Globular Domain Of Adiponectin Research Articles

Adiponectin's globular domain inhibits T cell activation by interacting with LAIR-1

Adiponectin (APN) is the most abundant adipokine in human plasma, and has insulin-sensitizing effect. Recent studies have reported that APN plays both anti- and pro-inflammatory roles under different circumstances. However, there is a lack of convincing evidence that decipher APN's anti-inflammatory role through the known receptors and their downstream signaling pathways. In this study, we evaluated a new molecular mechanism underlying APN's anti-inflammatory roles. Our results revealed that the globular domain of adiponectin (gAdp) interacted with the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). In vitro experiments showed that gAdp inhibited activation of the T cells via the LAIR-1, through a process that also involved downstream SHP-2. These findings indicate that LAIR-1 is a novel APN receptor, affirming APN's anti-inflammatory effect. In summary, we have identified a novel mechanism of peripheral immunoregulatory processes that provides baseline information for further studies on gAdp's role and its contribution to inflammation.

Globular adiponectin acts as a melanogenic signal in human epidermal melanocytes.

Adiponectin is an adipocyte-derived cytokine that circulates as a full-length protein and a fragment containing the globular domain of adiponectin (gAd). A recent study has reported the antimelanogenic effects of full-length adiponectin. To examine the involvement of gAd in melanogenesis and its mechanisms of action. The effects of gAd on melanogenesis and its mechanisms of action were investigated in human epidermal melanocytes and reconstructed epidermis, including melanin content, cellular tyrosinase activity, cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activity, expression and phosphorylation of signalling molecules. Exogenous gAd increased melanin content, and the mRNA levels of microphthalmia-associated transcription factor (MITF) and its downstream genes TRP1, but not TRP2, were increased by gAd. However, cAMP production and PKA activity were not affected by gAd. Moreover, attempts to elucidate the underlying mechanism behind the gAd-mediated effect revealed that gAd could regulate melanogenesis by upregulating MITF through phosphorylation of the cAMP response element-binding protein (CREB). In addition, upregulation of MITF was mediated by activation of adenosine monophosphate-activated protein kinase (AMPK)-p38 mitogen-activated protein kinase (MAPK) signalling. Taken together, these findings indicate that promotion of melanogenesis by gAd occurs through increased expression of MITF, which is mediated by activation of the AMPK-p38 MAPK-CREB pathway. These findings suggest that gAd contributes to epidermal homeostasis via its effect on melanocyte biology, and products of adipose tissue could affect epidermal biology.

Abstract 123: Perivascular Adiponectin Attenuates the Neointimal Response Potentially via Modulation of Hydrogen Sulfide

Objective: Intimal hyperplasia (IH) limits the durability of vascular interventions. Perivascular adipose serves as scaffolding for vessels and also as a source of adipokines, including adiponectin (APN). The globular domain of adiponectin (gAPN) has high affinity for endothelial AdipoR1 receptors and holds beneficial roles in vascular adaptations. Hydrogen sulfide (H 2 S) is a vasculo-protective gasotransmitter produced by cystathionine γ-lyase (CGL), and low circulating H 2 S levels associate with human vascular disease. We therefore hypothesized that APN knockout (KO) mice would be predisposed to IH, and that local administration of gAPN would protect APN KO mice from IH. Furthermore, we evaluated potential links between APN and H 2 S in these effects. Approach and Results: Wildtype (WT, n=7) and APN KO (n=8) mice maintained on high fat diet were subject to carotid focal stenosis. An additional group of APN KO mice (n=7) were treated by local injection of gAPN suspended in Matrigel. Carotids were harvested 28 days following surgery and analyzed 400-2800μm proximal to the stenosis microscopically. IH area was significantly increased at 400μm in APN KO mice compared to WT (p=<0.05, Fig. A), while gAPN significantly attenuated intimal area compared to untreated KO mice (p=<0.05, Fig. B). This finding was coupled with significant (p=<0.05, Fig. C) decrease in pre-operative baseline serum H 2 S production capacity in APN KO (lead acetate assay) compared to WT. Serum collected upon harvest trended toward increased H 2 S production capacity in APN KO mice treated with gAPN (Fig. D). Kidney CGL expression (Western) revealed a trend for increased in CGL expression in the gAPN group (Fig. E). Conclusion: In a focal stenosis model APN KO mice demonstrated increased distal IH; when administered locally, gAPN significantly attenuated IH. These data also uncover a reduction in H 2 S production capacity in APN KO mice, providing a novel potential link between these potent vasomediators.

Alleviation of high-fat diet-induced atherosclerosis and glucose intolerance by a novel GLP-1 fusion protein in ApoE−/− mice

We have previously constructed an engineered anti-diabetic fusion protein using glucagon-like peptide-1 and the globular domain of adiponectin. Herein, we evaluated the therapeutic effects of this fusion protein (GAD) on high-fat diet (HFD)-fed ApoE(-/-) mice. The lipid-lowering effect of GAD was determined in C57BL/6 mice using a lipid tolerance test. The effects of GAD on HFD-induced glucose intolerance, atherosclerosis, and hepatic steatosis were evaluated in HFD-fed ApoE(-/-) mice using glucose tolerance test, histological examinations and real-time quantitative PCR. The anti-inflammation activity of GAD was assessed in vitro on macrophages. GAD improved lipid metabolism in C57BL/6 mice. GAD treatment alleviated glucose intolerance, reduced blood lipid level, and attenuated atherosclerotic lesion in HFD-fed ApoE(-/-) mice, which was associated with a repressed macrophage infiltration in the vessel wall. GAD treatment also blocked hepatic macrophage infiltration and prevented hepatic inflammation. GAD suppressed lipopolysaccharide-triggered inflammation responses on macrophages, which can be abolished by H89, an inhibitor of protein kinase A. These findings demonstrate that GAD is able to generate a variety of metabolic benefits in HFD-fed ApoE(-/-) mice and indicate that this engineered fusion protein is a promising lead structure for anti-atherosclerosis drug discovery.

TNF-α antagonism ameliorates myocardial ischemia-reperfusion injury in mice by upregulating adiponectin.

Tumor necrosis factor-α (TNF-α) antagonism alleviates myocardial ischemia-reperfusion (MI/R) injury. However, the mechanisms by which the downstream mediators of TNF-α change after acute antagonism during MI/R remain unclear. Adiponectin (APN) exerts anti-ischemic effects, but it is downregulated during MI/R. This study was conducted to investigate whether TNF-α is responsible for the decrease of APN, and whether antagonizing TNF-α affects MI/R injury by increasing APN. Male adult wild-type (WT), APN knockout (APN KO) mice, and those with cardiac knockdowns of APN receptors via siRNA injection were subjected to 30 min of MI followed by reperfusion. The TNF-α antagonist etanercept or globular domain of APN (gAD) was injected 10 min before reperfusion. Etanercept ameliorated MI/R injury in WT mice as evidenced by improved cardiac function, and reduced infarct size and cardiomyocyte apoptosis. APN concentrations were augmented in response to etanercept, followed by an increase in AMP-activated protein kinase phosphorylation. Etanercept still increased cardiac function and reduced infarct size and apoptosis in both APN KO and APN receptors knockdown mice. However, its potential was significantly weakened in these mice compared with the WT mice. TNF-α is responsible for the decrease in APN during MI/R. The cardioprotective effects of TNF-α neutralization are partially due to the upregulation of APN. The results provide more insight into the TNF-α-mediated signaling effects during MI/R and support the need for clinical trials to validate the efficacy of acute TNF-α antagonism in the treatment of MI/R injury.

Adiponectin regulates SR Ca2 + cycling following ischemia/reperfusion via sphingosine 1-phosphate-CaMKII signaling in mice

The adipocyte-secreted hormone adiponectin (APN) exerts protective effects on the heart under stress conditions. Recent studies have demonstrated that APN induces a marked Ca2+ influx in skeletal muscle. However, whether APN modulates [Ca2+]i activity, especially [Ca2+]i transients in cardiomyocytes, is still unknown. This study was designed to determine whether APN modulates [Ca2+]i transients in cardiomyocytes. Adult male wild-type (WT) and APN knockout (APN KO) mice were subjected to myocardial ischemia/reperfusion (I/R, 30min/30min) injury. CaMKII-PLB phosphorylation and SR Ca2+-ATPase (SERCA2) activity were downregulated in I/R hearts of WT mice and further decreased in those of APN KO mice. Both the globular domain of APN and full-length APN significantly reversed the decrease in CaMKII-PLB phosphorylation and SERCA2 activity in WT and APN KO mice. Interestingly, compared with WT littermates, single myocytes isolated from APN KO mice had remarkably decreased [Ca2+]i transients, cell shortening, and a prolonged Ca2+ decay rate. Further examination revealed that APN enhances SERCA2 activity via CaMKII-PLB signaling. In in vivo and in vitro experiments, both APN receptor 1/2 and S1P were necessary for the APN-stimulated CaMKII-PLB-SERCA2 activation. In addition, S1P activated CaMKII-PLB signaling in neonatal cardiomyocytes in a dose dependent manner and improved [Ca2+]i transients in APN KO myocytes via the S1P receptor (S1PR1/3). Further in vivo experiments revealed that pharmacological inhibition of S1PR1/3 and SERCA2 siRNA suppressed APN-mediated cardioprotection during I/R. These data demonstrate that S1P is a novel regulator of SERCA2 that activates CaMKII-PLB signaling and mediates APN-induced cardioprotection.

GW24-e1276 Hypoadiponectinemia induces vascular insulin resistance in normotensive young spontaneously hypertensive rats and the underlying mechanisms

ObjectivesVascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. However, the mechanism responsible for inducing vascular insulin resistance in prehypertension remains elusive. The present study aimed to...

An In Vivo Screen of Secreted Proteins Identifies Adiponectin as a Regulator of Murine Cutaneous Wound Healing

Skin wounds comprise a serious medical issue for which few pharmacological interventions are available. Moreover, the inflammatory, angiogenic, and proliferative facets of a typical response to a wound each have broader relevance in other pathological conditions. Here we describe a genomics-driven approach to identify secreted proteins that modulate wound healing in a mouse ear punch model. We show that adiponectin, when injected into the wound edge, accelerates wound healing. Notably, adiponectin injection causes upregulation of keratin gene transcripts within hours of treatment, and subsequently promotes collagen organization, formation of pilosebaceous units, and proliferation of cells in the basal epithelial cell layer and pilosebaceous units of healing tissue. The globular domain of adiponectin is sufficient to mediate accelerated dorsal skin wound closure, and the effects are lost in mice that are homozygous null for the adiponectin receptor 1 gene. These findings extend recent observations of a protective role of adiponectin in other tissue injury settings, suggest modulation of AdipoR1 for the clinical management of wounds, and demonstrate a new approach to the identification of regulators of a wound healing response.

Effect of globular domain of adiponectin on glucose metabolism and insulin signal transduction in insulin resistant model adipocytes

Insulin resistance model of 3T3-L1 adipocytes were prepared with plamotic acid.Adipocytes with generated insulin resistance were cultured with different concentrations of globular domain of adiponectin(gAd:250,500,1 000 ng/ml).The cell culture medium glucose content was detected with the glucose oxidase method,the mRNA expressions of insulin receptor substrate-1 (IRS-1),phosphatidylinositol-3 kinase(PI3K),and protein kinase B(PKB) were detected with real-time quantitative PCR method.The phosphorylation of IRS-1 was detected by Western blot.Compared with the control group,the experimental group showed significantly increased glucose consumption (P ＜ 0.01),and with the increasing gAd concentration,glucose consumption was gradually increasing.IRS-1 phosphorylation was increased gradually with the increasing concentration of gAd.These results suggest that gAd can promote glucose uptake by 3T3-L1 adipocyte model with generated insulin resistance.This may be correlated with promoting insulin signal transduction and improving insulin resistance in adipocytes. Key words: Globular domain of adiponectin; 3T3-L1 adipocytes; Insulin signal transduction; Insulin resistant

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.

Protective effects of a novel trimerized sTNFRII on acute liver injury

TNF α plays a central role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and murine acute liver injury induced by injection of d-galactosamine and subsequent LPS. Recombinant Fc-fused soluble TNF receptor II (sTNFRII-Fc) has been used in the treatment of rheumatoid arthritis for a decade. We have recently constructed a novel fusion protein sTNFRII-gAD, which is composed of a soluble TNF receptor II and a globular domain of adiponectin. Utilizing the inclination of gAD to form homologous trimer naturally, we sought to explore TNFα antagonism of the novel trimerized sTNFRII-gAD and meantime compare TNFα-neutralizing effects in vitro and in vivo between sTNFRII-Fc and sTNFRII-gAD. Here, we evaluated the TNFα-antagonizing activity of sTNFRII-gAD with TNFα-induced L929 cytotoxicity assay. Furthermore, sTNFRII-Fc or sTNFRII-gAD was administered simultaneously with d-galactosamine 1h prior to LPS injection in the murine model of acute liver injury. Serum TNFα and TNFα-sTNFRII-gAD complex were measured by ELISA and the liver injury was assessed through alanine transaminase measurement and liver histological analysis. sTNFRII-gAD was shown to have higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in the L929 cytotoxicity assay. With a significant attenuation of murine lethality (p<0.05), sTNFRII-gAD showed more protective effects than sTNFRII-Fc in the murine model of acute liver injury. These results demonstrated that sTNFRII-gAD was more efficacious than sTNFRII-Fc as a TNFα antagonist, highlighting the potential of sTNFRII-gAD for the treatment of diseases associated with excessive TNFα.

Adiponectin and adiponectin receptor system in the rat adrenal gland: Ontogenetic and physiologic regulation, and its involvement in regulating adrenocortical growth and steroidogenesis

Adiponectin (ADN) is a regulatory peptide secreted mostly by adipose tissue and acting via two receptors: AdipoR1 and AdipoR2. Our aim was to investigate expression of adiponectin system genes in the rat adrenal gland as well as its ontogenetic and physiological control. Furthermore, we examined the effects of acute and prolonged activation of HPA axis on ADN system in adipose tissue. By means of QPCR, ADN and AdipoR1 expression was demonstrated in rat adrenal cortex both at mRNA and protein levels, while AdipoR2 could only be detected at mRNA levels. ADN expression level was significantly upregulated in a developing and regenerating adrenal cortex. Globular domain of adiponectin at 10 −9 M stimulated corticosterone output and BrdU incorporation by cultured rat adrenocortical cells. Moreover, both acute (ACTH and ether stress) and prolonged (ACTH) adrenal stimulation resulted in lowered ADN levels, while expression of AdipoR1 and AdipoR2 was upregulated by the acute treatment. Depending on its site of origin, visceral (VAT) or subcutaneous (SAT) adipose tissue responded differently to alterations in HPA axis. VAT expression of ADN and its receptors remained almost unchanged by experimental manipulations. In SAT, on the other hand, expression of ADN and AdipoR2 was markedly increased by ACTH treatment and stress, while dexamethasone suppressed ADN and AdipoR1 mRNA levels. The results of this study provide new evidence for direct and indirect interactions between adipokines and HPA axis.

Cardioprotective effect of adiponectin is partially mediated by its AMPK-independent antinitrative action

Adiponectin (APN) exerts its metabolic regulation largely through AMP-dependent protein kinase (AMPK). However, the role of AMPK in APN's antiapoptotic effect in ischemic-reperfused (I/R) adult cardiomyocytes remains incompletely understood. The present study was designed to determine the involvement of AMPK in the antiapoptotic signaling of APN. Cardiomyocytes from adult male mice overexpressing a dominant-negative alpha(2)-subunit of AMPK (AMPK-DN) or wild-type (WT) littermates were subjected to simulated I/R (SI/R) and pretreated with 2 microg/ml globular domain of APN (gAPN) or vehicle. SI/R-induced cardiomyocyte apoptosis was modestly increased in AMPK-DN cardiomyocytes (P < 0.05). Treatment with gAPN significantly reduced SI/R-induced apoptosis in WT cardiomyocytes as well as in AMPK-DN cardiomyocytes, indicating that the antiapoptotic effect of gAPN is partially AMPK independent. Furthermore, gAPN-induced endothelial nitric oxide synthase (eNOS) phosphorylation was significantly reduced in AMPK-DN cardiomyocytes, suggesting that the APN-eNOS signaling axis is impaired in AMPK-DN cardiomyocytes. Additional experiments demonstrated that treatment of AMPK-DN cardiomyocytes with gAPN reduced SI/R-induced NADPH oxidase overexpression, decreased superoxide generation, and blocked peroxynitrite formation to the same extent as that observed in WT cardiomyocytes. Collectively, our present study demonstrated that although the metabolic and eNOS activation effect of APN is largely mediated by AMPK, the superoxide-suppressing effect of APN is not mediated by AMPK, and this AMPK-independent antioxidant property of APN increased nitric oxide bioavailability and exerted significant antiapoptotic effect.

Abstract 212: Globular Domain of Adiponectin Attenuates Post-Ischemia Myocardial Oxidative/Nitrative Stress in an AMPK-Independent Fashion

Reduced plasma adiponectin (APN) level not only contributes to the increased incidence of MI in diabetic patients, but also plays a causative role in increased cardiac injury after MI in these patients. However, singling mechanisms by which APN achieves its cardioprotection remains incompletely understood. Here, we investigated the role of AMP activated protein kinase (AMPK), the molecule that transfers APN’s metabolic signaling, in APN’s cardioprotection. Male adult mice with cardiac specific overexpression of a dominant negative AMPK α 2 subunit (AMPK-DN) or wild type (WT) littermates controls were subjected to 30 min of MI followed by reperfusion (R) and treated with vehicle (V) or globular domain of APN (gAPN, 2 μg/g) 10 min before R. Comparing with WT mice, MI/R caused greater cardiac injury including larger infarct size, more apoptosis and poorer cardiac function (P&lt;0.05) in AMPK-DN mice. Treatment of WT mice with gAPN significantly attenuated MI/R injury (P&lt;0.01). Most interestingly, although the metabolic regulatory effect of gAPN is completely lost in AMPK-DN mice, a significant portion of cardioprotection of gAPN retained in the transgenic animals. Specifically, treatment of AMPK-DN mice with gAPN reduced infarct size (54±4.7% vs. 81±3.6% in V group, P&lt;0.01), decreased apoptosis (lesser TUNEL positive labeling, weaker DNA fragmentation, and lower caspase 3 activity. P value all &lt;0.01) and improved cardiac function after MI/R (higher LVEF, lower LVEDP and higher dP/dt max . P value all &lt;0.01). To further delineate the mechanisms responsible for gAPN’s AMPK-independent cardioprotection, effect of gAPN on cardiac oxidative/nitrative stress following MI/R were examined. Treatment of AMPK-DN mice with gAPN reduced superoxide and nitric oxide overproduction, decreased peroxynitrite formation (from 0.38±0.04 to 0.23±0.02 ng/mg protein in WT and from 0.50±0.06 to 0.32±0.02 ng/mg protein in AMPK-DN mice), and inhibited NADPH oxidase and iNOS expression to the same extent as that seen in WT mice. Taken together, our results demonstrated for the first time that a significant portion of gAPN cardioprotection is not mediated by the traditional AMPK signaling pathway and that the anti-oxidative/anti-nitrative effects of gAPN are AMPK-independent.

Adiponectin Protects Against Angiotensin II or Tumor Necrosis Factor α–Induced Endothelial Cell Monolayer Hyperpermeability

Angiotensin II (Ang II) and tumor necrosis factor (TNF)-alpha levels increase endothelial permeability, and we hypothesized that adiponectin suppressed these responses in a cAMP-dependent manner. The effect of adiponectin on transendothelial electric resistance (TEER) and diffusion of albumin through human umbilical vein and bovine aortic endothelial cell monolayers induced by Ang II (100 nmol/L) or TNF-alpha (5 ng/mL) was measured. Treatment with the globular domain of adiponectin (3 mug/mL) for 16 hours abrogated the adverse TEER effect of TNF-alpha (-35 versus -12 Omega/cm(2) at 45 minutes, P<0.05) and Ang II (-25 versus -5 Omega/cm(2) at 45 minutes, P<0.01) and partially suppressed the increased diffusion of albumin with Ang II (40% versus 10% change, P<0.05) or TNF-alpha (40% versus 20% change, P<0.05). Full-length adiponectin also suppressed Ang II-induced monolayer hyperpermeability. Adiponectin treatment also suppressed Ang II-induced increased actin stress fiber development, intercellular gap formation, and beta-tubulin disassembly. Adiponectin increased cAMP levels, and its effects were abrogated by inhibition of adenylyl cyclase or cAMP-dependent protein kinase signaling. Adiponectin protects the endothelial monolayer from Ang II or TNF-alpha-induced hyperpermeability by modulating microtubule and cytoskeleton stability via a cAMP/ PKA signaling cascade.

Adiponectin suppresses IκB kinase activation induced by tumor necrosis factor-α or high glucose in endothelial cells: role of cAMP and AMP kinase signaling

Adiponectin is a protein secreted from adipocytes that exhibits salutary effects in the vascular endothelium by signaling mechanisms that are not well understood. In obesity-related disease states and type 2 diabetes, circulating substances, including tumor necrosis factor-alpha (TNFalpha) and high glucose, activate IkappaB kinase (IKK)beta and reduce the abundance of its substrate, inhibitor of kappaB (IkappaB)alpha, leading to nuclear translocation of the transcription factor NF-kappaB and stimulation of an inflammatory signaling cascade closely associated with endothelial dysfunction. The present study demonstrates that the globular domain of adiponectin (gAd) potently suppresses the activation of IKKbeta by either TNFalpha or high glucose in human umbilical vein endothelial cells and ameliorates the associated loss of IkappaBalpha protein. Interestingly, activation of AMP kinase was substantially more effective than cAMP signaling in suppressing high glucose-induced IKKbeta activity, whereas both pathways were comparably active in suppressing the TNFalpha-induced increase in IKKbeta. Both cAMP/protein kinase A signaling and activation of the AMP kinase pathway played a role in the suppression by gAd of TNFalpha- and high glucose-mediated IKKbeta activation. These findings support an important role for adiponectin in anti-inflammatory signaling in the endothelium and also imply that multiple pathways are involved in the cellular effects of adiponectin.

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.

Functional expression of the globular domain of human adiponectin in Pichia pastoris

Adiponectin is an adipokine that predominantly synthesized and secreted from adipocytes mainly in the white adipose tissue. Here, we report that we have successfully expressed human gAdiponectin (the globular domain of adiponectin) in the methylotrophic yeast Pichia pastoris after codon optimization and established the purification procedure. The human gAdiponectin gene was designed and synthesized by PCR according to the P. pastoris preferred codons, and then inserted into the P. pastoris pPIC9K expression vector. The plasmid was electroporated into the P. pastoris strain GS115 and only the G418 resistance colonies could produce the gAdiponectin. After fermentation and purification, we could get 1.2 g of recombinant gAdiponectin (purity is ∼95%) from a 24 L culture media. The recombinant gAdiponectin is fully functional as evidenced by induction the phosphorylation of ACC in differentiated C2C12 myotubes, significantly lowering the blood glucose level and accelerating the clearance of free fatty acid in animal models.

Adiponectin counteracts cytokine- and fatty acid-induced apoptosis in the pancreatic beta-cell line INS-1.

Pancreatic beta-cell apoptosis is a common feature of Type 1 and Type 2 diabetes and leptin exerts an anti-apoptotic function in these cells. The beta-cell line INS-1 was used to test the hypothesis that the adipocyte hormone adiponectin might mediate an anti-apoptotic effect comparable to leptin. Apoptosis was induced by culturing cells with a cytokine combination (interleukin-1beta/interferon-gamma) or palmitic acid in absence or presence of leptin or the globular domain of adiponectin (gAcrp30), respectively. INS-1 cells had a prominent sensitivity towards cytokine- and fatty acid-induced apoptosis, resulting in about three- and six-fold increases in caspase 3 activation and DNA fragmentation, respectively. gAcrp30 strongly (50-60%) inhibited palmitic acid-induced apoptosis, with a weaker effect against cytokine-induced apoptosis (35%). The same result was observed for leptin with both adipokines being non-additive. Reduction of apoptosis by an inhibitor of IkappaB-kinase (IKK) indicated the involvement of the nuclear factor (NF)-kappaB pathway in both cytokine- and fatty acid-induced apoptosis, however, leptin and gAcrp30 were unable to block NF-kappaB activation. Cytokine- and fatty-acid-induced suppression of glucose/forskolin-stimulated insulin secretion was completely prevented through the action of gAcrp30, whereas leptin was only effective against lipotoxicity-mediated beta-cell dysfunction. Our data show that gAcrp30 partially rescues beta cells from cytokine- and fatty-acid-induced apoptosis and completely restores autoimmune- and lipotoxicity-induced dysfunction of insulin-producing cells. We suggest that gAcrp30 exerts its anti-apoptotic function without modulating NF-kappaB activation. This novel beta cell protective function of gAcrp30 might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.

Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes.

Adiponectin is an abundant adipocyte-derived plasma protein with anti-atherosclerotic and insulin-sensitizing properties that suppresses hepatic glucose production and enhances glucose uptake into skeletal muscle. To characterize the potential effects of adiponectin on glucose uptake into adipose cells, we incubated isolated epididymal rat adipocytes with the globular domain of recombinant adiponectin purified from an E. coli expression system. Globular adiponectin increased glucose uptake in adipocytes without stimulating tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1, and without enhancing phosphorylation of Akt on Ser-473. Globular adiponectin further enhanced insulin-stimulated glucose uptake at submaximal insulin concentrations and reversed the inhibitory effect of tumor necrosis factor-alpha on insulin-stimulated glucose uptake. Cellular treatment with globular adiponectin increased the Thr-172 phosphorylation and catalytic activity of AMP-activated protein kinase and enhanced the Ser-79 phosphorylation of acetyl CoA carboxylase, an enzyme downstream of AMP kinase in adipose cells. Inhibition of AMP kinase activation using two pharmacological inhibitors (adenine 9-beta-D-arabinofuranoside and compound C) completely abrogated the increase in glucose uptake stimulated by globular adiponectin, indicating that AMP kinase is integrally involved in the adiponectin signal transduction pathway. Coupled with recent evidence that the effects of adiponectin are mediated via AMP kinase activation in liver and skeletal muscle, the findings reported here provide an important mechanistic link in the signaling effects of adiponectin in diverse metabolically responsive tissues.