Relationship between the oxygen consumption rate and protein content.

<p>The correlations between the basal oxygen consumption rate and cytochrome oxidase complex 4 (COX4) content (A) and between the norepinephrine (NE)-induced oxygen consumption rate and uncoupling protein 1 (UCP1) content (B) in adipocytes isolated from interscapular brown adipose tissue (BA) of the control group (○), of the cold-acclimated group (•), or inguinal white adipose tissue (I-WA: ▴) are shown. Statistical data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084229#pone.0084229.s002" target="_blank">Table S2</a>. (C) The gradient of regression lines in (B). The difference between BA and I-WA was compared by ANCOVA. *<i>P</i><0.05, versus BA. (D) The ability of UCP1 to enhance oxygen consumption by NE stimulation was calculated by dividing the NE-induced oxygen consumption rate by the UCP1 content. (E) NE-induced oxygen consumption per COX4 was calculated. Values are means ± SE for 18 samples. *<i>p</i><0.05, versus BA.</p

Effect of norepinephrine on the oxygen consumption rate of adipocytes isolated from different fat depots.

<p>(A) Oxygen consumption was measured using adipocytes isolated from interscapular brown adipose tissue (BA), inguinal (I-WA) and perigonadal white adipose tissue (G-WA) of the control and cold-acclimated mice. At the arrow (5 min), 1 µM norepinephrine (NE) was added. (B) Basal oxygen consumption rate was calculated and expressed as a mean oxygen consumption rate for 5 min before NE stimulation. NE-induced oxygen consumption rate was calculated as a mean oxygen consumption rate for 10 min after NE stimulation subtracted by basal oxygen consumption rate. White and black columns indicate the control and cold-acclimated groups, respectively. Gray column shows NE-induced oxygen consumption rate of beige adipocytes estimated on the basis of their abundance in I-WA of the cold-acclimated group. Values are means ± SE for 9 mice. *<i>p</i><0.05, versus the control group of the same tissue.</p

Characteristics of adipocytes isolated from different fat depots of the control and cold-acclimated mice.

<p>Adipocytes were isolated from interscapular brown adipose tissue (BA), inguinal (I-WA) and perigonadal white adipose tissue (G-WA) of the control and cold-acclimated mice. (A) Typical photomicrographs of adipocytes isolated form each depot of the control or cold-acclimated mice. Arrows indicate multilocular beige adipocytes. (B) Expression of several genes in the isolated adipocytes was measured by quantitative real-time PCR. Expression levels normalized to ß-actin expression are shown. White and black columns indicate the control and cold-acclimated groups, respectively. Values are means ± SE for 9 mice. Different letters indicate significant differences between the groups (<i>p</i><0.05).</p

Effect of cold acclimation on the protein content in isolated adipocytes.

<p>Protein was extracted from isolated adipocytes from interscapular brown adipose tissue (BA), inguinal (I-WA) and perigonadal white adipose tissue (G-WA) of the control and cold-acclimated mice. (A) Protein content was measured and expressed as the amount of protein from 10⁵ cells. (B) Uncoupling protein 1 (UCP1) and cytochrome oxidase complex 4 (COX4) were detected by western blotting. A typical detection pattern is shown. Protein from BA (1 µg) and WA (10 µg) was loaded on the SDS-PAGE gel. (C) COX4 and UCP1 content per 10⁵ cells were calculated from the protein content (A) and the band density obtained by western blotting (B). White and black columns indicate the control and cold-acclimated groups, respectively. Gray column shows UCP1 content in beige adipocytes estimated on the basis of their abundance in I-WA of the cold-acclimated group. Values are means ± SE for 9 mice. *<i>p</i><0.05, versus the same depot of the control mice.</p

Effect of cold acclimation on cell morphology and uncoupling protein 1 expression in adipose tissues.

<p>Mice were housed in a cold environment (10°C; cold-acclimated mice) or a control environment (24°C; control mice) for 3 weeks. (A) Sections of interscapular brown adipose tissue (BAT), inguinal white adipose tissue (WAT) (I-WAT), and perigonadal WAT (G-WAT) were stained with hematoxylin and eosin (HE) or immunostained using antibody against uncoupling protein 1 (UCP1). (B) UCP1 protein expression was analyzed by western blotting. To detect UCP1, 2.5 µg (BAT) or 20 µg (I-WAT and G-WAT) of protein was used. Values are means ± SE for 4 mice. *<i>p</i><0.05, versus the control group.</p