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

Abstract

Key pointsr The molecular and cellular mechanisms involved in short-term regulation of white adipocyte adipokine release remain elusive.r Here we have examined effects of intracellular cAMP, Ca 2+ and ATP on exocytosis and adipokine secretion by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of secreted adipokines.r Our findings show that white adipocyte exocytosis is stimulated by cAMP/Epac (exchange proteins activated by cAMP)-dependent but Ca 2+ -and PKA-independent mechanisms and can largely be correlated to release of adiponectin vesicles residing in a readily releasable vesicle pool.r A combination of Ca 2+ and ATP augments exocytosis/adiponectin secretion via a direct action on the release process and by recruitment of new releasable vesicles.r 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.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 ß10 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 ß2-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 of the membrane-permeable PKA inhibitor . 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.

Functional expression of TRPM7 as a Ca2+influx pathway in adipocytes

“…Since TRPM7 is a Ca 2+ /Mg 2+ -permeable channel that contains a protein kinase domain, it likely exerts functions via Ca 2+ /Mg 2+ influx and/or its kinase activity. In adipocytes, Ca 2+ modulates lipolysis (Tebar et al 1996;Xue et al 1998), glucose uptake (Draznin et al 1987;Kelly et al 1989;Whitehead et al 2001;Worrall and Olefsky 2002), and the secretion of adipokines (Levy et al 2000;Cammisotto and Bukowiecki 2004;Sukumar et al 2012;Komai et al 2014;Schlottmann et al 2014;El Hachmane et al 2015). It has been reported that the reduction in intracellular Mg 2+ content is related to impaired glucose metabolism and insulin resistance (Kandeel et al 1996;Takaya et al 2004;Barbagallo and Dominguez 2007).…”

“…In contrast, it remains unclear whether TRPM7 is functionally expressed and plays physiological roles in mature adipocytes. It has been reported in mature adipocytes that [Ca 2+ ] i affects insulin signaling (Draznin et al 1987;Kelly et al 1989;Whitehead et al 2001;Worrall and Olefsky 2002), lipolysis (Tebar et al 1996;Xue et al 1998), and the secretion of adipokines, including leptin (Levy et al 2000;Cammisotto and Bukowiecki 2004), adiponectin (Sukumar et al 2012;Komai et al 2014;El Hachmane et al 2015), and fatty acid binding protein 4 (FABP4) (Schlottmann et al 2014). Calcium plays permissive roles in these physiological processes rather than triggers them; however, the regulation in [Ca 2+ ] i levels appears to be important for proper metabolic signaling in mature adipocytes.…”

Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis

“…To investigate the molecular and cellular mechanisms controlling adiponectin secretion, we have previously examined the role of the three intracellular mediators cAMP, Ca 2ϩ , and ATP in 3T3-L1 adipocyte adiponectin exocytosis (6). Those mediators are known to have important roles in regulation of (neuro)endocrine cell secretion (2).…”

“…Our previous work has demonstrated that adiponectin is released from an intracellular pool of adiponectincontaining vesicles (6,7). However, unlike most peptide/protein hormones (2), exocytosis of adiponectin is triggered by cAMP, and a combination of intracellular Ca 2ϩ and ATP potently augments the cAMP-stimulated secretion (6,7). Thus, the regulation of adiponectin secretion shows similarities with how hormone release is controlled in other endocrine cell types, although there are some fundamental differences (i.e.…”

Quantitative mapping of triacylglycerol chain length and saturation using broadband CARS microscopy

“…3T3-L1 adipocytes were cultured on glass-bottom petri-dishes and differentiated as previously described (42,43). RAW 246.7 and HEK-293 cells were cultured on glass and grown in medium supplemented with 2mg/ml low density lipoprotein for 24 and 48 hours, respectively.…”