PKA‐independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP
Authors: Komai Ali M. , Brännmark Cecilia , Musovic Saliha , Olofsson Charlotta S. 
Published: September 2014

Key points
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 (0.1 mm) was included in the intracellular (pipette‐filling) solution. Exocytosis typically plateaued within ∼10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mm ATP in combination with elevation of [Ca2+]i to ≥700 nm augmented the rate of cAMP‐evoked exocytosis ∼2‐fold and exocytosis proceeded for longer periods (≥20 min) 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 (1 mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp‐cAMPS (0.5 mm) to the cAMP‐containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10 μm) and the phosphodiesterase inhibitor IBMX (200 μm; forsk–IBMX) augmented adiponectin secretion measured over 30 min 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 (1 μm) 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 (200 μm). 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.