title: PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP.
abstract: We examined the effects of cAMP, Ca(2+) 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 Ca(2+) ([Ca(2+)]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 [Ca(2+)]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 Ca(2+) chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca(2+) 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 Ca(2+)- and PKA-independent release of vesicles residing in a readily releasable pool and that the release is augmented by a combination of Ca(2+) and ATP. We further suggest that secreted vesicles chiefly contain adiponectin.
authors.affiliation: Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 11, SE-405 30, Göteborg, Sweden.
authors.forename: Ali M
authors.initials: AM
authors.lastname: Komai
authors.affiliation: Discovery Sciences, AstraZeneca R&D, Pepparedsleden 1, SE43153, Mölndal, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Brännmark
authors.affiliation: Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 11, SE-405 30, Göteborg, Sweden.
authors.forename: Saliha
authors.initials: S
authors.lastname: Musovic
authors.affiliation: Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 11, SE-405 30, Göteborg, Sweden charlotta.olofsson@physiol.gu.se.
authors.forename: Charlotta S
authors.initials: CS
authors.lastname: Olofsson
mesh_terms: D041721:3T3-L1 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D052438:Adipocytes, White / Q000166:cytology / Q000502:physiology*; D054392:Adipokines / Q000378:metabolism*; D052242:Adiponectin / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D048429:Cell Size; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis; D005089:Exocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D018408:Patch-Clamp Techniques
chemical_list: D054392:Adipokines; D052242:Adiponectin; D020662:Guanine Nucleotide Exchange Factors; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium