Human Adrenomedullin Research Articles

PS-BPB09-2: DEVELOPMENT OF LONG-ACTING HUMAN ADRENOMEDULLIN FC FUSION PROTEINS

Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone with strong anti-inflammatory effects. AM consists of 52 amino acid residues with an amidated C-terminus and a ring structure formed by intramolecular disulfide bonds. Both of these structures are necessary for binding to receptors and to exert their biological functions. AM has also shown therapeutic effects in various animal disease models, including inflammatory bowel disease, ischemic heart disease, sepsis and stroke. However, the short half-life of native AM in the blood required continuous administration for treatment. To solve this, we have developed four human IgG1 and IgG4 Fc fusion proteins containing full-length human AM or AM(6–52). Methods: Recombinant Fc-AM derivatives were generated using mammalian cells. After that, we tested the biological activities of Fc-fusion AMs (Fc-AMs) to stimulate the intracellular accumulation of cAMP in HEK-293 cells stably expressing the AM1 receptor. Next, we compared IgG1-AM (6–52) and IgG4-AM (6–52) concentrations in the peripheral blood and tissues of rats after subcutaneous injection. In addition, the antihypertensive effect of Fc-AM was examined on spontaneously hypertensive rats (SHRs) which were given high salt diets. Results: We have developed four Fc-AMs, which are long-acting AM derivatives in mammalian cells. Fc-AMs produced in mammalian cells do not require refolding or amidation. Indeed, Fc-AMs could be measured by mAM assay, which recognizes the amidated form, indicating that Fc-AMs produced in mammalian cells were amidated. Fc-AMs, generated in a mammalian cell production system can therefore easily be produced in large amounts with few purification steps. The Fc-AMs stimulated intracellular cAMP production in cultured cells stably expressing the AM type I receptor in vitro. Fc-AM (6–52) induced higher cAMP levels for the receptor than Fc-AM. The pharmacokinetic study was performed using N-terminal deficient AM derivatives, which have strong receptor binding ability. Sufficient concentrations of IgG1-AM (6–52) and IgG4-AM (6–52) were observed in blood 14 days after a single subcutaneous administration. Tissue transfer to the kidney and small intestine was observed after administration of IgG1-AM (6–52) or IgG4-AM (6–52) to rats. In addition, the single subcutaneous administration of IgG4-AM (6–52) suppressed the increase in blood pressure in SHRs which were loaded with high salt diets. Conclusion: Fc-AM produced from mammalian cells is easily prepared and seems to be an effective new therapeutic agent for hypertension.

Plasma Clearance of Intravenously Infused Adrenomedullin in Rats with Acute Renal Failure.

Plasma adrenomedullin concentrations are reportedly elevated in patients with renal failure; however, the underlying mechanism is unclear. In this study, we investigated the plasma clearance of synthetic human adrenomedullin (AM) in two models of rats with renal dysfunction; one was induced by subcutaneous injection of mercury chloride (RD-Ag) and the other by completely blocking bilateral renal blood flow (RD-Bl). Sixty minutes after starting intravenous AM infusion, AM levels in RD-Ag, RD-Bl, and rats with normal renal function (NF) were still increased slightly; however, plasma AM levels in RD-Ag rats were approximately three times as high as in RD-Bl and NF rats. Plasma AM disappearance after the end of treatment was similar among the three groups. Pharmacokinetic analysis revealed that elevated plasma AM in RD-Ag rats may be caused by a reduced volume of distribution. The adrenomedullin functional receptor is composed of heterodimers, including GPCR, CLR (calcitonin receptor-like receptor, CALCRL), and the single transmembrane proteins, RAMP2 or RAMP3 (receptor activity modifying protein). Calcrl expression was downregulated in the lungs and kidneys of RD-Ag rats. Furthermore, the plasma concentration of exogenous AM was elevated in mice deficient in vascular endothelium-specific Ramp2. These results suggest that decreased plasma AM clearance in RD-Ag is not due to impaired renal excretion but to a decreased volume of distribution caused by a reduction in adrenomedullin receptors.

Development of Long-Acting Human Adrenomedullin Fc-Fusion Proteins.

Simple SummaryAdrenomedullin (AM) is a hypotensive peptide hormone that exerts anti-inflammatory effects and is involved in wound healing and embryogenesis. However, treatment requires continuous administration as the half-life of native AM is short in blood. To resolve this, we developed four human IgG1 and IgG4 Fc-fusion proteins containing full-length hAM or hAM residues 6-52 using mammalian cells. The Fc-AM produced were amidated and in the active form. All Fc-AMs stimulated cAMP production in HEK-293 cells stably expressing the AM1 receptor. The activities of IgG1-AM (6-52) and IgG4-AM (6-52) were higher than those of IgG1-AM and IgG4-AM. Sufficient concentrations of IgG1-AM (6-52) and IgG4-AM (6-52) were observed in blood 14 days after a single subcutaneous administration. Furthermore, after IgG1-AM (6-52) or IgG4-AM (6-52) administration, tissue transfer to the kidney and small intestine was observed. Treatment with IgG4-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. Fc-AM produced from mammalian cells can be easily prepared and might be an effective novel therapeutic agent.(1) Background: Human adrenomedullin (hAM) is a hypotensive peptide hormone that exerts powerful anti-inflammatory effects. AM also had therapeutic effects in various animal experimental models of disease. However, treatment required continuous administration as the half-life of native AM is short in blood. To resolve this, we developed four human IgG1 and IgG4 Fc-fusion proteins containing full-length hAM or hAM residues 6-52. (2) Methods: We used mammalian cells to produce recombinant Fc-AM derivatives and tested the pharmacokinetics and biological activity of Fc-AM. (3) Results: We developed four Fc-fusion AMs (Fc-AM), which are long-acting AM derivatives in mammalian cells. Fc-AM had a prolonged half-life in blood and retained its ability to bind to the AM1 receptor. Fc-AM (6-52) induced higher cAMP levels for the receptor than Fc-AM. After the administration of IgG1-AM (6-52) or IgG4-AM (6-52) to rats, tissue transfer to the kidney and small intestine was observed. In addition, treatment with IgG4-AM (6-52) inhibited blood pressure increase in spontaneously hypertensive rats. (4) Conclusions: Fc-AM produced from mammalian cells can be easily prepared and might be an effective novel therapeutic agent.

Plasma clearance of intravenously infused human adrenomedullin in rats with renal dysfunction

Plasma clearance of intravenously infused human adrenomedullin in rats with renal dysfunction

Human adrenomedullin and its binding protein attenuate tissue injury and inflammation following hepatic ischemia reperfusion in rabbits

BackgroundLiver injury caused by ischemia reperfusion (I/R) during surgical procedures, such as liver resection or liver transplantation, is a major cause of liver damage and graft failure. The current method of treatment is mostly preventative (i.e., ischemic preconditioning). While a number of pharmacological modalities have been studied to reduce hepatic I/R injury, none have been entirely successful. It has been demonstrated that the administration of adrenomedullin (AM) in combination with AM-binding protein (AM/AMBP-1) exerts significant protective effects in various pathological conditions. In an effort to develop AM/AMBP-1 as a novel therapeutic for hepatic I/R injury, the present study examined the effect of a low dose of human AM, which does not induce hypotension, in combination with human AMBP-1 in a rabbit model of hepatic I/R (i.e., non-rodent species). MethodsIschemia of 70% of the liver was induced by placing a microvascular clip across the hilum of the left and median lobes for 60 min. The clip was then removed to commence reperfusion. At 15 min following clip removal (i.e., reperfusion), human AM/AMBP-1 was administered intravenously via the ear marginal vein continuously for 30 min. At 20 h, blood and tissue samples were collected for various measurements. ResultsThe serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and lactate dehydrogenase, were elevated following hepatic I/R. The administration of AM/AMBP-1 significantly decreased these levels by 58, 44, 41%, respectively. Hepatic I/R increased the direct and total bilirubin levels, whereas treatment with human AM/AMBP-1 decreased these levels by 60% and 69%, respectively. Treatment with AM/AMBP-1 also inhibited interleukin-6 gene expression by 95%. There were no changes in tumor necrosis factor-α (TNF-α) gene expression and myeloperoxidase activity (MPO), lactate and Suzuki scores after treatment. The treatment, however, reduced apoptosis post-hepatic I/R in the ischemic portion of the liver. ConclusionAdditional experiments with AM and AMBP-1 alone are needed to completely interpret the experimental results in this non-rodent species of hepatic I/R injury. The present study suggests that human AM/AMBP-1 may be developed as a novel therapeutic to attenuate hepatic I/R associated inflammation and liver injury.

Development of a novel human adrenomedullin derivative: human serum albumin-conjugated adrenomedullin.

Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.

Effect of Adrenomedullin on Migraine-Like Attacks in Patients With Migraine

Objective To determine whether the IV infusion of adrenomedullin, a potent vasodilator belonging to calcitonin family of peptides, provokes attacks of migraine in patients. Methods Twenty patients with migraine without aura participated in a placebo-controlled and double-blind clinical study. In a randomized crossover design, the patients received an IV infusion of human adrenomedullin (19.9 pmol/kg/min) or placebo (saline) administrated via an automated IV pump (20 minutes). The patients participated in 2 study days with a washout period of minimum of 7 days. The primary outcome of the study was predefined as a difference in migraine incidence (0–12 hours), and the secondary outcomes were the area under curve (AUC0–12 hours) for the headache intensity score and AUC0–90 minutes for mean arterial blood pressure (MAP), flushing, and heart rate (HR). Results Eleven patients with migraine without aura (55%) fulfilled migraine attacks criteria after adrenomedullin infusion compared to only 3 patients who reported attack (15%) after placebo (p = 0.039). We found that patients reported in a period of 0 to 12 hours stronger headache intensity after adrenomedullin compared to placebo infusion (p = 0.035). AUC0–90 minutes value for HR and flushing (p < 0.05) was significant and for MAP (p = 0.502) remained unchanged. Common reported adverse events were facial flushing, heat sensation, and palpitation (p < 0.001). Conclusion Our data implicate adrenomedullin in migraine pathogenesis. This suggests that adrenomedullin or its receptors are novel therapeutic targets for the treatment of migraine. However, we cannot discount the possibility that adrenomedullin may be acting through the canonical calcitonin gene-related peptide receptor. Trial Registration Information ClinicalTrials.gov Identifier: NCT04111484.

20 kDa PEGylated Adrenomedullin as a New Therapeutic Candidate for Inflammatory Bowel Disease

Human adrenomedullin (AM), a hypotensive peptide, also has anti-colitis activity. We prepared a polyethylene glycol (PEG) ylated form of AM through the conjugation of PEG-AM (1–15) and AM (15–52). Highly pure monomeric 20 kDa PEG-AM (20kPEG-AM) stimulated cyclic adenosine monophosphate production in HEK-293 cells stably expressing the type 1 AM receptor in a dose-dependent manner. The half-life of 20kPEG-AM was 7.4 h following subcutaneous administration in mice. We assessed the anti-colitis effect of subcutaneous 20kPEG-AM administration in the dextran sodium sulfate murine colitis model. Single and double subcutaneous injection of 20kPEG-AM significantly reduced total inflammation scores. These results suggest that 20kPEG-AM is a promising therapeutic candidate for the treatment of human inflammatory bowel diseases.

Antagonism of CGRP Signaling by Rimegepant at Two Receptors.

The “gepants” are a class of calcitonin gene-related peptide (CGRP) receptor antagonist molecules that have been developed for the prevention and treatment of migraine. Rimegepant is reported to act at the CGRP receptor, has good oral bioavailability, and has had positive clinical trial results. However, there is very little data available describing its receptor pharmacology. Importantly, rimegepant activity at the AMY1 receptor, a second potent CGRP receptor that is known to be expressed in the trigeminovascular system, has not been reported. The ability of rimegepant to antagonize activation of human CGRP, AMY1, and related adrenomedullin receptors was determined in transfected in Cos7 cells. Rimegepant was an effective antagonist at both the CGRP and AMY1 receptor. The antagonism of both CGRP and AMY1 receptors may have implications for our understanding of the mechanism of action of rimegepant in the treatment of migraine.

Thrombin rapidly digests adrenomedullin: Synthesis of adrenomedullin analogs resistant to thrombin

Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13–44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.

Expression of human adrenomedullin gene in gastric adenocarcinoma and construction and identification of adrenomedullin overexpression vector and adrenomedullin-shRNA vector

Expression of human adrenomedullin gene in gastric adenocarcinoma and construction and identification of adrenomedullin overexpression vector and adrenomedullin-shRNA vector

Polyethylene glycol-conjugated human adrenomedullin as a possible treatment for vascular dementia

Adrenomedullin (AM) is a multifunctional bioactive peptide. Recent studies have shown that AM has protective effects against ischemic brain damage. We recently prepared a long-acting human AM derivative that was conjugated with a 60 kDa polyethylene glycol (PEG-AM), which had an effect similar to that of native AM. In this study, we examined the effect of PEG-AM on four-vessel occlusion model rats, which exhibit vascular dementia. From day 10 to day 14 after surgery, the learning and memory abilities of the rats were examined using a Morris water maze. The rats were treated with a single subcutaneous injection of 1.0 or 10.0 nmol/kg of PEG-AM. PEG-AM treatment reduced the escape latency in the hidden platform test. Furthermore, the treatment increased the time spent in the platform quadrant in the probe test. The data showed that PEG-AM injection prevented memory loss and learning disorders in dose-dependent manner. On day 14, the immunoreactive AM concentration in plasma was 9.749 ± 2.167 pM in the high-dose group (10.0 nmol/kg) and 0.334 ± 0.073 pM in the low-dose group (1.0 nmol/kg). However, even in the low-dose group, a significant effect was observed in both tests.The present data indicate that PEG-AM is a possible therapeutic agent for the treatment of ischemic brain injury or vascular dementia.

2037-P: Characterization of Novel Unimolecular Amylin-Adrenomedullin Dual Agonists

Amylin and Adrenomedullin both belong to the calcitonin peptide family. While amylin plays an important role in maintaining glucose and energy homeostasis, adrenomedullin is primarily known for its beneficial vasoactive effects. We hypothesize that combining the glucoregulatory and anti-obesity properties of amylin with the cardioprotective effects of adrenomedullin could be an effective treatment strategy to address type 2 diabetes, obesity and cardiovascular diseases in one molecule. Here we report the design and receptor potencies of unimolecular peptide agonists with dual activity at the amylin and adrenomedullin receptors. Using solid-phase peptide synthesis, an array of hybrid peptides was rationally designed by substituting amino acids essential for amylin activity into the native adrenomedullin sequence. The pharmacokinetic profile was optimized by lipidation with a C20 diacid. Functional activities of the analogues were measured using a cAMP accumulation assay in cells overexpressing the human amylin receptor subtype 3 (hAMY3-R) or human adrenomedullin receptor subtype 1 (hAM1-R). All analogues were full agonists on both receptors. hAMY3R potencies were comparable to human native amylin, whereas hAM1-R potencies ranged from equipotent to 100-fold less potent compared to human native adrenomedullin. In summary, we have synthesized novel hybrid amylin-adrenomedullin peptide agonists with dual activity at the hAMY3R and hAM1-R. Ongoing studies address in vivo efficacy and potency of amylin-adrenomedullin dual agonists in rodent models of obesity and diabetes. Disclosure L.S. Dalboege: None. P. Magotti: None. N. Buch-Månson: None. E.M. Bech: None. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. S.L. Pedersen: None. K. Fosgerau: Stock/Shareholder; Self; Gubra.

Human Milk Adrenomedullin Is Unstable During Cold Storage at 4°C

Under some circumstances human milk (HM) extraction and refrigerated storage may be necessary. Depending on the length and on the type of cold storage, milk may lose some important properties, but current advices on safe HM storage are discordant. Moreover until now no data in literature were present on the effect of prolonged cold storage on biologically active components of the HM such as adrenomedullin (AM). This important peptide is involved in response to hypoxia and inflammation, associated with neovascularization, in several tissues. The aim is to evaluate: (a) the presence of AM in preterm and term HM and (b) the concentration of AM in refrigerated milk at 4°C at 24-hour intervals, up to 96 hours of storage. The experiment was repeated four times. Immediately after collection, each HM sample deriving from each mother was divided into two parts as follows: "Pool" line and "Single Mother" line. One part (Pool line) was pooled and then divided into five aliquots. The other part (Single Mother line) was divided into five aliquots. From each line, one aliquot was analyzed within 3 hours, while the others were stored in the refrigerator for 24, 48, 72, and 96 hours, respectively, and then analyzed. AM levels were determined using a specific ELISA test. AM was detectable in all samples. Its concentration was significantly higher in preterm milk with respect to term milk (p < 0.05). Significant differences were observed during the cold storage: the AM levels decreased steadily during the storage and the remaining concentration at 96 hours is ∼2%. This study provides evidences regarding the presence of AM in HM, regardless of the gestational age. In particular, the refrigeration of fresh HM in controlled conditions significantly affected its bioactivity and nutritional quality related with AM, already at 24 hours.

Abstract P334: Persistent Adrenomedullin Derivative Inhibits Development of Hypertension

Human adrenomedullin (hAM) consists of a 52 amino acid peptide that is amidated and has a disulfide bond. The bioactive peptide hAM has a variety of physiological functions, such as vasodilatation, hormone secretion, neurotransmission, embryogenesis, wound healing and immunoregulation. hAM has shown several therapeutic effects in experimental models of various diseases, including ischemic heart disease, inflammatory bowel disease, stroke and retinochoroidal disease. However, these therapies required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve these issues, the aim of this study was to synthesize novel hAM derivative, and to examine its effect in spontaneously hypertensive rats (SHR). First, we conjugated the hAM N-terminal with 60kDa polyethylene glycol (PEG) (60 kDa PEG-hAM). To demonstrate that PEG binds covalently to α-amino residue of the N-terminal tyrosine of hAM, 60 kDa PEG-hAM was digested with cyanogen bromide and the AM(6-52) thus obtained was confirmed by ion exchange chromatography and MALDI-MS. Next, to determine plasma hAM concentrations, either 60 kDa PEG-hAM (10 nmol/kg) or native hAM was administrated subcutaneously with Wistar rat. We compared hAM concentrations in the peripheral blood of rats after injection with either 60 kDa PEG-hAM or native hAM. The hAM concentrations in the 60 kDa PEG-hAM group after 1, 7 and 10 days were 2600, 740 and 280 pM, respectively. In contrast, the hAM concentrations in the native hAM group after 1 day was 6.7pM, not detect 7 and 10 days. In addition, 60kDa PEG-hAM stimulated cAMP production in cultured human embryonic kidney cells expressing a specific AM receptor. Finally, we compared blood pressor after administrated subcutaneously with either 60 kDa PEG-hAM (20 nmol/kg) or saline in SHR with high salt diet. After 9 days, a single subcutaneous administration of 60 kDa PEG-hAM inhibited development of hypertension with SHR. In summary, the plasma half-life of 60 kDa PEG-hAM was much longer than native hAM, and subcutaneous injection of 60 kDa PEG-hAM in a single dose inhibited development of hypertension with SHR. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of hypertension.

Anti-Inflammatory Effects of PEGylated Human Adrenomedullin in a Mouse DSS-Induced Colitis Model.

Preclinical Research Human adrenomedullin (hAM), a hypotensive peptide, also has anti-inflammatory effects. hAM can reduce the severity of the dextran sulphate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in animal models. Furthermore, in a clinical study, hAM treatment reduced the Disease Activity Index in ulcerative colitis. However, these therapeutic effects required continuous administration of hAM as the half-life of native hAM is quite short in blood. To resolve this problem, hAM N-terminal was conjugated with two kinds of polyethylene glycol (PEG); 5 kDa PEG or 60 kDa PEG (5 kDa PEG-hAM and 60 kDa PEG-hAM respectively). In a previous study, 5 kDa PEG-hAM stimulated cAMP production and prolonged the plasma half-life compared with native hAM. Herein we examine the effect of PEG-hAM in the DSS colitis model. Treatment with both PEG-hAM preparations reduced the total inflammation score. In addition, the plasma half-life of 60 kDa PEG-hAM was much longer than 5 kDa PEG-hAM. In summary, a single subcutaneous administration of 60 kDa PEG-hAM reduced the total inflammation score in mice with DSS-induced colitis. Therefore, these results suggest that 60 kDa PEG-hAM is a possible therapeutic agent for the treatment of inflammatory bowel disease. Drug Dev Res 78 : 129-134, 2017. © 2017 Wiley Periodicals, Inc.

Prognostic value of human mature adrenomedullin in patients with acute myocardial infarction.

Adrenomedullin (ADM) correlates with adverse cardiovascular outcomes in patients with acute myocardial infarction (AMI) and in patients with heart failure. Measurement of human mature ADM (mADM) has been difficult, and recent studies have used its surrogate - the mid-regional pro-ADM (MRproADM). Our objective was to determine whether mADM measured by a novel sandwich immunoassay, using the anti-C-terminal and an anti-mid-regional monoclonal antibody, was prognostic of 30-day, 90-day, 1-year, and 2-year major adverse cardiovascular events (MACEs) in 1111 consecutive patients who have suffered an AMI. We also compared it with the effect of MRproADM in the same patient population. A total of 311 (27.0%) patients experienced the primary endpoint at 2 years follow-up. The median (inter-quartile range) of mADM was significantly higher in patients who experienced a 2-year MACE [60.90 (44.00-86.97)] pg/ml, compared to event-free survivors [49.59 (36.20-68.15)] pg/ml (P < 0.001). mADM, taken as 1 SD of the continuous variable, was predictive of MACEs in multivariate analysis, with hazard ratios [95% confidence intervals (CIs)] at 90 days [1.28 (1.01-1.62)], 1 year [1.31 (1.08-1.59)], and 2 years [1.42 (1.07-1.64)]. It was also independently predictive of death at 1-year [1.52 (1.12-2.05)] and 2-year [1.42 (1.07-1.89)] follow-up. mADM was a better predictor of these outcomes than MRproADM, apart from death at 90 days, and combined death and heart failure hospitalization at 1 and 2 years, respectively. Human mADM can be reliably measured and predicts MACE events at medium-term follow-up, and confirms the paradigm of risk stratification using MRproADM - a surrogate for the active hormone. The relationship between mADM and MACE appears to be a continuum.

Adrenomedullin and angiotensin II signaling pathways involved in the effects on cerebellar antioxidant enzymes activity.

Human adrenomedullin (AM) is a 52-amino acid peptide involved in cardiovascular control. AM has two specific receptors formed by the calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP) 2 or 3, known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the calcitonin gene-1 related peptide receptor (CGRP1), composed of CRLR/RAMP1. In brain, AM and their receptors are expressed in several localized areas, including the cerebellum. AM has been reported as an antioxidant. Little is known about the role of AM in the regulation of cerebellar reactive oxygen species (ROS) metabolism. We assessed the effect of AM on three antioxidant enzymes activity: catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) and on thiobarbituric acid reactive substances (TBARS) production in rat cerebellar vermis, as well the receptor subtypes involved in AM actions. Additionally, we evaluated the role of angiotensin II (ANG II), protein kinase A (PKA) activity, and protein kinase C/nicotinamide adenine dinucleotide phosphate oxidase (PKC/NAD(P)H) (oxidase) pathway. Sprague-Dawley rats were sacrificed by decapitation and cerebellar vermis was microdissected under stereomicroscopic control. CAT, GPx, SOD activity and TBARS production was determined spectrophotometrically. Our findings demonstrated that in cerebellar vermis, AM decreased and ANG II increased CAT, GPx and SOD activity and TBARS production. Likewise, AM antagonized ANG II-induced increase antioxidant enzyme activity. AM(22-50) and CGRP(8-37) blunted AM-induced decrease of antioxidant enzymes activity and TBARS production indicating that these actions are mediated through AM and CGRP1 receptors. Further, PKA inhibitor (PKAi) blunted AM action and apocynin and chelerythrine reverted ANG II action, suggesting that AM antioxidant action is mediated through stimulation of PKA activity, while ANG II-induced stimulation through PKC/NAD(P)H oxidase pathway. Our results support the role of AM in the regulation of cerebellar antioxidant enzymes activity and suggest a physiological role for AM in cerebellum.

Adrenomedullin 2 Improves Early Obesity-Induced Adipose Insulin Resistance by Inhibiting the Class II MHC in Adipocytes.

MHC class II (MHCII) antigen presentation in adipocytes was reported to trigger early adipose inflammation and insulin resistance. However, the benefits of MHCII inhibition in adipocytes remain largely unknown. Here, we showed that human plasma polypeptide adrenomedullin 2 (ADM2) levels were negatively correlated with HOMA of insulin resistance in obese human. Adipose-specific human ADM2 transgenic (aADM2-tg) mice were generated. The aADM2-tg mice displayed improvements in high-fat diet-induced early adipose insulin resistance. This was associated with increased insulin signaling and decreased systemic inflammation. ADM2 dose-dependently inhibited CIITA-induced MHCII expression by increasing Blimp1 expression in a CRLR/RAMP1-cAMP-dependent manner in cultured adipocytes. Furthermore, ADM2 treatment restored the high-fat diet-induced early insulin resistance in adipose tissue, mainly via inhibition of adipocyte MHCII antigen presentation and CD4(+) T-cell activation. This study demonstrates that ADM2 is a promising candidate for the treatment of early obesity-induced insulin resistance.

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [125I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM1 receptor and further determined the region of the CLR C-tail responsible for this GRK function.