Adiponectin Pretreatment Research Articles

Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection

Besides metabolic homeostasis regulation, adipokines are recently emerged as important players in regulating immunity and inflammation. Helminth infection has known to modulate circulating adipokine secretion; however, the regulation and function of adipokines in response to helminth infection is still unclear. Here, we investigated the regulation and function of adiponectin during T. spiralis infection. While there was no change in circulating level of adiponectin, we found an increased adiponectin, but not leptin expression in the small intestine. Interestingly, the intestinal adiponectin expression was strongly associated with the expression of epithelial cell-derived cytokines IL-25, IL-33, and TSLP following infection. Indeed, mice deficiency of IL-25 receptor exhibited no intestinal adiponectin induction upon helminth infection. Interestingly, IL-25-induced adiponectin modulated intestinal epithelial cell responses by enhancing occludin and CCL17 expression. Using LPS-induced intestinal epithelial barrier dysfunctions in a Caco-2 cell monolayer model, adiponectin pretreatment enhanced a Transepithelial electrical resistance (TEER) and occludin expression. More importantly, adiponectin pretreatment of Caco2 cells prevented T. spiralis larval invasion in vitro and its administration during infection enhanced intestinal IL-13 secretion and worm expulsion in vivo. Altogether, our data suggest that intestinal adiponectin expression induced by helminth infection through the regulation of IL-25 promotes worm clearance and intestinal barrier function.

Adiponectin inhibits TGF-β1-induced skin fibroblast proliferation and phenotype transformation via the p38 MAPK signaling pathway.

The aim of this study was to investigate the effects of adiponectin (APN) on the proliferation and phenotypic transformation of human skin fibroblasts (HSFs) induced by TGF-β1. Primary fibroblast cultures were collected from prepuce surgery, and the cell viability and proliferative activity of HSFs were detected by Cell Counting Kit-8 and EdU assays. In addition, cell migration was detected by Transwell assay. The protein levels of related genes in HSF were detected by Western blotting. The results showed that the proliferation and migration abilities of HSF in the TGF-β1 group were significantly improved, and the relative protein expression levels of PCNA, α-SMA, and Collagen I in the TGF-β1 group were greatly increased. Furthermore, TGF-β1 stimulated the phosphorylation of p38 in HSF, while APN pretreatment significantly inhibited the TGF-β1-induced phosphorylation of p38. Additionally, blocking the p38 MAPK signaling pathway relieved the injury in the HSF induced by TGF-β1 and enhanced the therapeutic effect of APN in the TGF-β1-treated HSF. In conclusion, APN inhibits TGF-β1-induced HSF proliferation and myofibroblast phenotypic transformation by activating the p38 MAPK signaling pathway. APN is expected to become a potential target for preventing and treating skin fibrosis and pathological scars.

Adiponectin Treatment Attenuates Cerebral Ischemia-Reperfusion Injury through HIF-1α-Mediated Antioxidation in Mice.

Adiponectin (ADPN) plays an important role in cerebral ischemia-reperfusion injury. Although previous studies have confirmed that ADPN pretreatment has a protective effect on ischemic stroke, the therapeutic effect of ADPN on ischemic stroke and the underlying mechanism are still unclear. In order to clarify these questions, focal transient cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in mice and ADPN was administered for three times at 6 h, 24 h, and 48 h after reperfusion. Meanwhile, a virus-delivered HIF-1α siRNA was used before ADPN administration. The infarct volume, neurological score, cellular apoptosis, and oxidative stress were assessed at 72 h after reperfusion. The long-term outcome of mice after stroke was recorded as well. The results indicated that ADPN treatment reduced the infarct volume (P = 0.032), neurological deficits (P = 0.047), cellular apoptosis (P = 0.041), and oxidative responses (P = 0.031) at 72 h after MCAO. Moreover, ADPN increased both the protein level and transcriptional activity of HIF-1α as evidenced by the transcription levels of VEGF (P = 0.046) and EPO (P = 0.043) at 72 h after MCAO. However, knockdown of HIF-1α partially reversed the antioxidant and treatment effect of ADPN after cerebral ischemia. In the observation of long-term outcome after ADPN treatment, it demonstrated that ADPN not only prevented the cerebral atrophy (P = 0.031) and the neurological function decline (P = 0.048), but also promoted angiogenesis (P = 0.028) after stroke. In conclusion, our findings suggest that ADPN is effective in treatment of ischemic stroke which could be attributed to the increased antioxidant capacity regulated by HIF-1α.

Adiponectin protects HL-1 cardiomyocytes against rotenone-induced cytotoxicity through AMPK activation

The relationship between mitochondrial dysfunction or ER stress with pathogenesis of cardiovascular disease is well documented, but the crosstalk between them in cardiovascular diseases is not clear. Adiponectin (APN) is reported to become a potential cardioprotective molecule, but whether and how APN regulates mitochondrial dysfunction and ER stress is not clear. In this study, we used rotenone-treated HL-1 atrial cardiomyocytes as an in vitro model of mitochondrial dysfunction to investigate the possible interactions between mitochondrial dysfunction and ER stress and explore the effects of APN on rotenone-induced cytotoxicity and the underlying mechanisms. It found that rotenone treatment significantly activated the ER stress PRK-like endoplasmic reticulum kinase (PERK)-dependent pathway, decreased autophagic flux and APN expression in a dose-dependent manner. Pretreatment of GSK2606414, an inhibitor of PERK kinase activity, attenuated the rotenone-induced decrease of APN expression. In return exogenous APN pretreatment inhibited rotenone-induced ER stress and activated autophagy via AMP-activated protein kinase (AMPK) activation and protected HL-1 cells against apoptosis and enhanced the viability after rotenone treatment. In conclusion, rotenone treatment induced significant cardiomyocyte cytotoxicity and ER stress, suppressed autophagy, and decreased APN expression in HL-1 cells. APN in return inhibited ER stress and activated autophagy through AMPK activation, thus alleviating rotenone induced HL-1 apoptosis.

Interactions between metabolism regulator adiponectin and intrinsic cardiac autonomic nervous system: A potential treatment target for atrial fibrillation

BackgroundPrevious studies indicated that inhibiting the cardiac autonomic nervous system (CANS) suppressed atrial fibrillation (AF). Clinical research revealed serum adiponectin (APN) exerted a beneficial influence on sympathetic and vagal tone in patients with type 2 diabetes. However, the effects of APN on CANS is unknown. This study aims to investigate whether APN could regulate CANS and suppress rapid atrial pacing (RAP)-induced AF. MethodsEighteen beagles were divided into the control group (saline plus sham RAP, N = 6), the RAP group (saline plus RAP, N = 6) and the APN + RAP group (APN plus RAP, N = 6). APN (10 μg, 0.1 μg/μL) or saline was microinjected into 4 major ganglionated plexi (GP) prior to RAP. Atrial electrophysiological parameters, anterior right GP (ARGP) function, neural activity and GP tissues were detected. ResultsCompared with the control treatment, RAP shortened effective refractory period (ERP) values at all sites and increased cumulative window of vulnerability (ΣWOV), ARGP function and neural activity, whereas APN injection reversed these changes. Mechanistically, APN ameliorated RAP-induced inflammatory response and down-regulated the expression of c-fos protein and nerve growth factor. Moreover, the APN receptors 1 and APN receptors 2 were detected both in neurons and in non-neuronal cells. APN pretreatment activated downstream adenosine monophosphate-activated protein kinase (AMPK) signaling, inhibited nuclear factor-kappa B signaling and promoted macrophage phenotype switching from proinflammatory to anti-inflammatory state. ConclusionsThis study demonstrates that administration of APN into GP can suppress RAP-induced AF by regulating the CANS. APN signaling may provide a potential therapeutic target to AF.

Adiponectin suppresses amyloid-\u03b2 oligomer (A\u03b2O)-induced inflammatory response of microglia via AdipoR1-AMPK-NF-\u03baB signaling pathway

BackgroundMicroglia-mediated neuroinflammation is important in Alzheimer’s disease (AD) pathogenesis. Extracellular deposition of β-amyloid (Aβ), 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.MethodsInflammatory response of cultured microglia (BV2 cells) to AβO and effects of APN were studied by measuring levels of proinflammatory cytokines (tumor necrosis factor α [TNFα] and interleukin-1β [IL-1β]) in cultured medium before and after exposure to AβO, 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 AβO-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.ResultsAdipoR1 and AdipoR2 were expressed in BV2 cells and microglia of mice. Pretreatment with APN for 2 h suppressed TNFα and IL-1β release induced by AβO in BV2 cells. Additionally, APN rescued the decrease of AMPK phosphorylation and suppressed nuclear translocation of nuclear factor kappa B (NF-κB) induced by AβO. 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 AβO. Moreover, pretreatment with APN inhibited the cytotoxicity of HT-22 cells co-cultured with AβO-exposed BV2 cells. Lastly, APN deficiency exacerbated microglia activation in 9-month-old APN−/−5xFAD mice associated with upregulation of TNFα and IL-1β in the cortex and hippocampus.ConclusionsOur findings demonstrate that APN inhibits inflammatory response of microglia to AβO 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.

Adiponectin protects palmitic acid induced endothelial inflammation and insulin resistance via regulating ROS/IKKβ pathways

Endothelial inflammation and insulin resistance (IR) has been closely associated with endothelial dysfunction. Adiponectin (APN), an adipocyte-secreted hormone from adipose tissues, showed cardioprotective effects. Here, the protective effect of APN on palmitic acid (PA)-induced endothelial inflammation and IR was investigated. Cultured human umbilical vein endothelial cells (HUVECs) were treated with PA without or without APN pretreatment. The expression of inflammatory cytokines TNF-α, IL-6, adhesion molecule ICAM-1 were determined by western blotting, ELISA, and real-time PCR. The protein expression and protein-protein interaction were determined by western blotting and immunoprecipitation. The intracellular reactive oxygen species (ROS) and nitric oxide (NO) production were monitored with fluorescence probes. PA-induced secretion of TNF-α, IL-6, and expression of ICAM-1 at protein and mRNA levels, which was significantly inhibited by APN. PA treatment caused increase of ROS generation, NOX2, p-IKKβ, p-IκBα, p-p65 expression, and p-IκBα-IKKβ interaction, which were all partly reversed by APN. ROS scavenger N-acetylcysteine (NAC) and NF-κB inhibitor PDTC showed similar effect on PA-induced secretion of TNF-α, IL-6, and expression of ICAM-1. Furthermore, APN and NAC pretreatment restored PA-induced increase of p-IRS-1(S307), decrease of p-IRS-1(Tyr). In addition, insulin-triggered expression of p-IRS-1(Tyr), p-PI3K, p-AKT, p-eNOS and NO generation were inhibited by PA, which were also restored by both APN and NAC. These results suggested that APN ameliorated endothelial inflammation and IR through ROS/IKKβ pathway. This study shed new insights into the mechanisms of APN’s cardiovascular protective effect.

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-β1, 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-β1, 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.

Abstract 268: Adiponectin Ameliorates Palmitate Acid Induced Endothelial Inflammation in Endothelial Cells

Endothelial dysfunction (ED) is considered an early event of cardiovascular diseases including hypertension, atherosclerosis and so on. Inflammation participates centrally in all stages of cardiovascular diseases and is considered as a hallmark of endothelial dysfunction. In this study, the effect of adiponectin (APN), an adipocytokine derived mainly from adipocytes, on palmitate acid (PA)-induced inflammation in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with PA with or without APN pretreatment. The mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and ICAM-1 were measured with RT-PCR. The protein expression of ICAM-1, NOX1, NOX2, NOX4, and phosphorylation of MAPKs (JNK, ERK, and p38MAPK), IKKβ, p65 NF-κB were determined by Western blotting. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) formation were determined with DCFH2-DA and DAF-FM respectively. APN significantly ameliorated PA-induced mRNA expression of TNF-α, IL-6 and ICAM-1 and protein expression of ICAM-1, NOX2, and phosphorylation of IKKβ, p65 NF-κB, p38MAPK, without affecting NOX2 and phosphorylation of JNK and ERK. APN also partly reversed PA induced ROS formation and NO decrease. NAC, a ROS scavenger, showed similar activities. The p38MAK inhibitor, SB203580, also reversed PA induced protein expression of ICAM-1 and mRNA expression of TNF-α, IL-6 and ICAM-1. Taken together, these results showed that APN improved PA induced endothelial dysfunction by regulating ROS/p38MAK/NF-κB pathways. Acknowledgement: This study was supported by the National Natural Science Foundation of China (No. 81160048) and the Science and Technology Development Fund of Macau Special Administrative Region (No. 021/2012/A1).

Adiponectin inhibits oxidative stress and modulates TGF-b1 and COL-1 expression via the AMPK pathway in HSC-T6 cells

To investigate the anti-oxidative stress and anti-fibrotic mechanisms of adiponectin by examining effects on oxidative stress levels and expression of fibrosis-related signaling factors, including transforming growth factor-beta 1 (TGFb1), collagen I (COL-1), and the adenosine monophosphate-activated protein kinase (AMPK) pathway by using an in vitro HSC-T6 cultured cell system. Activated HSC-T6 cells were pre-treated with 1.0 mug/mL adiponectin for 0, 30, 60 and 120 min, or left untreated to serve as controls, and both groups were then exposed to 5 mumol/L H2O2; a portion of the adiponectin-treated oxidative stress-induced cells were treated with an AMPK inhibitor (Compound C). The effects on mRNA levels of TGFb1. and COL-1 were analyzed by real-time PCR, in the levels of secreted TGF-b1 and COL-1 were detected by enzyme-linked immunosorbent assay of supernatants, and in the phosphoAMPK and AMPK protein expressions were detected by Western blotting. Compared to the H2O2 group without adiponectin pre-treatment, the H2O2 group with adiponectin pre-treatment showed significantly increased activity of superoxide dismutase (SOD), decreased content of malondialdehyde (MDA), and decreased gene and protein expressions of TGF-b1 and COL-1 (P less than 0.05). Moreover, inhibition of the AMPK pathway inhibited these adiponectin-mediated effects. The H2O2 group with adiponectin pre-treatment also showed increased levels of phospho-AMPK protein expression, with the maximum effect detected after 120 min of the adiponectin pre-treatment (P less than 0.01). Inhibition of oxidative stress is one of the mechanisms of the anti-fibrotic effects of adiponectin. Adiponectin can attenuate oxidative stress levels, resulting in down-regulation of TGFb1 and COL-1 expression through activation of the AMPK pathway.

Protective role of adiponectin in a rat model of intestinal ischemia reperfusion injury.

To determine the potential protective role of adiponectin in intestinal ischemia reperfusion (I/R) injury. A rat model of intestinal I/R injury was established. The serum level of adiponectin in rats with intestinal I/R injury was determined by enzyme-linked immunosorbent assay (ELISA). The serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were also measured by ELISA. Apoptosis of intestinal cells was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The production of malondialdehyde (MDA) and superoxide dismutase (SOD) and villous injury scores were also measured. Adiponectin was downregulated in the serum of rats with intestinal I/R injury compared with sham rats. No significant changes in the expression of adiponectin receptor 1 and adiponectin receptor 2 were found between sham and I/R rats. Pre-treatment with recombinant adiponectin attenuated intestinal I/R injury. The production of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, in rats with intestinal I/R injury was reduced by adiponectin pre-treatment. The production of MDA was inhibited, and the release of SOD was restored by adiponectin pre-treatment in rats with intestinal I/R injury. Adiponectin pre-treatment also inhibited cell apoptosis in these rats. Treatment with the AMP-activated protein kinase (AMPK) signaling pathway inhibitor, compound C, or the heme oxygenase 1 (HO-1) inhibitor, Snpp, attenuated the protective effects of adiponectin against intestinal I/R injury. Adiponectin exhibits protective effects against intestinal I/R injury, which may involve the AMPK/HO-1 pathway.

Adiponectin and resistin protect steatotic livers undergoing transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion. Controversial roles for adiponectin and related adipocytokines visfatin and resistin have been described in different liver pathologies, nevertheless it is unknown their possible implication in ischemia-reperfusion injury associated with liver transplantation. Our study aimed at characterizing the role of the adiponectin-derived molecular pathway in transplantation with steatotic and non-steatotic liver grafts. Steatotic and non-steatotic liver transplantation was carried out and the hepatic levels of adiponectin, visfatin and resistin were measured and modulated either pharmacologically or surgically. Steatotic liver grafts exhibited downregulation of both adiponectin and resistin when subjected to transplantation. Adiponectin pre-treatment only protected steatotic grafts and did it so through a visfatin-independent and resistin-dependent mechanism. Adiponectin-derived resistin accumulation activated the PI3K/Akt pathway, unravelling AMPK as an upstream mediator of adiponectin's actions in steatotic grafts. Strategies aimed at increasing adiponectin including either AMPK activators or the induction of ischemic preconditioning (which activates AMPK) increased resistin accumulation, prevented the downregulation of PI3K/Akt pathway and protected steatotic liver grafts. Conversely, PI3K/Akt pathway upregulation and hepatic protection induced by adiponectin were abolished when resistin action was inhibited. Our findings reveal a new protective pathway in steatotic liver transplantation, namely AMPK-adiponectin-resistin-PI3K/Akt, which may help develop new strategies aimed at increasing either adiponectin or resistin in the steatotic liver undergoing transplant to ultimately increase organ donor pool and reduce waiting list.

Adiponectin protects Leydig cells against proinflammatory cytokines by suppressing the nuclear factor‐κB signaling pathway

Adiponectin is an adipocyte hormone that is predominantly secreted by adipocytes, and has important roles in glucose and lipid homeostasis. Recent studies have shown that adiponectin is also involved in the regulation of many endocrine organs, such as the ovary, adrenal gland, and pituitary. However, its biological role in male testes is largely unexplored. The present findings demonstrate the presence of adeponectin receptors (adiponectin receptor 1 and adiponectin receptor 2) in TM3 cells derived from mouse Leydig cells. Proinflammatory cytokine treatment significantly downregulated mRNA and protein levels of adiponectin receptor 1 and adiponectin receptor 2. However, adiponectin pretreatment successfully inhibited the signaling pathway mediated by proinflammatory cytokines. At the molecular level, we provide compelling evidence that adeponectin achieves this by suppressing nuclear factor-κB activation through promotion of AMP-activated protein kinase phosphorylation. Thus, our data clearly indicate that adiponectin plays a protective role in Leydig cells through its anti-inflammatory actions.

Effects of adiponectin pretreatment on liver injury in endotoxemic rats

Objective To investigate the effect of adiponectin pretreatment on liver injury in endotoxemic rats.Methods Eighty male Wistar rats,aged 10-14 weeks,weighing 250-300 g,were randomly divided into 4 groups (n =20 each):control group (C group),lipopolysaccharide (LPS) group,adiponectin pretreatment group (APN group) and interlukin-10 (IL-10) neutralizing antibody intervention group (IL-10 antibody group).Endotoxemia was induced by intraperitoneal LPS 20 mg/kg in LPS,APN and IL-10 antibody groups.In groups APN and IL-10 antibody,intraperitoneal adiponectin 6 mg/kg was injected at 12 h before LPS administration.IL-10 neutralizing antibody 3 mg was injected intraperitoneally at 30 min before adiponectin administration in group IL-10 antibody.Ten rats in each group were chosen at 2 h after LPS administration and blood samples were collected from the hearts for determination of the serum levels of alanine aminotransferase (ALT),aspartate transaminase (AST),tumor necrosis factor (TNF)-α,IL-1β and IL-10.Auother 10 rats in each group were chosen at 2 h after LPS administration and sacrificed and the livers were removed for determination of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity and expression of IL-10 mRNA and heme oxygenase-1 (HO-1) mRNA in liver tissues.Results Compared with C group,the serum levels of ALT,AST,TNF-α,IL-1β and IL-10,MDA content and MPO activity in liver tissues were significantly increased,and HO-1 mRNA expression in liver tissues was upregulated in groups LPS,APN and IL-10 antibody and IL-10 mRNA in liver tissue was up-regulated in LPS and APN groups (P ＜ 0.05).Compared with LPS group,the serum levels of ALT,AST,TNF-α and IL-1β,MDA content and MPO activity in liver tissues were significantly decreased,and HO-1 mRNA expression in liver tissues was up-regulated in APN and IL-10 antiboby groups and IL-10 mRNA in liver tissue was up-regulated in APN group (P ＜ 0.05).Compared with APN group,the serum levels of ALT,AST,TNF-α and IL-1β,MDA content and MPO activity in liver tissues were increased,while the serum level of IL-10 and HO-1 mRNA expression in liver tissues were decreased in IL-10 antiboby group (P ＜ 0.05).Conclusion Adiponectin pretreatment can attenuate liver injury in endotoxic rats by inducing IL-10 production. Key words: Adiponectin; Endotoxemia; Liver injury

Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling

Reactive oxygen species (ROS) induce matrix metalloproteinase (MMP) activity that mediates hypertrophy and cardiac remodeling. Adiponectin (APN), an adipokine, modulates cardiac hypertrophy, but it is unknown if APN inhibits ROS-induced cardiomyocyte remodeling. We tested the hypothesis that APN ameliorates ROS-induced cardiomyocyte remodeling and investigated the mechanisms involved. Cultured adult rat ventricular myocytes (ARVM) were pretreated with recombinant APN (30 μg/ml, 18 h) followed by exposure to physiologic concentrations of H(2)O(2) (1-200 μM). ARVM hypertrophy was measured by [(3)H]leucine incorporation and atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) gene expression by RT-PCR. MMP activity was assessed by in-gel zymography. ROS was induced with angiotensin (ANG)-II (3.2 mg·kg(-1)·day(-1) for 14 days) in wild-type (WT) and APN-deficient (APN-KO) mice. Myocardial MMPs, tissue inhibitors of MMPs (TIMPs), p-AMPK, and p-ERK protein expression were determined. APN significantly decreased H(2)O(2)-induced cardiomyocyte hypertrophy by decreasing total protein, protein synthesis, ANF, and BNP expression. H(2)O(2)-induced MMP-9 and MMP-2 activities were also significantly diminished by APN. APN significantly increased p-AMPK in both nonstimulated and H(2)O(2)-treated ARVM. H(2)O(2)-induced p-ERK activity and NF-κB activity were both abrogated by APN pretreatment. ANG II significantly decreased myocardial p-AMPK and increased p-ERK expression in vivo in APN-KO vs. WT mice. ANG II infusion enhanced cardiac fibrosis and MMP-2-to-TIMP-2 and MMP-9-to-TIMP-1 ratios in APN-KO vs. WT mice. Thus APN inhibits ROS-induced cardiomyocyte remodeling by activating AMPK and inhibiting ERK signaling and NF-κB activity. Its effects on ROS and ultimately on MMP expression define the protective role of APN against ROS-induced cardiac remodeling.

Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat hindlimb

Recent studies have shown that adiponectin is able to increase nitric oxide (NO) production by the endothelium and relax preconstricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. Endothelin-1 (ET-1) is a potent vasoconstrictor, elevated levels of which are associated with obesity, type 2 diabetes, hypertension, and cardiovascular disease. We hypothesized that adiponectin has NO-dependent vascular actions opposing the vasoconstrictor actions of ET-1. We studied the vascular and metabolic effects of a physiological concentration of adiponectin (6.5 μg/ml) on hooded Wistar rats in the constant-flow pump-perfused rat hindlimb. Adiponectin alone had no observable vascular activity; however, adiponectin pretreatment and coinfusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1 nM) ET-1. Adiponectin was not able to oppose vasoconstriction when infusion was commenced after ET-1. This is in contrast to the NO donor sodium nitroprusside, which significantly reduced the pressure due to established ET-1 vasoconstriction, suggesting dissociation of the actions of adiponectin and NO. In addition, adiponectin had no effect on vasoconstriction caused by either high-dose (20 nM) ET-1 or low-dose (50 nM) norepinephrine. Our findings suggest that adiponectin has specific, apparently NO-independent, vascular activity to oppose the vasoconstrictor effects of ET-1. The hemodynamic actions of adiponectin may be an important aspect of its insulin-sensitizing ability by regulating access of insulin and glucose to myocytes. Imbalance in the relationship between adiponectin and ET-1 in obesity may contribute to the development of insulin resistance and cardiovascular disease.

Adiponectin Pretreatment Counteracts the Detrimental Effect of a Diabetic Environment on Endothelial Progenitors

OBJECTIVEIt has been shown that vascular progenitors from patients with diabetes are dysfunctional. However, therapeutic strategies to counteract their reduced functional capacity are still lacking. Because adiponectin has reported salutary effects on endothelial function, we investigated the functional effects of globular adiponectin (gAcrp), the active domain of adiponectin, on isolated endothelial colony-forming cells (ECFC).RESEARCH DESIGN AND METHODSECFC were isolated from peripheral blood of type 2 diabetic patients (dmECFC) and compared with ECFC of healthy young volunteers (yECFC) and nondiabetic age-matched control subjects (hECFC). Cells were treated with gAcrp for 48 h followed by assessment of cell counts, cell cycle analysis, and migration capacity. For in vivo evaluation, human ECFC were injected into normoglycemic or streptozotocin-induced hyperglycemic nu/nu mice after hind limb ischemia.RESULTSWhereas dmECFC were functionally impaired compared with yECFC and hECFC, gAcrp significantly enhanced their in vitro proliferation and migratory activity. In vitro effects were significantly stronger in hECFC compared with dmECFC and were mediated through the cyclooxygenase-2 pathway. Most important, however, we observed a profound and sustained increase of the in vivo neovascularization in mice receiving gAcrp-pretreated dmECFC compared with untreated dmECFC under both normoglycemic and hyperglycemic conditions.CONCLUSIONSPretreatment of ECFC with gAcrp enhanced the functional capacity of ECFC in vitro and in vivo in normoglycemic and hyperglycemic environments. Therefore, preconditioning of dmECFC with gAcrp may be a novel approach to counteract their functional impairment in diabetes.

The effects of adiponectin on interleukin-6 and MCP-1 secretion in lipopolysaccharide-treated 3T3-L1 adipocytes: Role of the NF-κB pathway

It was recently suggested that the transcription nuclear factor-kappaB (NF-kappaB) plays an important role in controlling the inflammation and metabolic alterations associated with obesity. In endothelial and monocytic cells, adiponectin acts as a modulator of the inflammatory response, suppressing NF-kappaB activation. The aim of this study was to assess the ability of different forms of adiponectin to modulate the inflammatory response in adipocytes. 3T3-L1 preadipocytes were cultured according to standard conditions. Fully differentiated adipocytes were stimulated with 1 microg/ml lipopolysaccharides (LPS) for 16 h, with or without pre-treatment with 10 microg/ml of globular (AdG) or full-length (AdFl) adiponectin. Both AdG and AdFl significantly suppressed LPS-induced expression of IL-6 mRNA in adipocytes and reduced the concentration of IL-6 in culture media. Adiponectin pre-treatment significantly reduced the increase in MCP-1 mRNA in adipocytes exposed to LPS. In culture media, the increase in MCP-1 detected after LPS stimulation was significantly attenuated after pre-treatment with AdG. In 3T3-L1, AdG and AdFl reduced NF-kappaB activity by 50 and 40%, respectively compared to the NF-kappaB activation induced by LPS alone. Moreover, both forms of adiponectin significantly attenuated IkappaB-alpha as well as IKK gene expression. Pre-treatment of adipocytes with AdG or AdFl significantly increased PPARgamma mRNA levels, taking its expression back to the basal level. Both AdG and AdFl exert anti-inflammatory activity suppressing IL-6 and MCP-1 production from inflamed adipocytes. This anti-inflammatory action may be mediated through inhibition of NF-kappaB activity as well as through increased PPARgamma expression.

Adiponectin inhibits insulin-like growth factor-1-induced cell migration by the suppression of extracellular signal-regulated kinase 1/2 activation, but not Akt in vascular smooth muscle cells

Adiponectin, an adipocyte-derived hormone, has been proposed to show antiatherogenic properties through the inhibitory effects against various growth factors. Insulin-like growth factor-1 (IGF-1) is one of the potent mitogens, which has been considered to play important roles in both atherogenesis and plaque stabilization in accordance to the phase of atherosclerosis. The aim of this study is to elucidate the adiponectin effects on IGF-1-induced cell migration and its intracellular signaling pathways in vascular smooth muscle cells (VSMCs). In this study, we assessed cell migration and several kinase activities in cultured rat aortic smooth muscle cells (RASMCs). Adiponectin pretreatment suppressed IGF-1-induced cell migration and extracellular signal-regulated kinase (ERK)1/2 activation, which is one of the major mediators for IGF-1-induced cell migration. In RASMCs, adiponectin and 5-aminoimidazole-4-carboxamide riboside (AICAR), a 5'-AMP-activated protein kinase (AMPK) activator, stimulated AMPK activation. AMPK activation by AICAR inhibited IGF-1-induced ERK1/2 activation and cell migration in RASMCs. On the other hand, phosphorylation of Akt and Bad, proapoptotic molecules of the Bcl-2 family, which were increased by IGF-1 stimulation, was not diminished by the pretreatment with adiponectin. It was shown that adiponectin inhibited IGF-1-induced VSMC migration through suppression of ERK1/2 activation, which might be implicated in AMPK activation. Furthermore, adiponectin selectively inhibited ERK1/2 pathway, not Akt-Bad pathway, stimulated by IGF-1. From these findings, it was implied that adiponectin suppressed IGF-1-induced VSMC migration and its signaling selectivity.

Abstract 981: Hypoxia Results in Exacerbation of Pulmonary Arterial Hypertension (PAH) in Male Mice with Deletion of PPARγin Smooth Muscle Cells (SMC) but Females are Protected by Adiponectin Mediated Induction of PPARα

Lung tissues of patients with PAH have reduced levels of the peroxisome proliferator activated receptor gamma (PPARγ) and we previously reported that transgenic male mice with deletion of PPARγ in arterial smooth muscle (SM22CrePPARγ flox/flox ) develop spontaneous PAH. Moreover, PPARγ is necessary in mediating bone morphogenetic protein (BMP-2) inhibition of PDGF-BB induced SMC proliferation. We therefore hypothesized that PAH would be exacerbated in SM22CrePPARγ flox/flox mice under conditions like chronic hypoxia (3 weeks, FiO 2 =0.10) that induce PDGF-BB mediated SMC proliferation. Results: Consistent with this hypothesis, we report a more severe PAH phenotype only in the male SM22CrePPARγ flox/flox mice, as measured by greater right ventricular systolic pressure (RVSP: 38.6 vs. 30.9 mmHg), right ventricular hypertrophy (RVH=right ventricle/left ventricle+septum: 0.58 vs 0.35), and arterial muscularization (90.6 vs 60.3 %), when compared to littermate controls (p&lt;0.05, n=6 – 8 per group, for all comparisons). Interestingly, female SM22CrePPARγ flox/flox mice were resistant to spontaneous or chronic hypoxia-induced PAH (RVSP: 18.5mmHg). We investigated the acute hypoxic vasoconstrictive response and found it exaggerated in the male but suppressed in the female (RVSP: 43.12 vs 20.42mmHg) SM22CrePPARγ flox/flox mice. We found no reduction in endothelin-1 (P&lt;0.05) in the female vs. male SM22CrePPARγ flox/flox mice but serum levels of adiponectin were twofold higher (14.5 vs 6.9 μg/ml; p&lt;0. 001) in females under normoxia and hypoxia. We further show through cell counts, that in pulmonary artery (PA) SMC from male SM22CrePPARγ flox/flox mice, BMP2 fails to suppress PA SMC proliferation in response to PDGF-BB (p&lt;0.01; n=3) but exogenous adiponectin can ‘rescue’ this phenotype (p&lt;0.01, n=3). The mechanism appears to be related to adiponectin mediated induction of PPAR alpha (PPARγ mRNA relative level: 0.37 vs 0.42 after adiponectin pretreatment, p&lt;0.01), which we report for the first time in PA SMC. Conclusion : These findings strengthen the potential efficacy of PPAR-α/ γ agonists in the treatment of PAH patients with low endogenous levels of PPAR-γ and/or adiponectin, such as those with BMP-RII dysfunction or insulin resistance.