PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP

The molecular and cellular mechanisms involved in short-term regulation of white adipocyte adipokine release remain elusive. Here we have examined effects of intracellular cAMP, Ca2+ and ATP on exocytosis and adipokine secretion by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of secreted adipokines. Our findings show that white adipocyte exocytosis is stimulated by cAMP/Epac (exchange proteins activated by cAMP)-dependent but Ca2+- and PKA-independent mechanisms and can largely be correlated to release of adiponectin vesicles residing in a readily releasable vesicle pool. A combination of Ca2+ and ATP augments exocytosis/adiponectin secretion via a direct action on the release process and by recruitment of new releasable vesicles. Our results elucidate several previously unknown cellular mechanisms involved in regulation of white adipocyte exocytosis/secretion. The well-established disturbances of adipokine secretion in obese individuals highlight the significance of understanding how white adipocyte adipokine release is controlled. AbstractWe examined the effects of cAMP, Ca2+ and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca2+ ([Ca2+](i); buffered with BAPTA) provided cAMP (0.1mm) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within approximate to 10min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3mm ATP in combination with elevation of [Ca2+](i) to 700nm augmented the rate of cAMP-evoked exocytosis approximate to 2-fold and exocytosis proceeded for longer periods (20min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac (exchange proteins activated by cAMP) agonist 8-Br-2-O-Me-cAMP (1mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp-cAMPS (0.5mm) to the cAMP-containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10m) and the phosphodiesterase inhibitor IBMX (200m; forsk-IBMX) augmented adiponectin secretion measured over 30min 3-fold and 2-fold in 3T3-L1 and human subcutaneous adipocytes, respectively. This effect was unaltered by pre-loading of cells with the Ca2+ chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca2+ ionophore ionomycin (1m) in the extracellular solution. Adiponectin release was also stimulated by the membrane-permeable Epac agonist 8-Br-2-O-Me-cAMP-AM but unaffected by inclusion of the membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (200m). The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1-6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk-IBMX. We propose that white adipocyte exocytosis is stimulated by cAMP/Epac-dependent but Ca2+- and PKA-independent release of vesicles residing in a readily releasable pool and that the release is augmented by a combination of Ca2+ and ATP. We further suggest that secreted vesicles chiefly contain adiponectin.