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Immunohistochemical and quantitative analysis of ghrelin in Syzygium aromaticum
Suleyman Aydin*1, Adile F Dagli†, Yusuf Ozkan‡, Yalcin Kendir*, İbrahim Sahin§, Aziz Aksoy║ and İbrahim H Ozercan†
*Department of Medical Biochemistry and Clinical Biochemistry Firat Hormones Research group, Firat University Hospital, Elazig, Turkey, †Department of Pathology, Firat University Hospital, Elazig, Turkey, ‡Deparment of Endocrinology and Metabolism, Firat University Hospital, Elazig, Turkey, §Department of Nutrition and Dietetic, Erzincan University, Erzincan, Turkey, and ║Department of Nutrition and Dietetic, Bitlis Eren University, Bitlis, Turkey
Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, has been identified in mammals, fish, amphibians, birds, reptiles and some plants. The present investigation was designed to determine whether ghrelin is present in the appetite-stimulating plants Syzygium aromaticum and Salvadora persica, using IHC (immunohistochemistry) to indicate the location of the peptide and ELISA to measure the concentration. ELISA demonstrated that a ghrelin-like substance was present at concentrations of 4070.75±664.67 and 75.25±24.49 pg/mg in the tissues of flower bud of S. aromaticum and branch of S. persica, respectively. The concentration of ghrelin in human salivary gland tissue was 436.00±95.83 pg/mg. Ghrelin was predominantly localized to the T (trachea) and PCs (parenchyma cells) in the flower bud of S. aromaticum. However, no ghrelin immunoreactivity was observed in the PC or T of the branch of S. persica. The evolutionary role of this peptide hormone in plants and animals suggests that they have evolved in a more similar way than previously thought.
Key words: ghrelin, immunohistochemistry, plant, Syzygium aromaticum
Abbreviations: AEC, 3-amino-9-ethyl carbazole, CVs, coefficients of variation, PCs, parenchyma cells, proANF, prohormone of atrial natriuretic factor, T, trachea
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Peptides, amino acids linked by peptide bonds with molecular weights of up to 10 kDa, are abundant in living organisms (Lodish et al., 2008). Not only are peptides present in animals and microorganisms where they carry out multiple functions, but they have also been identified in plants. For example, systemin, which is present in the leaves of tomato plants, was the first plant hormone identified as a peptide. It is an 18-amino acid peptide processed from the C-terminus of a 200-amino acid precursor called prosystemin (McGurl et al., 1992). Atrial natriuretic-like peptides have also been identified in the plant kingdom (Vesely and Giordano, 1991; Vesely et al., 1993). The 126-amino acid proANF (prohormone of atrial natriuretic factor), proANF-(1–30), proANF-(31–67) and ANF-like peptides are present in the roots, stems and leaves of a wide variety of Embryophyta and serve to increase the flow of solutes and/or water to the leaves and flowers of plants (Vesely and Giordano, 1991).
Ghrelin, characterized from extracts of rat stomach by Kojima and his co-workers in 1999, is a 28-amino acid peptide hormone in which the third amino acid at the N-terminal (normally serine but in some species threonine) is modified by a fatty acid. This modification is essential for ghrelin to function, and this is the first known example of a bioactive peptide modified by fatty acids (Kojima et al., 1999). Ghrelin is predominantly responsible for growth hormone release and the modulation of energy expenditure; its levels increase during hunger and decrease immediately postprandially (Kojima and Kangawa, 2005, review; Aydin, 2007). Administration of ghrelin to rats and humans increases appetite (Yukawa et al., 2008).
Ghrelin has been identified in several mammals including American bison, cats, cows, dogs, goats, humans, Mongolian gerbils, moose, pigs, pronghorns, pygmy sperm whales, rats, reindeer, rhesus monkeys, sheep, wapiti, water buffalo and white-tailed deer, and the amino acid sequences are well conserved. It has also been identified in birds (chicken, duck, emu, goose, Japanese quail and turkey) and fish (rainbow trout 1, rainbow trout 2, European sea bass, Black Sea bream, channel catfish, goldfish, Japanese eel, tilapia, zebrafish and shark), and fish ghrelin varies in terms of amino acid length and specific acyl modifications (Litwack, 2008). Evidence that ghrelin (appetite hormone) was present in plants came from radioimmunoassays and IHC (immunohistochemistry) carried out on Prunus x domestica L (common plum) and Morus alba (mulberry) (Aydin et al., 2006).
Ghrelin has a highly conserved amino acid sequence (particularly at the N-terminal region) across all animal species investigated and is an excellent example of a biopeptide with diverse functions. Given that it is ubiquitous in the animals and plants studied so far, the aim of the present study was to determine whether appetite-stimulating plants, namely Syzygium aromaticum (cloves) from the Myrtaceae family (Chaieb et al., 2007) and Salvadora persica (meswak: arak tree: toothbrush tree) from the Salvadoraceae family (Ronse De Craene and Wanntorp, 2009), also expressed this multifunctional hormone. Various tissues from both plants were investigated using highly sensitive IHC and ELISA.
2.1. Kits and reagents
Standard (pure) ghrelin and associated reagents for ghrelin assays were obtained from Phoenix Europe. All other reagents were purchased from Lab Vision Corporation (Cat: TA-125-HAS). Ghrelin-like immunoreactivity was measured using a specific (Cat. no. EZGRT-89K, Upstate Chemicon Linco, Millipore) and sensitive ELISA as previously described (Aydin et al., 2009), using an ELEXUS 800 ELISA plate reader.
2.2. Preparation of homogenates
Homogenates of flower bud of S. aromaticum and branch of S. persica were prepared by finely slicing 100 mg of tissue and then crushing it with an iron mold. Crushed samples were homogenized in PBS (5%, w/v) using a stainless steel mortar. Homogenized samples were centrifuged at 10000 rev./min for 20 min at 4°C, and the supernatant was stored at −20°C until required.
IHC was carried out using the ABC (streptavidin–avidin–biotin–peroxidase complex) procedure with minor modifications (Hsu et al., 1981; Aydin et al., 2006). Sections (4 μm) were mounted on silanized slices, and the solutions utilized were freshly prepared before staining. The chromogen solution [AEC (3-amino-9-ethyl carbazole)] was used within 15 s of being prepared. The IHC method was as follows: 4 μm sections from paraffin-embedded tissue samples were transferred to polylysine microscope slides and put in the drying oven (+80°C) for 20 min. The slides were processed through a pure xylol series four times and left in each solution for 5 min. They were then processed through an ethanol series (99.5, 96, 90, 80 and 70%) five times and left in each solution for 3 min. The slides were placed in distilled water for 10 min and then placed in 3% H2O2 diluted with methanol.
For staining, the samples were incubated with 10% citrate buffer, pH 6.0, for 15 min in a microwave oven (750 W) and left to cool for 20 min at room temperature before being placed in PBS (0.01 M, pH 7.4) for 5 min. They were incubated with horseradish peroxidase for 10 min to prevent non-specific antibody binding and then with rabbit anti-ghrelin (human) antibody® (generated against glycine-serine-Ser (octanoyl)-phenylalanine-leucine-serine-proline-glutamic acid-histidine-glutamine-arginine-valine-glutamine-glutamine-arginine-lysine-glutamic acid-serine-lysine-lysine-proline-proline-alanine-lysine-leucine-glutamine-proline-arginine) at 38°C for 30 min. The slides were washed with PBS for 5 min, incubated with biotinylated goat anti-polyvalent (Lab Vision Corporation) for 15 min in a water bath (+38°C) and washed again in PBS for 5 min. They were incubated with streptavidin peroxidase for 10 min in a water bath (+38°C), washed with PBS for 5 min, incubated with AEC for 10 min in a water bath (+38°C) and washed with distilled water for 1–2 min. They were subjected to contrast staining with Mayer haematoxylin for 1–2 min, washed in distilled water for 1–3 min and dried. The slides were covered with lamellae to obtain permanent preparations and examined under a light microscope and photographed. Salivary gland tissue, which is known to express ghrelin (Aydin et al., 2005), was used as a positive control. For preparing negative tissue samples, PBS was used instead of primary antibody (ghrelin tissue antibody).
2.4. Assessing the presence of ghrelin in plant tissues
Ghrelin immunoreactivity in plant tissues was investigated using a human ghrelin ELISA Kit (Cat. no. EZGRT-89K, Upstate Chemicon Linco, Millipore) according to the manufacturer's protocol. PBS was used as a zero control. The lower detection threshold (determined by interpolating the mean optical density plus 2 S.D. for 3 sets of duplicates at the 0 pg/mg tissue standard) was 100 pg/mg tissues. Within-assay CVs (coefficients of variation) at low (446.8 pg/mg tissues) and high (3999.62 pg/mg tissues) concentrations were 5.9 and 1.9%, respectively; between-assay CVs at low (538.14 pg/mg tissues) and high (4330.41 pg/mg tissues) concentrations were 11.2 and 3.7%, respectively. The linearity of dilution was assessed by serial dilutions (1:2, 1:4, 1:8 and 1:16). The observed values ranged from 79.4 to 110.4% of the expected values, with a mean of 96.8%.
2.5. Statistical analysis
Data obtained in this investigation are presented as means±SD. Statistical analysis of concentration differences between the respective plant tissues and between plant and animal tissues were evaluated by repeated ANOVA (analysis of variance). A probability value of less than 0.05 was considered statistically significant.
3. Results and discussion
ELISA (quantitative analysis) and a highly specific immunohistochemical analysis that utilized a human anti-ghrelin antibody demonstrated that ghrelin-like peptides were present in the flower bud of S. aromaticum but not in the tissues of the branch of S. persica. S. aromaticum contains ghrelin at concentrations higher than those in human salivary gland (Figure 1), rat kidney, human kidney, human stomach, P. x domestica L (common plum) and Morus alba (mulberry) (Aydin et al., 2006). From the calculated molecular mass of 3244.6 for the 28-amino acid sequence of the peptide, these concentrations are significantly higher than the average ghrelin levels (150 fmol) found in the plasma of fasting humans (reviewed, Aydin, 2007). Similarly, it has previously been shown that the leaves and stems of Florida beauty contain concentrations of ANP, proANP-(1–98) and proANP-(31–67) higher than in rat ventricles but lower than in rat atria (Vesely and Giordano, 1991). Atrial natriuretic peptides are present in Bryophyta and in Euglena, a single-cell, flagellated, chlorophyll-containing plant without leaves, stems or roots (Vesely et al., 1993). The high levels of ghrelin identified in this study are significantly different from those described by Aydin et al. (2006) in their assessment of the levels of ghrelin in plants, but it may be that P. x domestica L (common plum) and M. alba (mulberry) plants contain less ghrelin than S. aromaticum. In this study, the ghrelin-like substance present in S. aromaticum was measured using Linco's ghrelin, whereas previous studies have utilized Phoenix Pharmaceuticals kits to analyse the ghrelin-like substance present in P. x domestica L (common plum) and M. alba (mulberry). There is evidence in the literature to support the use of the Linco assay over the Phoenix assay, as it has been shown to be more sensitive (Groschl et al., 2004). Therefore, it is likely that the levels of ghrelin identified in S. aromaticum in this study were considerably lower than those measured by Aydin et al. (2006). However, different plants could contain different amounts of ghrelin-like substances, and the differing results may not be due to differences between the assay utilized in this study and the one utilized by Aydin et al. (2006). Another possible reason why more ghrelin was identified in S. aromaticum could be that the peptide is readily digested by proteases. In order to avoid protease digestion, aprotinin (protease inhibitor) was added to the plant supernatants in this study. However, we are not aware of any peptides in plants that are digestable by endogenous proteases at this time.
Ghrelin expression in salivary gland was used as a positive control, and immunoreactivity was observed in the excretory duct and striated duct (Figure 2B; red colour). Ghrelin is predominantly localized in PCs (parenchyma cells) of S. aromaticum (Figures 3B and 3D), which have functions including storage, secretion and tissue repair in plants, but not found in the T (trachea) and PCs (parenchymal cells) of S. persica using ELISA or the highly specific immunohistochemical analysis (Figures 4A, 4B, 4C, 4D). Therefore, ghrelin hormone is absent, or the gene is inactive in this plant, and it is likely that ghrelin has no effect on the physiology of S. persica. However, these results should be interpreted with caution as the S. persica used here was dry, and the ghrelin could have been degraded. S. persica does not grow in Turkey, and therefore, ghrelin levels in fresh branches of S. persica could not be assessed. We tentatively conclude that the observation of ghrelin activity in PCs is related to plant growth regulation through interaction with one or more plant growth hormones, particularly auxins (Garcia-Luis et al., 2002), which are predominantly transported in a stream of fluid in phloem vessels and in the T. The localization of ghrelin within the parenchyma is consistent with a role in growth and the transport of water and salt(s) within plants. Furthermore, this localization is ideal for nutrient uptake, an expected function of a hormone of this kind.
In conclusion, an amino acid sequence highly conserved (>90%) across species indicates an evolutionary role for the ghrelin hormone similar to those of atrial natriuretic peptides (Vesely et al., 1993) and melatonin (Murch et al., 2009), which have been demonstrated in animals and also in the common plum, mulberry and S. aromaticum plants using ELISA. Although the physiological relevance of this finding is as yet unclear, it strongly suggests that that plants and animals evolved in a more similar manner than previously thought and that ghrelin has essential biological functions. In humans and animals, ghrelin is predominantly produced in the stomach, and its main roles concern the regulation of energy balance, appetite stimulation and growth hormone release. When ingested, plant ghrelin can affect the GIT (gastrointestinal tract) structures, appetite stimulation, energy balance and growth hormone release in the same way that endogenous ghrelin does (Kotunia and Zabielski, 2006).
Suleyman Aydin was involved in the conception, design and manuscript writing. Ferda Dagli, Yalcin Kendir, İbrahim Sahin and Aziz Aksoy were involved in the collection and assembly of data. Suleyman Aydin, Adile F Dagli, Yusuf Ozkan, İbrahim Ozercan were responsible for the data analysis and interpretation. Final approval of manuscript was the responsibility of Suleyman Aydin, Adile Dagli, Yusuf Ozkan, Yalcin Kendir, İbrahim Sahin, Aziz Aksoy and İbrahim Ozercan.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
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Received 28 July 2010/30 August 2010; accepted 29 October 2010
Published as Cell Biology International Immediate Publication 29 October 2010, doi:10.1042/CBI20100565
© The Author(s) Journal compilation © 2011 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 1 Quantitative analysis of ghrelin (pg/mg tissue) in human salivary gland (SG), branch of S. persica (SP) and flower bud of S. aromaticum (SA)