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Cooling reduces cAMP-stimulated exocytosis and adiponectin secretion at a Ca2+-dependent step in 3T3-L1 adipocytes.

El Hachmane MF, Komai AM, Olofsson CS - PLoS ONE (2015)

Bottom Line: 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.Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C.At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

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°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca2+] to ~1.5 μM (9 mM Ca2+ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°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.

No MeSH data available.


Related in: MedlinePlus

Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca2+]i.Example traces of [Ca2+]i responses upon extracellular application of 1 μM ionomycin (A) or 1 mM 8-Br-cAMP (C). B Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.
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pone.0119530.g004: Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca2+]i.Example traces of [Ca2+]i responses upon extracellular application of 1 μM ionomycin (A) or 1 mM 8-Br-cAMP (C). B Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.

Mentions: 3T3-L1 adipocytes secrete substantial quantities of adiponectin [3,16] and we have previously documented a strong correlation between membrane capacitance increases and secretion of this adipokine [5]. In order to compare effects of temperature on exocytosis and adiponectin release, 3T3-L1 adipocytes were incubated in the presence of the membrane permeable cAMP agonist 8-Br-cAMP alone or in combination with the Ca2+-ionophore ionomycin (to maximally elevate [Ca2+]i). Incubations were carried out for 30 minutes at 23°C or 32°C. Effects of ionomycin and 8-Br-cAMP on intracellular Ca2+ levels were validated by use of Ca2+ imaging. As shown in Fig. 4A, external application of ionomycin (1 μM at 23°C or 32°C) elevated [Ca2+]i resulting in a peak at t = ~3 min, after which [Ca2+]i declined towards baseline before stabilising at a plateau at 10 min (Fig. 4B). The magnitudes of the responses were comparable although the peak value was slightly (20 nM) but significantly higher at 32°C (P<0.001). However, the measured minute difference is likely of negligible physiological importance. An ionomycin-induced peak [Ca2+]i of ~200 nM may appear too small to affect exocytosis but it has to be kept in mind that the measurements show the average [Ca2+]i in the cytoplasm and may be several-fold higher at the release sites [17]. Inclusion of 8-Br-cAMP (1 mM) was without effect on [Ca2+]i (Fig. 4C; [Ca2+]i averaged 108±2 nM before and 119±3 nM after agonist application).


Cooling reduces cAMP-stimulated exocytosis and adiponectin secretion at a Ca2+-dependent step in 3T3-L1 adipocytes.

El Hachmane MF, Komai AM, Olofsson CS - PLoS ONE (2015)

Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca2+]i.Example traces of [Ca2+]i responses upon extracellular application of 1 μM ionomycin (A) or 1 mM 8-Br-cAMP (C). B Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368704&req=5

pone.0119530.g004: Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca2+]i.Example traces of [Ca2+]i responses upon extracellular application of 1 μM ionomycin (A) or 1 mM 8-Br-cAMP (C). B Average responses to ionomycin at indicated time points between 0 and 15 min. Ionomycin or 8-Br-cAMP was added extracellularly to the dish of cells and remained present throughout the recording as indicated. Note that the peak response to ionomycin shown in (B) was slightly shifted at 23°C (peak at 3.6 min; 5 separate experiments and 122 cells) compared to 32°C (peak at 3.1 min; 4 experiments and 107 cells). The trace in (C) is representative for 101 analysed cells in 4 separate experiments.
Mentions: 3T3-L1 adipocytes secrete substantial quantities of adiponectin [3,16] and we have previously documented a strong correlation between membrane capacitance increases and secretion of this adipokine [5]. In order to compare effects of temperature on exocytosis and adiponectin release, 3T3-L1 adipocytes were incubated in the presence of the membrane permeable cAMP agonist 8-Br-cAMP alone or in combination with the Ca2+-ionophore ionomycin (to maximally elevate [Ca2+]i). Incubations were carried out for 30 minutes at 23°C or 32°C. Effects of ionomycin and 8-Br-cAMP on intracellular Ca2+ levels were validated by use of Ca2+ imaging. As shown in Fig. 4A, external application of ionomycin (1 μM at 23°C or 32°C) elevated [Ca2+]i resulting in a peak at t = ~3 min, after which [Ca2+]i declined towards baseline before stabilising at a plateau at 10 min (Fig. 4B). The magnitudes of the responses were comparable although the peak value was slightly (20 nM) but significantly higher at 32°C (P<0.001). However, the measured minute difference is likely of negligible physiological importance. An ionomycin-induced peak [Ca2+]i of ~200 nM may appear too small to affect exocytosis but it has to be kept in mind that the measurements show the average [Ca2+]i in the cytoplasm and may be several-fold higher at the release sites [17]. Inclusion of 8-Br-cAMP (1 mM) was without effect on [Ca2+]i (Fig. 4C; [Ca2+]i averaged 108±2 nM before and 119±3 nM after agonist application).

Bottom Line: 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.Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C.At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology/Metabolic Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

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°C, 27°C, 32°C and 37°C). However, the augmentation of exocytosis induced by an elevation of the free cytosolic [Ca2+] to ~1.5 μM (9 mM Ca2+ + 10 mM EGTA) was potent at 32°C or 37°C but less distinct at 27°C and abolished at 23°C. Adiponectin secretion stimulated by 30 min incubations with the membrane permeable cAMP analogue 8-Br-cAMP (1 mM) or a combination of 10 μM forskolin and 200 μM IBMX was unaffected by a reduction of temperature from 32°C to 23°C. At 32°C, cAMP-stimulated secretion was 2-fold amplified by inclusion of the Ca2+ ionophore ionomycin (1μM), an effect that was not observed at 23°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.

No MeSH data available.


Related in: MedlinePlus