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.

We investigated the physiological regulation of adiponectin exocytosis in health and metabolic disease by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of short-term (30-min incubations) adiponectin secretion. Epinephrine or the β-adrenergic receptor (AR) agonist CL 316,243 (CL) stimulated adiponectin exocytosis/secretion in cultured 3T3-L1 and in primary subcutaneous mouse adipocytes, and the stimulation was inhibited by the Epac (Exchange Protein directly Activated by cAMP) antagonist ESI-09. The βAR was highly expressed in cultured and primary adipocytes, whereas other ARs were detected at lower levels. 3T3-L1 and primary adipocytes expressed Epac1, whereas Epac2 was undetectable. Adiponectin secretion could not be stimulated by epinephrine or CL in adipocytes isolated from obese/type 2 diabetic mice, whereas the basal (unstimulated) adiponectin release level was elevated twofold. Gene expression of βAR and Epac1 was reduced in adipocytes from obese animals, and corresponded to a respective ∼35% and ∼30% reduction at the protein level. Small interfering RNA-mediated knockdown of βAR (∼60%) and Epac1 (∼50%) was associated with abrogated catecholamine-stimulated adiponectin secretion. We propose that adiponectin exocytosis is stimulated via adrenergic signaling pathways mainly involving βARs. We further suggest that adrenergically stimulated adiponectin secretion is disturbed in obesity/type 2 diabetes as a result of the reduced expression of βARs and Epac1 in a state we define as "catecholamine resistance."

SummaryChronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6.

Adiponectin is a hormone secreted from white adipocytes and takes part in the regulation of several metabolic processes. Although the pathophysiological importance of adiponectin has been thoroughly investigated, the mechanisms controlling its release are only partly understood. We have recently shown that adiponectin is secreted via regulated exocytosis of adiponectin-containing vesicles, that adiponectin exocytosis is stimulated by cAMP-dependent mechanisms, and that Ca and ATP augment the cAMP-triggered secretion. However, much remains to be discovered regarding the molecular and cellular regulation of adiponectin release. Here, we have used mathematical modeling to extract detailed information contained within our previously obtained high-resolution patch-clamp time-resolved capacitance recordings to produce the first model of adiponectin exocytosis/secretion that combines all mechanistic knowledge deduced from electrophysiological experimental series. This model demonstrates that our previous understanding of the role of intracellular ATP in the control of adiponectin exocytosis needs to be revised to include an additional ATP-dependent step. Validation of the model by introduction of data of secreted adiponectin yielded a very close resemblance between the simulations and experimental results. Moreover, we could show that Ca-dependent adiponectin endocytosis contributes to the measured capacitance signal, and we were able to predict the contribution of endocytosis to the measured exocytotic rate under different experimental conditions. In conclusion, using mathematical modeling of published and newly generated data, we have obtained estimates of adiponectin exo- and endocytosis rates, and we have predicted adiponectin secretion. We believe that our model should have multiple applications in the study of metabolic processes and hormonal control thereof.

The hormone adiponectin is secreted by white adipocytes and has been put forward as a key mediator of obesity-linked insulin resistance and the metabolic syndrome. Although adiponectin was discovered two decades ago, the knowledge about the molecular and cellular regulation of its secretion is incomplete. Here we have investigated the adrenergic regulation of adiponectin secretion in primary visceral (gonadal) adipocytes isolated from lean or obese/diabetic mice. We show that visceral adipocyte adiponectin release is triggered by cAMP/catecholamines via signalling pathways involving adrenergic beta-3-receptors (β 3 ARs) and E xchange P rotein directly A ctivated by c AMP, isoform 1 (Epac1). The adrenergically stimulated adiponectin secretion is blunted in visceral adipocytes isolated from obese and diabetic mice and our results suggest the existence of a secretory defect. We have previously shown that adiponectin secretion in subcutaneous adipocytes is abolished in the obese/diabetic state due to reduced abundance of β 3 ARs and Epac1. However, here we show that protein levels of β 3 ARs and Epac1 are maintained in visceral adipocytes from obese/diabetic mice proposing that other molecular defects underlie the blunted adiponectin release. Gene expression analysis indicate diabesity-associated disturbances of the signalling downstream of Epac1 and/or the exocytotic process itself. Our study proposes that visceral adipocytes partake in the regulated secretion of adiponectin and may thus influence circulating levels of the hormone, in health and in metabolic disease.