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The lipid mediators, platelet-activating factor (PAF) and lysophosphatidylcholine (LPC), play relevant pathophysiological roles in Trypanosoma cruzi infection. Several species of LPC, including C18:1 LPC, which mimics the effects of PAF, are synthesized by T. cruzi. The present study identified a receptor in T. cruzi, which was predicted to bind to PAF, and found it to be homologous to members of the progestin and adiponectin family of receptors (PAQRs). We constructed a three-dimensional model of the T. cruzi PAQR (TcPAQR) and performed molecular docking to predict the interactions of the TcPAQR model with C16:0 PAF and C18:1 LPC. We knocked out T. cruzi PAQR (TcPAQR) gene and confirmed the identity of the expressed protein through immunoblotting and immunofluorescence assays using an anti-human PAQR antibody. Wild-type and knockout (KO) parasites were also used to investigate the in vitro cell differentiation and interactions with peritoneal mouse macrophages; TcPAQR KO parasites were unable to react to C16:0 PAF or C18:1 LPC. Our data are highly suggestive that PAF and LPC act through TcPAQR in T. cruzi, triggering its cellular differentiation and ability to infect macrophages., (© 2021 John Wiley & Sons Ltd.)

The human adiponectin receptors, AdipoR1 and AdipoR2, are key anti-diabetic molecules. We previously reported the crystal structures of human AdipoR1 and AdipoR2, revealing that their seven transmembrane helices form an internal closed cavity (the closed form). In this study, we determined the crystal structure of the D208A variant AdipoR1, which is fully active with respect to the major downstream signaling. Among the three molecules in the asymmetric unit, two assume the closed form, and the other adopts the open form with large openings in the internal cavity. Between the closed- and open-form structures, helices IV and V are tilted with their intracellular ends shifted by about 4 and 11 Å, respectively. Furthermore, we reanalyzed our previous wild-type AdipoR1 diffraction data, and determined a 44:56 mixture of the closed and open forms, respectively. Thus, we have clarified the closed-open interconversion of AdipoR1, which may be relevant to its functional mechanism(s).

Background: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet., Objective and Methods: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2)., Results: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide., Conclusion: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this review article, we summarize the current knowledge on a large and diverse superfamily of seven-pass transmembrane proteins functionally independent from the GPCR superfamily. We include the newest research findings about their physiological roles and their mechanism of action. In particular, we concentrate on the structural basis for the newly discovered amide hydrolase activity, with a focus on adiponectin receptors for which structures are available. Finally, we discuss the remaining challenges in understanding the activation and signaling of these intramembrane proteins and suggest how regulation of the amide hydrolase activity may help in development of new therapeutic agents., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn 2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca 2+ binding site physically and functionally connected to the Zn 2+ providing a structural explanation for the known regulatory role of Ca 2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

As the close paralog of adiponectin, C1q/TNF-Related Protein 9 (CTRP9) has been reported to be involved in the regulation of glucose and fat metabolism, immunization and endothelial cell functions. However, information regarding the actions of Ctrp9 on reproduction is extremely limited in fish. As a first step, Ctrp9, adiponectin receptor 1 (Adipor1) and Adipor2 were identified from Nile tilapia. The open reading frame (ORF) of ctrp9 was 1020 bp which encoded a 339 amino acids. Moreover, the ORFs of adipor1 and adipor2 were 1131 bp and 1134 bp encoding 376 and 377 amino acids, respectively. Tissue distribution showed that ctrp9 mRNA levels were highest in the kidney in both sexes. And, the expression of adipor1 and adipor2 were widely distributed in all tissues examined, exhibiting high levels in the brain, gonad, gut and stomach. In addition, intraperitoneal (i.p.) injection of gCtrp9 (globular Ctrp9) suppressed the hypothalamic expression of gnrh2 (gonadotropin-releasing hormone 2) and gnrh3, as well as gthα (gonadotropic hormone α), fshβ (follicle-stimulating hormone β), lhβ (luteinizing hormone β), lhr (LH receptor) and fshr (FSH receptor) mRNA levels in the pituitary. The mRNA levels of adipor1, but not adipor2, in the gonads were also inhibited after injection. Moreover, the levels of serum E2 (estrogen) in female and T (testosterone) in male were significantly decreased after injection of gCtrp9. Overall, our data provides novel data indicating, for the first time, a regulatory effect of CTRP9 on teleost reproduction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of in vitro and in vivo studies over last two decades. These findings suggest that AdipoRs' agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs' agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1's peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions via surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins., Competing Interests: B.B.K. is a salaried employee of EncuraGen, S.K. is a salaried employee of Polus Inc., and Y.J.S. is a salaried employee of Samhyun Inc. With regards to the patent related to the submission, there is a registered patent in Korea. The patent number is 10-1838622 and the title is Agonist Peptide for Adiponectin Receptor. Also, a PCT application was filed as of April 10, 2017 and the application number is 10-2017-0037762. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1.

The adiponectin axis regulates cardiometabolic and inflammatory tone making it an attractive therapeutic focus. Rudimentary understanding of the adiponectin receptors, AdipoR1 and AdipoR2, constrains our ability to target these atypical seven trans-membrane proteins. Here, we aimed to further elaborate the molecular details governing cell-surface expression and signal transduction by transient expression of AdipoR1 or AdipoR2 in HEK293 cells. Following serum starvation, adiponectin reduced cell-surface expression of both receptors, consistent with internalisation, and promoted phosphorylation of downstream effectors. Temporal phosphorylation profiles differed with AdipoR1 and AdipoR2 transduced signals peaking at 15 min and 24 h. Analysis of receptor chimeras showed that the non-conserved N-terminal trunks (AdipoR1(1-70) and AdipoR2(1-81)) define the temporal signalling profiles and contain multiple regions that promote or inhibit cell-surface expression, respectively. These findings highlight the importance of the non-conserved N-terminal trunks and demonstrate that cell-surface expression of AdipoR1 and AdipoR2 is required for effective coupling to downstream effectors., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Adiponectin exerts an antidiabetic function through the adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2). The mechanism regulating the expression of adiponectin receptors remains to be elucidated. Bioinformatics analysis demonstrated that microRNA (miR)‑218 targets the 3' untranslated region (3'UTR) of the AdipoR2 mRNA. The present study aimed to investigate whether miR-218 regulated the expression of AdipoR2 using immunoblotting, reverse transcription quantitative polymerase chain reaction and luciferase assays. The protein level and the mRNA level of AdipoR2 were reduced when miR‑218 was expressed in HepG2 cells. Additionally, overexpression of miR‑218 repressed the activity of a luciferase reporter containing the 3'UTR of AdipoR2. Furthermore, the present study aimed to determine whether miR-218 regulated glucose metabolism through detecting signaling pathways and glucose uptake. The phosphorylation of AMP‑activated protein kinase and p38 mitogen‑activated protein kinase was reduced in miR‑218‑expressing cells. In addition, miR‑218 inhibited adiponectin‑induced glucose uptake. The present results suggested that miR‑218 targets AdipoR2 to inhibit adiponectin signaling.

Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5' AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89-375) and AdipoR2Δ99 (residues 100-386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.

Adiponectin receptor 1 (AdipoR1) mediates adiponectin's pleiotropic effects in muscle and liver and plays an important role in the regulation of insulin resistance and diabetes. Here, we demonstrate a pivotal role for microRNA-221 (miR-221) and the RNA-binding protein polypyrimidine tract-binding protein (PTB) in posttranscriptional regulation of AdipoR1 during muscle differentiation and in obesity. RNA-immunoprecipitation and luciferase reporter assays illustrated that both PTB and miR-221 bind AdipoR1-3'UTR and cooperatively inhibit AdipoR1 translation. Depletion of PTB or miR-221 increased, while overexpression of these factors decreased, AdipoR1 protein synthesis in both muscle and liver cells. During myogenesis, downregulation of PTB and miR-221 robustly induced AdipoR1 translation, providing a mechanism for enhanced AdipoR1 protein expression and activation in differentiated muscle cells. In addition, since both PTB and miR-221 are upregulated in liver and muscle of genetic and dietary mouse models of obesity, this novel translational mechanism may be at least partly responsible for the reduction in AdipoR1 protein levels in obesity. These findings highlight the importance of translational control in regulating AdipoR1 protein expression and adiponectin signaling. Given that adiponectin is reduced in obesity, induction of AdipoR1 could potentially enhance adiponectin beneficial effects and ameliorate insulin resistance and diabetes.

The discovery of adiponectin and subsequently the receptors it acts upon have lead to a great surge forward in the understanding of the development of insulin resistance and obesity-linked diseases. Adiponectin is a hormone that is derived from adipose tissue and is reduced in obesity-linked diseases including insulin resistance/type 2 diabetes and atherosclerosis. Adiponectin exerts its effects by binding to adiponectin receptors, two of which, AdipoR1 and AdipoR2, have been cloned. This has enabled researchers to carry out detailed studies elucidating the role played by these receptors and the metabolic pathways that are involved following their activation. Such studies have clearly shown that the stimulation of these receptors is associated with glucose homeostasis and ongoing research into their role will clarify the underlying molecular mechanisms of adiponectin. Such knowledge can then be used to provide therapeutic targets aimed at managing obesity-linked diseases including type 2 diabetes and metabolic syndrome., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to β2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including β1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2., (© 2013.)

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.

Adiponectin is a fat-derived hormone whose reduction plays central roles in obesity-linked diseases including insulin resistance/type 2 diabetes and atherosclerosis. The cloning of Adiponectin receptors AdipoR1 and AdipoR2 has stimulated adiponectin research, revealing pivotal roles for AdipoRs in pleiotropic adiponectin actions, as well as some postreceptor signaling mechanisms. Adiponectin signaling has thus become one of the major research fields in metabolism and clinical medicine. Studies on AdipoRs will further our understanding of the role of adiponectin in obesity-linked diseases and shortened life span and may guide the design of antidiabetic and antiaging drugs with AdipoR as a target., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Receptor-ligand interactions represent one of the most basic processes in biological systems. Receptor activation and deactivation induce or prevent a series of downstream signaling events that ultimately result in normal or abnormal cellular functions. Contemporary biology is in continuous search for the identification of novel receptors and their ligands. The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including leptin and adiponectin. A recent discovery and design process for leptin and adiponectin receptor response modifier peptides can be generalized to a series of transmembrane receptor ligands. A family of 11-13 amino acid residue-long leptin receptor (ObR) agonists has been identified by analyzing the effect of peptides corresponding to the three presumed active sites of leptin on the growth of leptin-responsive cancer cells. In the case of adiponectin, overlapping peptides were walked across the entire globular domain of the protein to identify the active site and derive adiponectin receptor (AdipoR) agonist peptides. In both sets, native residues were replaced by nonnatural analogs to improve the pharmacological properties including stability, efficacy and targeting. Later the ObR analogs were converted into true ObR antagonists that show antagonist-agonist selectivity of 1,000 in cellular assays. The design process of ObR antagonists included shortening of the peptide length and incorporating additional nonnatural residues. Here I take a look into this receptor agonist and antagonist discovery process from a practical point of view.

The anterograde trafficking of GPCR has been described as a tightly controlled process involving specific amino acid sequences that mediate the receptor transport. In this study, we investigated whether the cell surface delivery of the adiponectin receptor 1, a newly identified class of heptahelix receptors different from G protein-coupled receptors, is regulated. Sequential N-terminal deletion revealed that the export of the AdipoR1 from the endoplasmic reticulum (ER) is controlled by distinct parts of the receptor N-terminus. Strong evidence is provided that the ER exit is mediated by two specific sequences, a F(X)(3)F(X)(3)F and a D(X)(3)LL motif. Disruption of these motifs led to a substantial accumulation of the AdipoR1 in the ER. Mutation of similar motifs in the AdipoR1 C-terminus did not result in aberrant receptor localization, suggesting that these motifs are sequence and position specific to the AdipoR1 N-terminus. Further analysis of the regulation mechanism identified an interaction with the chaperone BiP and additionally, strong evidence is provided that both motifs exert different biological function in the AdipoR1 ER export. In conclusion, our data demonstrate that the receptor transport shares similar ER exit motifs although AdipoR are structurally different from GPCR. However, since even two specific sequences are identified, the anterograde trafficking of the AdipoR1 seems to be regulated in a more complex manner., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Adiponectin (Ad) is an abundant protein hormone regulatory of numerous metabolic processes. The 30 kDa protein originates from adipose tissue, with full-length and globular domain circulatory forms. A collagenous domain within Ad leads to spontaneous self-assemblage into various oligomeric isoforms, including trimers, hexamers, and high-molecular-weight multimers. Two membrane-spanning receptors for Ad have been identified, with differing concentration distribution in various body tissues. The major intracellular pathway activated by Ad includes phosphorylation of AMP-activated protein kinase, which is responsible for many of Ad's metabolic regulatory, anti-inflammatory, vascular protective, and anti-ischemic properties. Additionally, several AMP-activated protein kinase-independent mechanisms responsible for Ad's anti-inflammatory and anti-ischemic (resulting in cardioprotective) effects have also been discovered. Since its 1995 discovery, Ad has garnered considerable attention for its role in diabetic and cardiovascular pathology. Clinical observations have demonstrated the association of hypoadiponectinemia in patients with obesity, cardiovascular disease, and insulin resistance. In this review, we elaborate currently known information about Ad malfunction and deficiency pertaining to cardiovascular disease risk (including atherosclerosis, endothelial dysfunction, and cardiac injury), as well as review evidence supporting Ad resistance as a novel risk factor for cardiovascular injury, providing insight about the future of Ad research and the protein's potential therapeutic benefits.

Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm in vivo the molecular modelling data, osmotin has been purified from nicotiana tabacum seeds and its nine residues peptide synthesized. We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico.

Background: Although lipids transfer through placenta is very limited, modification in dietary fatty acids can lead to implications in fetal and postnatal development. Trans fatty acid (TFA) intake during gestation and lactation have been reported to promote dyslipidemia and increase in pro- inflammatory adipokines in offspring. The aim of this study was to evaluate whether the alterations on pro-inflammatory cytokines and dyslipidemia observed previously in 21-d-old offspring of rats fed a diet containing hydrogenated vegetable fat during gestation and lactation were related to alterations in TLR-4, TRAF-6 and adipo-R1 receptor in white adipose tissue and muscle. On the first day of gestation, rats were randomly divided into two groups: (C) received a control diet, and (T) received a diet enriched with hydrogenated vegetable fat, rich in trans fatty acids. The diets were maintained throughout gestation and lactation. Each mother was given eight male pups. On the 21st day of life the offspring were killed. Blood, soleus and extensor digital longus (EDL) muscles, and retroperitoneal (RET) white adipose tissue were collected., Results: 21-d-old of T rats had higher serum triacylglycerols, cholesterol, and insulin. The Adipo R1 protein expression was lower in RET and higher in EDL of T group than C. TLR-4 protein content in all studied tissues were similar between groups, the same was verified in TRAF-6 protein expression in soleus and EDL. However, TRAF-6 protein expression in RET was higher in T than C., Conclusion: These results demonstrated that maternal ingestion of hydrogenated vegetable fat rich in TFAs during gestation and lactation decrease in Adipo R1 protein expression and increase in TRAF-6 protein expression in retroperitoneal adipose tissue, but not in skeletal muscle, which could contributed for hyperinsulinemia and dyslipidemia observed in their 21-d-old offspring.

AdipoR1 and AdipoR2 are newly discovered members of the huge family of seven-transmembrane receptors, but both receptors are structurally and functionally different from G-protein-coupled receptors. Little is known about the oligomerization of the AdipoRs. Here, we show the presence of endogenous AdipoR1 dimers in various cell lines and human muscle tissue. To directly follow and localize the dimerization, we applied bimolecular fluorescence complementation (BiFC) in combination with flow cytometry. We visualized and quantified AdipoR1 homodimers in HEK293 cells. Moreover, we identified a GxxxG dimerization motif in the fifth transmembrane domain of the AdipoR1. By mutating both glycine residues to phenylalanine or glutamic acid, we were able to modulate the dimerization of AdipoR1, implicating a role for the GxxxG motif in AdipoR1 dimerization. Furthermore, we tested whether the AdipoR1 ligand adiponectin had any influence on receptor dimerization. Interestingly, we found that adiponectin decreases the receptor dimerization in a concentration-dependent manner. This effect is mainly mediated by segments of the collagen-like domain of full-length adiponectin. Accordingly, this is the first direct read-out signal of adiponectin at the AdipoR1 receptor, which revealed the involvement of specific amino acids of both the receptor and the ligand to modulate the quaternary structure of the AdipoR1.

Adiponectin is an adipokine with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Its effects on energy homeostasis, glucose and lipid metabolism are mediated by two ubiquitously expressed seven-transmembrane receptors, AdipoR1 and -R2. With the exception of APPL1 and RACK1, no intracellular binding partners of adiponectin receptors are reported and thus signaling pathways downstream of these receptors remain largely unknown. To incorporate adiponectins protective potential in drug development it is essential to understand adiponectin signaling cascades in detail. A yeast two-hybrid approach employing AdipoR1s cytoplasmatic N-terminus led to the identification of the regulatory subunit of protein kinase CK2. We confirmed the interaction in co-immunoprecipitation, ELISA experiments and co-localization analysis in mammalian cells. Furthermore we could localize the interaction site in an N-terminal basic region close to the transmembrane domain. In adiponectin stimulation experiments of C2C12 mouse myotubes and MCF7 cells incorporating CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benz-imidazole (DMAT) we found a modulator role of CK2 in adiponectin signaling. Accordingly we identified the regulatory subunit of protein kinase CK2 as a novel intracellular partner of AdipoR1 and have strong evidence of CK2 as an effector molecule in adiponectin signaling. Since CK2 is involved in signaling cascades of other adipokines and hormones, e.g. leptin and insulin, our findings suggest a possible key function in crosstalk between adiponectin and insulin signaling pathways and could provide further insight into the anti-diabetic effects of adiponectin.

To establish a method for preparation of anti-mouse adiponectin receptor-1 (AdipoR-1) polyclonal antibody, the polypeptide antigen corresponding to AdipoR-1 was designed by bioinformatics analysis. The possible physicochemical property and trans-membrane structure were predicted by ExPASy and TMHMM, respectively. The antigen epitopes of mouse AdipoR-1 and its immunogenicity were analyzed by Antigenic Prediction and AntigenProfiler, respectively. According to the similarity analysis between AdipoR-1 and AdipoR-2 by Clustal W, a 16-amino acid polypeptide was designed as the antigen corresponding to AdipoR-1. To ensure the specificity of the polypeptide antigen, similarity search was run in the protein databases such as SWISS-PROT, PDB and Prosite databases. The polypeptide synthesized by solid-phase synthesis was used as immunogen to immunize rats to obtain anti-mouse AdipoR-1 polyclonal antibodies, the specificity and titer of which was identified by Western blot and indirect ELISA. The antibodies were applied to detect the AdipoR-1 expression in the muscle tissue in normal and cholesterolemic mice. The results from bioinformatics analysis showed that the similarity of amino acid sequences between AdipoR-1 and AdipoR-2 in mouse was 66%, and the designed polypeptide antigen corresponding to AdipoR-1 exhibited excellent immunogenicity (score=3.1). Using the polypeptide as antigen for immunization, anti-mouse AdipoR-1 polyclonal antibodies with high titer and good specificity were obtained. The results of Western blot demonstrated that there was no statistical difference in AdipoR-1 expression in muscle tissue between normal (1.80±0.06) and cholesterolemic mice (1.71±0.11). These results suggest that the antigen epitopes of mouse AdipoR-1 are well predicted by bioinformatics analysis, and successful preparation of the specific anti-AdipoR-1 polyclonal antibodies provides a useful tool for identification and further functional study of AdipoR-1.

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.