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1.  Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway 
PLoS ONE  2013;8(1):e49915.
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.
doi:10.1371/journal.pone.0049915
PMCID: PMC3551944  PMID: 23349663
2.  Adiponectin and its receptors are expressed in adult ventricular cardiomyocytes and upregulated by activation of peroxisome proliferator-activated receptor γ 
Adiponectin is a protein hormone involved in maintaining energy homeostasis in metabolically active tissues. It enhances glucose and lipid metabolism via activation of AMP-dependent kinase (AMPK) in skeletal muscle and liver. Energy homeostasis is vital for the heart to work as a pump. In this study, we investigated whether adiponectin and its receptors are expressed in adult ventricular cardiomyocytes. We observed adiponectin transcript and protein in cultured ventricular cardiomyocytes isolated from adult rat, by quantitative real-time PCR, ELISA assays, Western blots, and immunofluorescent staining. In addition, we detected adiponectin receptor (AdipoR1 and AdipoR2) expression in the heart. AdipoR1 was expressed in rat myocardium at a level of about 50% of that in skeletal muscle; whereas adipoR2 was expressed at a similar level to that in liver. Rosiglitazone, a Peroxisome proliferator activated receptor γ (PPARγ) activator, substantially elevated expression of adiponectin in cultured cardiomyocytes and its secretion into cultured media. Rosiglitazone also increased adipoR1 and adipoR2 expression in cardiomyocytes. Treatment of recombinant globular adiponectin in cultured cardiomyocytes increased fatty acid oxidation and glucose uptake via activation of AMPK, suggesting a role for adiponectin in cardiac energy metabolism. Together, these data establish the existence of a local cardiac-specific adiponectin system that is regulated by PPARγ. Moreover, these findings indicate a role for adiponectin on normal myocardial energy homeostasis, in part, through the activation of AMPK.
doi:10.1016/j.yjmcc.2007.04.014
PMCID: PMC1950729  PMID: 17532004
adiponectin; adipoR1; adipoR2; rosiglitazone; AMPK; Fatty acid oxidation; glucose uptake; cardiomyocytes
3.  A Novel Role of Globular Adiponectin in Treatment with HFD/STZ Induced T2DM Combined with NAFLD Rats 
The Scientific World Journal  2014;2014:230835.
Aims. To evaluate the effects of globular adiponectin (gAd) on treatment of type 2 diabetic rats combined with NAFLD. Materials and Methods. Twenty-one male wistar rats were fed with normal diet (7 rats) or high fat diet (HFD) (14 rats) for 4 weeks, and then HFD-fed rats were injected with streptozotocin (STZ) to induce type 2 diabetes mellitus (T2DM). Half of T2DM rats were randomly injected with gAd intraperitoneally for 7 days. The expressions of adiponectin receptors (adipoR1/R2) in liver and skeletal muscle tissues were detected through western blotting or RT-qPCR, respectively. Results. Globular adiponectin alleviated the hepatic steatosis and increased insulin secretion. In liver, both the protein and mRNA expressions of adipoR2 in T2DM group decreased (P < 0.05, resp.) in contrast to NC group and increased (P < 0.05 and P < 0.001, resp.) after gAd treatment. But the protein and mRNA expressions of adipoR1 increased (P < 0.05, resp.) in T2DM group and no change was found in the gAd-treated group. In skeletal muscle, the protein and mRNA expressions of adipoR1 and adipoR2 were upregulated in T2DM group and were downregulated after gAd treatment. Conclusions. Globular adiponectin could ameliorate the hepatic steatosis and vary the expressions of adiponectin receptors in liver and skeletal muscle by stimulating insulin secretion.
doi:10.1155/2014/230835
PMCID: PMC3933409
4.  A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling 
PLoS ONE  2013;8(6):e65454.
Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides’ ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.
doi:10.1371/journal.pone.0065454
PMCID: PMC3676391  PMID: 23762377
5.  AdipoR1 and 2 are expressed on warm sensitive neurons of the hypothalamic preoptic area and contribute to central hyperthermic effects of adiponectin 
Brain research  2011;1423:1-9.
Adiponectin can act in the brain to increase energy expenditure and reduce body weight by mechanisms not entirely understood. We found that adiponectin type 1 and type 2 receptors (AdipoR1 and AdipoR2) are expressed in warm sensitive neurons of the hypothalamic preoptic area (POA) which play a critical role in the regulation of core body temperature (CBT) and energy balance. Thus, we tested the ability of adiponectin to influence CBT in wild-type mice and in mice deficient for AdipoR1 or AdipoR2. Local injection of adiponectin into the POA induced prolonged elevation of core body temperature and decreased respiratory exchange ratio (RER) indicating that increased energy expenditure is associated with increased oxidation of fat over carbohydrates. In AdipoR1 deficient mice, the ability of adiponectin to raise CBT was significantly blunted and its ability to decrease RER was completely lost. In AdipoR2 deficient mice, adiponectin had only diminished hyperthermic effects but reduced RER similarly to wild type mice. These results indicate that adiponectin can contribute to energy homeostasis by regulating CBT by direct actions on AdipoR1 and R2 in the POA.
doi:10.1016/j.brainres.2011.09.019
PMCID: PMC3215583  PMID: 22000082
Adiponectin; Adiponectin receptor; Temperature; Homeostasis; Preoptic area; Warm sensitive neurons
6.  Adiponectin Increases Secretion of Rat Submandibular Gland via Adiponectin Receptors-Mediated AMPK Signaling 
PLoS ONE  2013;8(5):e63878.
Adiponectin and adiponectin receptors (AdipoR1/2) are expressed in various tissues and are involved in the regulation of multiple functions such as energy metabolism and inflammatory responses. However, the effect of adiponectin and AdipoRs in submandibular glands has not been fully evaluated. In the present study, we found that mRNA and protein of both adiponectin and AdipoR1/2 were expressed in rat submandibular glands and in the SMG-C6 cell line, as evidenced by RT-PCR and Western blot analysis. Immunofluorescence staining showed that adiponectin was diffused in the cytoplasm, while AdipoR1/2 was concentrated in the membrane of acinar cells. Saliva flow was significantly increased by full length adiponectin (fAd) or globular adiponectin (gAd) perfusion in isolated rat submandibular glands. 5-Aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR), an adenosine monophosphate activated protein kinase (AMPK) activator, also increased saliva secretion. fAd, gAd, and AICAR all increased the average width of apical tight junctions in perfused submandibular glands, and decreased transepithelial electrical resistance (TER) in SMG-C6 cells, suggesting that adiponectin promoted secretion by modulating paracellular permeability. fAd and gAd increased p-AMPK levels, while AraA, an AMPK antagonist, abolished fAd- and gAd-induced changes in secretion, tight junction ultrastructure, and TER. Moreover, both AdipoR1 and AdipoR2 were required for fAd- or gAd-induced p-AMPK and TER responses, suggesting from their inhibition following AdipoR1 or AdipoR2 knockdown, and co-knockdown of AdipoRs by RNA interference. Our results suggest that adiponectin functions as a promoter of salivary secretion in rat submandibular glands via activation of AdipoRs, AMPK, and paracellular permeability.
doi:10.1371/journal.pone.0063878
PMCID: PMC3646765  PMID: 23667684
7.  Peroxisome Proliferator-Activated Receptor-Gamma Expression in the Lung Tissue of Obese Rats 
Yonsei Medical Journal  2011;52(3):495-501.
Purpose
Obesity is a risk factor for asthma and type II diabetes. Peroxisome proliferator-activated receptor (PPAR)-γ has been suggested to regulate inflammatory responses in diabetes and asthma. We investigated whether PPAR-α, PPAR-γ, adiponectin receptors (AdipoR1, AdipoR2), leptin, and tumor necrosis factor (TNF)-α are expressed in rat lung tissues and whether the expression differs between obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long Evans Tokushima Otsuka (LETO) rats.
Materials and Methods
Obese and lean rats were given with a high fat diet or a 30% restricted diet for 32 weeks, and their blood glucose levels and weights were monitored. After 32 weeks, mRNA levels of PPAR-α, PPAR-γ, AdipoR1, AdipoR2, leptin, and TNF-α in lung tissues were measured using real time PCR.
Results
PPAR-α, PPAR-γ, AdipoR1, AdipoR2, leptin, and TNF-α were expressed in both obese and lean rat lung tissues. Increased serum glucose levels on intraperitoneal glucose tolerance testing and a higher weight gain at 32 weeks were observed in OLETF control rats compared to OLETF diet restricted rats. PPAR-γ expression was markedly elevated in obese control and diet restricted rats compared to lean rats, although PPAR-γ expression in obese rats was not affected by diet restriction. Leptin was highly expressed in OLETF rats compared to LETO rats. TNF-α expression was enhanced in OLETF control rats compared LETO diet restricted rats, and decreased by diet restriction. PPAR-α, AdipoR1, and AdipoR2 expression were not significantly different between obese and lean rats.
Conclusion
PPAR-γ was highly expressed in the lung tissues of obese rats and may be a novel treatment target for regulating lung inflammation associated with obesity.
doi:10.3349/ymj.2011.52.3.495
PMCID: PMC3101042  PMID: 21488194
Obesity; peroxisome proliferator activated receptor; adiponectin receptor; lung; leptin; TNF-alpha
8.  Gene expression of adiponectin receptors in human visceral and subcutaneous adipose tissue is related to insulin resistance and metabolic parameters and is altered in response to physical training 
Diabetes care  2007;30(12):3110-3115.
Objective
Adiponectin receptors 1 and 2 (AdipoR1/R2) mediate the effects of adiponectin on glucose and lipid metabolism in vivo. We examined whether AdipoR1 and/or AdipoR2 mRNA expression in human adipose tissue is fat-depot specific. We also studied whether their expression in visceral and subcutaneous fat depots is associated with metabolic parameters and whether their expression is regulated by intensive physical exercise.
Research design and methods
We determined metabolic parameters and assessed AdipoR1 and R2 mRNA expression using quantitative real-time PCR in adipose tissue in an observational study of 153 subjects, and an interventional study of 60 subjects (20 each with normal glucose tolerance, impaired glucose tolerance, and type 2 diabetes) before and after intensive physical training for 4 weeks.
Results
AdipoR1 and R2 mRNA expression is not significantly different between omental and subcutaneous fat, but their expression is several fold lower in adipose tissue than in muscle. AdipoR2 mRNA expression in visceral fat is highly correlated with its expression in subcutaneous fat. AdipoR2 mRNA expression in both visceral and subcutaneous fat is positively associated with circulating adiponectin and HDL levels but negatively associated with obesity as well as parameters of insulin resistance, glycemia and other lipid levels before and after adjustment for fat mass. Physical training for 4 weeks resulted in increased AdipoR1 and AdipoR2 mRNA expression in subcutaneous fat.
Conclusions
AdipoR2 mRNA expression in fat is negatively associated with insulin resistance and metabolic parameters independently of obesity, and may mediate the improvement of insulin resistance in response to exercise.
doi:10.2337/dc07-1257
PMCID: PMC2732345  PMID: 17878241
Adiponectin; AdipoR1; AdipoR2; adipose tissue; obesity; diabetes; exercise training
9.  Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome 
Journal of Clinical Investigation  2006;116(7):1784-1792.
Adiponectin is an adipokine that is specifically and abundantly expressed in adipose tissue and directly sensitizes the body to insulin. Hypoadiponectinemia, caused by interactions of genetic factors such as SNPs in the Adiponectin gene and environmental factors causing obesity, appears to play an important causal role in insulin resistance, type 2 diabetes, and the metabolic syndrome, which are linked to obesity. The adiponectin receptors, AdipoR1 and AdipoR2, which mediate the antidiabetic metabolic actions of adiponectin, have been cloned and are downregulated in obesity-linked insulin resistance. Upregulation of adiponectin is a partial cause of the insulin-sensitizing and antidiabetic actions of thiazolidinediones. Therefore, adiponectin and adiponectin receptors represent potential versatile therapeutic targets to combat obesity-linked diseases characterized by insulin resistance. This Review describes the pathophysiology of adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
doi:10.1172/JCI29126
PMCID: PMC1483172  PMID: 16823476
10.  Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells 
Background
Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. However, the molecular mechanisms involved in the reduction of steroid production have not been described. Adiponectin is an adipocytokine that has insulin-sensitizing actions including stimulation of glucose uptake in muscle and suppression of glucose production in liver. Adiponectin acts via two receptor isoforms – AdipoR1 and AdipoR2 – that are regulated by hyperglycaemia and hyperinsulinaemia in liver and muscle. We have recently identified AdipoR1 and AdipoR2 in rat ovary. However, their regulation in ovaries of diabetic female rat remains to be elucidated.
Methods
We incubated rat primary granulosa cells in vitro with high concentrations of glucose (5 or 10 g/l) + or - FSH (10-8 M) or IGF-1 (10-8 M), and we studied the ovaries of streptozotocin-induced diabetic rats (STZ) in vivo. The levels of oestradiol and progesterone in culture medium and serum were measured by RIA. We used immunoblotting to assay key steroidogenesis factors (3beta HSD, p450scc, p450 aromatase, StAR), and adiponectin receptors and various elements of signalling pathways (MAPK ERK1/2 and AMPK) in vivo and in vitro. We also determined cell proliferation by [3H] thymidine incorporation.
Results
Glucose (5 or 10 g/l) impaired the in vitro production in rat granulosa cells of both progesterone and oestradiol in the basal state and in response to FSH and IGF-1 without affecting cell proliferation and viability. This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation. In contrast, glucose did not affect the abundance of AdipoR1 or AdipoR2 proteins. In vivo, as expected, STZ treatment of rats caused hyperglycaemia and insulin, adiponectin and resistin deficiencies. Plasma progesterone and oestradiol levels were also reduced in STZ rats. However, the amounts of 3beta HSD and p450 aromatase were the same in STZ rat ovary and controls, and the amounts of StAR and p450scc were higher. Streptozotocin treatment did not affect adiponectin receptors in rat ovary but it increased AMPK phosphorylation without affecting MAPK ERK1/2 phosphorylation.
Conclusion
High levels of glucose decrease progesterone and oestradiol production in primary rat granulosa cells and in STZ-treated rats. However, the mechanism that leads to reduced ovarian steroid production seems to be different. Furthermore, adiponectin receptors in ovarian cells are not regulated by glucose.
doi:10.1186/1477-7827-6-11
PMCID: PMC2277425  PMID: 18353182
11.  Differential Expression of Novel Adiponectin Receptor-1 Transcripts in Skeletal Muscle of Subjects With Normal Glucose Tolerance and Type 2 Diabetes 
Diabetes  2011;60(3):936-946.
OBJECTIVE
Adiponectin receptor-1 (AdipoR1) expression in skeletal muscle has been suggested to play an important role in insulin resistance and diabetes. We aimed at evaluating the presence of novel AdiopR1 splice variants in human muscle and their regulation under physiological and pathophysiological states.
RESEARCH DESIGN AND METHODS
AdipoR1 5′UTR mRNA transcripts, predicted from bioinformatics data, were evaluated in fetal and adult human tissues. Expression and function of the identified transcripts were assessed in cultured human skeletal muscle cells and in muscle biopsies obtained from individuals with normal glucose tolerance (NGT) and type 2 diabetes (n = 49).
RESULTS
Screening of potential AdipoR1 5′UTR splice variants revealed a novel highly abundant muscle transcript (R1T3) in addition to the previously described transcript (R1T1). Unlike R1T1, R1T3 expression was significantly increased during fetal development and myogenesis, paralleled with increased AdipoR1 protein expression. The 5′UTR of R1T3 was found to contain upstream open reading frames that repress translation of downstream coding sequences. Conversely, AdipoR1 3′UTR was associated with enhanced translation efficiency during myoblast-myotube differentiation. A marked reduction in muscle expression of R1T3, R1T1, and R1T3-to-R1T1 ratio was observed in individuals with type 2 diabetes compared with expression levels of NGT subjects, paralleled with decreased expression of the differentiation marker myogenin. Among NGT subjects, R1T3 expression was positively correlated with insulin sensitivity.
CONCLUSIONS
These results indicate that AdipoR1 receptor expression in human skeletal muscle is subjected to posttranscriptional regulation, including alternative splicing and translational control. These mechanisms play an important role during myogenesis and may be important for whole-body insulin sensitivity.
doi:10.2337/db09-0532
PMCID: PMC3046855  PMID: 21289205
12.  Adiponectin receptor 2 is negatively associated with lymph node metastasis of colorectal cancer 
Oncology Letters  2012;3(4):756-760.
Adiponectin is a hormone secreted by adipose tissue and has a variety of functions including the inhibition of tumor growth. The expression and function of the two major adiponectin receptors, AdipoR1 and AdipoR2, in malignant tissue have not been well characterized. In the present study, we evaluated the mRNA levels of AdipoR1 and AdipoR2 expression in 48 surgically resected colorectal cancer specimens, as well as normal colonic mucosa, by quantitative RT-PCR. The values obtained were standardized by β-actin mRNA, and the correlation between their relative expression levels and the clinicopathological characteristics of the patients was examined. The relative expression levels of AdipoR1 and AdipoR2 were significantly reduced in cancer tissue compared with normal tissue (AdipoR1: 0.97±0.39 vs. 1.37±0.41, P<0.0001; AdipoR2: 0.92±0.31 vs. 1.60±0.46, P<0.0001). AdipoR1 and AdipoR2 levels were further reduced in tumors with nodal metastases and the difference was statistically significant in the case of AdipoR2 (0.79±0.27 vs. 1.02±0.30, P=0.012). The results of this study demonstrated that the expression levels of adiponectin receptors are reduced in cancer specimens compared to normal tissue, indicating a downregulation in the course of the development and progression of colorectal cancer. Since adiponectin is abundantly present in the whole body and has inhibitory effects on cancer cells, this downregulation of the receptors may be an escape mechanism of malignant cells from the suppressive effects of adiponectin.
doi:10.3892/ol.2012.583
PMCID: PMC3362449  PMID: 22740988
colorectal cancer; adiponectin receptor; lymph node metastasis
13.  Regulation of oxidative stress and inflammation by hepatic adiponectin receptor 2 in an animal model of nonalcoholic steatohepatitis 
The pathogenesis of nonalcoholic steatohepatitis (NASH) is not well understood; however, the progression of fatty liver to NASH has been linked to oxidative stress and lipid peroxidation in the liver, leading to inflammation. Although the adiponectin receptor 2 (AdipoR2) has been identified as a modulator of oxidative stress and inflammation in the liver, it remains unclear whether the receptor has hepatic antioxidant and anti-inflammatory effects in NASH. In this study, we used an animal model of NASH to examine hepatic AdipoR2. Obese fa/fa Zucker rats fed a high-fat and high-cholesterol (HFC) diet spontaneously developed fatty liver with inflammation and fibrosis, characteristic of NASH, after 4, 8, or 12 weeks of HFC diet consumption. AdipoR2 expression was significantly decreased, whereas the expression of genes related to NADPH oxidase complex were increased. As a result of the decrease in AdipoR2 expression, the mRNA expression of genes located downstream of AdipoR2, i.e., Cu-Zn superoxide dismutase (Cu-Zn SOD) and Mn-SOD, also decreased. Furthermore, the expression of genes related to inflammation was increased. Increased oxidative stress and inflammation by down-regulation of AdipoR2 may contribute to the progression of NASH. Thus, the AdipoR2 might be a crucially important regulator of hepatic oxidative stress and inflammation in NASH.
PMCID: PMC2897102  PMID: 20606728
Nonalcoholic steatohepatitis; adiponectin receptor 2; inflammation; oxidative stress; Zucker rats
14.  Direct role of adiponectin and adiponectin receptors in endometrial cancer: in vitro and ex vivo studies in humans 
Molecular cancer therapeutics  2011;10(12):2234-2243.
Low adiponectin levels are an independent risk factor for, and mediate the effect of obesity on endometrial cancer in epidemiology studies. The direct or indirect mechanisms underlying these findings remain to be elucidated. We first examined the expression of adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) in normal human endometrium and in endometrial cancer tissues ex vivo. We then utilized KLE and RL95-2 human endometrial cancer cell lines in vitro to study relative expression of AdipoRs, to investigate the effect of adiponectin on activating intracellular signaling pathways, and to assess its potential to alter malignant properties. We report for the first time that the relative expression level of AdipoR1 is higher than AdipoR2 in human endometrial cancer tissue but the expression of AdipoRs is not statistically different from non-neoplastic tissues. We also demonstrate for the first time in endometrial cancer cell lines in vitro that adiponectin suppresses endometrial cancer proliferation acting through AdipoRs. Adiponectin also increases the expression of the adaptor molecule LKB1 which is required for adiponectin-mediated activation of AMPK/S6 axis and modulation of cell proliferation, colony formation, adhesion and invasion of KLE and RL95-2 cell lines. These novel mechanistic studies provide for the first time in vitro and ex vivo evidence for a causal role of adiponectin in endometrial cancer.
doi:10.1158/1535-7163.MCT-11-0545
PMCID: PMC3237794  PMID: 21980131
adiponectin; adiponectin receptor; intracellular signaling; LKB1; AMPK; endometrial adenocarcinoma
15.  Adiponectin Receptor 2 Deficiency Results in Reduced Atherosclerosis in the Brachiocephalic Artery in Apolipoprotein E Deficient Mice 
PLoS ONE  2013;8(11):e80330.
Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1-/-AdipoR2-/-) on atherosclerosis. However, we made the interesting observation that AdipoR1-/-AdipoR2-/- leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice. AdipoR2-/-ApoE-/- mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2+/+ApoE-/- littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2-/-ApoE-/- mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2-/-ApoE-/- mice compared with AdipoR2+/+ApoE-/- controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.
doi:10.1371/journal.pone.0080330
PMCID: PMC3855811  PMID: 24324556
16.  Effect of exenatide on the cardiac expression of adiponectin receptor 1 and NADPH oxidase subunits and heart function in streptozotocin-induced diabetic rats 
Background
This study investigated the effect of exenatide on the cardiac expression of adiponectin receptor 1 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and heart function in streptozotocin-induced diabetic rats.
Methods
Male Sprague–Dawley rats were randomly divided into four groups, i.e. control group, diabetic group, diabetic treated with low doses of exenatide (2 μg · kg−1.d−1) and diabetic treated with high doses of exenatide (10 μg · kg−1.d−1). Diabetes was induced by intraperitoneal injection of streptozotocin (65 mg/kg body weight). At the termination after exenatide treatment for eight weeks, following anesthesia of the rats, a catheter was inserted into the left ventricle through the right common carotid artery for measurement of left ventricular pressure, which included left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the maximal rate of rise and decline of ventricular pressure (±dp/dt[max]). Plasma and myocardial adiponectin levels, and the expressions of myocardial adiponectin receptor 1, p22phox, NADPH oxidase 4 (NOX4), glucose transporter type 4 (Glut4), AMPK-α, phosphorylated-AMPK-α, connective tissue growth factor (CTGF) and copper zinc superoxide dismutase (Cu-Zn-SOD) were assayed.
Results
Heart function, plasma adiponectin levels, the protein expression of myocardial phosphorylated-AMPK-α, the mRNA expression of myocardial Glut4, and the positive expression of myocardial Cu-Zn-SOD were significantly decreased in diabetic. The protein expression of myocardial adiponectin receptor 1, the mRNA expression of myocardial p22phox and NOX4, and the positive expression of myocardial CTGF were significantly increased in diabetic. Low and high doses of exenatide treatment significantly attenuated these changes in diabetic rats.
Conclusions
These results suggest that exenatide may contribute to the improvement of the heart function in diabetic rats by down-regulating the expression of myocardial adiponectin receptor 1, p22phox and NOX4, and up-regulating plasma adiponectin level and the expression of myocardial AMPK-α, Glut4 and Cu-Zn-SOD.
doi:10.1186/1758-5996-6-29
PMCID: PMC3942060  PMID: 24576329
Exenatide; Diabetic cardiomyopathy; Adiponectin receptor 1; Glucose transporter type 4; NADPH oxidase
17.  Adiponectin receptor-1 expression is associated with good prognosis in gastric cancer 
Background
Adiponectin is inversely related to BMI, positively correlates with insulin sensitivity, and has anti-atherogenic effects. In recent years, adiponectin has been well studied in the field of oncology. Adiponectin has been shown to have antiproliferative effects on gastric cancer, and adiponectin expression is inversely correlated with clinical staging of the disease. However, no studies have reported the correlation between serum adiponectin and receptor expression with disease progression.
Methods
In this study, we evaluated expression levels of 2 adiponectin receptors--AdipoR1 and AdipoR2--and attempted to correlate their expression with prognosis in gastric cancer patients. AdipoR1 and AdipoR2 expression in gastric cancer cell lines (MKN45, TMK-1, NUGC3, and NUGC4) was evaluated by western blotting analysis, and the antiproliferative potential of adiponectin was examined in vitro. Serum adiponectin levels were evaluated in 100 gastric cancer patients, and the expression of AdipoR1 and AdipoR2 was assessed by immunohistochemical staining.
Results
MKN45 and NUGC3 expressed higher levels of AdipoR1 compared to NUGC4, even though there was no significance in AdipoR2 expression. The antiproliferative effect of adiponectin was confirmed in MKN45 and NUGC3 at 10 μg/ml. No significant associations were observed between serum adiponectin levels and clinicopathological characteristics, but lymphatic metastasis and peritoneal dissemination were significantly higher in the negative AdipoR1 immunostaining group (24/32, p = 0.013 and 9/32, p = 0.042, respectively) compared to the positive AdipoR1 group (lymphatic metastasis, 33/68; peritoneal dissemination, 8/68). On the other hand, AdipoR2 expression was only associated with histopathological type (p = 0.001). In survival analysis, the AdipoR1 positive staining group had significantly longer survival rates than the negative staining group (p = 0.01). However, multivariate analysis indicated that AdipoR1 was not an independent prognostic factor on patient's survival on gastric cancer.
Conclusions
In gastric cancer, adiponectin has the possibility to be involved in cell growth suppression via AdipoR1. The presence of AdipoR1 could be a novel anticancer therapeutic target in gastric cancer.
doi:10.1186/1756-9966-30-107
PMCID: PMC3223499  PMID: 22078265
Adiponectin; AdipoR1; AdipoR2; gastric cancer; survival
18.  Up-Regulation of Adiponectin Expression in Antigravitational Soleus Muscle in Response to Unloading Followed by Reloading, and Functional Overloading in Mice 
PLoS ONE  2013;8(12):e81929.
The purpose of this study was to investigate the expression level of adiponectin and its related molecules in hypertrophied and atrophied skeletal muscle in mice. The expression was also evaluated in C2C12 myoblasts and myotubes. Both mRNA and protein expression of adiponectin, mRNA expression of adiponectin receptor (AdipoR) 1 and AdipoR2, and protein expression of adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1 (APPL1) were observed in C2C12 myoblasts. The expression levels of these molecules in myotubes were higher than those in myoblasts. The expression of adiponectin-related molecules in soleus muscle was observed at mRNA (adiponectin, AdipoR1, AdipoR2) and protein (adiponectin, APPL1) levels. The protein expression levels of adiponectin and APPL1 were up-regulated by 3 weeks of functional overloading. Down-regulation of AdipoR1 mRNA, but not AdipoR2 mRNA, was observed in atrophied soleus muscle. The expression of adiponectin protein, AdipoR1 mRNA, and APPL1 protein was up-regulated during regrowth of unloading-associated atrophied soleus muscle. Mechanical loading, which could increase skeletal muscle mass, might be a useful stimulus for the up-regulations of adiponectin and its related molecules in skeletal muscle.
doi:10.1371/journal.pone.0081929
PMCID: PMC3855747  PMID: 24324732
19.  Analysis of the association between adiponectin, adiponectin receptor 1 and diabetic cardiomyopathy 
The aim of this study was to explore the association between adiponectin (APN), APN receptors and insulin resistance (IR) using rats with type 2 diabetes mellitus (T2DM) as a model of diabetic cardiomyopathy (DC). Serum and cardiac APN levels were assessed using a double-antibody sandwich ELISA. In addition, the mRNA and protein expression of the myocardial APN receptor 1 (AdipoR1) was determined using the reverse transcription polymerase chain reaction and immunohistochemical staining. The results showed that the heart weight/body weight ratio, fasting plasma glucose (FPG) and lipid levels, and the homeostasis model assessment-estimated IR (HOMA-IR) index were elevated in the T2DM group compared with the control group. Cardiac function was significantly lower in the T2DM group compared with the control group (P<0.05). Furthermore, serum and cardiac APN levels were significantly reduced in the T2DM group compared with the control group, and mRNA and protein expression of AdipoR1 was lower in the T2DM group compared with the control group (P<0.05). Changes in the morphology of myocardial cells were observed under the light microscope using hematoxylin and eosin staining. Myocardial cell hypertrophy, a disordered cell arrangement and irregular nuclear size were observed in the T2DM group. By contrast, myocardial cells in the control group were arranged in neat rows with uniform cytoplasmic and nuclear staining. According to the correlation analyses, serum APN levels in the T2DM group were negatively correlated with FPG, triglyceride, total cholesterol and fasting insulin (FINS) levels, as well as with the HOMA-IR index. Myocardial AdipoR1 protein expression was positively correlated with myocardial APN levels, and negatively correlated with FINS and HOMA-IR. It may be concluded that myocardial and serum levels of APN are reduced in rats with DC. Metabolic disorders of blood glucose and lipid levels, as well as IR, are associated with low APN levels. Furthermore, low levels of myocardial Adipo1R mRNA and protein expression correlate with reduced insulin sensitivity.
doi:10.3892/etm.2014.1539
PMCID: PMC3964935  PMID: 24669271
diabetic cardiomyopathy; adiponectin; adiponectin receptor
20.  Activation of the LH receptor up regulates the type 2 adiponectin receptor in human granulosa cells 
Purpose
Adiponectin is a predominantly adipocyte-derived hormone which influences insulin sensitivity and energy homeostasis through at least two receptors, AdipoR1 and AdipoR2. In animal models, adiponectin may regulate ovarian steroidogenesis, folliculogenesis, and ovulation. The receptors AdipoR1 and AdipoR2 are present in the human ovary, but their regulation is unknown. In these studies, we determined the effects of LH receptor activation on the expression and function of the two adiponectin receptors in human granulosa cells.
Methods
Granulosa cells were obtained at the time of oocyte retrieval in women undergoing in vitro fertilization (IVF). Cells were isolated and cultured for 48 h in DMEM/F12 medium with 5 % FBS and 50 ug/ml gentamicin. Medium was changed to low serum for 12 h and cells were treated with hCG (100 ng/ml), forskolin (30 μMol/L), or FSH (1 IU/ml) for 24 h for mRNA experiments. mRNA was isolated and RT PCR was performed using Taqman assays and quantification with the delta delta CT method. For immunocytochemistry, cells were grown on chamber slides and treated with hCG for 1 to 24 h and fixed with acetone. ICC was performed with polyclonal rabbit primary antibodies followed by alexa fluor goat anti-rabbit antibody and imaging with a fluorescence microscope and Zeiss software analysis. 3β-hydroxysteroid dehydrogenase (3βHSD) enzyme activity was determined by measuring the progesterone produced when cells were provided with an excess of 22-hydroxy-cholesterol as substrate following an incubation with hCG (1 IU/ml) and/or adiponectin (10 ng/ml). Progesterone content in the media was determined by ELISA.
Results
Messenger RNA for the two Adiponectin receptors is differentially regulated by activation of LHR with hCG treatment. AdipoR2 was increased nearly 4-fold (p < 0.05), whereas AdipoR1 expression was not changed by hCG treatment. Treatment with either FSH or forskolin (an activator of cAMP) had similar effects. Basal AdipoR2 protein was fairly low in granulosa cells in culture however treatment of cells with hCG resulted in a discernible increase in immunodetectable cytoplasmic protein as early as 6 h after treatment and was maintained for at least 24 h. The number of cells positive for AdipoR2 at 6 h increased from a basal of 20 % to almost 60 % (p < 0.05). Adiponectin treatment of hCG-primed cells resulted in increased 3βHSD activity by approximately 60 % over hCG alone and more than 3-fold over basal levels.
Conclusions
AdipoR2 is regulated by the LH receptor function via a cAMP dependant mechanism. Increased expression of adipoR2 prior to and following ovulation may contribute to enhanced 3βHSD activity and increased progesterone secretion by the corpus luteum of the ovary. Dysregulation of adiponectin that may occur with PCOS may impair normal progesterone production.
doi:10.1007/s10815-013-0012-3
PMCID: PMC3725221  PMID: 23779096
Ovary; Granulosa; Adiponectin; Adiponectin receptor; PCOS; Progesterone
21.  Adiponectin and adiponectin receptors in the mouse preimplantation embryo and uterus 
BACKGROUND
Adiponectin (Adipoq), a protein secreted by adipocytes in inverse proportion to the adipose mass present, modulates energy homeostasis and increases insulin sensitivity. Tissue Adipoq signaling decreases in settings of maternal diabetes, polycystic ovary syndrome (PCOS) and endometriosis, conditions which are associated with reproductive difficulty. Our objective was to define the expression and hormonal regulation of Adipoq and its receptors in the mouse preimplantation embryo and uterus.
METHODS AND RESULTS
By real-time quantitative PCR, mRNA transcripts for Adipoq, AdipoR1, AdipoR2, Ppara, Ppard, FATP1 (SLC27A1) and acyl CoA oxidase (Acox1) were identified in mouse 2-cell and 8-cell embryos, while blastocyst stage embryos and trophoblast stem (TS) cells expressed mRNA for all genes except Adipoq. Protein expression of Adipoq, AdipoR1, AdipoR2, the insulin sensitive transporters GLUT8 (Slc2A8), GLUT12 (Slc2A12) and p-PRKAA1 was identified by immunofluorescence staining in all stages of preimplantation embryos including the blastocyst. In situ hybridization demonstrated the presence of Adipoq, AdipoR1 and AdipoR2 mRNA in the mouse decidual cells of the implantation site and in artificially decidualized cells, and the expression of these proteins was confirmed by western blotting. Flow cytometry confirmed cell surface expression of AdipoR1 and AdipoR2 in TS cells and decidual cells.
CONCLUSIONS
These results suggest for the first time that Adipoq signaling may play an important role in preimplantation embryo development and uterine receptivity by autocrine and paracrine methods in the mouse. Implantation failures and pregnancy loss, specifically those experienced in women with maternal metabolic conditions such as diabetes, obesity and PCOS, may be the result of aberrant Adipoq and AdipoR1 and AdipoR2 expression and suboptimal decidualization in the uterus.
doi:10.1093/humrep/deq292
PMCID: PMC3005998  PMID: 21106494
adiponectin; adiponectin receptor; preimplantation embryo; uterus; decidua
22.  The Impact of Full-Length, Trimeric and Globular Adiponectin on Lipolysis in Subcutaneous and Visceral Adipocytes of Obese and Non-Obese Women 
PLoS ONE  2013;8(6):e66783.
Contribution of individual adiponectin isoforms to lipolysis regulation remains unknown. We investigated the impact of full-length, trimeric and globular adiponectin isoforms on spontaneous lipolysis in subcutaneous abdominal (SCAAT) and visceral adipose tissues (VAT) of obese and non-obese subjects. Furthermore, we explored the role of AMPK (5'-AMP-activated protein kinase) in adiponectin-dependent lipolysis regulation and expression of adiponectin receptors type 1 and 2 (AdipoR1 and AdipoR2) in SCAAT and VAT. Primary adipocytes isolated from SCAAT and VAT of obese and non-obese women were incubated with 20 µg/ml of: A) full-length adiponectin (physiological mixture of all adiponectin isoforms), B) trimeric adiponectin isoform or C) globular adiponectin isoform. Glycerol released into media was used as a marker of lipolysis. While full-length adiponectin inhibited lipolysis by 22% in non-obese SCAAT, globular isoform inhibited lipolysis by 27% in obese SCAAT. No effect of either isoform was detected in non-obese VAT, however trimeric isoform inhibited lipolysis by 21% in obese VAT (all p<0.05). Trimeric isoform induced Thr172 p-AMPK in differentiated preadipocytes from a non-obese donor, while globular isoform induced Ser79 p-ACC by 32% (p<0.05) and Ser565 p-HSL by 52% (p = 0.08) in differentiated preadipocytes from an obese donor. AdipoR2 expression was 17% and 37% higher than AdipoR1 in SCAAT of obese and non-obese groups and by 23% higher in VAT of obese subjects (all p<0.05). In conclusion, the anti-lipolytic effect of adiponectin isoforms is modified with obesity: while full-length adiponectin exerts anti-lipolytic action in non-obese SCAAT, globular and trimeric isoforms show anti-lipolytic activity in obese SCAAT and VAT, respectively.
doi:10.1371/journal.pone.0066783
PMCID: PMC3689658  PMID: 23805277
23.  Endoplasmic Reticulum Protein ERp46 in Renal Cell Carcinoma 
PLoS ONE  2014;9(3):e90389.
An established inverse clinical correlation between serum adiponectin levels and renal cell carcinoma (RCC) aggressiveness exists. We have recently demonstrated that adiponectin suppresses clear cell RCC (ccRCC) progression through interaction with its receptor, adiponectin receptor 1 (AdipoR1). ERp46 has been shown to inhibit adiponectin signaling via interaction with AdipoR1 in HeLa cells. However, the expression of ERp46 in RCC has not been described thus far. The objectives of this study were to investigate ERp46 in RCC, its expression, its effects on RCC growth in a mouse model and whether it interacts with AdipoR1. We demonstrated a higher ERp46/AdipoR1 expression ratio in metastatic compared to non-metastatic ccRCC, as determined by immunohistochemistry of tissue microarrays and subsequent image analysis. When ERp46 was stably knocked down using shRNA or overexpressed in murine RCC RAG cells, RCC growth after subcutaneous injection in BALB/c nude mice was inhibited and accelerated, respectively. In vitro analysis to determine the molecular interaction between AdipoR1 and ERp46 included co-immunoprecipitation using human ccRCC 786-O cells and a bacterial adenylate cyclase-based two hybrid system and demonstrated no sustained AdipoR1-ERp46 interaction. This is the first report to suggest a role for ERp46 as a potential therapeutic target in RCC given its expression profile in human RCC samples and its effect on in vivo RCC growth. Since a stable interaction with AdipoR1 could not be established, we suggest that the tumorigenic properties of ERp46 in RCC cells are not related to an inhibitory modulation of AdipoR1.
doi:10.1371/journal.pone.0090389
PMCID: PMC3940878  PMID: 24594673
24.  Expression of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) in the porcine pituitary during the oestrous cycle 
Background
Adiponectin, protein secreted mainly by white adipose tissue, is an important factor linking the regulation of metabolic homeostasis and reproductive processes. The biological activity of the hormone is mediated via two distinct receptors, termed adiponectin receptor 1(AdipoR1) and adiponectin receptor 2 (AdipoR2). The present study analyzed mRNA and protein expression of AdipoR1 and AdipoR2 in the anterior (AP) and posterior (NP) pituitary of cyclic pigs.
Methods
The total of 20 animals was assigned to one of four experimental groups (n = 5 per group) as follows: days 2–3 (early-luteal phase), 10–12 (mid-luteal phase), 14–16 (late-luteal phase), 17–19 (follicular phase) of the oestrous cycle. mRNA and protein expression were analyzed using real-time PCR and Western Blot methods, respectively.
Results
The lowest AdipoR1 gene expression was detected in AP on days 10–12 relative to days 2–3 and 14–16 (p < 0.05). In NP, AdipoR1 mRNA levels were elevated on days 10–12 and 14–16 (p < 0.05). AdipoR2 gene expression in AP was the lowest on days 10–12, and an expression peak occurred on days 2–3 (p < 0.05). In NP, the lowest (p < 0.05) expression of AdipoR2 mRNA was noted on days 17–19. The AdipoR1 protein content in AP was the lowest on days 17–19 (p < 0.05), while in NP the variations in protein expression levels during the oestrous cycle were negligible. AdipoR2 protein in AP was most abundant on days 10–12, and it reached the lowest level on days 2–3 and 17–19 of the cycle (p < 0.05). The presence of AdipoR2 protein in NP was more pronounced on days 10–12 (p < 0.05).
Conclusions
Our study was the first experiment to demonstrate that AdipoR1 and AdipoR2 mRNAs and proteins are present in the porcine pituitary and that adiponectin receptors expression is dependent on endocrine status of the animals.
doi:10.1186/1477-7827-11-18
PMCID: PMC3608220  PMID: 23497348
Adiponectin; Adiponectin receptor; Pituitary; Oestrous cycle; Pig
25.  Inhibitory role of adiponectin peptide I on rat choroidal neovascularization 
Biochimica et biophysica acta  2012;1823(8):10.1016/j.bbamcr.2012.05.017.
Age-related macular degeneration (AMD) is a leading cause of central blindness in elderly population. Wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied effect of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density was investigated by microscopy. Immunohistochemical staining (IHC) for Von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels were decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 fold less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV.
doi:10.1016/j.bbamcr.2012.05.017
PMCID: PMC3845810  PMID: 22633972
Adiponectin; Adiponectin receptors; VEGF; Angiogenesis; Macular degeneration; Neovascularization; Proliferation

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