Arrhenius plots presenting the effect of Ca²⁺ on the rate of cAMP-triggered exocytosis.

<p>Absolute values of Δ<i>C</i>/Δ<i>t</i> at t = 2 min plotted against the inverse of temperature (T) in the absence <b>(A)</b> and presence <b>(B)</b> of cytosolic Ca²⁺. The curve represents a linear least-squares fit to the equation lnk = lnA—E_A/RT. E_A is the energy of activation, k is the change in Δ<i>C</i>/Δ<i>t</i> upon a temperature change, A is the pre-exponential factor. R represents the gas constant and T the absolute temperature as usual. The negative slope of the curve gives activation energies of 5.7 kJ mol^-1 (A) and 53 kJ mol^-1 (B).</p

Undifferentiated and mature 3T3-L1 adipocytes as well as demonstration of analysis performance.

<p><b>A</b> Example of 3T3-L1 adipocytes in the fibroblast-like state before differentiation (left) as well as when differentiated into mature adipocytes (right). <b>B</b> Representative capacitance recording with Δ<i>C</i>_m (delta membrane capacitance) plotted against time showing how analyses were carried out. Δ<i>C</i>/Δ<i>t</i> was measured at the time intervals indicated by the dotted lines by fitting straight functions to the data points (red lines superimposed on the black capacitance trace). Scale bar = 50μm.</p

Ca²⁺-augmented adiponectin secretion is abolished by cooling while secretion stimulated by cAMP alone is unaffected.

<p><b>A</b> Adiponectin secretion as fold-increase compared to control stimulated by 8-Br-cAMP (1 mM) alone or in combination with ionomycin (1 μM) during 30 min incubations at 23°C (blue) or 32°C (red). <b>B</b> As in <b>(A)</b> but using forskolin (10 μM) and IBMX (200 μM; forsk/IBMX) as a stimulator. Data are mean values ± S.E.M. of 11 experiments at each temperature in (A) and 7 (23°C) and 8 (32°C) in (B). <i>*P<0</i>.<i>05; **P<0</i>.<i>01</i>; <i>**P<0</i>.<i>001</i> vs. control at corresponding temperature. †<i>P<0</i>.<i>01</i> vs. 8-Br-cAMP or forsk/IBMX alone at 32°C; ǂ <i>P<0</i>.<i>05</i> vs. 8-Br-cAMP + ionomycin or forsk/IBMX + ionomycin at room temperature. The data in (B) at 32°C are the same as in Fig. 7B in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119530#pone.0119530.ref005" target="_blank">5</a>].</p

Ionomycin, but not 8-Br-cAMP, elevates adipocyte [Ca²⁺]_i.

<p>Example traces of [Ca²⁺]_i responses upon extracellular application of 1 μM ionomycin <b>(A)</b> or 1 mM 8-Br-cAMP <b>(C)</b>. <b>B</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.</p

Proposed model of cooling effects on white adipocyte exocytosis.

<p>White adipocytes release adiponectin containing vesicles belonging to two functionally distinct populations. cAMP stimulates release of vesicles residing in a readily releasable pool in a temperature-independent manner. The Ca²⁺-dependent augmentation of secretion is reduced/abolished by cooling. Intracellular ATP is necessary for the Ca²⁺ effect. See text for more details.</p