Neuroprotective effect of osmotin against ethanol-induced apoptotic neurodegeneration in the developing rat brain

Fetal alcohol syndrome is a neurological and developmental disorder caused by exposure of developing brain to ethanol. Administration of osmotin to rat pups reduced ethanol-induced apoptosis in cortical and hippocampal neurons. Osmotin, a plant protein, mitigated the ethanol-induced increases in cytochrome c, cleaved caspase-3, and PARP-1. Osmotin and ethanol reduced ethanol neurotoxicity both in vivo and in vitro by reducing the protein levels of cleaved caspase-3, intracellular [Ca²⁺]_cyt, and mitochondrial transmembrane potential collapse, and also upregulated antiapoptotic Bcl-2 protein. Osmotin is a homolog of adiponectin, and it controls energy metabolism via phosphorylation. Adiponectin can protect hippocampal neurons against ethanol-induced apoptosis. Abrogation of signaling via receptors AdipoR1 or AdipoR2, by transfection with siRNAs, reduced the ability of osmotin and adiponectin to protect neurons against ethanol-induced neurodegeneration. Metformin, an activator of AMPK (adenosine monophosphate-activated protein kinase), increased whereas Compound C, an inhibitor of AMPK pathway, reduced the ability of osmotin and adiponectin to protect against ethanol-induced apoptosis. Osmotin exerted its neuroprotection via Bcl-2 family proteins and activation of AMPK signaling pathway. Modulation of AMPK pathways by osmotin, adiponectin, and metformin hold promise as a preventive therapy for fetal alcohol syndrome.     

Receptor for activated C-kinase 1, a novel binding partner of adiponectin receptor 1

Adiponectin is an adipose tissue derived hormone with anti-diabetic and insulin-sensitizing properties. Two adiponectin receptors, AdipoR1 and AdipoR2, have recently been identified, yet the signaling pathways triggered through adiponectin receptors remain to be elucidated. Using a yeast two-hybrid screen, we identified an adaptor protein, receptor for activated protein kinase C1 (RACK1), as an interacting partner of human AdipoR1. RACK1 was confirmed to interact with AdipoR1 by co-immunoprecipitation and co-localization analysis in mammalian cells. The interaction was enhanced by adiponectin stimulation. In addition, the knockdown of RACK1 by RNA interference inhibited adiponectin-stimulated glucose uptake in HepG2 cells. These results suggest that RACK1 may act as a key bridging factor in adiponectin signaling transduction through interacting with AdipoR1.     

APPL1 binds to adiponectin receptors and mediates adiponectin signalling and function

Adiponectin, also known as Acrp30, is an adipose tissue-derived hormone with anti-atherogenic, anti-diabetic and insulin sensitizing properties. Two seven-transmembrane domain-containing proteins, AdipoR1 and AdipoR2, have recently been identified as adiponectin receptors, yet signalling events downstream of these receptors remain poorly defined. By using the cytoplasmic domain of AdipoR1 as bait, we screened a yeast two-hybrid cDNA library derived from human fetal brain. This screening led to the identification of a phosphotyrosine binding domain and a pleckstrin homology domain-containing adaptor protein, APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding (PTB) domain and leucine zipper motif). APPL1 interacts with adiponectin receptors in mammalian cells and the interaction is stimulated by adiponectin. Overexpression of APPL1 increases, and suppression of APPL1 level reduces, adiponectin signalling and adiponectin-mediated downstream events (such as lipid oxidation, glucose uptake and the membrane translocation of glucose transport 4 (GLUT4)). Adiponectin stimulates the interaction between APPL1 and Rab5 (a small GTPase) interaction, leading to increased GLUT4 membrane translocation. APPL1 also acts as a critical regulator of the crosstalk between adiponectin signalling and insulin signalling pathways. These results demonstrate a key function for APPL1 in adiponectin signalling and provide a molecular mechanism for the insulin sensitizing function of adiponectin.     

Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling

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.     

Insulin‐regulated expression of adiponectin receptors in muscle and fat cells

Adp (adiponectin), an adipocyte‐secreted hormone, exerts its effect via its specific receptors, AdipoR1 and AdipoR2 (adiponectin receptors 1 and 2), on insulin‐sensitive cells in muscle, liver and adipose tissues, and plays an important role in lipid and glucose metabolisms. The study has investigated the effect of insulin on AdipoRs expression in muscle and fat cells. Differentiated fat [3T3‐L1 (mouse adipocytes)], L6 (skeletal muscle) and vascular smooth muscle (PAC1) cells were serum starved and exposed to 100 nM insulin for 1–24 h. AdipoR1 and AdipoR2 mRNAs expression was monitored by real‐time PCR. The results demonstrate that insulin down‐regulates both AdipoR1 and AdipoR2 mRNAs levels in a biphasic manner in L6 and PAC1 cells. Insulin had little or no effect in the regulation of AdipoR1 expression in 3T3‐L1 cells, but significantly up‐regulated AdipoR2 mRNA level in a biphasic manner. The fact that insulin differentially regulates the expression of AdipoR1 and AdipoR2 in muscle and fat cells suggests this is also dependent on the availability of the endogenous ligand, such as Adp for AdipoR1 and AdipoR2 in fat cells. The effects of globular Adp were also tested on insulin‐regulated expression of AdipoRs in L6 cells, and found to up‐regulate and counter insulin‐mediated suppression of AdipoRs expression in L6 cells.     

Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.     

Adiponectin inhibits lymphotoxin-β receptor-mediated NF-κB signaling in human umbilical vein endothelial cells

Adiponectin exerts anti-diabetic and anti-atherogenesis properties through its 2 receptors (AdipoR1 and AdipoR2). However, the signaling pathways responsible for the anti-inflammatory effects of adiponectin are largely unknown. In this study, we identified the lymphotoxin (LT)-β receptor (LTBR) as an interacting partner of human AdipoR1 by using a yeast two-hybrid screening. The interaction between LTBR and AdipoR1 was confirmed by co-immunoprecipitation and co-localization analysis. Furthermore, adiponectin incubation inhibited lymphotoxin-induced NF-κB activation and the expression of adhesion molecules in human umbilical vein endothelial cells. These results indicated that AdipoR1 interacted with LTBR and mediated the inhibition of LTBR-activated NF-κB pathway.     

Adiponectin-Mediated Analgesia and Anti-Inflammatory Effects in Rat

The adipose tissue-derived protein, adiponectin, has significant anti-inflammatory properties in a variety of disease conditions. Recent evidence that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in central nervous system, suggests that it may also have a central modulatory role in pain and inflammation. This study set out to investigate the effects of exogenously applied recombinant adiponectin (via intrathecal and intraplantar routes; 10–5000 ng) on the development of peripheral inflammation (paw oedema) and pain hypersensitivity in the rat carrageenan model of inflammation. Expression of adiponectin, AdipoR1 and AdipoR2 mRNA and protein was characterised in dorsal spinal cord using real-time polymerase chain reaction (PCR) and Western blotting. AdipoR1 and AdipoR2 mRNA and protein were found to be constitutively expressed in dorsal spinal cord, but no change in mRNA expression levels was detected in response to carrageenan-induced inflammation. Adiponectin mRNA, but not protein, was detected in dorsal spinal cord, although levels were very low. Intrathecal administration of adiponectin, both pre- and 3 hours post-carrageenan, significantly attenuated thermal hyperalgesia and mechanical hypersensitivity. Intrathecal administration of adiponectin post-carrageenan also reduced peripheral inflammation. Intraplantar administration of adiponectin pre-carrageenan dose-dependently reduced thermal hyperalgesia but had no effect on mechanical hypersensitivity and peripheral inflammation. These results show that adiponectin functions both peripherally and centrally at the spinal cord level, likely through activation of AdipoRs to modulate pain and peripheral inflammation. These data suggest that adiponectin receptors may be a novel therapeutic target for pain modulation.     

Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed

The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.     

Yin-Yang Regulation of Adiponectin Signaling by APPL Isoforms in Muscle Cells

APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells.     

Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 μg/ml) or full-length (5 μg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.     

Insulin-sensitizing effects of thiazolidinediones are not linked to adiponectin receptor expression in human fat or muscle

Circulating adiponectin levels are increased by the thiazolidinedione (TZD) class of PPARgamma agonists in concert with their insulin-sensitizing effects. Two receptors for adiponectin (AdipoR1 and AdipoR2) are widely expressed in many tissues, but their physiological significance to human insulin resistance remains to be fully elucidated. We examined the expression patterns of AdipoR1 and AdipoR2 in fat and skeletal muscle of human subjects, their relationship to insulin action, and whether they are regulated by TZDs. Expression patterns of both AdipoRs were similar in subcutaneous and omental fat depots, with higher expression in adipocytes than in stromal cells and macrophages. To determine the effects of TZDs on AdipoR expression, subcutaneous fat and quadriceps muscle were biopsied in 14 insulin-resistant subjects with type 2 diabetes mellitus after 45 mg pioglitazone or placebo for 21 days. This duration of pioglitazone improved insulin's suppression of glucose production by 41% and enhanced stimulation of glucose uptake by 27% in concert with increased gene expression and plasma levels of adiponectin. Pioglitazone did not affect AdipoR expression in muscle, whole fat, or cellular adipose fractions, and receptor expression did not correlate with baseline or TZD-enhanced insulin action. In summary, both adiponectin receptors are expressed in cellular fractions of human fat, particularly adipocytes. TZD administration for sufficient duration to improve insulin action and increase adiponectin levels did not affect expression of AdipoR1 or AdipoR2. Although TZDs probably exert many of their effects via adiponectin, changes in these receptors do not appear to be necessary for their insulin-sensitizing effects.     

Structural and functional similarities between osmotin from Nicotiana tabacum seeds and human adiponectin

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.     

ATF3 negatively regulates adiponectin receptor 1 expression

Adiponectin is an adipocyte-derived hormone that has antidiabetic and antiatherogenic effects through two membrane receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). Although it has been reported that the expression of AdipoR1 and AdipoR2 is regulated under physiological and pathophysiological states, their regulation is largely unknown. Previously, we demonstrated that endoplasmic reticulum (ER) stress or obesity-inducible ATF3 negatively regulates the expression of adiponectin and AdipoR2. Here, we investigated the regulation of another adiponectin receptor, AdipoR1 by ATF3, to determine if ATF3 may contribute to impairment of adiponectin signaling by repressing the expression of both adiponectin and adiponectin receptors. We found that treatment with thapsigargin, a stimulator of ATF3 expression as an inducer of ER stress, decreased AdipoR1 expression in insulin-sensitive cells (HepG2, C2C12) and insulin secreting cells (MIN6N8). Furthermore, overexpression of lentivirus carrying-ATF3 decreased AdipoR1 expression in those cells, demonstrating that ATF3 downregulates AdipoR1 expression. Next, we investigated the effects of ATF3 on human AdipoR1 promoter activity and identified an ATF3-responsive region in the promoter. Both thapsigargin treatment and ATF3 expression repressed AdipoR1 promoter activity. Transfection studies using mutant constructs containing 5′-deletions in the human AdipoR1 promoter revealed that putative ATF/CRE site is located between the −248 and −224, TGACGCGG. Chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to human AdipoR1 promoter spanning from −248 to −224. Finally, deletion of the putative ATF/CRE site abrogated ATF3-mediated transrepression of the AdipoR1 promoter. Importantly, ATF3 expression was increased in hyperglycemia or TNF-α-treated C2C12 cells in which AdipoR1 expression was decreased, suggesting that ATF3 may contribute to downregulation of AdipoR1 by hyperglycemia and TNF-α. Collectively, these results demonstrate that ATF3 negatively regulates human AdipoR1 expression via binding to an ATF3-responsive region in the promoter, which plays an important role in attenuation of adiponectin signaling and induction of insulin resistance.     

Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors expressed in macrophages where they control cholesterol homeostasis and inflammation. In an attempt to identify new PPARalpha and PPARgamma target genes in macrophages, a DNA array-based global gene expression profiling experiment was performed on human primary macrophages treated with specific PPARalpha and PPARgamma agonists. Surprisingly, AdipoR2, one of the two recently identified receptors for adiponectin, an adipocyte-specific secreted hormone with anti-diabetic and anti-atherogenic activities, was found to be induced by both PPARalpha and PPARgamma. AdipoR2 induction by PPARalpha and PPARgamma in primary and THP-1 macrophages was confirmed by Q-PCR analysis. Interestingly, treatment with a synthetic LXR agonist induced the expression of both AdipoR1 and AdipoR2. Furthermore, co-incubation with a PPARalpha ligand and adiponectin resulted in an additive effect on the reduction of macrophage cholesteryl ester content. Finally, AdipoR1 and AdipoR2 are both present in human atherosclerotic lesions. Moreover, AdipoR1 is more abundant than AdipoR2 in monocytes and its expression decreases upon differentiation into macrophages, whereas AdipoR2 remains constant. In conclusion, AdipoR1 and AdipoR2 are expressed in human atherosclerotic lesions and macrophages and can be modulated by PPAR and LXR ligands, thus identifying a mechanism of crosstalk between adiponectin and these nuclear receptor signaling pathways.     

Endocytosis of adiponectin receptor 1 through a clathrin- and Rab5-dependent pathway

In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogenesis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Eps15 mutants or depleting K(+) trapped AdipoR1 at the plasma membrane, and K(+) depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoR1 and adiponectin is clathrin-dependent. Depletion of K(+) and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might downregulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoR1 is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.     

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2β, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.     

Atractylenolide III ameliorates Non-Alcoholic Fatty Liver Disease by activating Hepatic Adiponectin Receptor 1-Mediated AMPK Pathway

Background: Nonalcoholic fatty liver disease (NAFLD) is the most frequent cause of chronic liver diseases worldwide. At present, there are no effective pharmacological therapies for NAFLD except lifestyle intervention-mediated weight loss. Atractylenolide III (ATL III), the major bioactive component found in Atractylode smacrocephala Koidz, has been shown to exert anti-oxidant, anti-tumor, anti-allergic response, anti-bacterial effects and cognitive protection. Here we investigate the therapeutic potential and underlying mechanisms of ATL III for the treatment of NAFLD.Methods: Male C57BL/6J mice were fed a high-fat diet (HFD) and treated with ATL III. Lipid accumulation was analyzed by Oil Red O staining in liver tissues and free fatty acids (FFAs)-treated hepatocytes. AMP-activated protein (AMPK) and sirtuin 1(SIRT1) signaling pathways were inhibited by Compound C and EX527 in vitro, respectively. Small-interfering RNA (siRNA) was used to knockdown adiponectin receptor 1 (AdipoR1) expression in HepG2 cells.Results: ATL III treatment ameliorated liver injury and hepatic lipid accumulation in the HFD-induced NAFLD mouse model as demonstrated by that ATL III administration significantly reduced serum levels of alanine aminotransferase, glutamic oxaloacetic transaminase, triglycerides, total cholesterol and low-density lipoprotein. Furthermore, treatment with ATL III alleviated hepatic oxidative stress, inflammation and fibrosis in the HFD feeding model. To study the underlying mechanisms, we performed Computer Aided Design assay and found that open-formed AdipoR1 and adiponectin receptor 2 were the potential receptors targeted by ATL III. Interestingly, HFD feeding or FFAs treatment only reduced hepatic AdipoR1 expression, while such reduction was abolished by ATL III administration. In addition, in vitro treatment with ATL III activated the AdipoR1 downstream AMPK /SIRT1 signaling pathway and reduced lipid deposition in HepG2 cells, which was diminished by silencing AdipoR1. Finally, inhibition of AMPK or SIRT1, the AdipoR1 downstream signaling, abolished the protective effects of ATL III on lipid deposition and oxidative stress in FFAs-treated HepG2 cells.Conclusion: Our findings suggest that ATL III is a therapeutic drug for the treatment of NAFLD and such protective effect is mediated by activating hepatic AdipoR1-mediated AMPK/SIRT1 signaling pathway.     

Evidence for a protective role for adiponectin in osteoarthritis

Obesity has been associated with an increased risk of osteoarthritis (OA). However, the mechanism by which obesity contributes to OA remains uncertain. Adiponectin, an adipocyte-derived hormone, has shown anti-diabetic and anti-atherogenic properties. In the present study, we aimed to investigate the potential role of adiponectin in OA disease. We demonstrated that adiponectin was present in OA synovial fluid (SF) and its expression level was almost 100-fold decrease compared with that in OA plasma. FPLC and ELISA studies revealed the distribution and abundance of the adiponectin complexes in plasma and SF from patients with OA. The percentage of high molecular weight (HMW) per total adiponectin in OA SF was lower than in OA plasma, while that of the hexamer form was similar and the trimer form was higher. The expression levels of adiponectin receptors AdipoR1 and AdipoR2 were examined in human OA tissues by RT-PCR. AdipoR1 was abundantly expressed in cartilage, bone and synovial tissues, whereas AdipoR2 was rarely detected. Finally, the effects of adiponectin on primary chondrocyte functions were studied by using antibody-based protein array and RT-PCR. The patterns of mRNA expression and protein production strongly indicate that adiponectin is involved in the modulation of cartilage destruction in chondrocytes by up-regulating TIMP-2 and down-regulating IL-1beta-induced MMP-13. Together these findings clearly indicate that the adiponectin may act as a protective role in the progression of OA, and this also provide new thinking on the relationship between obesity and OA.