Komai Ali M, Brännmark Cecilia, Musovic Saliha, Olofsson Charlotta S
Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Medicinaregatan 11, SE-405 30, Göteborg, Sweden.
Discovery Sciences, AstraZeneca R&D, Pepparedsleden 1, SE43153, Mölndal, Sweden.
J Physiol. 2014 Dec 1;592(23):5169-86. doi: 10.1113/jphysiol.2014.280388. Epub 2014 Sep 5.
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.
我们通过膜电容膜片钳记录和脂肪因子分泌的生化测量相结合的方法，研究了环磷酸腺苷（cAMP）、钙离子（Ca(2+)）和三磷酸腺苷（ATP）对白色脂肪细胞胞吐作用和脂肪因子释放的影响。即使在细胞内完全没有钙离子（[Ca(2+)]i；用乙二醇双（2-氨基乙醚）四乙酸（BAPTA）缓冲）的情况下，只要细胞内（移液管灌注）溶液中含有cAMP（0.1 mM），3T3-L1脂肪细胞的胞吐作用仍可进行。胞吐作用通常在约10分钟内达到平稳状态，这可能意味着可释放囊泡池的耗尽。将3 mM ATP与细胞内钙离子浓度升高至≥700 nM相结合，可使cAMP诱导的胞吐作用速率增加约2倍，且胞吐作用持续的时间（≥20分钟）比单独使用cAMP时更长。用环磷酸腺苷（cAMP）活化交换蛋白（Epac）激动剂8-溴-2'-O-甲基-cAMP（移液管溶液中含有1 mM）替代cAMP时，胞吐作用受到类似程度的刺激。向含有cAMP的移液管溶液中添加Rp-cAMPS（0.5 mM）抑制蛋白激酶A（PKA）没有效果。腺苷酸环化酶激活剂福斯高林（10 μM）和磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX，200 μM；福斯-IBMX）联合使用，在30分钟内分别使3T3-L1和人皮下脂肪细胞中脂联素的分泌增加了3倍和2倍。预先用钙离子螯合剂BAPTA-AM处理细胞不会改变这种作用，而在细胞外溶液中加入钙离子载体离子霉素（1 μM）时，这种作用会增强2倍。膜通透性Epac激动剂8-溴-2'-O-甲基-cAMP-AM也刺激了脂联素的释放，但膜通透性PKA抑制剂Rp-8-溴-cAMPS（200 μM）的加入对其没有影响。脂肪因子瘦素、抵抗素和Apelin在孵育培养基中的含量较低（为测得的脂联素含量的1-6%）。脂肪酶大量分泌（为脂联素浓度的50%），但福斯-IBMX对这种脂肪因子的释放没有影响。我们提出，白色脂肪细胞的胞吐作用是由cAMP/Epac依赖但钙离子和PKA非依赖的方式刺激位于易释放池中的囊泡释放所介导的，并且钙离子和ATP的联合作用会增强这种释放。我们进一步表明，分泌的囊泡主要含有脂联素。
