AdipoR2 inhibits human glioblastoma cell growth through the AMPK/mTOR pathway.

BACKGROUND: AdipoR2, which belongs to the seven-transmembrane-domain receptor family, has been shown to play an important role in the development of human tumours, but the underlying mechanisms are poorly understood. In this study, we found that AdipoR2 expression correlates with glioma grade. In addition, we also investigated the mechanisms behind the antiproliferative effects of AdipoR2 in U251 cells (a human glioma cell line) using colony formation and WST-8 growth assays. METHODS: The U251 cell line was cultured in vitro. Western blotting was used to detect the expression of relevant proteins. Quantitative RT-PCR was used to detect AdipoR1 and AdipoR2 expression. Flow cytometry was used to detect cell cycle assay results. The gene expression profiles of glioma samples from the CGGA database were analysed by MATLAB and GSEA software. RESULTS: The AMPK/mTOR pathway plays a central role in the regulation of cell proliferation, differentiation and migration and may promote tumorigenesis. Therefore, we can control cancer progression by modulating the AMPK/mTOR pathway. However, there is no information on the relationship between AdipoR and AMPK/mTOR in central nervous system tumours such as GBM. In this study. We found 648 upregulated genes and 436 downregulated genes correlated with AdipoR2 expression in 158 glioma samples. GSEA suggested that AdipoR2 is a cell cycle-associated gene. The results of the flow cytometry analysis indicated that AdipoR2 induced G0/G1 cell cycle arrest in U251 cells. Furthermore, we identified the AMPK/mTOR signalling axis to be involved in AdipoR2-induced cell cycle arrest. CONCLUSIONS: Our results suggest that AdipoR2 may represent a novel endogenous negative regulator of GBM cell proliferation. These findings also suggest that AdipoR2 may be a promising therapeutic target in GBM patients.

Adiponectin inhibits cardiac arrest/cardiopulmonary resuscitation­induced apoptosis in brain by increasing autophagy involved in AdipoR1­AMPK signaling.

Emerging evidence suggests that both apoptosis and autophagy contribute to global cerebral ischemia­reperfusion (GCIR)­induced neuronal death, which results from cardiac arrest (CA). However, the mechanism of how GCIR may affect the balance between apoptosis and autophagy resulting from CA remains to be elucidated. Additionally, the role of adiponectin (APN) in reversing the apoptosis and autophagy induced by GCIR following cardiac arrest­cardiopulmonary resuscitation (CA­CPR) is unclear. Thus, the aim of the present study was to investigate how GCIR affect the apoptosis and autophagy in response to CA and to clarify whether APN may alter the apoptosis and autophagy of neuronal death in GCIR­injured brain post­CA­CPR. Using normal controls (Sham group) and two experimental groups [CA­CPR­induced GCIR injury (PCAS) group and exogenous treatment with adiponectin post­CA­CPR (APN group)], it was demonstrated that both apoptosis and autophagy were observed simultaneously in the brain subjected to GCIR, but apoptosis appeared to be more apparent. Exogenous administration of APN significantly reduced the formation of malondialdehyde, a marker of oxidative stress and increased the expression of superoxide dismutase, an anti­oxidative enzyme, resulting in the stimulation of autophagy, inhibition of apoptosis and reduced brain tissue injury (P<0.05 vs. PCAS). APN treatment increased the expression of APN receptor 1 (AdipR1) and the phosphorylation of AMP­activated protein kinase (AMPK; Ser182) in brain tissues. In conclusion, GCIR induced apoptosis and inhibited autophagy, contributing to brain injury in CA­CPR. By contrast, APN reduced the brain injury by reversing the changes of neuronal autophagy and apoptosis induced by GCIR. The possible mechanism might owe to its effects on the activation of AMPK after combining with AdipR1 on neurons, which suggests a novel intervention against GCIR injury in CA­CPR conditions.

Taste Receptor Cells in Mice Express Receptors for the Hormone Adiponectin.

The metabolic hormone adiponectin is secreted into the circulation by adipocytes and mediates key biological functions, including insulin sensitivity, adipocyte development, and fatty acid oxidation. Adiponectin is also abundant in saliva, where its functions are poorly understood. Here we report that murine taste receptor cells (TRCs) express specific adiponectin receptors and may be a target for salivary adiponectin. This is supported by the presence of all three known adiponectin receptors in transcriptomic data obtained by RNA-seq analysis of purified circumvallate (CV) taste buds. As well, immunohistochemical analysis of murine CV papillae showed that two adiponectin receptors, ADIPOR1 and T-cadherin, are localized to subsets of TRCs. Immunofluorescence for T-cadherin was primarily co-localized with the Type 2 TRC marker phospholipase C ß2, suggesting that adiponectin signaling could impact sweet, bitter, or umami taste signaling. However, adiponectin null mice showed no differences in behavioral lick responsiveness compared with wild-type controls in brief-access lick testing. AAV-mediated overexpression of adiponectin in the salivary glands of adiponectin null mice did result in a small but significant increase in behavioral lick responsiveness to the fat emulsion Intralipid. Together, these results suggest that salivary adiponectin can affect TRC function, although its impact on taste responsiveness and peripheral taste coding remains unclear.

Reciprocal Inhibition of Adiponectin and Innate Lung Immune Responses to Chitin and Aspergillus fumigatus.

Chitin is a structural biopolymer found in numerous organisms, including pathogenic fungi, and recognized as an immune-stimulating pathogen associated molecular pattern by pattern recognition molecules of the host immune system. However, programming and regulation of lung innate immunity to chitin inhalation in the context of inhalation of fungal pathogens such as Aspergillus fumigatus is complex and our understanding incomplete. Here we report that the systemic metabolism-regulating cytokine adiponectin is decreased in the lungs and serum of mice after chitin inhalation, with a concomitant decrease in surface expression of the adiponectin receptor AdipoR1 on lung leukocytes. Constitutive lung expression of acidic mammalian chitinase resulted in decreased inflammatory cytokine gene expression and neutrophil recruitment, but did not significantly affect lung adiponectin transcription. Exogenous recombinant adiponectin specifically dampened airway chitin-mediated eosinophil recruitment, while adiponectin deficiency resulted in increased airway eosinophils. The presence of adiponectin also resulted in decreased CCL11-mediated migration of bone marrow-derived eosinophils. In contrast to purified chitin, aspiration of viable conidia from the high chitin-expressing A. fumigatus isolate Af5517 resulted in increased neutrophil recruitment and inflammatory cytokine gene expression in adiponectin-deficient mice, while no significant changes were observed in response to the isolate Af293. Our results identify a novel role for the adiponectin pathway in inhibition of lung inflammatory responses to chitin and A. fumigatus inhalation.

Adiponectin suppresses amyloid-ß oligomer (AßO)-induced inflammatory response of microglia via AdipoR1-AMPK-NF-κB signaling pathway.

BACKGROUND: Microglia-mediated neuroinflammation is important in Alzheimer's disease (AD) pathogenesis. Extracellular deposition of ß-amyloid (Aß), a major pathological hallmark of AD, can induce microglia activation. Adiponectin (APN), an adipocyte-derived adipokine, exerts anti-inflammatory effects in the periphery and brain. Chronic APN deficiency leads to cognitive impairment and AD-like pathologies in aged mice. Here, we aim to study the role of APN in regulating microglia-mediated neuroinflammation in AD. METHODS: Inflammatory response of cultured microglia (BV2 cells) to AßO and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor α [TNFα] and interleukin-1ß [IL-1ß]) in cultured medium before and after exposure to AßO, with and without APN pretreatment. Adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2) were targeted by small interference RNA. To study the neuroprotective effect of APN, cultured HT-22 hippocampal cells were treated with conditioned medium of AßO-exposed BV2 cells or were co-cultured with BV2 cells in transwells. The cytotoxicity of HT-22 hippocampal cells was assessed by MTT reduction. We generated APN-deficient AD mice (APN-/-5xFAD) by crossing APN-knockout mice with 5xFAD mice to determine the effects of APN deficiency on microglia-mediated neuroinflammation in AD. RESULTS: AdipoR1 and AdipoR2 were expressed in BV2 cells and microglia of mice. Pretreatment with APN for 2 h suppressed TNFα and IL-1ß release induced by AßO in BV2 cells. Additionally, APN rescued the decrease of AMPK phosphorylation and suppressed nuclear translocation of nuclear factor kappa B (NF-κB) induced by AßO. Compound C, an inhibitor of AMPK, abolished these effects of APN. Knockdown of AdipoR1, but not AdipoR2 in BV2 cells, inhibited the ability of APN to suppress proinflammatory cytokine release induced by AßO. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with AßO-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APN-/-5xFAD mice associated with upregulation of TNFα and IL-1ß in the cortex and hippocampus. CONCLUSIONS: Our findings demonstrate that APN inhibits inflammatory response of microglia to AßO via AdipoR1-AMPK-NF-κB signaling, and APN deficiency aggravates microglia activation and neuroinflammation in AD mice. APN may be a novel therapeutic agent for inhibiting neuroinflammation in AD.

Expression of adiponectin receptors in the brain of adult zebrafish and mouse: Links with neurogenic niches and brain repair.

Adiponectin and its receptors (adipor) have been initially characterized for their role in lipid and glucose metabolism. More recently, adiponectin signaling was shown to display anti-inflammatory effects and to participate in brain homeostasis and neuroprotection. In this study, we investigated adipor gene expression and its regulation under inflammatory conditions in two complementary models: mouse and zebrafish. We demonstrate that adipor1a, adipor1b, and adipor2 are widely distributed throughout the brain of adult fish, in neurons and also in radial glia, behaving as neural stem cells. We also show that telencephalic injury results in a decrease in adipor gene expression, inhibited by an anti-inflammatory treatment (Dexamethasone). Interestingly, adiponectin injection after brain injury led to a consistent decrease (a) in the recruitment of microglial cells at the lesioned site and (b) in the proliferation of neural progenitors, arguing for a neuroprotective role of adiponectin. In a comparative approach, we investigate Adipor1 and Adipor2 gene distribution in the brain of mice and demonstrated their expression in regions shared with fish including neurogenic regions. We also document Adipor gene expression in mice after middle cerebral artery occlusion and lipopolysaccharide injection. In contrast to zebrafish, these inflammatory stimuli do no impact cerebral adiponectin receptor gene expression in mouse. This work provides new insights regarding adipor expression in the brain of fish, and demonstrates evolutionary conserved distribution of adipor with mouse. This is the first report of adipor expression in adult neural stem cells of fish, suggesting a potential role of adiponectin signaling during vertebrate neurogenesis. It also suggests a potential contribution of inflammation in the regulation of adipor in fish.

High-intensity interval training induced PGC-1∝ and AdipoR1 gene expressions and improved insulin sensitivity in obese individuals.

INTRODUCTION: High-intensity interval training (HIIT) has been found to improve cardiometabolic health outcome as compared to moderate-intensity continuous exercise. However, there is still limited data on the benefits of HIIT on the expression of regulatory proteins that are linked to skeletal muscle metabolism and insulin sensitivity in obese adults. This study investigated the effects of HIIT intervention on expressions of peroxisome proliferatoractivated receptor-γ coactivator 1-∝ (PGC-1∝) and adiponectin receptor-1 (AdipoR1), insulin sensitivity (HOMAIR index), and body composition in overweight/obese individuals. METHODS: Fifty overweight/obese individuals aged 22-29 years were assigned to either no-exercise control (n=25) or HIIT (n=25) group. The HIIT group underwent a 12-week intervention, three days/week, with intensity of 65-80% of age-based maximum heart rate. Anthropometric measurements, homeostatic model of insulin resistance (HOMA-IR) and gene expression analysis were conducted at baseline and post intervention. RESULTS: Significant time-by-group interactions (p<0.001) were found for body weight, BMI, waist circumference and body fat percentage. The HIIT group had lower body weight (2.3%, p<0.001), BMI (2.7%, p<0.001), waist circumference (2.4%, p<0.001) and body fat percentage (4.3%, p<0.001) post intervention. Compared to baseline, expressions of PGC-1∝ and AdipoR1 were increased by approximately three-fold (p=0.019) and two-fold (p=0.003) respectively, along with improved insulin sensitivity (33%, p=0.019) in the HIIT group. CONCLUSION: Findings suggest that HIIT possibly improved insulin sensitivity through modulation of PGC-1∝ and AdipoR1. This study also showed that improved metabolic responses can occur despite modest reduction in body weight in overweight/obese individuals undergoing HIIT intervention.

miR-449a induces EndMT, promotes the development of atherosclerosis by targeting the interaction between AdipoR2 and E-cadherin in Lipid Rafts.

EndMT plays an important role in the relationship between endothelial dysfunction and atherosclerosis. This work will elucidate the biofunction induced by miR-449a and lipid rafts in EndMT and development of atherosclerosis. The differential miRNA expression between atherosclerotic plaques and normal arteries were analyzed. The luciferase activities of AdipoR2 3' UTR treated with miR-449a were determined. ECs were dealt with miR-449a mimics or inhibitors, then cell proliferation and migration were assessed. Moreover, the expression of AdipoR2 and mesenchymal cell markers were analyzed. The influences of lipid rafts related to reciprocity between E-cadherin and AdipoR2 on TNF-α-induced damage in ECs were investigated. ApoE KO diabetic mice were used to explore the potential roles of miR-449a on atherosclerosis. Our results indicated that compared with normal arteries, 17 miRNAs were upregulated and 3 miRNAs were down-regulated in atherosclerotic plaques. The relative expression of miR-449a in plaques was significantly higher than that in normal arteries. MiR-449a suppressed AdipoR2 expression, additionally its interaction protein E-cadherin in ECs. MiR-449a enhanced expression of mesenchymal cell markers, induced cell proliferation and migration of ECs, regulated the interaction between E-cadherin and AdipoR2 interceded by lipid rafts. The miR-449a antagomir could protect against the development process of atherosclerosis in ApoE KO diabetic mice. In conclusion, miR-449a targeted to AdipoR2, and was a crucial mediator of EndMT and atherosclerosis in ECs through regulating E-cadherin bindability with AdipoR2 in lipid rafts. These results suggested that aim to lipid rafts and miR-449a in chronic EC inflammation response, was a feasible therapy strategy for atherosclerosis.

Expression of Adiponectin Receptor-1 and Prognosis of Epithelial Ovarian Cancer Patients.

BACKGROUND Adiponectin receptor-1 (AdipoR1) has been reported to be associated with the risk of obesity-associated malignancies, including epithelial ovarian cancer (EOC). The aim of this study was to determine if AdipoR1 could serve as a prognosis indicator for patients with EOC. MATERIAL AND METHODS In this study, expression of AdipoR1 in 73 EOC patients consecutively admitted to our hospital was detected by immunohistochemical staining. Univariate and multivariate analyses were performed to assess the relationship between AdipoR1 expression level and progression-free survival (PFS) and overall survival (OS) rates in patients. RESULTS A relatively lower expression of AdipoR1 in the cancerous tissues was detected compared to normal ovarian tissues, but the difference was not significant (p>0.05). AdipoR1 expression level in EOC patients was negatively correlated with advanced FIGO stages in patients and tumor differentiation, but had no correlation with pathological types, presenting of ascites, shorter platinum-free interval (PFI), diabetes, preoperative and postoperative body mass index (BMI), or platelet counts (p>0.05). Moreover, patients with AdipoR1 expression had a significantly longer PFS and OS compared to the negative expression group (p<0.001). CONCLUSIONS Our findings suggest that AdipoR1 expression level in cancerous tissues might serve as an independent prognostic indicator in EOC patients and is associated with longer PFS and OS.

Expression of adiponectin receptors in human and rat intervertebral disc cells and changes in receptor expression during disc degeneration using a rat tail temporary static compression model.

BACKGROUND: Adipose tissue is a large endocrine organ known to secret adiponectin, which has anti-diabetic, anti-atherogenic, and anti-inflammatory properties. Adiponectin is widely involved in systemic disease, diabetes mellitus, and cardiac infraction. This study aimed to investigate the involvement of adiponectin in intervertebral disc (IVD) degeneration. METHODS: Adipose and IVD tissues were obtained from human patients undergoing surgery (n = 4) and from skeletally mature Sprague-Dawley rats (n = 21). Tissues were stained immunohistochemically for adiponectin and adiponectin receptors AdipoR1 and AdipoR2. Changes in adiponectin receptor expression with IVD degeneration severity were then investigated using a rat tail temporary compression model. Rat IVD tissues were stained immunohistochemically with AdipoR1 or AdipoR2, and immunopositive cell percentages were calculated. Rat nucleus pulposus (NP) and annulus fibrosus (AF) tissues were isolated separately and treated with recombinant adiponectin (Ad 0.1 or 1.0 µg/ml) and/or interleukin-1 beta (IL-1ß) (0.2 µg/ml) for 24 h. The four groups were as follows: control group (no treatment), IL-1ß group (IL-1ß-only treatment), IL-1ß+Ad (0.1) group (IL-1ß and adiponectin [0.1 µg/ml] treatment), and IL-1ß+Ad (1.0) group (IL-1ß and adiponectin [1.0 µg/ml]). Real-time reverse transcription-polymerase chain reaction was performed to evaluate messenger-RNA (mRNA) expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). RESULTS: Adiponectin was widely expressed in human subcutaneous and epidural adipose tissue. In rat IVD tissue, adiponectin was not observed in NP and AF. However, both AdipoR1 and AdipoR2 were widely expressed in both human and rat IVD tissues, with no significant differences in expression levels between receptors. Furthermore, expression levels of AdipoR1 and AdipoR2 were gradually decreased with increased IVD degeneration severity. Interestingly, mRNA expression levels of TNF-α and IL-6 were significantly upregulated by IL-1ß stimulation. TNF-α expression in the IL-1ß+Ad 1.0 group was significantly lower than that in the IL-1ß group in both NP and AF cells (P < 0.05). Finally, IL-6 expression was not affected by adiponectin treatment in IVD cells. CONCLUSIONS: This study investigated for the first time the expression of adiponectin receptors in human and rat IVD cells. The findings indicate that adiponectin produced by the systemic or epidural adipose tissue may be involved in the pathomechanism of IVD degeneration.

Dietary α-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice.

Low levels of n-3 polyunsaturated fatty acids (PUFAs) in serum and liver tissue biopsies are the common characteristics in patients with alcoholic liver disease. The α-linolenic acid (ALA) is a plant-derived n-3 PUFA and is rich in flaxseed oil. However, the impact of ALA on alcoholic fatty liver is largely unknown. In this study, we assessed the potential protective effects of ALA-rich flaxseed oil (FO) on ethanol-induced hepatic steatosis and observed that dietary FO supplementation effectively attenuated the ethanol-induced hepatic lipid accumulation in mice. Ethanol exposure stimulated adipose lipolysis but reduced fatty acid/lipid uptake, which were normalized by FO. Our investigations into the corresponding mechanisms demonstrated that the ameliorating effect of FO might be associated with the lower endoplasmic reticulum stress and normalized lipid metabolism in adipose tissue. In the liver, alcohol exposure stimulated hepatic fatty acid uptake and triglyceride synthesis, which were attenuated by FO. Additionally, dietary FO upregulated plasma adiponectin concentration, hepatic adiponectin receptor 2 expression, and the activation of hepatic adenosine monophosphate-activated protein kinase. Collectively, dietary FO protects against alcoholic hepatic steatosis by improving lipid homeostasis at the adipose tissue-liver axis, suggesting that dietary ALA-rich flaxseed oil might be a promising approach for prevention of alcoholic fatty liver.

Weight Gain Alters Adiponectin Receptor 1 Expression on Adipose Tissue-Resident Helios+ Regulatory T Cells.

Adipose tissue produces multiple mediators that modulate the immune response. Adiponectin is an adipocyte-derived cytokine that exhibits metabolic and anti-inflammatory effects. Adiponectin acts through binding to adiponectin receptor 1 and 2 (AdipoR1/AdipoR2). AdipoR1 is ubiquitously expressed, whereas AdipoR2 is restricted to skeletal muscle and liver. AdipoR1 expression has been reported on a small percentage of T cells; nevertheless, it is still unknown whether Foxp3(+) regulatory T cells (Tregs) express AdipoR1. Recently, it has been shown that Tregs accumulate in adipose tissue and that they play a potential role in modulating adipose tissue inflammation. Our aim was to evaluate AdipoR1 expression in adipose tissue-resident Tregs and to evaluate the effect of weight gain on this expression. Male C57BL/6 mice were fed with a high-fat diet for 14 weeks (to develop overweight) or 21 weeks (to develop obesity). Mice on a standard diet were used as age-matched controls. Helios expression was evaluated as a marker to discriminate thymic-derived from peripherally induced Tregs. The majority of Tregs in both adipose tissue and the spleen expressed Helios. Adipose tissue Tregs expressed higher levels of AdipoR1 than Tregs in the spleen. AdipoR1 expression on adipose tissue Helios(+) Tregs was negatively correlated with epididymal fat. Overall, we show that AdipoR1 is expressed on adipose tissue-resident Tregs, mainly Helios(+) Tregs, and that this expression is dependent on weight and fat accumulation. Because both adiponectin and Tregs play roles in anti-inflammatory mechanisms, our data propose a new mechanism through which weight gain might alter immunoregulation.

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression.

BACKGROUND: Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors. METHODS: In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48 or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student's t-test, and P<0.05 was considered statistically significant. RESULTS: Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05). CONCLUSIONS: Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.

Role of adiponectin in adipose tissue wound healing.

The purpose of this study was to investigate the mechanism behind adipose tissue wound healing (ATWH). The preadipocyte cell line 3T3-L1 was cultured and expression of adiponectin receptors (AdipoR1/2) was detected by immunohistochemistry and reverse transcription polymerase chain reaction. The concentration of adiponectin secreted at different cell densities was measured by enzyme-linked immunosorbent assay, while preadipocyte proliferation and migration were determined in vitro by MTT and wound closure assays. AdipoR1/2 were found to be expressed in 3T3-L1 preadipocytes. There were no statistically significant differences in the concentrations of adiponectin secreted by cell solutions of different densities (P > 0.05). In addition, adiponectin was seen to promote the growth and migration of preadipocytes. In conclusion, adiponectin may regulate ATWH by promoting preadipocyte proliferation and migration, and its systemic and/or local application is proposed as a promising therapeutic approach for the treatment of wounds incurred as a result of surgery.

Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-ß1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-ß1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose-dependent manner, via suppression of lung fibroblast activation. Functional AdipoR1 are expressed by human WI-38 lung fibroblasts, suggesting potential future clinical applicability of APN against pulmonary fibrosis.

Tumor expression of adiponectin receptor 2 and lethal prostate cancer.

To investigate the role of adiponectin receptor 2 (AdipoR2) in aggressive prostate cancer we used immunohistochemistry to characterize AdipoR2 protein expression in tumor tissue for 866 men with prostate cancer from the Physicians' Health Study and the Health Professionals Follow-up Study. AdipoR2 tumor expression was not associated with measures of obesity, pathological tumor stage or prostate-specific antigen (PSA) at diagnosis. However, AdipoR2 expression was positively associated with proliferation as measured by Ki-67 expression quartiles (P-trend < 0.0001), with expression of fatty acid synthase (P-trend = 0.001), and with two measures of angiogenesis (P-trend < 0.1). An inverse association was observed with apoptosis as assessed by the TUNEL assay (P-trend = 0.006). Using Cox proportional hazards regression and controlling for age at diagnosis, Gleason score, year of diagnosis category, cohort and baseline BMI, we identified a statistically significant trend for the association between quartile of AdipoR2 expression and lethal prostate cancer (P-trend = 0.02). The hazard ratio for lethal prostate cancer for the two highest quartiles, as compared to the two lowest quartiles, of AdipoR2 expression was 1.9 (95% confidence interval [CI]: 1.2-3.0). Results were similar when additionally controlling for categories of PSA at diagnosis and Ki-67 expression quartiles. These results strengthen the evidence for the role of AdipoR2 in prostate cancer progression.

Adipo-R1 and adipo-R2 expression in colorectal adenomas and carcinomas.

BACKGROUND: Human adiponectin (ApN), a 30 kDa glycoprotein of 244-amino acids which is predominantly produced by adipocytes, exerts its effects via two receptors, namely adiponectin receptor-1 (adipo-R1) and adiponectin receptor-2 (adipo-R2) with differential binding affinity to globular adiponectin. Adiponectin receptor expression has been studied in several cancer tissues. However, there are no studies of colorectal adenomas which are considered to be precursors for colorectal carcinoma (CRC). OBJECTIVES: In the present study, the expression of adipo-R1 and adipo-R2 was investigated immunohistochemically in colorectal adenomas and colorectal carcinoma tissues in an attempt to determine associations with these tumors. MATERIALS AND METHODS: The study enrolled 50 CRC patients with tumor resection and 82 patients who were diagnosed with adenomatous polyps, classified as negative for neoplasia, low-grade dysplasia (L-GD) or high- grade dysplasia (H-GD). RESULTS: Expression of both adipo-R1 and adipo-R2 was found to be significantly lower in the CRCs than in colorectal adenomas (tubular and tubulovillous, p=0.009 and p<0.001, respectively). Adipo-R1 and adipo-R2 expression was also significantly lower in the CRC group when compared with the groups of patients with low grade dysplasia, high-grade dysplasia or no neoplasia (p=0.012 and p<0.001, respectively). In addition, it was observed that adipo-R2 expression was generally positive in the non-neoplastic group irrespective of the adipo-R2 expression. In the L-GD, H-GD and CRC groups, the adipo-R2 result was positive whenever adipo-R1 result was positive but some patients with negative adipo-R1 had positive adipo-R2 (p<0.001, p=0.004, p<0.001, respectively). CONCLUSIONS: This study indicated that ApN may play a role in the progression of colorectal adenomatous polyps to carcinoma through actions on adipo-R1 and adipo-R2 receptors.

Adiponectin inhibits VEGF-A in prostate cancer cells.

A role of adiponectin in tumorigenesis has recently been appreciated. Although plasma adiponectin levels in subjects with prostate cancer have been found to be significantly lower than in subjects with benign prostatic hyperplasia or in normal healthy controls, the underlying molecular mechanisms remain unknown. Here, we not only detected significant decreases in plasma adiponectin levels in prostate cancer patients, but also showed significant decreases in adiponectin receptor I (AdipoR1) levels in the resected prostate cancer specimen. Prostate cancer cell lines examined in the current study had all lower levels of adiponectin and AdipoR1, compared to normal healthy prostate tissue. Moreover, overexpression of adiponectin in prostate cancer cells decreased production of vascular endothelial growth factor A (VEGF-A), while adiponectin depletion increased VEGF-A. Furthermore, adiponectin seemed to activate AMPK/TSC2 to inhibit mTor-mediated activation of VEGF-A. Taken together, our data suggest that adiponectin may play an essential role in suppressing growth of prostate cancer cells through inhibition of VEGF-A-mediated cancer neovascularization.

Adiponectin receptor and adiponectin signaling in human tissue among patients with end-stage renal disease.

BACKGROUND: Adiponectin plasma levels in chronic kidney disease (CKD) are two to three times higher than in individuals with normal kidney function. Despite adiponectin's anti-diabetic, anti-inflammatory and anti-atherogenic properties, patients with CKD have insulin resistance, systemic inflammation and accelerated atherogenesis. Hence, although adiponectin production is increased by adipose tissue in end-stage renal disease (ESRD), it is unclear if its effects on metabolism remain intact. METHODS: To determine if there is adiponectin resistance in ESRD, we measured tissue levels of adiponectin receptor-1 (AdipoR1) and adiponectin downstream effectors in ESRD patients compared with normal kidney function controls. Blood and tissue samples were obtained from participants at the time of kidney transplantation or kidney donation. A follow-up blood sample was obtained 3-6 months after transplantation. RESULTS: AdipoR1 was higher in muscle and peripheral blood mononuclear cells collected from ESRD patients. There was also a nonsignificant increase in AdipoR1 in visceral fat of ESRD compared with controls. Compared with controls, phosphorylation of the adiponectin downstream effector adenosine monophosphate-activated protein kinase (AMPK) was higher in ESRD while acetyl-CoA carboxylase phosphorylation (ACC-P) and carnitine palmitoyl transferase-1 (CPT-1) levels were lower. In vitro, exposure of C2C12 cells to uremic serum resulted in upregulation of AdipoR1 and increased phosphorylation of AMPK but decreased ACC-P and CPT-1 expression. CONCLUSION: Both our in vivo and in vitro observations indicate that uremia results in upregulation of AdipoR1 but adiponectin resistance at the post-receptor level.

The effects of LPS-induced endotoxemia on the expression of adiponectin and its receptors in female rats.

Adiponectin (APN), secreted by white adipose tissue (WAT), acts as a protective factor against inflammatory conditions. However, the changes in the expression levels of endogenous APN and the two types of APN receptor (AdipoR1 and AdipoR2) induced by acute inflammatory conditions have not been fully elucidated. In this study, the changes in peripheral and/or central APN and AdipoR expression caused by lipopolysaccharide (LPS)-induced sepsis were examined in gonadal-intact (Sham) and ovariectomized (OVX) female rats. As it has been reported that APN and AdipoR suppress the production of inflammatory cytokines to prevent excessive inflammation, the mRNAs of these molecules were also examined. LPS injection induced increases in visceral WAT APN mRNA without affecting the serum APN level in both the Sham and OVX rats. OVX rats exhibited higher serum APN levels than Sham rats. LPS injection increased the subcutaneous WAT APN mRNA in OVX rats. In both Sham and OVX rats, LPS injection led to a decrease in hepatic AdipoR2 mRNA and an increase in hypothalamic AdipoR2 mRNA. Hypothalamic AdipoR2 mRNA was upregulated 24 h after LPS injection in OVX but not Sham rats. Serum TNF-α level at 6 h after LPS injection and hypothalamic and hepatic IL-6 and TNF-α mRNA at 24 h after LPS injection were significantly higher in Sham than OVX rats. These results suggest that APN and AdipoR play roles in modulating inflammation under septic conditions in female rats.