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Adiponectin is an adipokine with profound insulin-sensitizing, anti-inflammatory and anti-atherogenic properties. Plasma levels of adiponectin are reduced in insulin resistant states such as obesity, type 2 diabetes and cardiovascular diseases. Additionally, studies have shown that endothelin-1 (ET-1), a vasoconstrictor peptide, acutely stimulates and chronically inhibits adiponectin secretion. However, the mechanism by which ET-1 regulates adiponectin secretion is unknown. Therefore, we investigated the mechanism(s) responsible for the effects of ET-1 on adiponectin secretion. In order to determine the chronic effect of ET-1 on adiponectin secretion, 3T3-L1 adipocytes were treated for 24 hrs with ET-1 (10nM) and then stimulated with vehicle or insulin (100nM) for a period of 1-2 hrs. Chronic ET-1 (24 hrs) treatment significantly decreased basal and insulin stimulated adiponectin secretion by 66% and 47%, respectively. Inhibition of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by the PLC? inhibitor, U73122, or exogenous addition of PIP2: histone carrier (1.25µM: 0.625µM) ameliorated the decrease in basal and insulin-stimulated adiponectin secretion observed with ET-1. However, treatment with exogenous PIP2: histone carrier complex and the actin depolymerizing agent latrunculin B (20µM) did not reverse the ET-1-mediated decrease in adiponectin secretion. In conclusion, we demonstrate that ET-1 inhibits basal and insulin-stimulated adiponectin secretion through PIP2 modulation of the actin cytoskeleton. These studies provided evidence that actin cytoskeleton plays an important role in adiponectin secretion from adipocytes. In order to further elucidate the role of F-actin in the regulation of adiponectin secretion, we investigated the role of myosin II, an actin-based motor, in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Myosin IIA and IIB isoforms were colocalized with adiponectin as determined by immunofluorescence and immunogold electron microscopy. Immunofluorescent and immunogold microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the cell while exhibiting perinuclear localization Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes appears to occur by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB.