Full-length Adiponectin Research Articles

Thrombin-Mediated Formation of Globular Adiponectin Promotes an Increase in Adipose Tissue Mass.

A decrease in the circulating levels of adiponectin in obesity increases the risk of metabolic complications, but the role of globular adiponectin, a truncated form produced by proteolytic cleavage, has not been defined. The objective of this investigation was to determine how globular adiponectin is generated and to determine whether this process impacts obesity. The cleavage of recombinant full-length adiponectin into globular adiponectin by plasma in vitro was used to identify Gly-93 as the N-terminal residue after proteolytic processing. The amino acid sequence of the cleavage site suggested thrombin was the protease responsible for cleavage, and inhibitors confirmed its likely involvement. The proteolytic site was modified, and this thrombin-resistant mutant protein was infused for 4 weeks into obese adiponectin-knockout mice that had been on a high-fat diet for 8 weeks. The mutation of the cleavage site ensured that globular adiponectin was not generated, and thus did not confound the actions of the full-length adiponectin. Mice infused with the mutant adiponectin accumulated less fat and had smaller adipocytes compared to mice treated with globular adiponectin, and concurrently had elevated fasting glucose. The data demonstrate that generation of globular adiponectin through the action of thrombin increases both adipose tissue mass and adipocyte size, but it has no effect on fasting glucose levels in the context of obesity.

Chicken recombinant adiponectin enhances fatty acid metabolism in oleic acid- and palmitic acid-treated LMH cells

The objective of this study was to investigate the effects of recombinant adiponectin on chicken liver cells. The full-length chicken adiponectin gene was amplified by PCR and cloned into the vector pET-32a, followed by the transformation of the vector into Escherichia coli BL21. SDS-PAGE was used to detect and analyze the purity of the expressed recombinant protein. Induction was performed with 1 mM IPTG at 30 °C for 3 h, and the recombinant thioredoxin–adiponectin fusion protein was purified using [...]

Adiponectin suppresses tumor growth of nasopharyngeal carcinoma through activating AMPK signaling pathway

BackgroundAdiponectin is an adipocyte-secreted cytokine that enhances insulin sensitivity and attenuates inflammation. Although circulating adiponectin level is often inversely associated with several malignancies, its role in the development of nasopharyngeal carcinoma (NPC) remains unclear. Here, we investigated the clinical association between circulating adiponectin level and NPC, and examined the impact of adiponectin, as well as the underlying mechanisms, on NPC growth both in vitro and in vivo.MethodsThe association between circulating adiponectin level and the risk of developing NPC was assessed in two different cohorts, including a hospital-based case–control study with 152 cases and 132 controls, and a nested case–control study with 71 cases and 142 controls within a community-based NPC screening cohort. Tumor xenograft model, cell proliferation and cycle assays were applied to confirm the effects of adiponectin on NPC growth in cultured cells and in xenograft models. We also investigated the underlying signaling mechanisms with various specific pharmacological inhibitors and biochemistry analysis.ResultsHigh adiponectin levels were associated with a monotonic decreased trend of NPC risk among males in both the hospital-based case–control study and a nested case–control study. In vitro, recombinant human full-length adiponectin significantly inhibited NPC cell growth and arrested cell cycle, which were dependent on AMPK signaling pathway. The growth of xenograft of NPC tumor was sharply accelerated in the nude mice carrying genetic adiponectin deficiency. An adiponectin receptor agonist, AdipoRon, displayed strong anti-tumor activity in human xenograft models.ConclusionsThese findings demonstrated for the first time that circulating adiponectin is not only inversely associated with NPC, but also controls the development of NPC via AMPK signaling pathway. Stimulation of adiponectin function may become a novel therapeutic modality for NPC.

Adiponectin deficiency induces hepatic steatosis during pregnancy and gestational diabetes in mice.

Obesity and hepatic steatosis are risk factors for gestational diabetes mellitus (GDM), a common complication of pregnancy. Adiponectin is a fat-derived hormone that improves hepatic steatosis and insulin sensitivity. Low levels of circulating adiponectin are associated with GDM development. We hypothesised that adiponectin deficiency causes fatty liver during pregnancy, contributing to the development of GDM. To determine the role of adiponectin in fatty liver development during pregnancy, we compared pregnant (third week of pregnancy) adiponectin knockout (KO) mice (strain B6;129-Adipoqtm1Chan/J) with wild-type mice and assessed several variables of hepatic lipid metabolism and glucose homeostasis. The impact of adiponectin supplementation was measured by administering adenovirus-mediated full-length adiponectin at the end of the second week of pregnancy and comparing with green fluorescent protein control. In the third week of pregnancy, fasted pregnant adiponectin KO mice were hyperglycaemic on a low-fat diet (9.2mmol/l vs 7.7mmol/l in controls, p<0.05) and were glucose and pyruvate intolerant relative to wild-type mice. Pregnant adiponectin KO mice developed hepatic steatosis and a threefold elevation in hepatic triacylglycerols (p<0.05) relative to wild-type mice. Gestational weight gain and food consumption were similar in KO and wild-type mice. Adenoviral-mediated adiponectin supplementation to pregnant adiponectin KO mice improved glucose tolerance, prevented fasting hyperglycaemia and attenuated fatty liver development. Adiponectin deficiency increased hepatic lipid accumulation during the period of pregnancy associated with increased fat utilisation. Consequently, adiponectin deficiency contributed to glucose intolerance, dysregulated gluconeogenesis and hyperglycaemia, all of which are characteristic of GDM. Increasing adiponectin in the last week of pregnancy alleviated hepatic steatosis and restored normal glucose homeostasis during pregnancy.

Mechanisms of Adiponectin in Regulation of Proinflammatory Cytokine Production and Migration in Macrophages.

IntroductionEvidence indicate that adiponectin may exert pro-inflammatory effects on inflammatory cells. We have found that adiponectin knockout decreased inflammatory reaction and tubular damage in the ischemia-reperfusion kidney in APN-knockout mice. Globular adiponectin and full-length adiponectin (g-APN and f-APN) were used in this study to investigate the effects of adiponectin on proinflammatory cytokines production and migration in Raw 264.7 cells.MethodsProinflammatory cytokines production was detected by real-time RT-PCR. NF-kappaB activation was interrupted through Ad-DN-IκBα or SN-50 to confirm how g-APN induces proinflammatory cytokines production. The siRNA against AdipoR1 and AdipoR2 was investigated to uncover the signaling pathway that may involve in NF-kappaB activation and migration in Raw 264.7 cells.Resultsg-APN, not f-APN, was found triggering the production of inflammatory cytokine MIP-2, IL-6, TNFα, and MCP-1 in Raw 264.7 cells. NF-kappaB Inhibition by Ad-DN-IκBα expression or cell-permeable NF-κB inhibitor SN-50 could decrease NF-kappaB nuclear translocation and subsequently decrease inflammatory cytokine expression triggered by globular ANP. However, AdipoR1 and AdipoR2 were not found involved in NF-kappaB activation in this study. Full-length APN, not g-APN, was involved in the promotion of macrophage migration. The migration induced by full-length APN could be inhibited by knockdown of AdipoR1 expression with siRNA. The migration effect could also be inhibited by PI3Kγ inhibitor, AS-605240.DiscussionThese results suggested that full-length adiponectin increases macrophage migration through Adiponectin-AdipoR1-PI3Kgamma signaling pathway. However, NF-κB activation induced by g-APN in this study was independent of AdipoR1 or AdipoR2. The exact signaling pathway of NF-κB activation by adiponectin should be further studied to find a new anti-inflammatory target.

Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane sn-1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.

Adiponectin stimulates glucose uptake in mouse blastocysts and embryonic carcinoma cells

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

Interaction of Nerve Growth Factor β with Adiponectin and SPARC Oppositely Modulates its Biological Activity.

Both adiponectin and secreted protein, acidic and rich in cysteine (SPARC) inhibit platelet-derived growth factor-BB (PDGF-BB)-induced and basic fibroblast growth factor (FGF2)-induced angiogenic activities through direct and indirect interactions. Although SPARC enhances nerve growth factor (NGF)-dependent neurogenesis, the physical interaction of NGFβ with adiponectin and SPARC remains obscure. Therefore, we first examined their intermolecular interaction by surface plasmon resonance method. NGFβ bound to immobilized SPARC with the binding constant of 59.4 nM, comparable with that of PDGF-BB (24.5 nM) but far less than that of FGF2 (14.4 µM). NGFβ bound to immobilized full length adiponectin with the binding constant of 103 nM, slightly higher than those of PDGF-BB (24.3 nM) and FGF2 (80.2 nM), respectively. Treatment of PC12 cells with SPARC did not cause mitogen-activated protein kinase (MAPK) activation and neurite outgrowth. However, simultaneous addition of SPARC with NGFβ enhanced NGFβ-induced MAPK phosphorylation and neurite outgrowth. Treatment of the cells with adiponectin increased AMP-activated protein kinase (AMPK) phosphorylation but failed to induce neurite outgrowth. Simultaneous treatment with NGFβ and adiponectin significantly reduced cell size and the number of cells with neurite, even after silencing the adiponectin receptors by their siRNA. These results indicate that NGFβ directly interacts with adiponectin and SPARC, whereas these interactions oppositely regulate NGFβ functions.

Ca2+-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland.

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca2+ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca2+ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca2+ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca2+, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca2+ modulation in rat submandibular gland acinar cells. Ca2+-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.

Adiponectin suppression of late inflammatory mediator, HMGB1-induced cytokine expression in RAW264 macrophage cells.

High-mobility group protein B1 (HMGB1) is a late inflammatory mediator released from inflammatory cells when stimulated, resulting in exaggerating septic symptoms. We recently demonstrated that full-length adiponectin, a potent anti-inflammatory adipokine, inhibits lipopolysaccharide-induced HMGB1 release. However, the effects of adiponectin on HMGB1-induced exaggerating signals currently remain unknown. This study aimed to investigate the effects of adiponectin on the pro-inflammatory function of HMGB1 in RAW264 macrophage cells. The treatment of RAW264 cells with HMGB1 significantly up-regulated the mRNA expression of tumour necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10. HMGB1-induced cytokine expression was markedly suppressed by a toll-like receptor 4 (TLR4) antagonist and slightly suppressed by an antagonist of the receptor for advanced glycation end products. A prior treatment with full-length or globular adiponectin dose-dependently suppressed all types of HMGB1-induced cytokine expression, and this suppression was abolished by compound C, an AMPK inhibitor, but not by the haem oxygenase (HO)-1 inhibitor, zinc protoporphyrin IX. Both forms of adiponectin also reduced the mRNA expression of TLR4. These results suggest that full-length and globular adiponectin suppress HMGB1-induced cytokine expression through an AMPK-mediated HO-1-independent pathway.

Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44

Adiponectin is a collagenous adipokine with anti-diabetic, anti-inflammatory and anti-tumor properties. Mediated by the conserved Cys39 located in the variable region of the N-terminus, the trimeric (low-molecular weight; LMW) adiponectin subunit assembles into different higher-order complexes e.g. hexamers (MMW) and 12-18mers (HMW) - the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. In obese patients, adiponectin levels are greatly reduced with an accompanying loss of its profound protective effects. This reduction is partly due to obesity-induced dysregulation of endoplasmic reticulum (ER) chaperones such as ERp44, which mediate adiponectin assembly and secretion. In this study, we sought molecular-level understanding of the role of ER chaperone ERp44 in adiponectin assembly and secretion. Such investigations are mainly hampered by the complexity of adiponectin assemblies and its structural polymorphism. To overcome this problem, we developed multiple model peptides, which mimic the different oligomeric states of the N-terminal region of adiponectin containing the conserved Cys39. Using these model peptides in in vitro assays and combined with cellular studies using full-length adiponectin we show that ERp44 specifically recognizes the LMW and MMW but not the HMW form. Our binding assays for short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW, into reduced trimeric precursors of the HMW form. Once the ERp44-precursors-complex is formed in the acidic environment of the cisGolgi, the cargo-complex is transported back to the ER and the trimeric precursor is released triggered by the more alkaline pH there. Using such a process, we suggest that ERp44 enhances the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control.

Adiponectin Inhibits LPS-Induced HMGB1 Release through an AMP Kinase and Heme Oxygenase-1-Dependent Pathway in RAW 264 Macrophage Cells.

High mobility group protein B1 (HMGB1) is a late inflammatory mediator that exaggerates septic symptoms. Adiponectin, an adipokine, has potent anti-inflammatory properties. However, possible effects of adiponectin on lipopolysaccharide- (LPS-) induced HMGB1 release are unknown. The aim of this study was to investigate effects of full length adiponectin on HMGB1 release in LPS-stimulated RAW 264 macrophage cells. Treatment of the cells with LPS alone significantly induced HMGB1 release associated with HMGB1 translocation from the nucleus to the cytosol. However, prior treatment with adiponectin suppressed LPS-induced HMGB1 release and translocation. The anti-inflammatory cytokine interleukin- (IL-) 10 similarly suppressed LPS-induced HMGB1 release. Adiponectin treatment decreased toll-like receptor 4 (TLR4) mRNA expression and increased heme oxygenase- (HO-) 1 mRNA expression without inducing IL-10 mRNA, while IL-10 treatment decreased TLR2 and HMGB1 mRNA expression and increased the expression of IL-10 and HO-1 mRNA. Treatment with the HO-1 inhibitor ZnPP completely prevented the suppression of HMGB1 release by adiponectin but only partially inhibited that induced by IL-10. Treatment with compound C, an AMP kinase (AMPK) inhibitor, abolished the increase in HO-1 expression and the suppression of HMGB1 release mediated by adiponectin. In conclusion, our results indicate that adiponectin suppresses HMGB1 release by LPS through an AMPK-mediated and HO-1-dependent IL-10-independent pathway.

Adipocyte-Derived Adiponectin Positively Regulates Exit from Quiescence of Hematopoietic Stem Cells By Potentiating mTORC1 Activation after Myelotoxic Injury

Myelotoxic injury unlocks the vigorous power of hematopoietic stem cells (HSCs) to replenish the hematopoietic system, making quiescent HSCs enter the cell cycle. Microscopically, it is well known that adipose tissue replaces cellular components in bone marrow (BM) after myeloablation by chemotherapeutic agents or irradiation. In a steady-state hematopoiesis, both HSC-intrinsic and -extrinsic mechanisms enforce quiescence of HSCs, and the interaction between HSCs and BM microenvironment has been drawing much attention in maintenance of the quiescence. In this meaning, it is supposed that the drastic change in BM microenvironment by myeloablation might trigger and promote the cell cycle entry of HSCs. We have previously reported that adiponectin, adipocyte-derived anti-diabetic hormone, indirectly enhances proliferation of murine immature myeloid progenitors upon granulocyte-colony stimulating factor treatment (emergency granulopoiesis) in Socs3-Stat3 dependent fashion by suppressing TNF-α production from macrophages (ASH meeting 2013, Abstract 221), however, its direct effect against HSCs in vivo is needed to be elucidated. Additionally, we have shown that both genetic loss and high-fat diet-induced reduction of adiponectin have no impact on steady-state hematopoiesis. Considering BM adipose tissue is an endocrine organ and adipocytes are the major cellular component in ablated marrow, we hypothesized that adiponectin derived from adipocytes might be implicated in HSC activation and subsequent hematopoietic recovery.Adiponectin-null (adipo-/-) mice showed significantly delayed hematopoietic recovery after 5-fluorouracil (5-FU) administration. In 5-FU-treated BM, adipo-/- SLAM-HSCs (CD150+ CD48- Lin- Sca-1+) and CD34- SLAM-HSCs were more quiescent than adipo+/+ counterparts. Adipo-/- mice survived longer than adipo+/+ control mice after serial 5-FU treatment. Taken into account our previous data showing that impaired emergency granulopoiesis of adipo-/- mice is Socs3-Stat3 dependent and Socs3 haploinsufficiency ameliorated the defect, we further investigated whether activation of adipo-/- HSCs on 5-FU treatment was potentiated by genetic loss of Socs3. But Socs3 haploinsufficiency had no capacity to revert impaired activation of adipo-/- HSCs, suggesting some mechanisms other than that of impaired emergency granulopoiesis in adipo-/- mice.Strikingly, adipo-/- HSCs were shown to be defective in mTORC1 activation, phosphorylation of S6 and mitochondrial activity after 5-FU treatment. In vivo rapamycin treatment cancelled the effect of adiponectin upon HSC activation by 5-FU, suggesting that adiponectin enhances HSC activation through mTORC1-dependent mechanism. Physiological isoform of adiponectin (full-length adiponectin) enhanced not only 5-FU-induced mTORC1 activation in vivo but also cytokine-induced activation in vitro, shortened the time to first division, without affecting subsequent proliferation of HSCs, in contrast to the previous report using non-physiological isoform (globular adiponectin) in vitro.The concentration of adiponectin in BM had a 4-fold increase after 5-FU treatment while the level in plasma remained unchanged. In a steady state, adiponectin level in adipocyte-rich tibia is higher than femur, suggesting local production of adiponectin constitutes a significant portion of BM adiponectin. Every BM cell components examined expressed adiponectin mRNA, and adipocytes had the highest. As 5-FU treatment had little effect on adiponectin expression in adipocytes, it was suggested that increased adipocytes in BM contributed to increased adiponectin upon myelotoxic injury. Furthermore, reciprocal transplants with adipo+/+ and adipo-/- mice demonstrated that adiponectin from BM environment of recipient mice plays a major role in the activation of HSC after in vivo 5-FU treatment and in vitro cytokine stimulation.These data reveal that adiponectin, produced mainly from increased adipocytes after myelotoxic injury, positively regulates HSC activation and subsequent hematopoietic recovery. Our data also highlight adipocytes as an essential source of adiponectin to ensure the proliferative burst of hematopoietic cells in myeloablated marrow. Adiponectin treatment could be clinically applied to relieve myelosuppression by chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Globular adiponectin enhances invasion in human breast cancer cells

Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer.

Abstract 989: Regulation of metastatic potential through adiponectin-stimulated induction of autophagy

Abstract Each year, an unacceptable number of deaths occur in women because of the lack of curative approaches for metastatic breast cancer. Some of the key regulators of breast cancer progression are molecules within the tumor microenvironment including adipokines, a broad array of autocrine-, endocrine-, and paracrine-acting bioactive molecules secreted by adipocytes. Adiponectin is one of the most abundant adipokines and, while multiple widely cited epidemiological studies have indicated that low levels of circulating plasma adiponectin portend poorer prognosis, recent work has reported that elevated adiponectin expression in breast tissue is, in fact, correlated with more advanced disease. Thus, the purpose of this work is to better understand how adiponectin in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. Our hypothesis is that adiponectin alters metastatic potential of breast cancer cells via induction of autophagy. To begin to test this premise, we discerned the effect of globular versus full-length adiponectin on invasive and migratory phenotypes of a human metastatic breast cancer cell line (MDA-MB-231). In transwell assays with and without Matrigel, globular adiponectin increased invasion (91%; p &amp;lt; 0.001) and migration (222%; p &amp;lt; 0.05) compared to untreated cells, whereas full length adiponectin had no significant effect. Rapamycin, an established autophagy inducer, elicited effects similar to globular adiponectin (increase of 210%; p &amp;lt; 0.001 in invasion and 238%; p &amp;lt; 0.05 in migration). Likewise, three-dimensional growth in Matrigel revealed that cells treated with globular adiponectin and rapamycin developed extended spikes indicative of a more invasive phenotype, whereas those treated with full-length adiponectin maintained a less invasive grape-like structure. Neither adiponectin isoform altered proliferation. Biochemical assays of autophagic induction supported these observations, demonstrating increases in LC3B-II expression (immunoblot) and in the number of intracellular LC3B puncta (immunofluorescence) upon treatment with globular, but not full-length, adiponectin. Together, these results suggest a plausible model linking a specific adiponectin isoform with induction of autophagy to stimulate breast cancer metastasis. Our findings will advance the field by revealing distinct, novel roles for key microenvironmental regulatory molecules, potentially opening up new avenues for therapeutic development. This work is timely considering recent interest in adiponectin and adiponectin receptor agonists as therapeutic strategies. In addition, noting that an aberrant level of adiponectin is a putative mechanism linking obesity to poor breast cancer prognosis and metastasis, our results may provide mechanistic insight to guide lifestyle interventions that will reduce breast cancer's heavy morbidity and mortality burden. Citation Format: Emily Falk Libby, Jianzhong Liu, Monica J. Lewis, Andra R. Frost, Wendy Demark-Wahnefried, Douglas R. Hurst. Regulation of metastatic potential through adiponectin-stimulated induction of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 989. doi:10.1158/1538-7445.AM2015-989

UNDERSTANDING THE ROLE OF ADIPONECTIN PROCESSING IN AMELIORATING INSULIN RESISTANCE AND RELATED COMPLICATIONS

BACKGROUND: The rising prevalence of obesity is recognized as one of the most important risk factors for the development of diabetes and associated cardiovascular complications. The precise mechanisms that link obesity to diabetes and increased atherosclerotic vascular diseases are not yet fully understood. Insulin resistance, a decreased sensitivity to the actions of insulin, results in impaired glucose metabolism and contributes to the cardiovascular complications associated with diabetes. Secretion of adiponectin, an antiatherogenic and anti-diabetic adipokine is decreased in obesity, as fat accumulates and adipocytes enlarge. Even though a link between adipose dysfunction, insulin resistance and diabetes has been identified, the direct physiological effects of adiponectin processing on obesity induced diabetes have yet to be investigated. Recently, it was hypothesized that full-length adiponectin (fAcrp) is inactive and that globular adiponectin (gAcrp) is the functional adipokine. Our group has identified thrombin and not trypsin or leukocyte elastase as generally believed, as the protease responsible for generating gAcrp. Preliminary studies showed that the gAcrp and not fAcrp inhibited the proliferation of primary smooth muscle cells in vitro in response to PDGF. Hence we hypothesize that the generation of gAcrp from fAcrp is essential for eliciting the beneficial actions of adiponectin in reversing insulin resistance. Our aim is to compare the effects of different forms of adiponectin namely fAcrp, gAcrp and prAcrp (protease resistant mutant of fAcrp that cannot be cleaved into gAcrp) on insulin resistance. METHODS AND RESULTS: In in vivo studies, we will use a dietinduced obese adiponectin knockout mouse model and compare the ability of different adiponectin forms to rescue the metabolic phenotype of these mice. In in vitro studies, human umbilical vein endothelial cells (HUVEC) and hepatoma cells (HepG2) will be serum starved prior to treatment with different forms of adiponectin and activation of downstream molecular signatories such as AMPKa, MAPK p44/42, p38 MAPK, ACC and Akt will be measured by Western blotting. The purified recombinant fAcrp and prAcrp proteins were tested for sensitivity to thrombin in an in vitro cleavage assay. Recombinant fAcrp was digested in a dose dependent manner. CONCLUSION: Treatment of fAcrp but not prAcrp with thrombin resulted in the formation of gAcrp. The expression level of adiponectin receptor1, receptor2 and T-cadherin was similar in different primary human endothelial cells and hepatoma cells. Serum starvation of HUVECs for 6hours in 0% FBS and 16hours in 0.5% FBS was optimum for MAPK p44/42 and AMPK regulation by adiponectin. 428 MAXIMAL CCA IMT AND CARDIOVASCULAR OUTCOMES

SNPs in the Adiponectin Receptor 2 Gene and Their Associations with Chicken Performance Traits

The adiponectin receptor 2 (ADIPOR2) is a receptor for both globular and full-length adiponectin. In the current study, two genetic variations in ADIPOR2 gene were identified in an F2 resource population of Gushi chicken and Anka broiler. Association analysis between the two SNPs and chicken performance traits were determined using the linear mixed model. The data revealed that the g.34490C > T mutation in intron 3 was significantly associated with liver weight and globulin, the g.35363T > C polymorphism in exon 5 was significantly associated with body weights at 6, 10, and 12 weeks of age. Both polymorphisms have no significant effects on serum glucose and fat-related traits. The g.34490C > T mutation might play an important role in regulating liver weight. The g.35363T > C polymorphism does contribute in a significant manner to growth traits at the medium and later development stage but it is uncertain whether it could be a molecular marker for liver disease.

Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes

ObjectiveAdiponectin is known to confer its cardioprotective effects in obesity and type 2 diabetes, mainly by regulating glucose and fatty acid metabolism in cardiomyocytes. Dynamic actin cytoskeleton remodeling is involved in regulation of multiple biological functions, including glucose uptake. Here we investigated in neonatal cardiomyocytes whether adiponectin induced actin cytoskeleton remodeling and if this played a role in adiponectin-stimulated glucose uptake. Materials/methodsPrimary cardiomyocytes were treated with full-length and globular adiponectin (fAd and gAd, respectively). ResultsBoth fAd and gAd increased RhoA activity, phosphorylation of the Rho/ROCK signaling target cofilin and actin polymerization to form filamentous actin as determined by rhodamine–phallodin immunofluorescence and quantitative analysis of filamentous to globular actin ratio. Scanning electron microscopy also demonstrated structural remodeling. Adiponectin stimulated glucose uptake, was significantly abrogated in the presence of inhibitors of actin cytoskeleton remodeling (cytochalasin D) and Rho/ROCK signaling (C3 transferase, Y27632). We showed that adiponectin increased colocalization of actin and APPL1 and that actin remodeling, phosphorylation of AMPK, p38MAPK and cofilin, glucose uptake and oxidation were all attenuated after siRNA-mediated knockdown of APPL1. ConclusionWe show that adiponectin mediates Rho/ROCK-dependent actin cytoskeleton remodeling to increase glucose uptake and metabolism via APPL1 signaling.

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.

Increased myocardial ischemia-reperfusion injury in renal failure involves cardiac adiponectin signal deficiency

Plasma levels of adiponectin (APN) are significantly increased in patients with renal dysfunction and are inversely related to the risk of cardiovascular mortality. The present study was designed to determine the role of APN in myocardial ischemia-reperfusion (MI/R) injury in mice with renal failure and delineate the underlying mechanisms. Renal failure was induced by subtotal nephrectomy (SN). Human recombinant globular domain of adiponectin (gAd) or full-length adiponectin (fAd) was administered via intraperitoneal injection once daily for 7 consecutive days after SN, and in vivo MI/R was introduced 3 wk later. Both plasma and urinary levels of APN increased significantly in SN mice. Compared with sham-operated mice, cardiac function was significantly depressed, and myocardial infarct size and apoptosis increased in SN mice following MI/R. The aggravated MI/R injury was further intensified in APN-knockout mice and markedly ameliorated by treatment with gAd but not fAd. Moreover, SN increased myocardial NO metabolites, superoxide, and their cytotoxic reaction product peroxynitrite, upregulated inducible NO synthase expression, and decreased endothelial NOS phosphorylation. In addition, SN mice also exhibited reduced APN receptor-1 (AdipoR1) expression and AMPK activation. All these changes were further amplified in the absence of APN but reversed by gAd treatment. The present study demonstrates that renal dysfunction increases cardiac susceptibility to ischemic-reperfusion injury, which is associated with downregulated APN/AdipoR1/AMPK signaling and increased oxidative/nitrative stress in local myocardium, and provides the first evidence for the protective role of exogenous supplement of gAd on MI/R outcomes in renal failure.