In vivo emergence of beige-like fat in chickens as physiological adaptation to cold environments

Abstract

While it has been hypothesized that brown adipocytes responsible for mammalian thermogenesis are absent in birds, the existence of beige fat has yet to be studied directly. The present study tests the hypothesis that beige fat emerges in birds as a mechanism of physiological adaptation to cold environments. Subcutaneous neck adipose tissue from cold-acclimated or triiodothyronine (T₃)-treated chickens exhibited increases in the expression of avian uncoupling protein (avUCP, an ortholog of mammalian UCP2 and UCP3) gene and some known mammalian beige adipocyte-specific markers. Morphological characteristics of white adipose tissues of treated chickens showed increased numbers of both small and larger clusters of multilocular fat cells within the tissues. Increases in protein levels of avUCP and mitochondrial marker protein, voltage-dependent anion channel, and immunohistochemical analysis for subcutaneous neck fat revealed the presence of potentially thermogenic mitochondria-rich cells. This is the first evidence that the capacity for thermogenesis may be acquired by differentiating adipose tissue into beige-like fat for maintaining temperature homeostasis in the subcutaneous fat 'neck warmer' in chickens exposed to a cold environment.

Keywords: Avian models; Beige fat; Cold adaptation; Thermogenesis; avUCP.

Fig. 1

Increased mRNA expression levels of avian UCP (avUCP) and of beige adipose tissue markers in chickens exposed to 4 °C or fed a T₃-supplemented diet. a Weights of neck subcutaneous fat (left) and abdominal fat (right) of control, T₃-treated and cold-exposed chickens. Values are means ± S.E., n = 8 chickens in each group. b mRNA levels of avUCP in neck subcutaneous fat (left) and abdominal fat (right) of control, T₃-treated and cold-exposed chickens. Values are means ± S.E., n = 8 chickens in each group. c mRNA levels of markers known to be enriched in mammalian beige adipocytes in neck subcutaneous fat (left) and abdominal fat (right) of control, T₃-treated and cold-exposed chickens. Values are means ± S.E., n = 8 chickens for CIDEA, TBX1 and TMEM26 in each group; n = 5–8 chickens for CAR4, SLC27A1, CD137, EAR2 and CPT1b in each group. d Dependency of avUCP expression on avPGC-1α in neck subcutaneous fat of control, T₃-treated, and cold-exposed chickens (n = 24) and effects of T₃- and cold-treatments on mRNA levels of avPGC-1α. (n = 8 chickens in each group). All data for mRNA levels are shown as fold changes relative to control values. Quantitative real-time RT-PCR was used to quantify mRNA levels, and the results were normalized to 18S mRNA levels. ND means not appropriately detected for too low expression. Value in panel D represents Pearson correlation coefficient for the relationship between avUCP and PGC-1α gene levels. Differences between control, T₃- and cold-treated groups were assessed using the Shirley-Williams test. *p < 0.05 compared to the control group

Fig. 2

Emergence of multilocular fat cells in chickens exposed to cold and fed a T₃-supplemented diet. a H&E stains showing multilocular fat cells within the neck subcutaneous fat (upper) and abdominal fat (lower) depots in control, T₃-treated and cold-acclimated chickens. b Process for the quantitative evaluation of multilocular adipocytes. Fat depots containing small adipocytes were regarded as one cluster, such as large, medium, or small-sized cluster. c Scatter plots displaying data points of the number of clusters on the y-axis versus the cluster size on the x-axis within neck subcutaneous fat (upper) and abdominal fat (lower) tissues of control, T₃-treated and cold-acclimated chickens

Fig. 3

Clusters of beige-like adipocytes densely emerge in a mitochondria-rich cell of neck subcutaneous fat in T₃-treated chickens. a mRNA levels of avUCP in neck subcutaneous fat and abdominal fat tissues of control and T₃-treated chickens. Quantitative real-time RT-PCR was used to quantify mRNA levels, and the results were normalized to 18S rRNA levels. Values are means ± S.E., n = 5–6 chickens in each group. b Protein levels of avUCP in neck subcutaneous fat of control and T₃-treated chickens. avUCP protein levels were assessed by Western blot of tissue protein (80 μg) using an anti-avUCP antibody. Band intensities of avUCP shown as semi-quantified by densitometric tracing. Values are means ± S.E., n = 5 chickens in each group. Uncropped data and observed band size are depicted in Supplement A, Supporting Information. c VDAC protein content in neck subcutaneous fat of control and T₃-treated chickens. VDAC protein levels were assessed by Western blot analysis of tissue protein (10 μg) using an anti-VDAC antibody. Band intensities of VDAC shown as semi-quantified by densitometric tracing. Values are means ± S.E., n = 6 chickens in each group. Uncropped data are depicted in Supplement B, Supporting Information. d Immunohistochemical analysis using anti-VDAC antibody for neck subcutaneous fat of control and T₃-treated chickens. Data for mRNA and protein levels of avUCP or VDAC protein are shown as fold changes relative to control values for the neck subcutaneous fat. Differences between control and T₃-treated chickens were assessed using the Student’s t-test for unpaired data. *p < 0.05 compared to the control group