| 1. |
- Lissek, T, et al.
(författare)
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Building Bridges through Science
- 2017
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Ingår i: Neuron. - : Elsevier BV. - 1097-4199 .- 0896-6273. ; 96:4, s. 730-735
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Tidskriftsartikel (refereegranskat)
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| 2. |
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| 3. |
- Brännmark, Cecilia, et al.
(författare)
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Mathematical modeling of white adipocyte exocytosis predicts adiponectin secretion and quantifies the rates of vesicle exo- and endocytosis
- 2017
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 292:49, s. 20032-20043
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Tidskriftsartikel (refereegranskat)abstract
- 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 Ca2+ 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 Ca2+-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.
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| 4. |
- El Hachmane, Michael, et al.
(författare)
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Cooling Reduces cAMP-Stimulated Exocytosis and Adiponectin Secretion at a Ca2+-Dependent Step in 3T3-L1 Adipocytes
- 2015
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- We investigated the effects of temperature on white adipocyte exocytosis (measured as increase in membrane capacitance) and short-term adiponectin secretion with the aim to elucidate mechanisms important in regulation of white adipocyte stimulus-secretion coupling. Exocytosis stimulated by cAMP (included in the pipette solution together with 3 mM ATP) in the absence of Ca2+ (10 mM intracellular EGTA) was equal at all investigated temperatures (23 degrees C, 27 degrees C, 32 degrees C and 37 degrees C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca2+] to similar to 1.5 mu M (9mMCa(2+) + 10 mM EGTA) was potent at 32 degrees C or 37 degrees C but less distinct at 27 degrees C and abolished at 23 degrees C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 mu M forskolin and 200 mu M IBMX was unaffected by a reduction of temperature from 32 degrees C to 23 degrees C. At 32 degrees C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1 mu M), an effect that was not observed at 23 degrees C. We suggest that cooling affects adipocyte exocytosis/adiponectin secretion at a Ca2+-dependent step, likely involving ATP-dependent processes, important for augmentation of cAMP-stimulated adiponectin release.
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| 5. |
- Komai, Ali, 1987, et al.
(författare)
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An Approach to Monitor Exocytosis in White Adipocytes
- 2021
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Ingår i: Exocytosis and Endocytosis. Florence Niedergang, Nicolas Vitale, Stéphane Gasman (red.). - New York, NY : Springer. - 1064-3745. - 9781071610435 ; , s. 203-222
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Exocytosis, the fusion of vesicles with the plasma membrane, can be measured with the patch-clamp technique as increases in membrane capacitance. Here we provide detailed information on how to monitor white adipocyte exocytosis using this method. We describe how to isolate the stromal vascular fraction of cells (SVF) within adipose tissue and how to differentiate SVF and cultured 3T3-L1 cells into adipocytes suitable for patch-clamp studies. We also give detailed protocols of how to record and analyze exocytosis in the differentiated cells. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
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| 6. |
- Komai, Ali, 1987, et al.
(författare)
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PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: Modulations by Ca2+ and ATP
- 2014
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 592:23, s. 5169-5186
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Tidskriftsartikel (refereegranskat)abstract
- We 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.
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| 7. |
- Komai, Ali, 1987, et al.
(författare)
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White Adipocyte Adiponectin Exocytosis Is Stimulated via beta(3)-Adrenergic Signaling and Activation of Epac1: Catecholamine Resistance in Obesity and Type 2 Diabetes
- 2016
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 65:11, s. 3301-3313
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Tidskriftsartikel (refereegranskat)abstract
- 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 beta(3)-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 beta(3)AR 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 beta(3)AR and Epac1 was reduced in adipocytes from obese animals, and corresponded to a respective similar to 35% and similar to 30% reduction at the protein level. Small interfering RNA-mediated knockdown of beta(3)AR (similar to 60%) and Epac1 (similar to 50%) was associated with abrogated catecholamine-stimulated adiponectin secretion. We propose that adiponectin exocytosis is stimulated via adrenergic signaling pathways mainly involving beta(3)ARs. We further suggest that adrenergically stimulated adiponectin secretion is disturbed in obesity/type 2 diabetes as a result of the reduced expression of beta(3)ARs and Epac1 in a state we define as "catecholamine resistance."
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| 8. |
- Lövfors, William, et al.
(författare)
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A systems biology analysis of adrenergically stimulated adiponectin exocytosis in white adipocytes
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 297:5
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Tidskriftsartikel (refereegranskat)abstract
- Circulating levels of the adipocyte hormone adiponectin are typically reduced in obesity, and this deficiency has been linked to metabolic diseases. It is thus important to understand the mechanisms controlling adiponectin exocytosis. This understanding is hindered by the high complexity of both the available data and the underlying signaling network. To deal with this complexity, we have previously investigated how different intracellular concentrations of Ca2+, cAMP, and ATP affect adiponectin exocytosis, using both patch-clamp recordings and systems biology mathematical modeling. Recent work has shown that adiponectin exocytosis is physiologically triggered via signaling pathways involving adrenergic beta(3) receptors (beta(3)ARs). Therefore, we developed a mathematical model that also includes adiponectin exocytosis stimulated by extracellular epinephrine or the beta(3)AR agonist CL 316243. Our new model is consistent with all previous patch-clamp data as well as new data (collected from stimulations with a combination of the intracellular mediators and extracellular adrenergic stimuli) and can predict independent validation data. We used this model to perform new in silico experiments where corresponding wet lab experiments would be difficult to perform. We simulated adiponectin exocytosis in single cells in response to the reduction of beta(3)ARs that is observed in adipocytes from animals with obesity-induced diabetes. Finally, we used our model to investigate intracellular dynamics and to predict both cAMP levels and adiponectin release by scaling the model from single-cell to a population of cells-predictions corroborated by experimental data. Our work brings us one step closer to understanding the intricate regulation of adiponectin exocytosis.
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| 9. |
- Saliha, Musovic, 1990, et al.
(författare)
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Noradrenaline and ATP regulate adiponectin exocytosis in white adipocytes: Disturbed adrenergic and purinergic signalling in obese and insulin-resistant mice
- 2022
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Ingår i: Molecular and Cellular Endocrinology. - : Elsevier BV. - 0303-7207. ; 549
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Tidskriftsartikel (refereegranskat)abstract
- White adipocyte adiponectin exocytosis is triggered by cAMP and a concomitant increase of cytosolic Ca2+ potentiates its release. White adipose tissue is richly innervated by sympathetic nerves co-releasing noradrenaline (NA) and ATP, which may act on receptors in the adipocyte plasma membrane to increase cAMP via adrenergic receptors and Ca2+ via purinergic receptors. Here we determine the importance of NA and ATP for the regulation of white adipocyte adiponectin exocytosis, at the cellular and molecular level, and we specifically detail the ATP signalling pathway. We demonstrate that tyrosine hydroxylase (enzyme involved in catecholamine synthesis) is dramatically reduced in inguinal white adipose tissue (IWAT) isolated from mice with diet induced obesity; this is associated with diminished levels of NA in IWAT and with a reduced ratio of high molecular-weight (HMW) to total adiponectin in serum. Adiponectin exocytosis (measured as an increase in plasma membrane capacitance and as secreted product) is triggered by NA or ATP alone in cultured and primary mouse IWAT adipocytes, and enhanced by a combination of the two secretagogues. The ATP-induced adiponectin exocytosis is largely Ca2+-dependent and activated via purinergic P2Y2 receptors (P2Y2Rs) and the Gq11/PLC pathway. Adiponectin release induced by the nucleotide is abrogated in adipocytes isolated from obese and insulin-resistant mice, and this is associated with ~70% reduced abundance of P2Y2Rs. The NA-triggered adiponectin exocytosis is likewise abolished in "obese adipocytes ", concomitant with a 50% lower gene expression of beta 3 adrenergic receptors (beta 3ARs). An increase in intracellular Ca2+ is not required for the NA-stimulated adiponectin secretion. Collectively, our data suggest that sympathetic innervation is a principal regulator of adiponectin exocytosis and that disruptions of this control are associated with the obesity-associated reduction of circulating levels of HMW/total adiponectin.
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| 10. |
- Saliha, Musovic, 1990, et al.
(författare)
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Resistin is co-secreted with adiponectin in white mouse adipocytes
- 2021
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 534, s. 707-713
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Tidskriftsartikel (refereegranskat)abstract
- In the current work we have investigated the cellular and molecular regulation of resistin secretion in cultured and primary mouse adipocytes. Resistin is an adipose tissue hormone proposed to contribute to metabolic disease. In rodents, resistin is secreted from white adipocytes whereas it is in humans synthesised and released from other cell types within white adipose tissue. The metabolic importance of resistin has been studied in both mouse and man, but the regulation of its release remains poorly investigated. Here we define that, in mouse adipocytes, resistin secretion is triggered by an intracellular elevation of cAMP and/or Ca2+. Resistin release is stimulated via activation of beta 3 adrenergic receptors (β3ARs) and the downstream signalling protein exchange protein activated by cAMP (Epac). The secretion of resistin is markedly abrogated in adipocytes isolated from obese and diabetic mice. Immunocytochemical staining demonstrates a significant overlap between signals for resistin and the adipocyte hormone adiponectin. Our data propose that resistin and adiponectin are contained within the same vesicles in mouse adipocytes and that the two hormones are co-secreted in response to the same exocytosis-triggering signals. © 2020 Elsevier Inc.
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