Teses / dissertações sobre o tema "Ghrelin"

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Ghrelin is a 28 amino-acid peptide produced predominantly by the stomach. Two main isoforms of ghrelin are currently known (octanoyl- and desoctanoyl ghrelin). It functions as a circulating orexigenic hormone In addition, it has an effect on the nervous, cardiovascular and immune system. Current data suggest that ghrelin may have beneficial anti-inflammatory effects. Chapter 3 in this thesis primarily examines the relationship between ghrelin and inflammation in Crohn’s disease (CD). Modulation of inflammation with infliximab, a powerful anti-TNFα antibody therapy, can increase total ghrelin concentration by 25%. In addition, a normal physiological post-prandial decrease in ghrelin following a meal is restored when infused with infliximab, suggesting a dysregulation of ghrelin in CD patients with active inflammation. At cellular level, there is evidence that ghrelin may have an immunosuppressive effect on activated T-lymphocytes. Chapter 4 of this thesis examines the effect of ghrelin, a manufactured agonist and des-octanoyl ghrelin on NFκB activation on a human Blymphocyte cell line. This study demonstrated that exposure to octanoyl ghrelin confers an initial increase of NFκB activation in inactivated cells of up to 50% which suggests a pro-inflammatory effect. However, NFκB activation appears to decrease at much higher concentrations of octanoyl ghrelin, which may indicate toxicity at supra-physiological levels. Ghrelin is also involved in the regulation of gastric motility and has structural similarities to motilin. Symptoms of delayed gastric emptying can occur long after cancer chemotherapy has ended. Chapter 5 of this thesis compares the contractility and pro-motility neurotransmitter expression in chemotherapy and non-chemotherapy exposed stomach tissues obtained from patients undergoing surgery for oesophagogastric cancers. Chemotherapy exposed tissues have reduced contractility to carbachol and apparent destruction of the cholinergic activity. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. In conclusion, ghrelin can be altered by inflammation and may have beneficial effects on gastric motility.

Ghrelin is a hormone that regulates energy homeostasis and is expressed in the stomach, hypothalamus and pituitary of humans and rodents. Ghrelin circulates as acylated (AG) and unacylated (UAG) ghrelin. Acylation is mediated by ghrelin O-acyltransferase (GOAT) and both forms of ghrelin are degraded by plasma proteases. Maintenance of euglycemia is important during pregnancy, where transplacental transfer of glucose is required for optimal fetal outcome. Maternal malnutrition is associated with decreased substrate availability and is a risk for hypoglycemia. AG prevents hypoglycemia in calorie-restricted (CR) mice by stimulating growth hormone (GH) release however its role in pregnancy remains unknown. I hypothesize that CR causes lower blood glucose in pregnant (P) compared to non-pregnant (NP) mice and that the GOAT-ghrelin axis is part of the response to this hypoglycemia. To determine ghrelin’s role in glucose homeostasis during pregnancy, wild-type (WT) and GOAT-KO (KO) mice were time-mated and fed-freely (FF) or CR by 50% for one week beginning at day 10.5 after conception. KO mice showed reduced fertility as conception was rare compared to WT animals. Unexpectedly, KO-CR mice also showed pregnancy termination early after CR was started and failed to survive to day 18 (=sacrifice), suggesting that AG plays a critical role in maintaining energy homeostasis during pregnancy. To further investigate the effect of ghrelin during pregnancy, several parameters of energy metabolism were analyzed: body composition, blood glucose, plasma AG, UAG, GH, GOAT/ghrelin expression, hepatic glycogen and PCK1 expression. I demonstrate that CR affects glucose metabolism more severely in WT-P and KO-NP mice (which cannot produce AG) compared to WT-NP mice. The additive effect of pregnancy and CR in GOAT-KO mice increases the severity of hypoglycemia. I propose that in WT animals, an increase in AG and UAG levels, whether due to increased production, decreased degradation, or both, serves to mitigate the decrease in blood glucose. The mechanisms of action of AG remain unclear but may involve stimulation of glycogenolysis and/or gluconeogenesis directly or indirectly (via stimulation of GH). My work supports a physiological role for the AG/UAG pathway in the regulation of blood glucose concentrations during pregnancy.

This project characterised the bone microarchitecture of adult mice lacking the hormone, acyl ghrelin, by high resolution micro-computed tomography; and investigated the expression of the ghrelin axis in cells of human and mouse fetal cartilage. This thesis highlights for the first time the physiological role of the ghrelin axis in the bone microenvironment of aged mice. Furthermore it improves our understanding of the complex expression patterns of the ghrelin axis in cartilage cells of human and mouse fetal skeletons.

Acylated and unacylated ghrelin (AG and UnAG) are circulating peptide hormones generated by the ghrelin gene mainly in the stomach in consequence of fasting or caloric restriction. AG, through binding to growth hormone secretagogue receptor type 1a (GHSR1a), induces strong release of growth hormone (GH) and other central and peripheral activities. UnAG is not able to bind and activate GHSR1a but features several biological functions, such as improvement of insulin sensitivity in skeletal muscle and enhancement of skeletal muscle regeneration induced by hindlimb ischemia. Moreover, it has been demonstrated that both peptides share common activities, mediated by an unknown receptor, on skeletal muscle where they protect against atrophy and promote proliferation and fusion of myoblast. Giving the multiple activities of this hormone on skeletal muscle, we assessed involvement of ghrelin in muscle regeneration, cachexia and sarcopenia. We showed that UnAG enhances SCs function and stimulates Par polarity complex/P38-mediated asymmetric division, sustaining both SCs self-renewal and myoblast differentiation. We also showed that upregulation of plasmatic levels of UnAG results in an improvement of pathologic phenotype and in the ablation of self-renewal defect of dystrophin-null SCs with a consequent preservation of SCs pool in the later stage of the pathology. Moreover, we show that ghrelin analogs recognize the alternative receptor and protect against cytokines-induced skeletal muscle atrophy. We demonstrate that anamorelin protects against fasting-induced adipose tissue loss without inhibiting muscle wasting in WT mice. Finally, preliminary data show that upregulation of UnAG improves muscle functionality and slightly protects against muscle atrophy and intramyocellular fat deposition. Surprisingly, deletion of ghrelin gene differently affects muscle function, weight, food intake and glucose metabolism, indicating an important role of ghrelin during the aging process.

Abstract The increasing prevalence of metabolic syndrome and the consequent cardiovascular diseases, including atherosclerotic diseases and type 2 diabetes, are a cause of public concern worldwide. This development has stimulated an active search for novel risk factors and new candidate genes. The hormones regulating energy balance and the polymorphisms associated with them are of special interest as potential risk factors for metabolic syndrome. Ghrelin is a novel peptide hormone from stomach with strong growth hormone releasing activity. It is also able to modify glucose and insulin metabolism, blood pressure levels, cardiac function, adipogenesis and inflammatory processes in experimental conditions. Whether ghrelin and ghrelin gene variations have a role in the development of metabolic syndrome and the associated diseases, is not known. In the present study, the associations between fasting plasma ghrelin concentrations, ghrelin gene mutations (Arg51Gln and Leu72Met), features of metabolic syndrome, type 2 diabetes and carotid artery atherosclerosis were analysed. In addition, the relationship between ghrelin and insulin-like growth factor I (IGF-I) concentrations was studied. The study population consisted of 1045 middle-aged subjects of the hypertensive and the control cohorts of the OPERA project from the City of Oulu, Finland. Low ghrelin concentrations were found to be associated with several components of metabolic syndrome: adiposity, low HDL cholesterol levels, high insulin concentrations and high blood pressure levels. The prevalence of insulin resistance and type 2 diabetes was increased amongst the subjects with low ghrelin concentrations. Out of the individual factors tested, IGF-I concentration was the most significant predictor of ghrelin concentrations. This negative association was strongest in the subjects with insulin resistance and type 2 diabetes, which suggests that changes in ghrelin/IGF-I interactions might be involved in the development of these conditions. The subjects with the Gln51 allele of the ghrelin gene had lower ghrelin concentrations and, consistent with the findings mentioned above, higher prevalence of type 2 diabetes and hypertension compared with the subjects homozygous for the Arg51 allele. No correlation between ghrelin and C-reactive protein concentrations was seen. However, there was a positive association between ghrelin concentrations and carotid artery intima-media thickness. This association was independent of the commonly recognised risk factors of atherosclerosis and was only seen in men, who also had more advanced atherosclerosis than women. These observations call for further studies to clarify the potential causative role of ghrelin in the etiology of metabolic syndrome, type 2 diabetes and atherosclerotic cardiovascular diseases
Tiivistelmä Metaboliseen oireyhtymään liittyy kohonnut riski sairastua sydän- ja verisuonisairauksiin kuten tyypin 2 diabetekseen ja sepelvaltimotautiin. Metabolisen oireyhtymän nopea esiintyvyyden kasvu on johtanut aktiiviseen uusien riskitekijöiden etsintään. Erityisen kiinnostuksen kohteena ovat olleet energia-aineenvaihduntaa säätelevät hormonit ja niihin liittyvät polymorfiat. Greliini on ensisijaisesti vatsalaukusta erittyvä hormoni, joka lisää voimakkaasti kasvuhormonin eritystä. Koeolosuhteissa sillä on myös vaikutuksia sokeriaineenvaihduntaan, verenpaineeseen, sydämen toimintaan, rasvakudoksen kehittymiseen ja tulehduksellisiin tapahtumiin, minkä perusteella on syytä epäillä greliinillä olevan osuutta metabolisen oireyhtymän ja siihen liittyvien sairauksien synnyssä. Tässä tutkimuksessa selviteltiin greliinin paastoplasmapitoisuuksien ja greliinipolymorfioiden (Arg51Gln ja Leu72Met) yhteyksiä metabolisen oireyhtymän piirteisiin, tyypin 2 diabetekseen ja kaulavaltimoiden ateroskleroosiin. Lisäksi tutkittiin greliinin ja insuliinin kaltaisen kasvutekijän (IGF-I) pitoisuuksien yhteyksiä. Tutkimusväestö koostui 1045 oululaisesta keski-ikäisestä OPERA tutkimukseen kuuluvasta henkilöstä. Tutkimuksessa matalien greliinipitoisuuksien havaittiin olevan yhteydessä useisiin metabolisen oireyhtymän piirteisiin: lihavuuteen, alhaisiin HDL kolesterolin pitoisuuksiin, korkeisiin insuliinipitoisuuksiin ja kohonneeseen verenpaineeseen. Matala greliinipitoisuus yhdistyi myös tyypin 2 diabeteksen ja verenpainetaudin esiintyvyyteen. Tutkituista tekijöistä IGF-I -pitoisuudet selittivät parhaiten greliinipitoisuuksia. Tämä käänteinen yhteys oli erityisen vahva tyypin 2 diabeetikoilla ja insuliiniresistenteillä henkilöillä viitaten greliinin ja IGF-I:n mahdollisen vuorovaikutukseen liittyvän näiden tilojen kehittymiseen. Lisäksi havaittiin, että greliinigeenin Gln51-alleelia kantavien henkilöiden greliinipitoisuudet olivat alhaiset, ja että he sairastivat enemmän diabetesta ja verenpainetautia kuin henkilöt jotka olivat homotsygootteja Arg51-alleelin suhteen. Greliinipitoisuudet ja C-reaktiivisen proteiinin pitoisuudet eivät korreloineet keskenään. Kaulavaltimon seinämäpaksuus korreloi positiivisesti greliinipitoisuuksien kanssa miehillä riippumatta perinteisistä ateroskleroosin riskitekijöistä. Tutkimustulokset tukevat olettamusta, että greliinillä saattaa olla merkitystä metabolisen oireyhtymän, tyypin 2 diabeteksen ja ateroskleroosin kehittymisessä. Jatkotutkimukset ovat tarpeen tämän yhteyden osoittamiseksi

Abstract Atherosclerosis is the major cause of cardiovascular diseases and the leading cause of death globally. Atherosclerosis is a complex, chronic disease characterized by lipid accumulation and inflammation within the intima layer of vessel wall. Novel biomarkers and therapeutics are still being sought to provide both better diagnosis and treatment. Ghrelin represents an attractive target for studies into atherosclerosis. Ghrelin is a gastric peptide hormone, which has multiple functions, including regulation of appetite and energy metabolism. Emerging evidence suggests that it may also have a role in the cardiovascular and immune systems. The aim of the present study was to explore the role of ghrelin in atherosclerosis. The specific aims were 1) to investigate the association between the plasma ghrelin level and early atherosclerosis as determined by carotid artery intima media thickness (IMT) in a large (n = 1024) cross-sectional population-based study of middle-aged subjects, 2) to measure the associations between plasma ghrelin levels and already established risk factors of atherosclerosis in human subjects, 3) to assess the effects of ghrelin on atherogenesis in vitro by analyzing monocyte adhesion to endothelial cells, oxidized low density lipoprotein (LDL) binding and acetylated LDL uptake using macrophages, and 4) to study the influence of ghrelin on atherosclerosis using ghrelin vaccination in a mouse model of atherosclerosis. Plasma total ghrelin levels were positively associated with carotid IMT in male subjects. Association studies demonstrated plasma ghrelin levels to be negatively associated with total and LDL cholesterol, and triglyceride concentrations as well as with body mass index (BMI), and positively assocated with high density lipoprotein (HDL) cholesterol concentration in postmenopausal women and in a population-based study. In addition, estrogen increased plasma acylated ghrelin levels in postmenopausal women. Cell culture studies demonstrated that ghrelin could increase the binding of oxidized LDL and monocytes to endothelial cells. Interestingly, when endothelial cells were stimulated with tumor necrosis factor α (TNFα), then ghrelin prevented monocyte adhesion. The study with LDL receptor knockout mice, revealed that ghrelin vaccination could increase plasma ghrelin levels but had no effects on the development of atherosclerosis. However, the plasma MCP-1 level decreased in mice immunized with ghrelin vaccine. In conclusion, these studies suggest that ghrelin has modulatory functions in the vascular system and atherogenesis though the effect may not be as dominant as that of the known traditional risk factors. Whether this effect of ghrelin is positive or negative in atherogenesis will be clarified in further studies
Tiivistelmä Sydän- ja verisuonitaudit ovat suurin kuolinsyy niin Suomessa kuin useimmissa länsimaissakin. Näiden sairauksien taustalla on yleensä valtimonkovettumatauti eli ateroskleroosi, joka voi kliinisesti ilmentyä mm. sepelvaltimotautina, aivoveritulppana ja laskimotautina. Ateroskleroosissa tulehdussoluja ja kolesterolia kertyy verisuonen seinämään muodostaen ahtauman eli ateroomaplakin valtimoon. Valtimonkovettumataudin riskitekijäitä tunnetaan jo hyvin, mutta uusia tautia ennustavia merkkiaineita sekä hoitomuotoja tarvitaan yhä. Greliini on mahalaukusta eritettävä peptidihormoni, joka osallistuu elimistössä mm. ruokahalun, energiametabolian, tulehdustekijöiden sekä sydän- ja verenkiertoelimistön toiminnan säätelyyn. Tämän työn tavoitteena oli tutkia greliinin yhteyttä ihmisen valtimonkovettumatautiin. Tutkimus toteutettiin käyttämällä kahta eri potilasaineistoa, soluviljelykokeita sekä valtimonkovettumataudin hiirimallia. Laajassa väestöpohjaisessa potilasaineistossa tutkittiin veren greliinipitoisuuden yhteyttä kaulavaltimon seinämän paksuuteen, jota pidetään valtimonkovettumista kuvaavana tekijänä. Veren greliinipitoisuuden yhteyttä valtimonkovettumataudin tunnettuihin riskitekijöihin tutkittiin myös laajassa potilasaineistossa sekä vaihdevuosi-ikäisillä naisilla, joille annettiin estrogeenikorvaushoitoa. Solukokeilla selvitettiin greliinin vaikutusta tärkeisiin valtimonkovettumataudin syntyvaiheisiin käyttäen monosyytti-, endoteelisolu- sekä makrofaagi-soluviljelmiä. Greliinin vaikutusta ateroskleroosiin in vivo selvitettiin rokottamalla LDL-reseptoripuutteiset hiiret greliini-rokotteella. Tutkimuksessa havaittiin yhteys veren korkean greliinipitoisuuden ja kaulavaltimon seinämän paksuuden välillä miehillä laajassa potilasaineistossa (n = 1024). Tulosta tukivat soluilla tehdyt kokeet, joissa greliini lisäsi hapettuneen LDL:n sitoutumista makrofaageihin sekä monosyyttien tarttumista endoteelisolujen pinnalle. Greliinin vaikutukset monosyyttien tarttumiseen endoteelisolujen pinnalle olivat päinvastaiset silloin, kun endoteelisolut käsiteltiin tulehdusta stimuloivalla tekijällä. Matalat veren greliinipitoisuudet olivat myös yhteydessä korkeisiin LDL-kolesteroli- ja triglyseriditasoihin sekä painoindeksiin ja matalaan HDL-kolesterolitasoon potilasaineistoissa. Estrogeeni nosti veren greliinipitoisuutta vaihdevuosi-ikäisillä naisilla. Greliinirokote ei vaikuttanut ateroskleroosin kehittymiseen hiirimallissa. Tutkimustulosten perusteella greliinillä näyttäisi osallistuvan valtimonkovettumataudin kehitykseen, vaikkakin sen vaikutus on pienempi kuin aiemmin tunnetuilla taudin riskitekijöillä

The ghrelin receptor (GRLN-R) was cloned in 1996 after the discovery that a series of synthetic growth hormone-releasing compounds (the growth hormone secretagogues) acted through a receptor distinct from the growth hormone-releasing hormone receptor. In 1999 the endogenous ligand of the receptor, ghrelin, was discovered. As well as stimulating growth hormone release, ghrelin has been shown to be involved in many other processes such as appetite stimulation and the regulation of energy homeostasis, making the ghrelin/GRLN-R system an attractive pharmaceutical target for the treatment of disorders such as growth hormone deficiency, cachexia and obesity. The GRLN-R displays a high level of ligand-independent (constitutive) activity and has been suggested to couple to Gaq/11, Gai/o, Gas and Ga12/13 G protein pathways, although little is known about the signalling of ghrelin and the growth hormone secretagogues in all but the Gaq/11 pathway. Two of the growth hormone secretagogues, GHRP-6 and L-692,429, have been described as ‘ago-allosteric modulators’ of the GRLN-R as, when co-administered with ghrelin, GHRP-6 and L-692,429 were reported to act both as co-agonists (increasing the efficacy of the ghrelin response) and as negative or positive (respectively) regulators of the potency of ghrelin. This study sought to investigate the pharmacology of the GRLN-R through the Gai/o pathway. [35S]GTPyS binding assays were used to measure activation of the Gai/o pathway, demonstrating that GHRP-6, L-692,585 (a commercially available analogue of L-692,429) and a third growth hormone secretagogues, MK-677, acted with higher efficacy than ghrelin. At least in the system tested, upon co-administration with ghrelin each of the growth hormone secretagogues acted in a simple competitive fashion with ghrelin. Radioligand binding experiments showed that the dissociation kinetics of [His[125I]]- ghrelin from the GRLN-R were not altered by co-administration of the growth hormone secretagogues. Fitting data to a modified operational model of allosterism demonstrated that GHRP-6, L-692,585 and MK-677 were not ago-allosteric modulators of the GRLN-R but simple orthosteric agonists. In order to further examine the receptor-specific effects of ghrelin and the growth hormone secretagogues, a Flp-In™ T-REx™ HEK293 cell line expressing the GRLN-R was constructed. [35S]GTPyS binding assays confirmed that the GRLN-R was constitutively active through both the Gaq/11 and Gai/o pathways, demonstrated by an increase in [35S]GTPyS loading upon receptor expression which could be reduced by the administration of the GRLN-R inverse agonist SPA. Upon expression of the GRLN-R a considerable level of cell detachment was observed with the remaining cells appearing rounded compared to parental HEK293 cells, an affect that appeared to be mediated by the constitutive activity of the receptor. In contrast to the [35S]GTPyS assays used to measure activation of Gai/o, [35S]GTPyS assays with a Gaq-immunoprecipitation step demonstrated that the growth hormone secretagogues acted with equal efficacies to that of ghrelin, demonstrating functional selectivity at the GRLN-R. Intact cell assays were also used to measure Gaq/11 and Gai/o responses, however, a Gai/o-mediated response could not be measured in cAMP accumulation assays, suggesting that the GRLN-R could signal via activation of Gao but not Gai1-3. Although the activation of the Gas pathway by the GRLN-R remains controversial, in this study ghrelin and the growth secretagogues could evoke a Gas-mediated cAMP response (although L-692,585 acted with a lower efficacy than ghrelin). Finally, two naturally occurring missense mutations of the GRLN-R (A204E in the second extracellular loop and I134T in the third transmembrane helix) were analysed to investigate whether these mutations led to retention of the receptor within the endoplasmic reticulum and to investigate whether the mutations affected the ability of the GRLN-R to signal to the growth hormone secretagogues. The A204E mutation caused partial retention of the GRLN-R within the endoplasmic reticulum, whilst receptor that was transported to the plasma membrane did not display any measurable constitutive activity. In contrast, the I134T mutation did not alter receptor localisation, nor did it have any effect on the constitutive or ligand-induced activation of the GRLN-R, however, it appeared to lower the efficacy of the inverse agonist SPA.

The peptide hormone, ghrelin, exerts its physiological effects through a G-protein-coupled receptor called the ghrelin-R. The ghrelin-R displays a high degree of constitutive activity, signalling through the inositol phosphate pathway in the absence of bound agonist. TMs III and VI have been reported to be central to the activation of Family A GPCRs, with interactions between the two helices stabilising the ground state. During activation conformational rearrangements result in these interactions being broken, with new contacts forming and stabilising the active state. Investigation of the ghrelin-R constitutive activity gives an insight into the mechanisms involved in receptor activation. In this study the role of specific individual residues in the ghrelin-R has been investigated and the effect of disrupting or introducing intramolecular interactions was addressed. Site-directed mutagenesis and functional assays revealed that ghrelin-R constitutive activity can be increased and decreased with mutation of residues within the TM domains, specifically TMs III, VI and VII. The extracellular loops have been found to be involved in ligand binding and activation in a number of Family A GPCRs. The residues within ECL2 of the ghrelin-R were systematically mutated to alanine to determine their role. In particular, one residue, Asn196, was identified as being critical in ghrelin-R function and may be forming stabilising interactions which maintain ghrelin-R constitutive activity. The data presented in this thesis provide an insight into the structure and function of the ghrelin-R and the underlying molecular mechanisms of ghrelin-R constitutive activity.

This thesis investigated the role of ghrelin in children with Prader-Willi Syndrome (PWS) - a complex genetic neurodevelopmental disorder which often results in obesity. The data presented in this project provide evidence of new mechanisms of ghrelin regulation in PWS and will form a basis for future work that has potential therapeutic benefit in the areas of hyperphagia (excessive appetite) and obesity in children with PWS.

Abstract Ghrelin is a peptide hormone with anabolic functions. It increases growth hormone secretion and appetite, decreases fat utilisation as a metabolic fuel and increases fat storage in the adipose tissue. In addition, ghrelin exerts effects on glucose metabolism, heart function and inflammatory processes. Due to these characteristics ghrelin has become a hot topic for research focusing on obesity and its co-morbidities such as hypertension, type 2 diabetes and atherosclerosis. The aims of this study were to detect new sequential variations in the genes coding for the ghrelin receptor and ghrelin and to study whether these variations associate with obesity and metabolic risk factors for atherosclerosis. The roles of genetic and environmental factors in the determination of plasma ghrelin levels were also examined. In addition, the association of plasma ghrelin concentrations with disordered glucose regulation and type 2 diabetes was assessed in a longitudinal study. Five single nucleotide polymorphisms (SNPs) in the exons of the ghrelin receptor and 11 SNPs in the ghrelin gene 5´flanking area were found. The SNPs in the ghrelin receptor gene did not associate with plasma IGF-1 concentrations, but two of them did reveal an association with insulin and/or lipid metabolism related parameters. The SNPs found in the ghrelin gene 5´flanking area did not associate with plasma ghrelin levels, but one of them associated with body mass index (BMI). In monozygotic twins discordant for obesity, ghrelin levels were higher in the lean compared with the obese co-twins. Serum ghrelin levels at baseline did not differ between those who maintained normal glucose tolerance and those who developed impaired glucose regulation or type 2 diabetes during the follow up. In conclusion, the results suggest that two variations in the ghrelin receptor gene might be associated with glucose and lipid metabolism but not with IGF-1 levels. One SNP in the ghrelin gene might be associated with obesity. The results also indicate that plasma ghrelin levels are influenced by acquired obesity rather than genetic determinants. Finally, fasting serum ghrelin concentrations do not seem to have a significant predictive value on the incidence of impaired glucose tolerance and type 2 diabetes
Tiivistelmä Greliini on peptidihormoni, jolla on osoitettu olevan anabolisia vaikutuksia. Se voimistaa kasvuhormonin eritystä, toimii ruokahalua ja ravinnonottoa lisäävänä signaalina, vähentää rasvahappojen käyttöä energian lähteenä ja lisää rasvakudoksen määrää. Lisäksi greliinillä on osoitettu olevan vaikutuksia sokeriaineenvaihduntaan, sydämen toimintaan ja tulehduksellisiin prosesseihin. Näiden ominaisuuksiensa ansiosta greliinistä on tullut tärkeä tutkimuskohde lihavuuteen ja sen liitännäissairauksiin, kuten verenpainetautiin, tyypin 2 diabetekseen ja ateroskleroosiin liittyvässä tutkimuksessa. Tässä väitöskirjatutkimuksessa oli tavoitteina etsiä uusia geneettisiä poikkeamia greliiniä ja greliinireseptoria koodaavista geeneistä ja tutkia löytyneiden poikkeamien kytkeytymistä lihavuuteen sekä ateroskleroosin aineenvaihdunnallisiin riskitekijöihin. Tavoitteina oli myös selvittää geneettisten- ja ympäristötekijöiden roolia greliinipitoisuuksien säätelyssä. Lisäksi greliinipitoisuuksien yhteyttä tyypin 2 diabetekseen ja sokeriaineenvaihdunnan häiriöihin tutkittiin seurantatutkimuksessa. Greliinireseptorin geenin eksoneista löytyi viisi ja greliinigeenin 5´reunustavilta alueilta yksitoista yhden nukleotidin geneettistä poikkeamaa. Löytyneillä greliinireseptorin geenin poikkeamilla ei havaittu olevan yhteyttä plasman IGF-1 pitoisuuksiin, mutta kahdella niistä oli yhteyttä insuliini- ja/tai lipidiaineenvaihduntaan liittyviin muuttujiin. Greliinigeenin 5´reunustavan alueen geneettiset poikkeamat eivät liittyneet plasman greliinipitoisuuksiin, mutta yhdellä variaatiolla oli yhteyttä kehon painoindeksiin. Kaksostutkimukimuksissa greliinipitoisuudet olivat korkeammat hoikilla kaksosilla verrattuna lihaviin kaksospareihinsa, huolimatta samasta geeniperimästä. Seurantatutkimuksen alussa mitatut seerumin greliinipitoisuudet eivät ennustaneet tyypin 2 diabeteksen tai muun sokeriaineenvaihdunnan häiriön ilmaantumista. Yhteenvetona voidaan sanoa, että tutkimuksessa löytyneet greliinireseptorin geneettiset poikkeamat saattavat liittyä sokeri- ja rasva-aineenvaihduntaan, mutta eivät niinkään muihin tunnettuihin ateroskleroosin riskitekijöihin. Yksi greliinigeenin variaatioista näyttäisi liittyvän lihavuuteen. Tutkimukset osoittavat, että geneettiset tekijät säätelisivät plasman greliinipitoisuutta vähemmän kuin hankittu lihavuus. Lisäksi serumin greliinipitoisuuksilla ei ole merkittävää ennusteellista arvoa tyypin 2 diabeteksen ilmaantuvuuden suhteen

The thesis is divided in two parts. Part I describes effects of unacyated ghrelin, a small peptide mainly produced by stomach with multiple biological effects, on muscle regeneration in dystrophic condition using the murine mdx model. This study shows that unacylated ghrelin stimulates the polarity complex-mediated assymetric division of satellite cells, stem cells of muscle, leading to increase regenerative potential of satellite cells that in turn leads to the reduction of muscle degeneration and overall improvement of muscle function. These data were published in "Unacylated ghrelin enhances satellite cell function and relieves the dystrophic phenotype in Duchenne muscular dystrophy mdx model" (Reano et al., 2017, Stem cells) and a detailed protocol describing satellite cell handling in "Mouse satellite cell isolation and transplantaion" (Angelino et al., 2018, Bio-protocol). Moreover, Part I contains a study of the effect of unacylated ghrelin on cardiac muscle in mdx mice willing to establish if unacylated ghrelin treatment improved cardiac function in dystrophic mice. To complete information about the endogenous role of ghrelin peptides in muscle regeneration, Ghrl-/- mice, which lack all ghrelin gene products, have been investigated resulting in "Ghrelin knockout mice display defective skeletal muscle regeneration and impaired satellite cell self-renewal" (Angelino et al., Endocrine, under revision). Part II describes the in vitro study of the effect of vitamin D metabolites 25(OH)D and 1,25(OH)2D on skeletal muscle, both in normal condition and upon cytokine-induced cachexia, showing that these two metabolites have opposite effects: in basal condition, 25(OH)D induces hypertrophy while 1,25(OH)2D causes atrophy, and upon cytokine-induced atrophy, only 25(OH)D has a protective effect.

Abstract

G-protein coupled receptors (GPCRs) are having the high medical importance since almost half of the medicinal drugs are designed as modulators of receptor molecules. Crystal structure or NMR structures of GPCRs are very difficult to determine because all GPCRs are typically bound to the cell membrane and thus their molecular activation mechanism is still unclear. The recent publication of the crystal structure of the 2-adrenoreceptor will provide new insights in the field of GPCR research.

Ghrelin is a peptide growth hormone which binds to the growth hormone secretagogue receptor (GHS-R) and stimulates the release of growth hormone. Based on the known ghrelin receptor binding core sequences wFwLL (upper letter and lower letter representative for L-form and D-form of the amino acids respectively), we prepared two novel peptide analogs with terminal S-(2-aminoethylsulfenyl) cysteine residues. These peptides were tested for their ability to suppress the binding of ghrelin to transfected COS7 cell-line (Kidney fibroblast line from the green African monkey) cells expressing the ghrelin wild-type receptor or certain mutants thereof. As a result we observed a significant reduction of the total number of binding sites accessible for ghrelin, which increased with the time the cells were incubated with our test compounds. This observations support our hypothesis that the peptides we tested form a covalent bond with free thiols located closely to the ligand binding-site of the receptor protein by disulfide thiol exchange which is an interesting target for development of anti-obesity drugs.

Ghrelin is an orexigenic peptide that binds to the growth hormone secretagogue receptor (GHS-R). While it is known for regulating homeostatic feeding, recent research has revealed a role for ghrelin in reward-seeking. Missing from this research, however, is a systematic investigation into the role of ghrelin in reward-seeking: it is unclear whether ghrelin provides a reward signal, energises instrumental actions that earn palatable foods, or affects the ability of Pavlovian food-paired cues or food itself to invigorate instrumental responding, either generally or through the retrieval of specific associations. Given these phenomena promote overeating and weight gain, this thesis examined the role of ghrelin in Pavlovian and instrumental learning and performance. Overall, it was found that while ghrelin does not participate in Pavlovian or instrumental learning, it does modulate effort/response vigour: GHS-R antagonism attenuated established instrumental and progressive ratio responding for palatable food in rats. GHS-R antagonism had no effect on outcome devaluation, confirming that ghrelin affects instrumental responding by modulating effort/response vigour and not a change in outcome value. GHS-R antagonism had no effect on the return of responding driven by re-exposure to food after extinction (outcome-induced and outcome-specific reinstatement), and attenuated the general but not specific form of Pavlovian-instrumental transfer (PIT). Thus, ghrelin is also involved in the ability of Pavlovian food-paired cues to enhance instrumental responding based on a general excitatory relationship with food reward. As ghrelin levels fluctuate with changes in hunger/satiety, this thesis also investigated whether changes in ghrelin signalling recapitulate the effects of hunger/satiety. Generally, satiety had similar effects but was a more consistent modulator of instrumental performance and general PIT.

Ghrelin is a hormone produced mainly by the cells lining the gastric mucosa. Ghrelin was first extracted from human and rat stomachs, and identified as an endogenous stimulator of growth hormone release. Ghrelin is synthesized and produced in several tissues, but the gastric mucosa remains the major source of circulating ghrelin. Besides growth hormone release, ghrelin stimulates appetite and plays some major roles in different organs. In several studies, ghrelin was described as a hormone with multiple functions and diverse biological actions. Ghrelin exists in two major forms, active ghrelin and inactive ghrelin, and only the active from binds to the receptor. The majority of total circulating ghrelin is inactive ghrelin with no identified function. The aims of the present study were to measure active and total ghrelin in dairy cow’s milk and plasma during early lactation, and to observe changes in the ghrelin concentrations over time. We are interested in this period of time since the milk during early lactation contains a variety of biologically active hormones that are vital for newborn calves. In this study, fifteen Holstein dairy cows were selected randomly from different lactations. Milk and blood samples were taken daily from cows at early lactation for 10 days, and from some cows in mid-lactation. A laboratory test was used to measure active and total ghrelin in milk and plasma samples. Supplementary measurements such as milk fat, lactose, protein, and milk yield were recorded. Active and total milk ghrelin concentrations were found to be significantly higher in the first day of lactation during colostrum production. Interestingly, the percentage of active to total ghrelin in milk and blood was constant in all days tested, suggesting that this constant percentage can be used to estimate active or total ghrelin, if one of them is know, from the same sample. However, no correlation was observed between the percentage of milk ghrelin and plasma ghrelin or with other milk components. In conclusion, the presence of ghrelin in colostrum and milk in measurable amounts of both active and total form suggests that it is a critical compound for the metabolic activity of newborn calves and functions transiently to regulate the activity of some physiological processes until the endocrine system of the new calves starts to function independently.

Recent data suggest that ghrelin exerts its orexigenic action through regulation of hypothalamic AMP-activated protein kinase pathway, leading to a decline in malonyl-CoA levels and desinhibition of carnitine palmitoyltransferase 1A (CPT1A), which increases mitochondrial fatty acid oxidation and ultimately enhances the expression of the orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY). However, it is unclear whether the brain-specific isoform CPT1C, which is located in the endoplasmic reticulum of neurons, may play a role in this action. Here, we demonstrate that the orexigenic action of ghrelin is totally blunted in CPT1C knockout (KO) mice, despite having the canonical ghrelin signaling pathway activated. We also demonstrate that ghrelin elicits a marked upregulation of hypothalamic C18:0 ceramide levels mediated by CPT1C. Notably, central inhibition of ceramide synthesis with myriocin negated the orexigenic action of ghrelin and normalized the levels of AgRP and NPY, as well as their key transcription factors phosphorylated cAMP-response element-binding protein and forkhead box O1. Furthermore, central treatment with ceramide induced food intake and orexigenic neuropeptides expression in CPT1C KO mice. Third, we demonstrate that ceramides act increasing the BSX expression, the transcription factor that works coordinately with pCREB and FoxO1 to increase orexigenic neuropeptides. Finally we link CPT1C and longevity as we have seen that CPT1CKO mice have reduced lifespan. Overall, these data indicate that, in addition to formerly reported mechanisms, ghrelin also induces food intake through regulation of hypothalamic CPT1C and ceramide metabolism

Ingested lipid releases the gut peptide cholecystokinin (CCK) which limits food intake by modulating gut function and signalling to the brain. Current understanding of this pathway and its interaction with other gut-brain signalling peptides such as ghrelin is incomplete. To clarify the roles of lipid, CCK and ghrelin in gut-brain signalling pathway in man, a series of human studies were performed in order to answer the following questions.

Ghrelin is a gastric hormone circulating in acylated (AG) and unacylated (UnAG) forms. AG represents ~10% of total plasma ghrelin, has an appetite-stimulating effect and is the only form for which a receptor has been identified. UnAG has no orexigenic effects, and its circulating levels are positively associated with insulin sensitivity in metabolic syndrome patients. Skeletal muscle oxidative stress and inflammation are key negative modulators of tissue and whole-body insulin action, and UnAG was recently reported to reduce oxidative stress in non-muscle cells in-vitro. Its potential direct involvement in the regulation of muscle intermediate metabolism in vivo and its clinical impact remain however largely unknown. In the current studies, we first investigated potential associations between plasma AG, UnAG and HOMA insulin resistance index in the general population. In 719 individuals from the North-East-Italy MoMa epidemiological study, TG and UnAG but not of AG were negatively associated with HOMA after adjusting for gender and body mass both at baseline and at 5-year follow-up, and changes in TG and UnAG but not AG were negatively associated with changes in HOMA independently of potential confounders. We next tested the hypothesis that UnAG increases insulin sensitivity by modulating oxidative stress, inflammation and insulin action in skeletal muscle tissue. In healthy male rats, UnAG administration consistently reduced muscle mitochondrial ROS production, reduced inflammation and activated insulin-anabolic signalling. Analogous findings were observed in transgenic mice with systemic UnAG overexpression, and in UnAG-treated myotubes in vitro, thereby supporting a potential direct hormone effect. Importantly, these findings were not observed with AG administration nor in liver samples, thus further indicating independent and tissue specific UnAG actions. Autophagy, the removal of dysfunctional organelles, is an emerging protective mechanism in both cardiac and skeletal muscle. We therefore also hypothyzed that UnAG could reduce ROS production by inducing muscle autophagy. In cardiomiocytes UnAG reduced ROS production in association with increased dysfunctional mitochondria removal and in UnAG but not AG treated myotubes silencing of autophagy promoter ATG5 restored ROS generation. We next investigated the potential clinical relevance of UnAG actions in disease models characterized by muscle oxidative stress, inflammation and insulin resistance. In high-fat diet (HFD)-induced obese and diabetic mice, chronic UnAG overexpression prevented hyperglycemia and whole-body insulin resistance, as well as muscle oxidative stress, inflammation and altered insulin signalling. In rodent chronic kidney disease induced by 5/6 nephrectomy, protein-energy wasting, enhanced skeletal muscle ROS production, increased tissue inflammation and impaired insulin signalling were also completely normalized by UnAG treatment. Importantly, these findings were associated with a recovery of gastrocnemious muscle mass. While a specific receptor for UnAG needs to be identified, our combined findings consistently point towards a novel independent role of UnAG as a regulator of muscle metabolic pathways maintaining energy status and tissue anabolism. Underlying mechanism appear to involve the modulation of mitochondrial function with reduced ROS generation, which could be mediated at least in part by autophagy induction. Data from disease models also suggests that modulation of ghrelin acylation to enhance UnAG availability is a potential novel target in the treatment of metabolic derangements in disease states characterized by metabolic and nutritional complications.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Ghrelin is a recently discovered peptide, mainly produced in the stomach and involved in body's energy-maintenance processes. Ghrelin exerts its actions by activating the growth hormone secretagogue receptor (GHS-R). A number of studies have investigated the involvement of ghrelin in the modulation of emotional behavior. In this context, evidence shows the presence of GHS-R receptors in the dorsal raphe nucleus (DRN), the main source of serotonergic neurons that innervate encephalic structures involved in emotional control. Our study aims to evaluate the effects of the pharmacological manipulation of ghrelin receptors located in the DRN on the expression of the behavioral responses of Wistar rats. Such responses were assessed in the elevated T maze (ETM), an experimental model that allows the measurement, in the same animal, of two defensive behavioral strategies, inhibitory avoidance and escape. Our results showed that the intra-DRN infusion of ghrelin impaired the acquisition of inhibitory avoidance, an anxiolytic-like effect, and facilitated the expression of escape response in the ETM, indicating a panicogenic-like effect. The intra-DRN administration of the ghrelin receptor (GHS-R1a) antagonist PF-04628935 did not alter the behavioral tasks assessed in the ETM. Finally, our results revealed that intra-DRN infusions of PF-04628935 prior to the administration of ghrelin into this area neutralized the behavioral effects obtained in the ETM. Taken together, our data reveal an important involvement of the ghrelin central signaling system in the mediation of defensive behavioral responses that have been associated with generalized anxiety and panic disorders.
A grelina é um peptídeo recentemente descoberto, produzido principalmente no estômago e envolvido nos processos de manutenção de energia do organismo. A grelina exerce suas ações ao ativar o receptor secretagogo do hormônio do crescimento (GHS-R). Uma série de estudos tem investigado o envolvimento da grelina na modulação de comportamentos emocionais. Neste contexto, evidências mostram a presença de receptores GHS-R no núcleo dorsal da rafe (NDR), principal fonte de neurônios serotonérgicos que inervam estruturas encefálicas envolvidas no controle emocional. O objetivo deste trabalho foi avaliar os efeitos obtidos com a manipulação farmacológica dos receptores de grelina localizados no NDR sobre a expressão das respostas comportamentais de esquiva inibitória e fuga de ratos. Tais respostas foram avaliadas no labirinto em T elevado (LTE), modelo experimental que possibilita a medida, em um mesmo animal, de duas estratégias comportamentais defensivas, a esquiva inibitória e a fuga. Os dados de nosso estudo mostraram que a infusão intra-NDR de grelina prejudicou a aquisição da resposta de esquiva inibitória, um efeito ansiolítico, e facilitou a expressão da resposta de fuga dos animais no LTE, um efeito panicogênico. A administração intra-NDR do antagonista de receptores de grelina (GHS-R1a) PF-04628935 não modificou as respostas comportamentais defensivas avaliadas no LTE. Por fim, nossos dados mostraram que a infusão de PF-04628935 previamente à injeção de grelina no NDR foi capaz de bloquear os efeitos comportamentais obtidos no LTE. Tomadas em conjunto, as evidências obtidas em nosso estudo apontam para um importante envolvimento do sistema central de sinalização do peptídeo grelina na mediação de respostas comportamentais defensivas que têm sido associadas aos transtornos de ansiedade generalizada e do pânico.

La maladie de Parkinson (MP) est une maladie neurodégénérative caractérisée par trois symptômes moteurs principaux : la bradykinésie, la rigidité et le tremblement de repos. Son diagnostic définitif repose sur l’identification post-mortem d’une importante mort des neurones dopaminergiques de la substance noire (SN) et la présence de corps de Lewy dans les neurones survivants. Cette maladie progresse lentement et les premiers symptômes moteurs n’apparaissent qu’après la dégénérescence de plus de 50% de la SN. Le diagnostic clinique de MP est donc établi tardivement, réduisant ainsi la fenêtre d’action thérapeutique. De plus, les traitements actuels ne soulagent que temporairement les symptômes moteurs. Les défis de la recherche actuelle pour la MP sont donc : 1) d’anticiper le diagnostic de la MP à un stade où la SN est encore suffisamment intacte pour mettre en place des stratégies neuroprotectrices, et 2) d’améliorer les traitements actuels et/ou développer de nouvelles stratégies thérapeutiques pour stopper la progression de la maladie avant que le phénotype moteur ne soit installé. Le stade clinique de la MP est précédé d’une phase prémotrice durant laquelle les patients présentent souvent des symptômes non moteurs tels que l’anosmie, la dépression ou la constipation. Des travaux récents suggèrent que les lésions caractéristiques de la MP pourraient d’abord apparaître dans le système nerveux périphérique puis progresser lentement jusqu’au cerveau. Ces stades précoces de la MP sont cependant mal connus et leurs caractéristiques méritent d’être étudiées dans des modèles expérimentaux appropriés. Ainsi, des études récentes ont montré que la ghréline, un peptide gastro-intestinal, protège les neurones dopaminergiques de la SN contre la mort dans des modèles in vivo et in vitro de syndrome parkinsonien. De plus, dans un modèle animal de syndrome parkinsonien, la ghréline prévient l’aggravation des symptômes gastro intestinaux par la L-DOPA, traitement médicamenteux principal de la MP. Enfin, des altérations des concentrations plasmatiques de ghréline ont également été observées chez les patients aux stades précoces de la maladie. Dans ce contexte, nous avons émis l’hypothèse que la ghréline pourrait jouer un rôle important aux stades précoces de la maladie et donc être utilisée comme biomarqueur et/ou agent neuroprotecteur dans la MP. Ainsi, l’objectif de ma thèse était d’étudier les rôles de la ghréline aux stades précoces de la MP par des approches in vitro et in vivo.La première étape a consisté à déterminer les effets de la ghréline dans des cultures primaires de cellules mésencéphaliques exposées au pesticide roténone, un inhibiteur du complexe I mitochondrial connu pour son association avec la MP. Contrairement aux données de la littérature, nous montrons un effet délétère en fonction de la dose et du temps sur les cellules exposées à la roténone. Nous ne confirmons donc pas l’effet neuroprotecteur de la ghréline dans nos conditions expérimentales. En parallèle, nous avons étudié le potentiel de la ghréline en tant que biomarqueur dans un modèle murin de syndrome parkinsonien reproduisant les stades précoces de la maladie après exposition orale chronique à de faibles doses de roténone. Nous avons d’abord validé ce modèle et confirmé le développement des altérations non motrices et l’absence de mort neuronale au sein de la SN après 1.5 mois de ce régime. En revanche, nos résultats ne montrent pas de modification des taux plasmatiques de ghréline chez les souris exposées 1.5 mois à la roténone. Cependant, des facteurs tels que l’anxiété pourraient avoir affecté les taux de ghréline. Ces données devront donc être confirmées avec des animaux stratifiés selon leur niveau d’anxiété et/ou de plus longues expositions. En conclusion, nos résultats interrogent le rôle neuroprotecteur de la ghréline dans la MP et posent les bases pour de futures recherches sur l’implication de cette hormone orexigène dans la MP
Parkinson’s disease (PD) is the second most frequent neurodegenerative disease in the world. It is characterized by motor symptoms such as bradykinesia, rigidity and resting tremor. Its definite diagnosis relies on the identification of specific neuropathological hallmarks at autopsy including severe neuronal death within the substantia nigra (SN) and the presence of Lewy bodies in the surviving neurons. PD progresses slowly and the first motor symptoms appear when more than 50% of the SN has degenerated. Therefore, the clinical diagnosis is established late in the course of the disease, thus restricting the therapeutic window for clinicians. In addition, the currently available therapeutic options can only temporarily alleviate PD motor symptoms. The challenges of current PD research are: 1) to anticipate the diagnosis and be able to identify the disease as early as possible, when the SN is still intact enough to implement a disease-modifying/neuroprotection strategy to prevent the appearance of motor symptoms, and 2) to improve current medications and/or develop new therapeutic strategies able to stop the disease before the motor phenotype is installed. The decade preceding PD clinical diagnosis is of particular interest since patients often complain about non-motor symptoms such as anosmia, depression or constipation. Moreover, recent evidences suggest that PD-characteristic lesions could first appear in the peripheral nervous system and slowly progress towards the brain. Thus PD earlier stages and their characteristics deserve better investigations using appropriate experimental models. In this regard, recent studies realized in animal and cellular models of advanced parkinsonism have suggested that ghrelin, an orexigenic peptide mainly produced in the stomach, could play a neuroprotective role in PD. Indeed, exposure to ghrelin has shown a protective effect against the neuronal death in animal and cellular models of parkinsonism. In addition, in a rodent model of parkinsonism, ghrelin was shown to alleviate the L-DOPA-induced worsening of gastro-intestinal symptoms, L-DOPA being the current main therapeutic option in PD. Moreover, ghrelin plasma concentrations have shown alterations in early stages of the disease in small cohorts of PD patients. We therefore hypothesized that ghrelin might play an important role in PD early stages and could serve as a biomarker and a neuroprotective agent in PD. In this context, the aim of my PhD was to investigate the roles of ghrelin in PD early stages using both in vitro and in vivo approaches.We first studied the effects of ghrelin in primary mesencephalic cells exposed to the pesticide rotenone, a potent inhibitor of mitochondrial complex I known for its association with PD. Contrary to the data of the literature, we show a dose and time-dependant deleterious effect of ghrelin on mesencephalic cells exposed to rotenone. This does not confirm the neuroprotective potential of ghrelin in our experimental conditions. In parallel, we investigated the potential of ghrelin as a biomarker in a rodent model of parkinsonism mimicking early stages of the disease after chronic oral exposure to low doses of rotenone. We first validated this model in our animal facility and confirmed that mice exposed to such a regimen develop progressive non-motor alterations but no dopaminergic neuronal death in the SN after 1.5 months. Our initial results do not show a modification of plasma ghrelin levels in rotenone-exposed mice at early stages of the pathological condition. However, confounding factors such as anxiety might have altered ghrelin levels. This should therefore be further ascertained in animals stratified for their anxiety levels and/or in longer exposures. In conclusion, these results challenge the suggested role of ghrelin as a disease-modifying agent in PD and set the bases for future investigations of ghrelin in the context of PD