hormones in the offspring might be crucial for energy metabolism and growth. In our sheep study and as in our previous research in mice, we have shown that ghrelin and obestatin peptides do not work in opposition but complement each other according to energy metabolism.