Cold Exposure Induces Proliferation of Mature Brown Adipocyte in a beta 3-Adrenergic Receptor-Mediated Pathway

Hyperplasia of brown adipose tissue (BAT) is a fundamental mechanism for adaptation to survive in the cold environment in rodents. To determine which cell types comprising BAT contribute to tissue hyperplasia, immunohistochemical analysis using a proliferative marker Ki67 was performed on the BAT from 6-week-old C57BLJ6J mice housed at 23 degrees C (control) or 10 degrees C (cold) for 5 days. Interestingly, in the control group, the cell proliferative marker Ki67 was detected in the nuclei of uncoupling protein 1-positive mature brown adipocytes (7.2% +/- 0.4% of brown adipocyte), as well as in the non-adipocyte stromal-vascular (SV) cells (19.6% +/- 2.3% of SV cells), which include preadiopocytes. The percentage of Ki67-positive brown adipocytes increased to 25.6% +/- 1.8% at Day 1 after cold exposure and was significantly higher than the non-cold acclimated control until Day 5 (21.8%+/- 1.7%). On the other hand, the percentage of Ki67-positive SV cells gradually increased by a cold exposure and peaked to 42.1%+/- 8.3% at Day 5. Injection of a beta 3-adrenergic receptor (beta 3-AR) agonist for continuous 5 days increased the number of Ki67-positive brown adipocytes even at Day 1 but not that of SV cells. In addition, the beta 3-AR antagonist, but not beta 1-AR antagonist, attenuated the cold exposure-induced increase in the number of Ki67-positive brown adipocytes. These results suggest that mature brown adipocytes proliferate immediately after cold exposure in a beta 3-AR-mediated pathway. Thus, proliferation of mature brown adipocytes as well as preadipocytes in SV cells may contribute to cold exposure-induced BAT hyperplasia

Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24 degrees C (control group) or 10 degrees C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes

Proinsulin C-peptide activates α-enolase : implications for C-peptide-cell membrane interaction

Proinsulin C-peptide shows beneficial effects on microvascular complications of type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins, and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in Km for 2-phosphoglycerate without affecting Vmax. The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase via a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo

Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

We previously reported brown adipocytes can proliferate even after differentiation. To test the involvement of mature adipocyte proliferation in cell number control in fat tissue, we generated transgenic (Tg) mice over-expressing cell-cycle inhibitory protein p27 specifically in adipocytes, using the aP2 promoter. While there was no apparent difference in white adipose tissue (WAT) between wild-type (WT) and Tg mice, the amount of brown adipose tissue (BAT) was much smaller in Tg mice. Although BAT showed a normal cellular morphology, Tg mice had lower content of uncoupling protein 1 (UCP1) as a whole, and attenuated cold exposure-or beta 3-adrenergic receptor (AR) agonist-induced thermogenesis, with a decrease in the number of mature brown adipocytes expressing proliferation markers. An agonist for the beta 3-AR failed to increase the number of proliferating brown adipocytes, UCP1 content in BAT, and oxygen consumption in Tg mice, although the induction and the function of beige adipocytes in inguinal WAT from Tg mice were similar to WT mice. These results show that brown adipocyte proliferation significantly contributes to BAT development and adaptive thermogenesis in mice, but not to induction of beige adipocytes

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

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