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. 2008 Apr 21:6:17.
doi: 10.1186/1741-7007-6-17.

The brown adipocyte differentiation pathway in birds: an evolutionary road not taken

Nadejda V Mezentseva  1 , Jaliya S Kumaratilake, Stuart A Newman
Affiliations

The brown adipocyte differentiation pathway in birds: an evolutionary road not taken

Nadejda V Mezentseva et al. BMC Biol. .

Abstract

Background: Thermogenic brown adipose tissue has never been described in birds or other non-mammalian vertebrates. Brown adipocytes in mammals are distinguished from the more common white fat adipocytes by having numerous small lipid droplets rather than a single large one, elevated numbers of mitochondria, and mitochondrial expression of the nuclear gene UCP1, the uncoupler of oxidative phosphorylation responsible for non-shivering thermogenesis.

Results: We have identified in vitro inductive conditions in which mesenchymal cells isolated from the embryonic chicken limb bud differentiate into avian brown adipocyte-like cells (ABALCs) with the morphological and many of the biochemical properties of terminally differentiated brown adipocytes. Avian, and as we show here, lizard species lack the gene for UCP1, although it is present in amphibian and fish species. While ABALCs are therefore not functional brown adipocytes, they are generated by a developmental pathway virtually identical to brown fat differentiation in mammals: both the common adipogenic transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma), and a coactivator of that factor specific to brown fat differentiation in mammals, PGC1alpha, are elevated in expression, as are mitochondrial volume and DNA. Furthermore, ABALCs induction resulted in strong transcription from a transfected mouse UCP1 promoter.

Conclusion: These findings strongly suggest that the brown fat differentiation pathway evolved in a common ancestor of birds and mammals and its thermogenicity was lost in the avian lineage, with the degradation of UCP1, after it separated from the mammalian lineage. Since this event occurred no later than the saurian ancestor of birds and lizards, an implication of this is that dinosaurs had neither UCP1 nor canonically thermogenic brown fat.

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Figures

Figure 1
Figure 1
Appearance of cells with mammalian brown fat-like phenotype in embryonic chicken mesenchyme in vitro. (a) Oil-red and hematoxylin staining of light micrographs of limb bud mesenchyme (LBM) cells grown in adipocyte differentiation medium (ADM) for 22 days with 10% horse serum. (b) Transmission electron micrograph of a single cell from 22-day culture as in (a); L, lipid droplet. (c)-(f) Oil-red and hematoxylin staining of light micrographs of 8-day cell cultures.(c) Adipocytes from LBM cells grown in ADM with 10% horse serum. (d) Adipocytes from vascular stromal cells (VSCs) grown in ADM with 10% horse serum. (e) Adipocytes from LBM grown in ADM with 10% chicken serum. (f) LBM cells grown in ADM with 10% FBS. Scale bars: (a) 10 μm, (b) 1 μm; (c)-(f) 50 μm
Figure 2
Figure 2
Transmission electronmicrographs of adipocytes. (a) Adipocyte from limb bud mesenchyme (LBM) grown in adipocyte differentiation medium (ADM) with 10% horse serum for 22 days. (b) Adipocyte from the abdominal fat of a 20-day chicken embryo. Cy, cytoplasm; C, capillary; N, nucleus; M, mitochondrion; L, lipid droplet. Scale bars: 1 μm.
Figure 3
Figure 3
Avian brown adipocyte-like cells express common adipocyte markers and specific brown adipocyte markers. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of relative expression of genes common to differentiated phenotype of white and brown adipocytes and specific to brown fat differentiation in limb bud mesenchyme (LBM), limb bud preadipocytes (LBPAs), avian brown adipocyte-like cells (ABALCs) in 8-day cell culture, vascular stromal cells adipocytes (VSCAs) and white adipocytes (WAs) from abdominal fat of 20-day chicken embryos. (a) Fatty acid binding protein, FABP4. (b) Peroxisome proliferator activated receptor γ, PPARγ. (c) Peroxisome proliferator-activated receptor γ coactivator 1α, PGC-1α. (d) Type II iodothyronine deiodinase, DiO2 and peroxisome proliferator activated receptor α, PPARα. Each treatment was repeated three times. Measurements were in triplicate and normalized to β-actin mRNA levels. Relative mRNA levels were analyzed by one-way analysis of variance and means were compared using the Tukey test. Results are shown as means ± standard error of the mean.
Figure 4
Figure 4
ABALC cellular phenotype is associated with increased mitochondrial volume. FACScan of cells stained with JC-1. FL2-H is red fluorescence. Black line, unstained mesenchymal cells; blue line, stained mesenchymal cells; red line, limb bud preadipocytes (LBPAs); pink line, limb bud mesenchyme (LBM) adipocytes grown in chicken serum; green line, avian brown adipocyte-like cells (ABALCs).
Figure 5
Figure 5
Quantitative polymerase chain reaction analysis of relative mtDNA levels. Each measurement was normalized to β-actin DNA levels. Relative mtDNA levels were analyzed using one-way analysis of variance comparing means by the Tukey test. Results are shown as means ± standard error (N = 3)
Figure 6
Figure 6
Genomic comparison of human, mouse, chicken, lizard, amphibian and fish genomes reveals absence of UCP1 gene in chicken and lizard. Genomic sequences and contexts were analyzed using the Ensembl [42] and University of California Santa Cruz [43] genome browsers.
Figure 7
Figure 7
Mouse uncoupling protein 1 promoter is activated in avian brown adipocyte-like cells but not in vascular stromal cell adipocytes. Firefly luciferase activity was normalized with Renilla luciferase. Normalized Firefly luciferase activities were compared using one-way analysis of variance using the Tukey test. Results are shown as means ± standard error (N = 8).
Figure 8
Figure 8
Partial cladogram of the vertebrates. Taxa in which UCP1 are known to be present are indicated by a red dot. Those in which UCP1 is known to be absent are indicated by a black dot. The question mark indicates the span within the evolutionary history of the amniotes during which UCP1 and the capacity to produce functional brown fat was lost.
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