68Ga‐labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators

The dual interaction with integrins and neuropilin‐1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with ⁶⁸GaCl₃. Negatively charged ⁶⁸Ga‐DOTAGA‐iRGD and neutral ⁶⁸Ga‐NODAGA‐iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral ⁶⁸Ga‐NODAGA‐iRGD, but ⁶⁸Ga‐DOTAGA‐iRGD exhibited better tumor‐to‐blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.     

Gallium-68 complex of a macrobicyclic cage amine chelator tethered to two integrin-targeting peptides for diagnostic tumor imaging

Tumor-targeting peptides radiolabeled with positron-emitting (68)Ga are promising candidates as new noninvasive diagnostic agents for positron emission tomography (PET). The targeting peptides are tethered to a chelator that forms a stable coordination complex with Ga(3+) that is inert to dissociation of Ga(3+)in vivo. Metal complexes of macrobicyclic hexaamine "sarcophagine" (sar = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) ligands exhibit remarkable stability as a result of the encapsulating nature of the cage amine ligand. A Ga(3+) sarcophagine complex, [Ga-(1-NH(3)-8-NH(2)-sar)](4+), has been characterized using X-ray crystallography, demonstrating that Ga(3+) is coordinated to six nitrogen atoms in a distorted octahedral complex. A bifunctional derivative of (NH(2))(2)sar, possessing two aliphatic linkers with carboxylic acid functional groups has been attached to two cyclic-RGD peptides that target the α(v)β(3) integrin receptor that is overexpressed in some types of tumor tissue. This dimeric species can be radiolabeled with (68)Ga(3+) in >98% radiochemical yield and (68)Ga(3+) does not dissociate from the ligand in the presence of transferrin, an endogenous protein with high affinity for Ga(3+). Biodistribution and micro-PET imaging studies in tumor-bearing mice indicate that the tracer accumulates specifically in tumors with high integrin expression. The high tumor uptake is coupled with low nonspecific uptake and clearance predominantly through the kidneys resulting in high-quality PET images in animal models.     

Detailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides

The interest in using small peptides for therapeutic and diagnostic in vivo applications is based on several advantageous features such as good penetration into tissues and rapid clearance from the body. Because of their size, they can easily be synthesized chemically. The recently discovered cell-penetrating peptides (CPP) and among them CPP derived from the native peptide hormone human calcitonin (hCT) could meet these requirements. Therefore, they are nowadays widely used as delivery vectors for a variety of bioactive molecules. However, the knowledge about the distribution and metabolism of CPP in vivo is very limited. Hence, evaluation of the pharmacological features of any promising peptide is a crucial challenge in its development process. Herein, we studied the in vivo radiopharmacology of (68)Ga radiolabeled DOTA-modified, hCT-derived CPP in rats using small animal PET. Furthermore, the arterial blood at different time points and urine were analyzed for radio-metabolites. It was shown that d-amino acid modifications of the sequence hCT(9-32) resulted in an increased in vivo stability and lower retention in the kidney cortex of this peptide.     

Comparison study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2, and [68Ga]Ga-NOTA-PRGD2 for PET imaging of U87MG tumors in mice

[(18)F]FPPRGD2, an F-18 labeled dimeric cyclic RGDyK peptide, has favorable properties for PET imaging of angiogenesis by targeting the α(v)β(3) integrin receptor. This radiotracer has been approved by the FDA for use in clinical trials. However, the time-consuming multiple-step synthetic procedure required for its preparation may hinder the widespread usage of this tracer. The recent development of a method using an F-18 fluoride-aluminum complex to radiolabel peptides provides a strategy for simplifying the labeling procedure. On the other hand, the easy-to-prepare [(68)Ga]-labeled NOTA-RGD derivatives have also been reported to have promising properties for imaging α(v)β(3) integrin receptors. The purpose of this study was to prepare [(18)F]FPPRGD2 [corrected] , [(18)F]FAl-NOTA-PRGD2, and [(68)Ga]Ga-NOTA-PRGD2 and to compare their pharmacokinetics and tumor imaging properties using small animal PET. All three compounds showed rapid and high tracer uptake in U87MG tumors with high target-to-background ratios. The uptake in the liver, kidneys, and muscle were similar for all three tracers, and they all showed predominant renal clearance. In conclusion, [(18)F]FAl-NOTA-PRGD2 and [(68)Ga]Ga-NOTA-PRGD2 have imaging properties and pharmacokinetics comparable to those of [(18)F]FPPRGD2. Considering their ease of preparation and good imaging qualities, [(18)F]FAl-NOTA-PRGD2 and [(68)Ga]NOTA-PRGD2 are promising alternatives to [(18)F]FPPRGD2 for PET imaging of tumor α(v)β(3) integrin expression.     

Long-term effects of ghrelin and ghrelin receptor agonists on energy balance in rats

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is the only circulating agent to powerfully promote a positive energy balance. Such action is mediated predominantly by central nervous system pathways controlling food intake, energy expenditure, and nutrient partitioning. The ghrelin pathway may therefore offer therapeutic potential for the treatment of catabolic states. However, the potency of the endogenous hormone ghrelin is limited due to a short half-life and the fragility of its bioactivity ensuring acylation at serine 3. Therefore, we tested the metabolic effects of two recently generated GHS-R agonists, BIM-28125 and BIM-28131, compared with ghrelin. All agents were administered continuously for 1 mo in doses of 50 and 500 nmol x kg(-1) x day(-1) using implanted subcutaneous minipumps in rats. High-dose treatment with single agonists or ghrelin increased body weight gain by promoting fat mass, whereas BIM-28131 was the only one also increasing lean mass significantly. Food intake increased during treatment with BIM-28131 or ghrelin, whereas no effects on energy expenditure were detected. With the lower dose, only BIM-28131 had a significant effect on body weight. This also held true when the compound was administered by subcutaneous injection three times/day. No symptoms or signs of undesired effects were observed in any of the studies or treated groups. These results characterize BIM-28131 as a promising GHS-R agonist with an attractive action profile for the treatment of catabolic disease states such as cachexia.     

Effect of Ghrelin Administration on Serum Corticostrone,T<Subscript>3</Subscript>, T<Subscript>4</Subscript> and Some Biochemical Indices in the Turkey (<Emphasis Type="Italic">Meleagridis gallopova</Emphasis>)

Ghrelin is a multifunctional regulatory peptide that has widespread endocrine and metabolic effects in mammals and birds. The present study aimed to investigate the possible effect of ghrelin on blood hormone and biochemical indices in turkey. A total of forty-eight 28 day-old turkeys were divided into three groups for tests. Ghrelin was injected at the onset of the experiment (28-day old birds). Treatment doses were as follows: treatment 1 (control) without injection; treatment 2—50 ng ghrelin/kg body weight (BW); and treatment 3—100 ng ghrelin/kg BW. Two blood samples were taken from the birds, one at 12 h (short-term effect) and the other at 40 days (long-term effect). Blood analyses showed that level of corticosterone increased in response to ghrelin treatments G50 and G100 in samples taken on days 28 and 68 (p?<?0.01). There was an increase in T₄ concentration in the G50 and G100 groups in comparison with the control. Blood glucose increased in response to ghrelin administration, and total cholesterol and triglyceride concentrations decreased in the two samples in response to higher ghrelin dosage (p?<?0.01). In conclusion, the peripheral administration of ghrelin in turkeys may increase levels of serum corticosterone, glucose and T₄. Therefore, total cholesterol and triglyceride may decrease in birds following ghrelin administration. Ghrelin may increase metabolic rate (due to increases in T4) and regulate lipogenesis in poultry species such as turkey.     

Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.     

Novel DOTA-neurotensin analogues for 111In scintigraphy and 68Ga PET imaging of neurotensin receptor-positive tumors

Overexpression of the high affinity neurotensin receptor 1 (NTSR1), demonstrated in several human cancers, has been proposed as a new marker for human ductal pancreatic carcinoma and as an independent factor for poor prognosis for ductal breast cancer, head and neck squamous cell carcinoma, and non-small cell lung cancer. The aim of the present study was to develop new DOTA-neurotensin analogues for positron emission tomography (PET) imaging with (68)Ga and for targeted radiotherapy with (90)Y or (177)Lu. We synthesized a DOTA-neurotensin analogue series. Two of these peptides bear two sequence modifications for metabolic stability: DOTA-NT-20.3 shares the same peptide sequence as the previously described DTPA-NT-20.3. In the sequence of DOTA-NT-20.4, the Arg(8)-Arg(9) bond was N-methylated instead of the Pro(7)-Arg(8) bond in DOTA-NT-20.3. An additional sequence modification was introduced in DOTA-LB119 to increase stability. A spacer was added between DOTA and the peptide sequence to increase affinity. Binding to HT29 cells, which express NTSR1, in vivo stability, and biodistribution of the various analogues were compared, and the best candidate was used to image tumors of various sizes with the microPET in mice. (111)In-DOTA-NT-20.3, in spite of a relatively high uptake in kidneys, showed specific tumor uptake and elevated tumor to other organ uptake ratios. High contrast images were obtained at early time points after injection that allowed tumor detection at a time interval postinjection appropriate for imaging with the short-lived radionuclide (68)Ga. (111)In-DOTA-NT-20.4 displayed inferior binding to HT29 cells and reduced tumor uptake. (111)In-DOTA-LB119 displayed at early time points a significantly lower renal uptake but also a lower tumor uptake than (111)In-DOTA-NT-20.3, although binding to HT29 cells was similar. (68)Ga-DOTA-NT-20.3 displayed higher tumor uptake than (68)Ga-DOTA-LB119 and allowed the detection of very small tumors by PET. In conclusion, DOTA-NT-20.3 is a promising candidate for (68)Ga-PET imaging of neurotensin receptor-positive tumors. DOTA-NT-20.3 may also be considered for therapy, as the yttrium-labeled peptide has higher affinity than that of the indium-labeled one. A prerequisite for therapeutic application of this neurotensin analogue would be to lower kidney uptake, for example, by infusion of basic amino acids, gelofusin, or albumin fragments, to prevent nephrotoxicity, as with radiolabeled somatostatin analogues.     

Le DOTATOC-(68Ga) pour l’imagerie TEP des tumeurs endocrines digestives : présentation d’un cas et revue de la littérature

⁶⁸Ga is a positron emitter, obtained from a ⁶⁸Ge/⁶⁸Ga generator, which can be used to label peptides of clinical interest. DOTATOC is a tracer of high affinity for the type 2 somatostatin receptors and is used for imaging of tumours which are expressing them, including endocrine tumours. We report on the case of a patient with a history of small bowel carcinoid tumour with hepatic metastases, treated by somatostatin analogues, in whom somatostatin receptor scintigraphy showed three liver foci and DOTATOC-(⁶⁸Ga) PET/CT highlighted much more liver lesions. Two recent studies emphasised on the superiority of DOTATOC-(⁶⁸Ga) PET over somatostatin receptor scintigraphy, and its complementarity with CT, especially for the diagnosis of bone metastases. DOTATOC-(⁶⁸Ga) PET/CT, which associates the specificity of somatostatin receptor scintigraphic detection with the spatial resolution of PET and the anatomical precision of CT, seems to be promised to a brilliant future, the more so as it offers many advantages to the patient: a shorter waiting time, one single image acquisition and a satisfying dosimetry.     

The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser³. The previous studies have revealed that N-terminal part of ghrelin including modified Ser³ is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH₂ exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH₂ was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser³ from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.     

Synthesis and evaluation of macrocyclic amino acid derivatives for tumor imaging by gallium-68 positron emission tomography

(68)Ga PET imaging in clinical oncology represents a notable development because the availability of (68)Ga is not dependent on a cyclotron. Furthermore, labeled amino acid derivatives have been proven to be useful for the imaging many tumor types. In the present study, we synthesized β-aminoalanine, γ-aminohomoalanine, and lysine conjugates of macrocyclic bifunctional chelating agents, such as, NOTA (1a-c) and DOTA (2a-c). The compounds produced were found to be potential useful as (68)Ga-PET imaging agents. In particular, they showed high tumor uptakes in vitro and in vivo, and had high labeling yields and excellent stabilities. The co-ordination chemistry of NOTA-monoamide compound 1a was studied by multinuclear NMR. In vitro studies showed that the synthesized compounds were taken up by cancer cells more than controls ((68)Ga-NOTA and (68)Ga-DOTA). Furthermore, in vivo studies showed that they have high tumor to muscle and tumor to blood ratios, and small-animal PET imaging revealed high tumor uptakes as compared with other organs, and high bladder activities, indicating rapid renal excretion. These results might motivate the use of (68)Ga amino acid PET for tumor diagnosis.     

Transient increase of plasma ghrelin after laparoscopic adjustable gastric banding in morbid obesity.

Ghrelin is a peptide hormone with orexigenic properties that is produced by the stomach. Ghrelin and leptin are thought to be the main regulators of appetite and body weight. The present study was aimed at evaluating the effect of weight reduction after laparoscopic adjustable gastric banding (LAGB) on metabolic parameters and energy balance regulatory peptides. Patients were evaluated before and 6, 12, 24 or 36 months after the procedure, and a blood sample was obtained. Ghrelin rose 6 and 12 months after LABG, and then returned to near-baseline levels. In our study, the correlation between ghrelin and BMI was weak, but a strong significant correlation was maintained between leptin and BMI. We conclude that ghrelin is mainly stimulated by the negative caloric balance, and hypothesize that ghrelin is involved in maintaining a stable body weight, while leptin signals the body energy store; both hormones together are part of a more complex feedback mechanism.     

In vitro and in vivo characterization of three 68Ga- and 111 In-labeled peptides for cholecystokinin receptor imaging

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], and DOTA-MG0, labeled with (111)In or (68)Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.     

Ghrelin stimulates insulin-induced glucose uptake in adipocytes

The gastric and hypothalamic hormone ghrelin is the endogenous agonist of the growth hormone secretagogue receptor GHS-R1(a). Ghrelin stimulates growth hormone release and appetite via the hypothalamus. However, putative direct peripheral effects of ghrelin remain poorly understood. Rat adipose tissue expresses GHS-R1(a) mRNA, suggesting ghrelin may directly influence adipocyte function. We have investigated the effects of ghrelin on insulin-stimulated glucose uptake in isolated white adipocytes in vitro. RT-PCR confirmed the expression of GHS-R1(a) mRNA in epididymal adipose tissue. However, GHS-R1(a) expression was not detected in the peri-renal fat pads. Ghrelin increased insulin-stimulated deoxyglucose uptake in isolated white adipocytes extracted from the epididymal fat pads of male Wistar rats. Ghrelin 1000 nM significantly increased deoxyglucose uptake by 55% in the presence of 0.1 nM insulin. However, ghrelin administration in the absence of insulin had no effect on adipocyte deoxyglucose uptake, suggesting that ghrelin acts synergistically with insulin. Des-acyl ghrelin, a major circulating non-octanylated form of ghrelin, had no effect on insulin-stimulated glucose uptake. Furthermore, acylated ghrelin had no effect on deoxyglucose uptake in adipocytes from peri-renal fat pads suggesting that ghrelin may influence glucose uptake via the GHS-R1(a). Ghrelin therefore appears to directly potentiate adipocyte insulin-stimulated glucose uptake in selective adipocyte populations. Ghrelin may play a role in adipocyte regulation of glucose homeostasis.     

Identification of potent,selective, CNS-targeted inverse agonists of the ghrelin receptor

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine–piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.     

Microwave-supported preparation of (68)Ga bioconjugates with high specific radioactivity

The generator-produced positron-emitting (68)Ga (T(1/2) = 68 min) is of potential interest for clinical PET. (68)Ga as a metallic cation is suitable for complexation reactions with chelators, naked or conjugated, with peptides or other macromolecules. Large (68)Ga generator eluate volumes, metal traces from the generator column material, or reaction reagents, however, disturb a fast, reliable, and quantitative labeling procedure. In this paper we describe a simple technique, based on anion exchange, aiming first, to increase the (68)Ga concentration, second to purify it from competing impurities, and third to obtain a fast and quantitative (68)Ga-labeled peptide conjugate that can be applied in humans without further purification. Within 5 min one can obtain from the original 6 mL generator eluate a 200 microL (68)Ga preparation (volume reduction by a factor 30) that is suitable for direct and quantitative labeling of peptide conjugates. DOTATOC (DOTA-D-Phe(1)-Tyr(3)-octreotide, DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was used as a test tracer for comparing the labeling properties of the different (68)Ga preparations. In combination with microwave heating, peptide conjugates of 0.5-1 nmol quantities could be labeled within 10 min with the full (68)Ga activity of a generator. Further purification of the (68)Ga-labeled peptide conjugate was no longer required since the nuclide incorporation was quantitative. The specific radioactivity (with respect to the peptide) was improved by a factor approximately 100 compared to the previously applied techniques using the original generator eluate. The commercial (68)Ge/(68)Ga generator from Obninsk in combination with this system for purification and concentration with an integrated microwave-supported labeling technology resulted in a kitlike technology for (68)Ga-tracer production. The first automated prototype using this technology is being tested.     

Structures and molecular forms of the ghrelin-family peptides

Ghrelin is an acylated peptide hormone produced mainly from the stomach. The major active products of the ghrelin gene in the stomach of rats, mice and humans are 28-amino acid peptides acylated at the serine-3 position with an n-octanoyl group (C8:0), called simply ghrelin. However, recent studies have revealed that the ghrelin gene can generate a variety of bioactive molecules besides ghrelin. These include acyl forms of ghrelin other than C8:0-ghrelin (i.e., n-decanoyl ghrelin or n-decenoyl ghrelin), des-acyl ghrelin, obestatin and ghrelin-associated peptides originated from the ghrelin gene. This review surveys the structures of the ghrelin peptides and molecular forms of ghrelin gene-derived products, and summarizes the knowledge about the functions of these peptides, with an emphasis on the acyl forms of the ghrelin peptide.     

Hindbrain ghrelin receptor signaling is sufficient to maintain fasting glucose

The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have examined the roles of ghrelin receptor (GHSR) expression in the mouse hindbrain. Notably, selective hindbrain ghrelin receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action.     

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.