Adipose tissue synthesizes and secretes various adipokines, such as adiponectin and leptin. Adiponectin is likely to play a protective role against atherosclerosis and cardiovascular risk. The expression of adiponectin is inversely correlated with obesity, insulin resistance, and development of early atherosclerosis. Recent studies confirms that adiponectin and atherosclerosis are significantly related in both genders, independently of conventional cardiovascular risk factors.
Structural features and post-translational modifications of adiponectin
The circulating level of adiponectin ranges from 5 to 30 microg/ml in humans, which represents up to 0.05% of total plasma proteins. The gene that codes for human adiponectin is located on chromosome 3q27, a locus linked with susceptibility to diabetes and CVD. The protein consists of 247 aminoacids and contains an NH2-terminal hyper-variable region, a conserved collagen-like domain comprising 22 Gly-X-Y repeats and a COOH terminal C1q-like globular domain.
Adiponectin is secreted from adipocytes into the bloodstream as three oligomeric complexes, including trimer, hexamer and high molecular weight (HMW) multimer comprising at least 18 monomers.
The trimeric adiponectin is the basic building block of adiponectin multimers. The trimer is formed via hydrophobic interactions within its globular heads and is stabilized by the non-covalent interactions of the collagen-like domains in a triple-helix stalk. The assembly of hexameric and HMW forms of adiponectin requires the formation of an intermolecular disulfide bond between a highly conserved cysteine residue within the hyper-variable region. The post-translational modifications, especially hydroxylation and subsequent glycosylation of several conserved lysine residues within its collagen-like domain are crucial for the intracellular assembly and secretion of HMW oligomeric adiponectin. The biosynthesis and secretion of adiponectin oligomers in adipocytes are tightly controlled by several molecular chaperones in the endoplasmic reticulum, including Erp44 (ER protein of 44 kDa), Ero1-La (ER oxidoreductase 1-La) and DsbA-L (disulfide-bond A oxidoreductase-like protein). The three different oligomeric forms of adiponectin possess distinct biological activities. Among them, the HMW oligomer is the major active form mediating the cardiovascular protective effects of the adipokine. In adipose tissue of obese subjects, both the intracellular assembly and the secretion of HMW adiponectin are impaired, which may in turn contribute to insulin resistance and cardiovascular dysfunction.
Adiponectin is also modified by sialic acids through O-linked glycosylation situated on threonine residues within the hyper-variable region, which determines the half-life in circulation of the adipokine by modulating its clearance from the bloodstream. Furthermore, the highly conserved cysteine residue (Cys36) within the hyper-variable region of adiponectin is succinylated, thereby blocking its oligomerization through inhibition of disulphide bond formation. The extent of succination of adiponectin is elevated in diabetes, suggesting that this modification contributes to impaired adiponectin secretion in obesity-related disorders. Thus, extensive post-translational modifications are required for efficient maturation, oligomerization and secretion of adiponectin, and are also important for maintaining its stability in the circulation.
Intracellular signalling activated by adiponectin
Two structurally related seven transmembrane receptors for adiponectin have been identified, adiponectin receptor (AdipoR) 1 and 2. They are structurally and functionally distinct from classical G-protein coupled receptors. AdipoR1 and AdipoR2 mediate adiponectin-evoked activation of AMP kinase (AMPK), peroxisome-proliferator activated receptor alpha (PPARa) and P38 MAP kinase in liver, skeletal muscle and endothelial cells. APPL1, an adaptor protein containing a pleckstrin homology domain, a phosphotyrosine binding domain and a leucine zipper motif, is a direct interacting partner of both AdipoR1 and AdipoR2. Upon stimulation by adiponectin, the cytoplasmic domain of AdipoR1 and AdipoR2 bind to APPL1, which in turn promotes the translation of the protein kinase LKB1 from nuclei to cytosol, thereby leading to the activation of AMPK.
Anti-atherosclerotic effects of adiponectin
Adiponectin exerts its anti-atherosclerotic effects through multiple actions on almost each vascular cell type.
Most beneficial effects of adiponectin on endothelial functions are mediated by its ability to activate AMPK. Indeed, both globular and full-length adiponectin increase eNOS activity and NO production via AMPK-mediated phosphorylation of eNOS at Ser1177 and Ser633. Endothelium-derived NO protects the vascular system by enhancing vasodilation and inhibiting platelet aggregation, monocyte adhesion and SMC proliferation.
Both subtypes of adiponectin receptors (AdipoR1 and AdipoR2) are expressed in endothelial cells and mediate adiponectin-induced phosphorylation of AMPK and eNOS in a complementary manner. Adiponectin also promotes the complex formation between heat shock protein 90 and eNOS, which is required for the maximal activation of the enzyme. Although the mechanism is poorly understood, the multiple domain protein APPL1 may be a key mediator. APPL1 binds to both AdipoR1 and AdipoR2, and mediates adiponectin-induced activation of AMPK possibly by promoting the translocation of its upstream kinase LKB from nuclei to cytosol.
Adiponectin inhibits the production of reactive oxygen species (ROS) induced by high glucose, oxidized LDL and palmitate in cultured endothelial cells. The anti-oxidant activity of adiponectin appears to be mediated by cAMP-dependent protein kinase A (PKA) and AMPK.
In addition to its effects on eNOS activity and ROS production, adiponectin suppresses endothelial activation, characterized by increased expression of adhesion molecules (such as ICAM-1, VCAM-1 and E-selectin) and monocyte attachment, an early step of the inflammatory reaction leading to atherosclerosis. Indeed, adiponectin suppresses TNFa and resistin-induced expression of adhesion molecules as well as interleukin (IL)-8. This anti-inflammatory effect of adiponectin in endothelial cells appears to be mediated by PKA-dependent suppression of NF-kB activation, through both an AMPKdependent and -independent mechanism. In addition, adiponectin inhibits the interaction between leucocytes and endothelial cells by reducing the expression of E-selectin and vascular cell adhesion molecule-1( VCAM-1)
Then adiponectin has a determinant role in endothelial repair. Impairment in this process is a hallmark of vascular dysfunction and an early step of the atherosclerotic process. Endothelial progenitor cells (EPCs) are important contributors to endothelial repair following vascular injury. Both animal and clinical investigations suggest that adiponectin promotes endothelial repair and angiogenesis by increasing the number and function of EPCs. The endothelial repair mediated by EPCs involves multiple steps, including mobilization of EPCs from the bone marrow or the spleen into the bloodstream, recruitment and adhesion of EPCs to the injured blood vessel wall, followed by differentiation and tubule formation. Adiponectin modulates almost every step involved in endothelial repair of EPCs.
In addition to its beneficial effects on endothelial function and EPCs-mediated endothelial repair, adiponectin inhibits neointimal formation by suppressing proliferation and migration of vascular smooth muscle cells, and blocks inflammation and foam cell formation from macrophages. Adiponectin in physiological concentrations suppresses the proliferation and migration of human vascular smooth muscle cells induced by platelet-derived growth factor-BB(PDGF-BB), basic fibroblast growth factor (basic FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF). This effect of adiponectin is attributed to its ability of interacting with these atherogenic growth factors in an oligomerization-dependent manner, thereby blocking binding to their respective cell membrane receptors for further activation of the mitogenic pathways. http://www.jbc.org/content/280/18/18341.long
In addition, adiponectin inhibits insulin-like growth factor-1 induced migration and proliferation of vascular smooth muscle cells through AMPK-dependent suppression of P44/P42 MAP kinase.
Consistent with these in vitro findings, mechanically injured arteries of adiponectin-deficient mice exhibit severe neointimal thickening and increased proliferation of vascular smooth muscle cells, and this change can be prevented by supplementation of recombinant adiponectin.
In macrophages, chronic treatment with both globular and full-length adiponectin inhibits the production of proinflammatory cytokines induced by several stimuli, including lipopolysaccharide, leptin and resistin. The anti-inflammatory effect of adiponectin is associated with its ability to suppress NF-kB and p42/p44 MAP kinase-dependent signalling. In human monocyte-derived macrophages, adiponectin also induces a sequential up-regulation of the anti-inflammatory cytokine IL-10 (that renders macrophages tolerant to further stimulation by endotoxin or other pro-inflammatory cytokines) and tissue inhibitor of metalloproteinase-1. Acute treatment with adiponectin triggers the release of TNFα and IL-6 via NF-kB and ERK1/2 activation, which subsequently causes an induction of IL-10.
In addition, adiponectin may exert its anti-inflammatory activity by regulating macrophage polarization. Indeed, upon stimulation with adiponectin, human monocytes are primed into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Furthermore, macrophages isolated from adiponectin knockout mice exhibits diminished levels of markers for M2 macrophages, and this phenomenon can be prevented by adiponectin treatment.
Several independent mechanisms have been proposed to explain the inhibitory effects of adiponectin on the conversion of macrophage to foam cells.
First, adiponectin suppress class A scavenger receptor expression, thereby reducing uptake of acetylated LDL particles into macrophages. Second, it decreases the activity of acylcoenzyme A cholesterol acyltransferase: (ACAT) , a key enzyme that catalyses cholesteryl ester formation. Third, it increases cholesterol efflux by enhancing the expression of the ATP-binding cassette transporter ABCA1 in macrophages.
In addition to its direct actions on blood vessels, adiponectin acts in an autocrine manner to inhibit obesity-induced macrophage infiltration and production of pro-inflammatory cytokines in adipose tissue. This may also contribute to its anti-atherosclerotic properties by preventing the ‘inflammatory signal’ from adipose tissue to the vasculature.