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.
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.
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.
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.
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.
Exenatide; Diabetic cardiomyopathy; Adiponectin receptor 1; Glucose transporter type 4; NADPH oxidase
Adiponectin is an adipokine that exerts anti-inflammatory and anti-atherogenic effects during macrophage transformation into foam cells. To further understand the signaling pathways of adiponectin involved in macrophage foam cell transformation, we investigated the roles of two adiponectin receptors (AdipoR1 and AdipoR2) and their downstream adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) in mediating adiponectin action on foam cell transformation.
Methods and Results
Transfections were performed to overexpress or knockdown AdipoR1 or AdipoR2 genes in human THP-1 monocytes. Lentiviral-shRNAs were also used to knockdown APPL1 gene in these cells. Foam cell transformation was induced via exposure to oxidized low-density lipoprotein (oxLDL). Our results showed that both AdipoR1 and AdipoR2 were critical for transducing the adiponectin signal that suppresses lipid accumulation and inhibits transformation from macrophage to foam cell. However, AdipoR1 and AdipoR2 were found to have differential effects in diminishing proinflammatory responses. While AdipoR1 was required by adiponectin to suppress tumor necrosis factor alpha (TNF) and monocyte chemotactic protein 1 (MCP-1) gene expression, AdipoR2 served as the dominant receptor for adiponectin suppression of scavenger receptor A type 1 (SR-AI) and upregulation of interleukin-1 receptor antagonist (IL-1Ra). Knockdown of APPL1 significantly abrogated the ability of adiponectin to inhibit lipid accumulation, SR-AI and nuclear factor- B (NF- B) gene expression, and Akt phosphorylation in macrophage foam cells.
In current studies, we have demonstrated that adiponectin’s abilty to suppress macrophage lipid accumulation and foam cell formation is mediated through AdipoR1 and AdipoR2 and the APPL1 docking protein. However, AdipoR1 and AdipoR2 exhibited a differential ability to regulate inflammatory cytokines and SR-A1. These novel data support the idea that the adiponectin-AdipoR1/2-APPL1 axis may serve as a potential therapeutic target for preventing macrophage foam cell formation and atherosclerosis.
adiponectin; adiponectin receptor; APPL1; macrophages; foam cells; atherosclerosis; inflammation
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.
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.
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.
adiponectin; adiponectin receptor; preimplantation embryo; uterus; decidua
Hyperglycemia-induced oxidative stress plays a central role in the development of diabetic myocardial complications. Adiponectin (APN), an adipokine with anti-diabetic and anti-ischemic effects, is decreased in diabetes. It is unknown whether or not antioxidant treatment with N-acetylcysteine (NAC) and/or allopurinol (ALP) can attenuate APN deficiency and myocardial ischemia reperfusion (MI/R) injury in the early stage of diabetes.
Control or streptozotocin (STZ)-induced diabetic rats were either untreated (C, D) or treated with NAC (1.5 g/kg/day) or ALP (100 mg/kg/day) or their combination for four weeks starting one week after STZ injection. Plasma and cardiac biochemical parameters were measured after the completion of treatment, and the rats were subjected to MI/R by occluding the left anterior descending artery for 30 min followed by 2 h reperfusion. Plasma and cardiac APN levels were decreased in diabetic rats accompanied by decreased cardiac APN receptor 2 (AdipoR2), reduced phosphorylation of Akt, signal transducer and activator of transcription 3 (STAT3) and endothelial nitric oxide synthase (eNOS) but increased IL-6 and TNF-α (all P<0.05 vs. C). NAC but not ALP increased cardiac APN concentrations and AdipoR2 expression in diabetic rats. ALP enhanced the effects of NAC in restoring cardiac AdipoR2 and phosphorylation of Akt, STAT3 and eNOS in diabetic rats. Further, NAC and ALP, respectively, decreased postischemic myocardial infarct size and creatinine kinase-MB (CK-MB) release in diabetic rats, while their combination conferred synergistic protective effects. In addition, exposure of cultured rat cardiomyocytes to high glucose resulted in significant reduction of cardiomyocyte APN concentration and AdipoR2 protein expression. APN supplementation restored high glucose induced AdipoR2 reduction in cardiomyocytes.
NAC and ALP synergistically restore myocardial APN and AdipoR2 mediated eNOS activation. This may represent the mechanism through which NAC and ALP combination greatly reduces MI/R injury in early diabetic rats.
Recent studies have suggested that adiponectin (APN) is associated with several retinal diseases. We studied the expression of APN and its receptors (AdipoRs) in the human retina and in a mouse model of type 1 diabetes mellitus (T1DM).
Human eyeball specimens were obtained from the Chongqing Eye Bank. eNOS-knockout (eNOS−/−) mice were randomly divided into a T1DM group and a control group. The T1DM model was induced with an intraperitoneal injection of streptozotocin. To locate the AdipoRs in the retina, immunofluorescence was performed. Total APN protein and RNA were extracted from the neural retina and the retinal pigment epithelium (RPE)-choroid complex, and the APN protein was detected with enzyme-linked immunosorbent assay (ELISA). The mRNA and the protein of AdipoRs in the retina were detected with qRT-PCR and western blotting, respectively. The unpaired Student t test was used to assess the significance between the T1DM and the control groups, with p<0.05 regarded as statistically significant.
APN, AdipoR1, and AdipoR2 were identified in the neural retina and in the RPE-choroid of humans and mice. AdipoR1 was found in the internal limiting membrane and in the outer segments of the photoreceptors in human and mouse retinas, whereas no noticeable AdipoR2 expression was seen in the retinal frozen sections of human and mouse eyes. Compared to the control group, APN and AdipoR1 expression in the retina was elevated in the T1DM group, but AdipoR2 expression remained unchanged.
We demonstrated that APN, AdipoR1, and AdipoR2 exist in human and mouse retinas and that retinal APN and AdipoR1 protein levels are elevated in T1DM mice, implying that the APN-AdipoR1 axis may be activated in the diabetic retina. In contrast, AdipoR2 appears to play a minor role in this pathological process.
Adiponectin is the most abundant plasma protein synthesized for the most part in adipose tissue, and it is an insulin-sensitive hormone, playing a central role in glucose and lipid metabolism. In addition, it increases fatty acid oxidation in the muscle and potentiates insulin inhibition of hepatic gluconeogenesis. Two adiponectin receptors have been identified: AdipoR1 is the major receptor expressed in skeletal muscle, whereas AdipoR2 is mainly expressed in liver. Consumption of high levels of dietary fat is thought to be a major factor in the promotion of obesity and insulin resistance. Excessive levels of cortisol are characterized by the symptoms of abdominal obesity, hypertension, glucose intolerance or diabetes and dyslipidemia; of note, all of these features are shared by the condition of insulin resistance. Although it has been shown that glucocorticoids inhibit adiponectin expression in vitro and in vivo, little is known about the regulation of adiponectin receptors. The link between glucocorticoids and insulin resistance may involve the adiponectin receptors and adrenalectomy might play a role not only in regulate expression and secretion of adiponectin, as well regulate the respective receptors in several tissues.
Feeding of a high-fat diet increased serum glucose levels and decreased adiponectin and adipoR2 mRNA expression in subcutaneous and retroperitoneal adipose tissues, respectively. Moreover, it increased both adipoR1 and adipoR2 mRNA levels in muscle and adipoR2 protein levels in liver. Adrenalectomy combined with the synthetic glucocorticoid dexamethasone treatment resulted in increased glucose and insulin levels, decreased serum adiponectin levels, reduced adiponectin mRNA in epididymal adipose tissue, reduction of adipoR2 mRNA by 7-fold in muscle and reduced adipoR1 and adipoR2 protein levels in muscle. Adrenalectomy alone increased adiponectin mRNA expression 3-fold in subcutaneous adipose tissue and reduced adipoR2 mRNA expression 2-fold in liver.
Hyperglycemia as a result of a high-fat diet is associated with an increase in the expression of the adiponectin receptors in muscle. An excess of glucocorticoids, rather than their absence, increase glucose and insulin and decrease adiponectin levels.
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.
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.
adiponectin; adipoR1; adipoR2; rosiglitazone; AMPK; Fatty acid oxidation; glucose uptake; cardiomyocytes
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.
diabetic cardiomyopathy; adiponectin; adiponectin receptor
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.
Membrane protein; Adiponectin receptors AdipoR1 and AdipoR2; Purification; Antibody; Crystallization; Lipidic mesophase
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.
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.
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).
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.
Adiponectin; Adiponectin receptor; Pituitary; Oestrous cycle; Pig
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.
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.
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.
Obesity; peroxisome proliferator activated receptor; adiponectin receptor; lung; leptin; TNF-alpha
AIM: To determine circulating and hepatic adiponectin in rodents with fatty liver disease or liver cirrhosis and investigate expression of the adiponectin receptors AdipoR1 on the mRNA and protein level and AdipoR2 on the mRNA level.
METHODS: Fat fed rats were used as a model for fatty liver disease and bile duct ligation in mice to investigate cirrhotic liver. Expression of AdipoR1 and AdipoR2 mRNA was determined by real time RT-PCR. AdipoR1 protein was analysed by immunoblot. Adiponectin was measured by ELISA.
RESULTS: Systemic adiponectin is reduced in fat-fed rats but is elevated in mice after bile duct ligation (BDL). Hepatic adiponectin protein is lower in steatotic liver but not in the liver of BDL-mice when compared to controls. Adiponectin mRNA was not detected in human liver samples or primary human hepatocytes nor in rat liver but recombinant adiponectin is taken up by isolated hepatocytes in-vitro. AdipoR1 mRNA and AdipoR1 protein levels are similar in the liver tissue of control and fat fed animals whereas AdipoR2 mRNA is induced. AdipoR2 mRNA and AdipoR1 mRNA and protein is suppressed in the liver of BDL-mice.
CONCLUSION: Our studies show reduced circulating adiponectin in a rat model of fatty liver disease whereas circulating adiponectin is elevated in a mouse model of cirrhosis and similar findings have been described in humans. Diminished hepatic expression of adiponectin receptors was only found in liver cirrhosis.
Hepatic steatosis; Adiponectin; Liver cirrhosis; Adiponectin receptor 1; Adiponectin receptor 2
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.
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.
colorectal cancer; adiponectin receptor; lymph node metastasis
Although altered levels of adiponectin have been reported as a potential risk factor in colorectal cancer (CRC), the importance of the role played by adiponectin in colorectal carcinogenesis has not been established. We sought to examine the expression pattern of adiponectin and adiponectin receptors (AdipoRs) in the normal-adenoma-carcinoma sequence and to assess the implications of adiponectin in colorectal carcinogenesis.
Serum adiponectin concentrations, and the mRNA and protein expression of adiponectin and AdipoRs were examined using serum and tissues from patients with CRC, advanced adenoma, and a normal colon. mRNA expression of AdipoRs and epithelial-mesenchymal transition regulators including E-cadherin, cyclooxygenase-2 (COX-2) and T-cadherin were examined in HCT116 cells treated with adiponectin.
Serum adiponectin concentrations in patients with advanced adenoma and CRC were lower than those in controls. Adiponectin mRNA was not detected in colonic tissue, whereas AdipoRs mRNA was lower in advanced adenoma and CRC than that in normal colon tissues. Immunohistochemical staining demonstrated that adiponectin was expressed in spindle-shaped cells of the subepithelial layer in normal colon tissues, whereas ill-defined overexpression of adiponectin was seen in the stroma of advanced adenoma and CRC tissues. AdipoRs expression was strong in normal epithelium, but weak to negative in the epithelia of CRC tissues. Adiponectin downregulated COX-2 mRNA expression in vitro, but upregulated T-cadherin in HCT116 cells.
Systemic adiponectin and local AdipoRs expression in the colon may be associated with anti-tumorigenesis during the early stages of CRC. These findings offer new insight into understanding the relationship between adiponectin and colorectal carcinogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-811) contains supplementary material, which is available to authorized users.
Adiponectin; Adiponectin receptors; Colorectal cancer; Carcinogenesis
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.
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.
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.
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.
Adiponectin; AdipoR1; AdipoR2; gastric cancer; survival
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.
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.
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.
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.
Ovary; Granulosa; Adiponectin; Adiponectin receptor; PCOS; Progesterone
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.
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.
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.
Adiponectin; AdipoR1; AdipoR2; adipose tissue; obesity; diabetes; exercise training
Adiponectin has been associated with increased risks of microvascular complications in diabetes; however, its role in the development of diabetic retinopathy (DR) is unknown. Fenofibrate is a lipid-lowering agent that has been shown to be capable of preventing DR progression. We investigated the expression of adiponectin and its receptors in DR and evaluated the effects of fenofibrate on their expression. The mRNA and protein levels of adiponectin and its receptors were elevated in retinas of streptozotocin-induced diabetic rats and were suppressed following fenofibrate treatment. Immunofluorescence staining demonstrated that adiponectin and adipoR1 were expressed in cells located within blood vessels, the retinal ganglion, and the inner nuclear layer. AdipoR1 was strongly expressed whereas adipoR2 was only weekly expressed in vascular endothelial cells. The in vitro experiments showed that adiponectin expression was induced by high glucose concentrations in RGC-5 and RAW264.7 cells and was suppressed following fenofibrate treatment. AdipoR1 and adipoR2 levels in RGC-5 cells were elevated in high glucose concentrations and suppressed by fenofibrate. Our results demonstrated that adiponectin may be a proinflammatory mediator in diabetic retinas and fenofibrate appears to modulate the expression of adiponectin and its receptors in diabetic retinas, effectively reducing DR progression.
Chronic alcohol intake decreases adiponectin and sirtuin 1 (SIRT1) expressions, both of which have been implicated in various biological processes including inflammation, apoptosis and metabolism. We have previously shown that moderate consumption of alcohol aggravates liver inflammation and apoptosis in rats with pre-existing nonalcoholic steatohepatitis (NASH). This study investigated whether moderate alcohol intake alters SIRT1 activity, adiponectin/Adiponectin receptor (AdipoR)-related signaling and lipid metabolism in a pre-existing NASH status. Sprague-Dawley rats were fed with a high-fat diet (71% energy from fat) for 6 weeks to induce NASH then subsequently divided into 2 sub-groups: fed either a modified high-fat diet (HFD, 55% energy from fat) or a modified high-fat alcoholic diet (HFA, 55% energy from fat and 16% energy from ethanol) for an additional 4 weeks. We observed in comparison to HFD group, HFA increased hepatic nuclear SIRT1 protein but decreased its deacetylase activity. SREBP-1c protein expression and FAS mRNA levels were significantly upregulated, while DGAT1/2 and CPT-I mRNA levels were downregulated in the livers of HFA compared to HFD. Although hepatic AdipoR1 decreased, HFA did not alter AdipoR2 and their downstream signaling. There were no significant changes in plasma adiponectin and free fatty acids (FFA), as well as adiponectin expression in adipose tissue between the two groups. The present study indicates that suppression in SIRT1 deacetylase activity contributes to alcohol-exacerbated hepatic inflammation and apoptosis in rats with pre-existing NASH. In addition, moderate alcohol intake did not modulate adiponectin/AdipoR signaling axis in this model.
Adiponectin; alcohol consumption; nonalcoholic steatohepatitis (NASH); rats; sirtuin-1 (SIRT1)
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.
Nonalcoholic steatohepatitis; adiponectin receptor 2; inflammation; oxidative stress; Zucker rats
AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.
METHODS: The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064.
RESULTS: The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064.
CONCLUSION: The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.
Farnesoid X receptor; Adipokines; Adipokine receptors; 3T3-L1 cells; HepG2 cells; Nonalcoholic fatty liver disease