Content uploaded by Viv E.A. Perry
Author content
All content in this area was uploaded by Viv E.A. Perry on Mar 26, 2018
Content may be subject to copyright.
RESEARCH ARTICLE
Ontogeny and Thermogenic Role for Sternal
Fat in Female Sheep
Belinda A. Henry,
1
Mark Pope,
2
Mark Birtwistle,
2
Rachael Loughnan,
1
Reham Alagal,
2
John-Paul Fuller-Jackson,
1
Viv Perry,
3
Helen Budge,
2
Iain J. Clarke,
4
and Michael E. Symonds
2,5
1
Metabolic Disease and Obesity Program, Monash Biomedicine Discovery Institute and Department of
Physiology, Monash University, Clayton, Victoria 3800, Australia;
2
Early Life Research Unit, Division of Child
Health, Obstetrics & Gynaecology, School of Medicine, University of Nottingham, Nottingham NG7 2UH,
United Kingdom;
3
School of Veterinary Medicine and Science, The University of Nottingham, Sutton
Bonington LE12 5RD, United Kingdom;
4
Neuroscience Program, Monash Biomedicine Discovery Institute
and Department of Physiology, Monash University, Clayton, Victoria 3800, Australia; and
5
Nottingham
Digestive Disease Centre and Biomedical Research Unit, School of Medicine, Queen’s Medical Centre, The
University of Nottingham, Nottingham NG7 2UH, United Kingdom
Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in
preventing hypothermia in newborn sheep but is then thought to rapidly disappear during
postnatal life. The extent to which the anatomical location of fat influences postnatal development
and thermogenic function in adulthood, particularly following feeding, is unknown, and we ex-
amined both in our study. Changes in gene expression of functionally important pathways (i.e.,
thermogenesis, development, adipogenesis, and metabolism) were compared between sternal and
retroperitoneal fat depots together with a representative skeletal muscle over the first month of
postnatal life, coincident with the loss of brown fat and the accumulation of white fat. In adult sheep,
implanted temperature probes were used to characterize the thermogenic response of fat and muscle
to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant in sternal fat
than in retroperitoneal fat and was retained only in the sternal depot of adults. Distinct differences in
the abundance of gene pathway markers were apparent between tissues, with sternal fat exhibiting
some similarities with muscle that were not apparent in the retroperitoneal depot. In adults, the
postprandial rise in temperature was greater and more prolonged in sternal fat than in retroperitoneal
fat and muscle, a difference that was maintained with altered adiposity. In conclusion, sternal adipose
tissue retains UCP1 into adulthood, when it shows a greater thermogenic response to feeding than do
muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that
promote thermogenesis in large mammals. (Endocrinology 158: 2212–2225, 2017)
In the majority of large mammals studied to date, birth is a
critical period for the rapid recruitment of nonshivering
thermogenesis in brown adipose tissue (BAT), and this
coincides with the maximal appearance of uncoupling
protein (UCP1) (1, 2). This is followed by a transformation
of fat from a brown to a white appearance, although the rate
and magnitude of this process can vary between depots (3).
For example, in humans the supraclavicular (or neck) depot
retains UCP1 into adulthood (4), whereas the peri-adrenal
depot does not (5). Consequently, in adults the supra-
clavicular depot has the capacity to exhibit a major ther-
mogenic response to both cold exposure (6) and diet (7).
The extent to which the retention of UCP1 through the life
cycle, and thus the associated thermogenic potential, is
determined by a fat depot’s early development and/or an-
atomical location is currently unknown.
Studies in rodents initially suggested that brown and
white adipocytes arise from different lineages and that
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in USA
Copyright © 2017 Endocrine Society
Received 18 January 2017. Accepted 14 April 2017.
First Published Online 19 April 2017
Abbreviations: au, ar bi tr ar y un it ; BAT, brow n ad ip os e ti ss ue; mRN A, m es se ng er
RNA; NEFA, nonesterified fatty acid; PCR, polymerase chain reaction; UCP1,
uncoupling protein.
2212 https://academic.oup.com/endo Endocrinology, July 2017, 158(7):2212–2225 doi: 10.1210/en.2017-00081
brown adipocytes may originate from the same precursor
as skeletal muscle (8). This relationship now appears to be
more complex and depot specific because white adipo-
cytes can have diverse and mixed origins (9, 10), po-
tentially overlapping with brown adipocytes (11).
Additional populations of adipocytes have been identi-
fied as beige or “brite”(i.e., brown-in-white) and are
characterized as small populations of UCP1-expressing
cells surrounded by large numbers of white adipocytes
(12, 13). The thermogenic relevance of these cells remains
to be established, as their UCP1 content is only 10%
of that of classic BAT (14). To date, most studies in-
vestigating beige fat have been confined to adult rodents
in which “almost everything”examined was able to
“brown”white adipose tissue (15). Furthermore, a di-
verse range of molecular markers for beige adipocytes has
been suggested, but their applicability across species (16),
as well as the optimal conditions in which these classi-
fications are defined (17), is being questioned.
Our study therefore had two aims. The first was to
compare the gene-expression profiles for the primary
thermogenic, metabolic, and functional markers of brown,
beige, and white adipose tissue in retroperitoneal fat [the
most abundant depot in fetal sheep and a “classic”adipose
tissue depot (18)], sternal (or neck) fat, and hind limb
muscle, a representative skeletal muscle. Sheep, like many
large mammals, do not possess the interscapular BAT (19)
that is present in rodents. The current comparison, un-
dertaken in young sheep, spanned the period from birth to
1 month of postnatal life, examining three important time
points that in retroperitoneal fat are coincident with the
peak abundance of UCP1 after birth (i.e., 1 day of age), the
age at which UCP1 has declined to basal amounts before
the onset of rapid growth (i.e., 7 days of age), and the
subsequent loss of UCP1 (i.e., 28 days of age (1). We also
conducted detailed molecular analyses to establish whether
each depot could have a different developmental origin and
if adipose tissue in the sternal depot is more similar to
skeletal muscle than to classic (i.e., retroperitoneal) adipose
tissue. The second aim was to examine whether the sternal
depot responds to the thermogenic stimulus of feeding in
adulthood (20) and if this is comparable to responses in
skeletal muscle rather than those in classic adipose tissue. In
addition, we examined whether diet-induced obesity or low
body weight modulated this response and thus whether
sternal fat could be a potential target for promoting
thermogenesis.
Methods
Animal experimentation
All animal work was approved by the relevant animal
ethics committees at The University of Nottingham or Monash
University. To avoid any confounding effects of sex and/or a
disproportionate increase in muscle mass following overfeeding
[as seen in males (21)], only females were studied.
Study 1. The effects of postnatal age on sternal
adipose tissue development
Ten triplet-bearing sheep of mixed breed that all gave birth
naturally to appropriately grown offspring at term (over a 4-
week period) were entered into the study. Triplets were chosen
for the study because this meant there would be no confounding
maternal influences between each sampling date. One lamb
from each mother was therefore randomly selected at either 1, 7,
or 28 days of age, had blood sampled from the jugular vein, and
then was euthanized by injection of sodium pentobarbital
(0.5 mL/kg). The sternal and retroperitoneal adipose tissue
depots were dissected and weighed, together with a represen-
tative sample from the hind limb muscle (vastus lateralis).
Samples were immediately placed in liquid nitrogen and were
then stored at 280°C until analyzed.
Study 2. The effects of altered body weight and fat
mass on the temperature response to feeding in
sternal adipose tissue
Manipulation of adult body weight
Fifteen adult female Corriedale sheep aged between 3 and
5 years and of normal body weight (;55 kg) were all ovari-
ectomized (to avoid any confounding effects of the reproductive
cycle on tissue temperature) and were then randomly divided
into three different body weight groups. Five animals were made
lean (32.5 61.5 kg) by feeding them a restricted diet of ;500 g
of lucerne chaff per day, and five became obese (79.9 63.7 kg)
after receiving a supplemented diet of lucerne hay ad libitum and
;300 g daily of high-energy food supplement (i.e., oats and
lupin grain) (22, 23). The remaining five “control”animals
(52.6 61.1 kg) were maintained on pasture. Differential body
weights were then maintained for 1 year before experimenta-
tion. To assess the temperature response to feeding, the diets
were standardized across all groups, and the high-energy sup-
plementation of the obese group stopped. Visceral adiposity was
determined at the time of euthanasia (lean: 0.04 60.01 kg;
control: 1.58 60.18 kg; obese: 4.11 60.19 kg).
Profiling postprandial changes in temperature
and metabolites
For tissue-specific temperature recordings, customized
dataloggers with 10-cm or 20-cm download leads (SubCue,
Calgary, AB, Canada) were inserted into the skeletal muscle of
the hind limb (vastus lateralis) and the sternal (midline) and
retroperitoneal fat and were set to record temperatures at
15-minute intervals as previously published (20, 24). After
surgery, the animals were housed indoors to enable precisely
timed “meal”feeding and were exposed to natural variations
in photoperiod and ambient temperature. To entrain post-
prandial thermogenesis, animals were placed on a temporal
program-feeding regimen, in which they had access to food at
set mealtimes between 1100 and 1600 hours each day (20, 24,
25). Animals were program-fed for 2 weeks before the onset of
experimentation. To characterize changes in plasma metabolite
and insulin levels with feeding, blood samples (6 mL) were
collected into heparinized tubes at 30-minute intervals between
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2213
1000 and 1600 hours. The samples were centrifuged to obtain
plasma, which was stored at 220°C until assayed.
Food intake was recorded after offering the obese and standard
animals 2 kg of lucerne chaff and monitoring any refusals, whereas
the lean animals received and consumed 500 g of chaff per day.
Food intake, once corrected to body weight, was similar in the
obese and lean animals, whereas the control group ate slightly
more (P,0.01) than the other two groups (data not shown).
Temperature data were downloaded, and the diurnal ther-
mogenic pattern and the postprandial response were then an-
alyzed in each tissue. After 3 weeks of program feeding, all
animals were euthanized, as described previously, between 830
and 1030 hours. Representative fat and muscle samples were
collected and stored as described previously for study 1. At this
time, it was confirmed that each temperature probe was still
located within the same anatomical position as at surgery.
Laboratory analysis
Gene expression
Total RNA was extracted, and following confirmation of
RNA integrity, gene expression was determined using real-time
polymerase chain reaction (PCR) (1). The specificity of each
ovine primer was confirmed by classic PCR with ovine com-
plementary DNA from suitable tissue samples, negative con-
trols, and ovine genomic DNA and by analyzing the products by
agarose gel electrophoresis. The primers were used only when
there was a clear single band on the gel corresponding to the
expected amplicon size, negative control lanes were clear, and
any products from amplification of genomic DNA could be
easily distinguished from the target. The PCR products of the
selected primers were analyzed using high-sensitivity Sanger
dideoxy sequencing, and the returned sequences were verified
by alignment with the predicted on-line sequence to ensure that
they were specific to the intended target. The primers used
are summarized in Table 1. The amount of messenger RNA
(mRNA) was calculated relative to the geometric mean of the
most stable reference genes as determined by geNorm and/or
NormFinder analysis. For the postnatal tissues, housekeeping
genes included IPO8,KDM2B,RPLP0, and TBP, and cyclo-
philin,bactin,b
2
-microglobulin, and malate dehydrogenase 1
were used for the adult tissue.
Histology
Tissue sections were prepared as previously published (1)
and were stained using hematoxylin and eosin and for UCP1.
The number of adipocytes was counted in randomly positioned
grids using a counting frame area of 62,500 mm
2
. The Schaffer
method was used, and ;120 adipocytes were examined, with
the coefficient of variation ,2%. Adipocyte size was measured
using the nucleator method (26, 27), and cell area was calculated by
using orthogonal lines originating from the midpoint of the cell,
which was taken as the center of the lipid droplet within complete
adipocytes. For UCP1 immunohistochemistry, adjacent sections
were collected. Sections were deparaffinized, and endogenous
peroxide activity was blocked with 0.3% hydrogen peroxide in
methanol. Sections were then washed and blocking serum (normal
goat serum in 0.1 M phosphate-buffered saline) was added;
rewashed; and incubated with primary antibody (1:100 rabbit
anti-UCP1) for 24 hours at room temperature. Slides were
then washed and incubated for 1 hour with secondary antibody
(1:200 biotinylated anti-rabbit antibody; Antibodies Australia,
Melbourne, Australia). Immunostaining was revealed using 3,30-
diaminobenzidine color reagent. Rat BAT was used as a positive
control (primary antibody 1:1000), and staining without primary
antibody was used to determine staining specificity.
Mitochondrial content and immunoblotting
The relative abundance of UCP1 and the total mitochondrial
protein content were determined in the postnatal samples as
previously described (28). In the adult samples, the relative
abundance of UCP1, UCP3, SERCA1, and SERCA2a was de-
termined using antibodies as previously published (20, 29, 30)
and is summarized in Table 2. All data were corrected against
the density of staining for total protein. Each antibody gave a
signal at the correct molecular weight (see Supplemental Fig. 1),
and the specificity of binding for each antibody was confirmed
using nonimmune rabbit serum.
Plasma metabolite and hormone analyses
Plasma glucose and lactate were analyzed using an auto-
analyzer (YSI, Inc., Yellow Springs, OH), and nonesterified
fatty acid (NEFA) enzymatically (31). Plasma insulin (32) and
irisin (33) were analyzed by enzyme-linked immunosorbent
assay (Kit no. EK-067-29; Phoenix Pharmaceuticals, Inc.,
Burlingame, CA) in single assays.
Statistical analyses
Differences in gene expression and protein abundance be-
tween depots with age and/or different body weights were
analyzed using Kruskal-Wallis nonparametric tests with Bon-
ferroni correction for multiple analyses. In study 2, longitudinal
data for temperature and plasma analysis were analyzed by
repeated-measures analysis of variance. Differences in the
temperature response to feeding and in adipocyte size were
analyzed by one-way analysis of variance using Fisher’s least
significant differences for post hoc analyses.
Results
Changes in UCP1 and gene expression profile
between depots during early development
As expected, the abundance of the UCP1 gene and a key
regulator of BAT function, DIO2, was highest in both fat
depots examined at 1 day of age and then declined (Fig. 1).
This adaptation in gene expression occurred as white ad-
ipose tissue massincreased substantially, with growth up to
28 days of age being greater in terms of relative body
weight in the retroperitoneal depot (sternal 4.7 60.5 g/kg;
retroperitoneal 9.6 61.2 g/kg; P,0.05). At 1 day of age,
the total mitochondrial protein content was greater in
sternal fat than in retroperitoneal fat, which resulted in the
total amount of UCP1 protein being higher in the sternal
depot (sternal 1.4 60.5 arbitrary units (au) per depot;
retroperitoneal 0.3 60.1 au per depot; P,0.05).
However, the rate of decline in UCP1 was greater between
1 and 7 days of age in the sternal depot (i.e., 7 days: sternal
0.7 60.1 au per depot; retroperitoneal 0.5 60.1auper
depot). UCP1 protein was undetectable in muscle at any
time point.
2214 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
Very low amounts of UCP1 and DIO2 mRNA were
detected in skeletal muscle (Fig. 1). As each fat depot lost
UCP1, there was a parallel decrease in gene expression for
PDK4 [which is present in murine BAT (34)], a finding that
was also apparent in muscle. In contrast, the gene that
encodes for irisin (i.e., FNDC5) was highly abundant in all
three tissues examined, with a clear peak in muscle at 7 days of
age. Although there were no changes in FNDC5 expression
with age in retroperitoneal adipose tissue, it decreased in
sternal fat. No change in plasma irisin concentration was
Table 2. Antibodies Used
Peptide/
Protein
Target
Antigen
Sequence
(if known)
Name of
Antibody
Manufacturer, Catalog No.,
or Name of Source
Species Raised
in Monoclonal
or Polyclonal Dilution Used
Research
Resource
Identifier
UCP1 Anti-UCP1 Antibodies Australia Rabbit 0.1111111111 AB_2304253
UCP3 RALMKVQVLRESPF Anti-UCP3 Abcam Rabbit 0.7361111111 Ab34677
SERCA1 CaF2-5D2 Developmental Studies Hybridoma Bank Mouse 0.7361111111 AB_531812
SERCA2a CaS/C1 Developmental Studies Hybridoma Bank Mouse 3.5138888889 AB_2061452
Table 1. Summary of Specific Ovine Sequence of Forward and Reverse Oligonucleotide Primers Used for
Real-Time PCR
Gene Accession Number Forward Primer Reverse Primer
Amplicon
Length
(bp)
ACSM5 XM_015469322.1 CCACCATATGATGTGCAGGT TGTCTTCTCAGGGTTGTCCA 138
ADIPOQ NM_174742.2 ATCAAACTCTGGAACCTCCTATCTAC TTGCATTGCAGGCTCAAG 232
ADIRF NM_001114513.2 CCACAGAAGCAGGGCAGA AAACCCGAGAAAGCCTCA 100
ATF2 XM_004004570.1 TCCCACTTGTTCGACCAGTCA TTGACAGTATCGCCGTTGGT 151
C/EBPaXM_004015623.1 CTGGAGCTGACCAGTGACA GGGCAGCTGACGGAAGAT 96
C/EBPbNM_176788.1 ACGACTTCCTCTCCGACCTC CCCAGACTCACGTAGCCGTA 85
CIDEA NM_001083449.1 AAGGCCACCATGTACGAGAT GGTGCCCATGTGGATAAGACA 138
CPT1b NM_001034349.2 TGATCACGTATCGCCGTAAA GAGCACATCTGTGTCCTTCC 137
DIO2 NM_001010992.3 AGCCGCTCCAAGTCCACTC TTCCACTGGTGTCACCTCCT 175
En-1 XM_003581845.4 AACCCGGCCATACTGCTAAT TTCTTCTTCAGCTTCCTGGTG 152
Eva-1 XM_004016067.3 GGAATTTCCGTCCTCGAGAT AGGATGGAGACGTCATACCG 139
FABP4 NM_174314.2 TGAAATCACTCCAGATGACAGG TGGTGGTTGATTTTCCATCC 98
GPR120 XM_002698388.1 CCTGGGACGTGTCATTTGCTA CTGGTGGCTCTCCGAGTAGG 140
HOXC8 XM_002704245.5 TGTAAATCCTCCGCCAACAC TGATACCGGCTGTAAGTTTGC 140
HOXC9 XM_002704244.2 GACCTGGACCCCAGCAAC GCTCGGTGAGGTTGAGAAC 175
INSR XM_002688832.3 CTGCACCATCATCAACGGAA CGTAACTTCCGGAAGAAGGA 162
LEP NM_173928.2 CCAGGATGACACCAAAACC TGGACAAACTCAGGAGAGG 140
LHX8 XM_004003563.1 AGAGCACGCCACAAGAAACA AGGGCTGGAGTCCAAGAGTT 199
NR3C1 NM_001206634.1 ACTGCCCCAAGTGAAAACAGA ATGAACAGAAATGGCAGACATTTTATT 151
PGC1aNM_177945.3 GATTGGCGTCATTCAGGAGC CCAGAGCAGCACACTCGAT 84
PPARgNM_181024.2 GACCCGATGGTTGCAGATTA TGAGGGAGTTGGAAGGCTCT 145
PPARgNM_181024.2 GACCCGATGGTTGCAGATTA TGAGGGAGTTGGAAGGCTCT 145
PRDM16 XM_003583245.1 TGGCAGCTGGCTCAAGTACA CGGAACGTGGGCTCCTCATC 198
PRLR NM_174155.3 CTCCACCCACCATGACTGAT CAGCGAATCTGCACAAGGTA 169
RIP140 XM_002684642.2 CGAGGACTTGAAACCAGAGC TCTTAGGGACCATGCAAAGG 179
RyR1 NM_001206777 GGGATATGGGTGACACGAC TCTCAGCATCAGCTTTCTCC 158
Serca 1a XM_004020863.3 GCTGCTGTGGGCAATAAGAT GCCAGTACCCCACTCTTTGA 150
Serca 2a XM_012097784.2 CAGGTGTACCCACATTCGAG TTCCCGAATGACAGACATGA 85
SHOX2 NM_001205527.1 CGCCTTTATGCGTGAAGAAC TTGGCTGGCAGCTCCTAT 142
SREBF1 XM_004013336.1 AGGGGGACAAGGAGTTCTCA CTCCGGCCATATCCGAACAG 72
Tbx15 NM_001079775.1 AATGGACATTGTACCTGTGGAC TGACCACCTGTCTCATCCAA 158
TCF21 XM_014480981.1 ATCCTGGCCAACGACAAGTA TCAGGTCACTCTCGGGTTTC 94
UCP1 XM_003587124.1 GGGCTTTGGAAAGGGACTACT CAGGGCACATCGTCTGCTAAT 128
UCP2 NM_001033611 AAGGCCCACCTAATGACAGA CCCAGGGCAGAGTTCATGT 128
UCP3 NM_001308581.1 ACCTGCTCACCGACAACTTC CATATACCGCGTCTTCACCA 107
b
2
-Microglobulin AY549962 CCAGAAGATGGAAAGCCAAA CAGGTCTGACTGCTCCGATT 117
b-Actin U39357 GCAAAGACCTCTACGCCAAC TGATCTTGATCTTCATCGTGCT 120
Cyclophilin JX534530 GCATACAGGTCCTGGCATCT CATGCCCTCTTTCACTTTGC 136
IPO8 NM_001206120.1 GCCCTTGCTCTTCAGTCATT GTGCAACAGCTCCTGCATAA 93
KDM2B XM_004017579.1 CGGTCCTACCTCACTCAGGA CCGTCTATGCTGGGCTTTCT 74
Malate dehydrogenase 1 AF233351 CGTTGCAGAGCTGAAGGATT GGTGCACTGAGAGATCAAGG 100
RPLP0 NM_001012682.1 CAACCCTGAAGTGCTTGACAT AGGCAGATGGATCAGCCA 227
TBP NM_001075742.1 CTTGGACTTCAAGATTCAGAACA CCAGGAAATAACTCTGGCTCA 120
YWHAZ NM_174814.2 CCGGACACAGAACATCCAGTC TCAGCTCCTTGCTCAGTTACAG 125
Shaded rows at the bottom of the table designate housekeeping genes.
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2215
observed with age (7 days: 117 67ng/mL;28days1296
11 ng/mL). The lipid droplet protein CIDEA exhibited
high transcript expression in both fat depots and did not
change with age, whereas expression was very low in
muscle. RIP140 was equally abundant in both fat and
muscle, and although it showed a clear rise at 28 days in
Figure 1. Summary of the changes in gene expression for putative markers of brown, beige, or white adipose tissue or skeletal muscle in the
sternal and retroperitoneal fat depots and hind limb muscle over the first 28 days of life in young sheep. Values are means with their standard
errors; n = 4 to 6 per age group. Significant differences between age groups are indicated by *P,0.05.
2216 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
both fat depots, there were no significant age-related
changes in muscle.
To establish a clearer overview of the differences in
relative gene expression of a range of markers previously
considered to be indicative of brown, beige, and white ad-
ipose tissue or skeletal muscle, a more exhaustive analysis
was undertaken on the samples obtained at 7 days of age
(Fig. 2). This demonstrated that other BAT-related genes
(e.g., Eva1) were highly expressed in both fat depots, as were
those genes primarily involved in either lipid metabolism
(i.e., FABP4) or adipogenesis (i.e., PPARgand CEBPaand
b), whereas ADIRF was more abundant in sternal fat than in
retroperitoneal fat. None of these genes were present in
muscle. Other genes thought to potentially regulate adipose
development, such as HOXC8 and HOXC9,andthe
“white fat marker”gene TCF21 were also highly expressed
in retroperitoneal adipose tissue but not in muscle or, more
surprisingly, in sternal adipose tissue. mRNA for En-1 was
highly abundant in sternal fat but not in the other two
tissues. In contrast, SHOX2 mRNA was abundant in sternal
fat and muscle but was barely detectable in the retroperi-
toneal depot. Specific muscle marker genes CPT1b and
Tbx15, which have been reported to be expressed during the
differentiation or induction of BAT (35, 36), were highly
expressed in muscle but minimally expressed in fat. Finally,
ACSM5 mRNA was more abundant in muscle than in
retroperitoneal fat but was hardly detectable in sternal fat.
To further understand the development of sternal fat,
the expressions of additional genes were examined
(Table 3) and divided into four categories: developmental
genes previously considered to be markers of brown or
beige fat and those that regulate thermogenesis, meta-
bolism, or adipogenesis. For thermogenic genes, we
observed a reduction in PRLR and PGC1aexpression
between 7 and 28 days, whereas ATF2 expression in-
creased. Expressions of the beige/white marker gene
HOXC9 and the classic BAT marker gene LHX8 both
rose substantially at each age, whereas expression of the
BAT fate-determining gene PRDM16 showed a small
decrease by 28 days and the beige marker gene SHOX2
was unchanged. Surprisingly, changes in the expression
profiles of adipogenic genes varied. The mRNA abun-
dance of both PPARgand NR3C1 increased, whereas
that of CEBPatransiently increased at 7 days of age and
that of SREBF1 declined. A majority of other metabolic
genes (i.e. adiponectin, leptin, and GPR120) also showed
increased expression with age. However, mRNA abun-
dance for FABP4 and the INSR was unchanged.
Figure 2. Summary heat map comparison of quantitative PCR gene expression between sternal and retroperitoneal adipose tissue and skeletal
muscle from six individual sheep sampled at 7 days of age. All normalized data were made relative to the highest expressing sample and are
given in arbitrary units between 0 and 1. Each column contains data from an individual animal for the three tissues examined, and each row
contains data for a specific gene. Red squares represent the highest expression (1) and blue squares the lowest (0).
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2217
Differences in tissue temperature and metabolites in
response to feeding and altered fat mass in adults
In adult sheep, the temperature of retroperitoneal fat
was consistently higher than that of sternal fat and
skeletal muscle (Fig. 3), which might reflect its deep-body
location. Nevertheless, retroperitoneal fat and skeletal
muscle displayed comparable temperature responses to
feeding. The greatest feeding-induced temperature rise
was in sternal fat (Fig. 4). There was no effect of increased
fat mass on the temperature of the three tissues studied
(Figs. 3 and 4). Although low body weight/adiposity was
associated with reduced temperature of both adipose
tissue and skeletal muscle, this effect was less pronounced
in retroperitoneal fat (Fig. 3). Plasma glucose and insulin
levels were lower in the lean animals, but there was little
effect of altered adiposity on plasma NEFA and lactate
levels (Fig. 5).
Differences in gene profile with altered fat mass
in adults
The abundance of UCP1 mRNA was very low in all
adult tissues, and UCP1 was consistently detectable by
immunohistochemistry only in sternal fat (Fig. 6). UCP3
was highly abundant in skeletal muscle but was very low
in sternal and retroperitoneal fat, being expressed
100-fold more in muscle than in fat (i.e., skeletal muscle
561 au; adipose tissue 0.05 60.1 au). Gene expression
for UCP3 was increased in sternal adipose tissue in lean
animals compared with obese animals (lean 18 64 au;
obese 0.3 60.2 au; P,0.05), whereas protein abun-
dance was reduced (Table 4). In contrast, neither UCP1
mRNA nor protein were altered by changes in adiposity
in either fat depot (data not shown). UCP2 mRNA was
lower in sternal and retroperitoneal fat of lean animals
than in control animals (i.e., retroperitoneal: lean 1.0 6
0.3 au; control 3.3 60.5 au; P,0.05). There were also
no effects of body weight on UCP3,RyR1, or SERCA2a
in skeletal muscle. Expression of SERCA1 mRNA was
lower in skeletal muscle of lean animals, but protein
concentrations were again unaffected by fat mass. Fi-
nally, as expected, adipocyte cell size was influenced by
altered body weight and adiposity. In retroperitoneal fat,
adipocyte size changed in proportion to increased body
weight. On the other hand, adipocyte size was decreased
in the sternal fat of lean animals, but there was no effect of
obesity on adipocyte size in this depot (Fig. 6).
Table 3. Changes in Gene Expression of Putative Markers of Thermogenic, Developmental, Adipogenic, and
Metabolic Pathways in Sternal Adipose Tissue Over the First Month of Postnatal Life in Sheep
Animal Age
1 Day 7 Days 28 Days
Thermogenesis
PRLR 400 680
a
390 650
b
100 610
a,b
PGC1a11,270 61970
a
10,800 61540
c
2220 6650
a,c
ATF2 4770 6280
d
4300 6180
b
7090 6510
b,d
CIDEA 304 656 400 630 310 670
Development
HOXC9 65 610
a
110 610 190 630
a
LHX8 10 61
d,e
120 640
d
210 670e
PRDM16 120 640
d
80 610 60 610
d
SHOX2 1190 6420 990 690 1420 6210
Adipogenesis
PPARg21,810 6440
a
34,670 61890 46,480 66110
a
C/EBPa12,300 63070
a
27,130 61920
a,c
12,820 61180
c
NR3C1 17,820 62590
d
15,870 668
b
26,450 6910
b,d
SREBF1 8850 62860
a
4440 6270
d
3350 6260
a,d
Metabolism
LEP 110 640
b,d
1970 6470
d
6100 61200
b
ADIPOQ (310
3
)130 630
a,d
370 630
d
530 680
a
FABP4 (310
3
)1450 6390 1730 6110 2090 6300
GPR120 10 65
b
60 620
a
560 6150
a,b
INSR 5150 61960 7240 6860 5290 6580
Values are mean copy number with their standard errors; n = 5 or 6 per group.
Significant differences with age indicated by similar superscripts:
a
P,0.01.
b
P,0.001.
c
P,0.01.
d
P,0.05.
e
P,0.05.
2218 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
Discussion
We have shown that during postnatal development in
sheep, the sternal and retroperitoneal fat depots exhibit
contrasting gene expression profiles that could be
indicative of divergent prenatal origins. These differ-
ences potentially contribute to the enhanced temper-
ature responses seen in sternal fat compared with
Figure 3. Summary of the effect of altered body weight and fat mass on changes in the temperature of sternal and retroperitoneal (RP) adipose
tissue and skeletal muscle as measured continuously over a 24-hour period. Black arrow indicates when food was first made available. Gray
boxes indicate the time during which food was available. Obese animals are shown as white squares, controls as black squares, and lean animals
as black triangles. Values are means with standard errors of the mean, and n = 4 or 5 per age group. **P,0.01 lean compared with obese and
control;
a
P,0.05 lean compared with obese.
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2219
retroperitoneal fat and skeletal muscle following
feeding in adulthood. Although the abundance of
UCP1 within the sternal depot declines with increased
fat mass during both postnatal and adult life, it does
not appear to compromise the ability of this depot
to increase its temperature in response to feeding in
adulthood. Furthermore, adipocyte cell size in the
sternal fat depot appears unresponsive to increased
adiposity, suggesting that it serves a function other
than storing surplus lipids during nutrient excess
and obesity.
Divergent patterns of development between fat and
muscle in early life
It is becoming apparent that identifying functional
markers in BAT and/or beige adipocytes is a complex
process that is influenced by the depot and whether in
vivo or in vitro methodologies are used (17, 37, 38). As
Figure 4. Summary of the effect of altered body weight and fat mass on changes in postprandial rise in tissue temperature of sternal and RP
adipose tissue and skeletal muscle as measured after feeding. This was calculated as the amplitude of the temperature change that occurred
within the feeding window (1100 hours to 1600 hours). Values are means with standard errors of the mean, and n = 4 or 5 per group.
Significant differences between depots for each body weight group of sheep are indicted by *P,0.05; **P,0.01 sternal fat compared with
muscle and RP fat.
Figure 5. (a–d) Summary of the effect of increased body weight and fat mass on changes in plasma metabolite and insulin levels with feeding
(represented by shaded regions). Values are means with their standard errors, and n = 4 or 5 per group. Significant differences between depots
for each body weight group of sheep are indicted by **P,0.01; ***P,0.001 lean compared with control and obese.
2220 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
we have shown previously in ovine retroperitoneal fat,
there are at least three distinct phases of postnatal de-
velopment (1). The major functionally related changes
are seen between 1 and 28 days of postnatal age, co-
incident with the loss of UCP1 and the transition of
brown to white adipose tissue (1). In the current study, we
confirmed that this critical stage of development extends
to both sternal fat and muscle and is coincident with rapid
growth and functional changes within
each tissue (39, 40). These findings are
in accord with those recently described
within epicardial fat during develop-
ment of humans who have undergone
heart surgery (41). Consideration of
each gene or group of genes examined,
the accepted function of each, and
the known developmental ontogeny
in other species is presented in the
following sections, thereby providing
insights into the pronounced differ-
ences in the molecular signatures of fat
and muscle.
The most notable characteristic of
sternal fat compared with both retro-
peritoneal fat and muscle was the
very high abundance of En-1 mRNA.
Lineage-tracing studies in mice indicate
that cells showing early expression of
En-1 during development give rise to
dermis and epaxial muscle, but not to
other muscles, and interscapular BAT
“bundles”(42), which is in accordance
with our findings postnatally. Ana-
tomical location during early devel-
opment is determined along three axes:
anterior-posterior, proximal-distal,
and dorsal-ventral (43), but the pri-
mary regulators are not fully eluci-
dated, with a variety of gene families
such as Wnt (44, 45), HOX (46), and
Pax playing roles. HOX genes are
important regulators of development
(47, 48), for which expression of spe-
cific combinations of the paralogous
HOX gene sets specify a particular
anatomical location along the anterior-
posterior axis. Our data show that
HOXC8 and HOXC9 are highly
expressed in the more posteriorly lo-
cated retroperitoneal depot but not in
the more anterior sternal depot and
that this pattern of gene expression
persists to 28 days. In mice, HOXC8
and HOXC9 gene expression is higher in retroperitoneal
than in interscapular adipose tissue (49).
TCF21 was originally proposed as a marker for white
preadipocytes (50), but its gene expression differs be-
tween fat depots (51, 52). Therefore, our finding that
TCF21 mRNA was abundant in only retroperitoneal fat
supports the hypothesis that tissue-specific patterns of
TCF21 gene expression are indicative of fundamental
Figure 6. The effects of body weight on adipocyte size and histological appearance in
sternal and RP fat of adult sheep. (a) Mean adipocyte cell size with sternal fat represented by
black bars and RP fat by white bars. (b–g) Representative photomicrographs of hematoxylin
and eosin–stained sections. (h) Example of UCP1 immunostaining from sternal adipose tissue.
(i) Rat BAT was used as the positive control, and (j) staining specificity was determined with
rat BAT in the absence of primary antibody. Scale bar represents 50 mm. Values are means
with their standard errors, and n = 4 or 5 per group. -ve control indicates the negative
control. Significant differences between depots for each body weight group of sheep are
indicted by *P,0.05 compared with sternal fat;
a
P,0.01 compared with obese animals
(within a fat depot).
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2221
differences between depots that are dependent on ana-
tomical location. Further indirect evidence of depot-
specific rates of development comes from examining
SHOX2. Its pattern of gene expression was opposite to
that of the HOXC genes measured, being expressed in
both muscle and sternal fat, which is anteriorly located to
the retroperitoneal depot, where there is little if any
detectable expression. SHOX2 is able to interact with
CEBPato modulate ADRB3 and, by extension, to reg-
ulate lipolysis in adipose tissue (53). In addition, ablation
of SHOX2 promotes lipolysis in mice (53). The low
expression of this gene within retroperitoneal adipose
tissue could therefore be indicative of a more rapid
mobilization of NEFAs as well as a capacity for greater
growth through adulthood than in sternal fat. Differences
in metabolic capacity with respect to medium-chain fatty
acid synthesis between tissues could also explain the
much higher mRNA abundance of ACSM5 in retroper-
itoneal adipose tissue than in sternal adipose tissue.
ACSM5 is also considered a characteristic of white fat
rather than BAT (34), and its paucity reflects the retention
of UCP1 within sternal fat. Our finding of greater gene
expression for ACSM5 within muscle contrasts with
findings in adult rodents (54) and could be indicative of
the significant changes seen within muscle during early
postnatal development. In sheep, this is coincident with
the recruitment of shivering thermogenesis (40), which
could be accompanied by the increased utilization of
intramuscular fat as suggested in pigs (55). Gene ex-
pression for FNDC5 also peaked at 7 days in muscle,
coincident with maximal recruitment of shivering ther-
mogenesis as UCP1 declined (40). There was no parallel
change in plasma concentrations of irisin at this stage.
This was not entirely unexpected given the current
controversy regarding the measurement of irisin (56) and
its potential functionality or existence (57).
Some changes in gene expression with age were similar
in both muscle and fat depots (e.g., PDK4), which may
reflect the overall decline in basal metabolic rate (40), loss
of UCP1, and pronounced fat deposition up to 1 month of
age (39). At the same time, there is a transition from lipid
to glucose metabolism (58) that would be facilitated by a
decline in PDK4 activity (59), whereas increases in leptin,
adiponectin, RIP140, and GPR120 gene expression are
indicative of increased adiposity. In adults, however, fat
mass and plasma adiponectin and its gene expression are
normally negatively correlated (60). A different type of
relationship during early life, coincident with the rapid
growth of fat, is not unexpected, as has been seen for
plasma leptin and the loss of its positive correlation with
fat mass (61). At the same time, both LHX8 and ATF2
gene expression increased with age and fat mass, which
was not expected given their putative “BAT identity
marker roles,”as described by others in ovine retro-
peritoneal adipose tissue with development (62). As
suggested by rodent studies, both ATF2 and GRP120
may therefore have greater roles in stimulating adipo-
genesis than thermogenesis (63, 64), whereas raised
RIP140 would facilitate the loss of UCP1 (65). In sum-
mary, sternal fat development shares some characteristics
with skeletal muscle that may also affect the retention of
UCP1 and its thermogenic capacity in adulthood.
Functional consequences of UCP1 in sternal fat
The contribution of BAT to diet-induced thermo-
genesis in rodents remains contentious (66), although it
does appear to have a role in young sheep (58), children
(67), and adults (7). There is good evidence from de-
velopmental studies in both rodents and young sheep that
muscle is recruited to generate heat when UCP1 is absent
(68) and/or nonshivering thermogenesis is compromised
(40). In adult sheep, temperature excursions in skeletal
muscle in response to feeding and central infusion of
leptin are consistent with increased thermogenesis (24,
29). The basal temperature of muscle also appears to be
more sensitive to total fat mass than that of either fat
depot studied. This could reflect its anatomical position
and/or the impact of an increase in the surrounding fat
and its insulating properties. It should also be noted that
in large mammals, such as sheep, the thermogenic re-
sponse to feeding is an entrained response (20), whereas
in rodents, there is an influence of circadian rhythm (69).
Furthermore, rodents are normally active in the dark
phase, and the sensitivity of UCP1 to further stimulation
is modulated by these diurnal activity patterns (70). These
Table 4. Effects of Altered Adult Body Weight on
Protein Abundance of UCP1 and UCP3 in Fat and
Skeletal Muscle and of Potential Thermogenic
Proteins (i.e., SERCA) in Muscle
Lean Control Obese
Skeletal muscle
UCP3 0.63 60.30 1.0 60.26 1.00 60.25
SERCA1 1.26 60.16 1.0 60.14 1.06 60.17
SERCA2a 1.18 60.11 1.0 60.08 1.06 60.10
Sternal adipose tissue
UCP1 1.22 60.16 1.0 60.18 0.99 60.20
UCP3 0.68 60.17
a
1.0 60.08 1.35 60.10
b
Retroperitoneal adipose tissue
UCP1 1.05 60.16 1.0 60.09 1.04 60.10
UCP3 0.73 60.08 1.0 60.16 1.02 60.10
All results expressed in arbitrary units relative to controls and corrected
against the density of staining for total protein.
Abbreviation: SERCA, sarcoplasmic reticulum calcium-transporting
ATPase.
Significant differences between body weight groups indicated by dif-
ferent superscript letters:
a
vs
b
,P,0.01.
2222 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
are dependent in part on both light exposure and activity
of the sympathetic nervous system (71). A range of other
factors may be critical in determining the thermogenic
role of muscle in rodents, including sarcolipin (72) and
UCP3 (73). In adult sheep, gene expression of UCP3 is
increased in skeletal muscle after central infusion of leptin
and is associated with increased heat production and a
switch toward uncoupled respiration in isolated mito-
chondria (24). In addition, increased expression of RyR1
mRNA and SERCA2a protein in skeletal muscle co-
incides with dietary-induced thermogenesis (29). How-
ever, we found no difference in protein abundance for
either SERCA1 or 2a or UCP3 in the muscle of animals of
differing body weights.
In contrast to the acute effects of feeding, prolonged
food restriction caused a marked decrease in temperature
in skeletal muscle and sternal adipose tissue, an effect
attenuated in the retroperitoneal fat. Consistent with
decreased temperature in muscle, UCP3 gene expression
declined markedly in the skeletal muscle of lean animals,
but there was no associated change in protein. On the
other hand, altered adiposity had no effect on UCP1 gene
or protein abundance in sternal fat, but UCP3 mRNA
was reduced in the lean group. Because of the much larger
mass of muscle than BAT, its contribution to metabolic
homeostasis is appreciably greater in sheep and humans
(74), especially when plasma glucose concentrations are
raised. Notably, glucose concentrations were consider-
ably lower in the lean group in the current study. The
reduced temperature in both skeletal muscle and sternal
adipose tissue of lean animals is indicative of homeostatic
reduction in thermogenesis in response to chronic food
restriction and weight loss. This may be a mechanism to
reduce energy expenditure in order to maintain body
weight in states of negative energy balance and/or in the
lean condition and may be mediated by lower thyroid
hormone secretion (75).
In summary, sternal and retroperitoneal fat depots
have distinct developmental profiles that are different
from those seen in muscle. The different developmental
profiles are associated not only with early adipose growth
but also with thermogenesis in these tissues later in life.
The extent to which sternal fat expansion and particu-
larly UCP1 abundance can be modulated in early life may
inform new strategies to manipulate energy balance,
especially following feeding or in response to chronic
food restriction during adulthood.
Acknowledgments
The authors thank Dr. Robyn Murphy (La Trobe University),
who gifted the SERCA antibodies. In addition, we thank Bruce
Doughton, Lynda Morrish, and Elaine Chase for animal
husbandry and assistance with animal work. We thank Professor
Matthew Watt and Dr. Ruth Meex (Monash University) for
advice and guidance with regard to Western blotting.
Address all correspondence and requests for reprints to:
Michael E. Symonds, PhD, Academic Division of Child Health
Obstetrics& Gynaecology, Schoolof Medicine, Queen’s Medical
Centre, The University of Nottingham, Nottingham NG7 2UH,
UK. E-mail: michael.symonds@nottingham.ac.uk.
This work was supported by National Health and Medical
Research Council Project Grant 1005935 (B.A.H).
Disclosure Summary: The authors have nothing to disclose
References
1. Pope M, Budge H, Symonds ME. The developmental transition of
ovine adipose tissue through early life. Acta Physiol (Oxf). 2014;
210(1):20–30.
2. Lean MEJ. Brown adipose tissue in humans. Proc Nutr Soc. 1989;
48(2):243–257.
3. Symonds ME, Pope M, Sharkey D, Budge H. Adipose tissue and
fetal programming. Diabetologia. 2012;55(6):1597–1606.
4. Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi
T, Taittonen M, Laine J, Savisto NJ, Enerb ¨ack S, Nuutila P.
Functional brown adipose tissue in healthy adults. N Engl J Med.
2009;360(15):1518–1525.
5. Vergnes L, Davies GR, Lin JY, Yeh MW, Livhits MJ, Harari A,
Symonds ME, Sacks HS, Reue K. Adipocyte browning and higher
mitochondrial function in periadrenal but not SC fat in pheo-
chromocytoma. J Clin Endocrinol Metab. 2016;101(11):
4440–4448.
6. van Marken Lichtenbelt WD, Vanhommerig JW, Smulders NM,
Drossaerts JM, Kemerink GJ, Bouvy ND, Schrauwen P, Teule GJ.
Cold-activated brown adipose tissue in healthy men. N Engl J Med.
2009;360(15):1500–1508.
7. Scotney H, Symonds ME, Law J, Budge H, Sharkey D, Man-
olopoulos KN. Glucocorticoids modulate human brown adipose
tissue thermogenesis in vivo. Metabolism. 2017;70:125–132.
8. Seale P, Bjork B, Yang W, Kajimura S, Chin S, Kuang S, Scim `eA,
Devarakonda S, Conroe HM, Erdjument-Bromage H, Tempst P,
Rudnicki MA, Beier DR, Spiegelman BM. PRDM16 controls a brown
fat/skeletal muscle switch. Nature. 2008;454(7207):961–967.
9. Shan T, Liang X, Bi P, Zhang P, Liu W, Kuang S. Distinct pop-
ulations of adipogenic and myogenic Myf5-lineage progenitors in
white adipose tissues. J Lipid Res. 2013;54(8):2214–2224.
10. Sanchez-Gurmaches J, Hung CM, Sparks CA, Tang Y, Li H,
Guertin DA. PTEN loss in the Myf5 lineage redistributes body fat
and reveals subsets of white adipocytes that arise from Myf5
precursors. Cell Metab. 2012;16(3):348–362.
11. Sanchez-Gurmaches J, Guertin DA. Adipocytes arise from multiple
lineages that are heterogeneously and dynamically distributed. Nat
Commun. 2014;5:4099.
12. Petrovic N, Walden TB, Shabalina IG, Timmons JA, Cannon B,
Nedergaard J. Chronic peroxisome proliferator-activated receptor
gamma (PPARgamma) activation of epididymally derived white
adipocyte cultures reveals a population of thermogenically com-
petent, UCP1-containing adipocytes molecularly distinct from
classic brown adipocytes. J Biol Chem. 2010;285(10):7153–7164.
13. Harms M, Seale P. Brown and beige fat: development, function and
therapeutic potential. Nat Med. 2013;19(10):1252–1263.
14. Nedergaard J, Cannon B. UCP1 mRNA does not produce heat.
Biochem Biophys Acta. 2013;1831(5):943–949.
15. Nedergaard J, Cannon B. The browning of white adipose tissue:
some burning issues. Cell Metab. 2014;20(3):396–407.
16. Scheele C, Larsen TJ, Nielsen S. Novel nuances of human brown fat.
Adipocyte. 2014;3(1):54–57.
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2223
17. de Jong JM, Larsson O, Cannon B, Nedergaard J. A stringent
validation of mouse adipose tissue identity markers. Am J Physiol
Endocrinol Metab. 2015;308(12):E1085–E1105.
18. Alexander G. Quantitative development of adipose tissue in foetal
sheep. Aust J Biol Sci. 1978;31(5):489–503.
19. Alexander G, Bell AW. Quantity and calculated oxygen con-
sumption during summit metabolism of brown adipose tissue in
new-born lambs. Biol Neonate. 1975;26(3-4):214–220.
20. Henry BA, Dunshea FR, Gould M, Clarke IJ. Profiling postprandial
thermogenesis in muscle and fat of sheep and the central effect of
leptin administration. Endocrinology. 2008;149(4):2019–2026.
21. Bloor ID, S´ebert SP, Saroha V, Gardner DS, Keisler DH, Budge H,
Symonds ME, Mahajan RP. Sex differences in metabolic and ad-
ipose tissue responses to juvenile-onset obesity in sheep. Endocri-
nology. 2013;154(10):3622–3631.
22. Henry BA, Tilbrook AJ, Dunshea FR, Rao A, Blache D, Martin GB,
Clarke IJ. Long-term alterations in adiposity affect the expression of
melanin-concentrating hormone and enkephalin but not proo-
piomelanocortin in the hypothalamus of ovariectomized ewes.
Endocrinology. 2000;141(4):1506–1514.
23. Kurose Y, Iqbal J, Rao A, Murata Y, Hasegawa Y, Terashima Y,
Kojima M, Kangawa K, Clarke IJ. Changes in expression of the
genes for the leptin receptor and the growth hormone-releasing
peptide/ghrelin receptor in the hypothalamic arcuate nucleus
with long-term manipulation of adiposity by dietary means.
J Neuroendocrinol. 2005;17(6):331–340.
24. Henry BA, Andrews ZB, Rao A, Clarke IJ. Central leptin activates
mitochondrial function and increases heat production in skeletal
muscle. Endocrinology. 2011;152(7):2609–2618.
25. Henry BA, Blache D, Rao A, Clarke IJ, Maloney SK. Disparate
effects of feeding on core body and adipose tissue temperatures in
animals selectively bred for nervous or calm temperament. Am J
Physiol Regul Integr Comp Physiol. 2010;299(3):R907–R917.
26. Møller A, Strange P, Gundersen HJG. Efficient estimation of cell
volume and number using the nucleator and the disector. J Microsc.
1990;159(1):61–71.
27. Horvath TL, Erion DM, Elsworth JD, Roth RH, Shulman GI, Andrews
ZB. GPA protects the nigrostriatal dopamine system by enhancing
mitochondrial function. Neurobiol Dis. 2011;43(1):152–162.
28. Symonds ME, Bryant MJ, Clarke L, Darby CJ, Lomax MA. Effect
of maternal cold exposure on brown adipose tissue and thermo-
genesis in the neonatal lamb. J Physiol. 1992;455:487–502.
29. Clarke SD, Lee K, Andrews ZB, Bischof R, Fahri F, Evans RG,
Clarke IJ, Henry BA. Postprandial heat production in skeletal
muscle is associated with altered mitochondrial function and al-
tered futile calcium cycling. Am J Physiol Regul Integr Comp
Physiol. 2012;303(10):R1071–R1079.
30. Lee TK, Clarke IJ, St John J, Young IR, Leury BL, Rao A, Andrews
ZB, Henry BA. High cortisol responses identify propensity for
obesity that is linked to thermogenesis in skeletal muscle. FASEB J.
2014;28(1):35–44.
31. Sechen SJ, Dunshea FR, Bauman DE. Somatotropin in lactating
cows: effect on response to epinephrine and insulin. Am J Physiol.
1990;258(4 Pt 1):E582–E588.
32. Clarke SD, Clarke IJ, Rao A, Cowley MA, Henry BA. Sex dif-
ferences in the metabolic effects of testosterone in sheep. Endo-
crinology. 2012;153(1):123–131.
33. Nygaard H, Slettaløkken G, Vegge G, Hollan I, Whist JE, Strand T,
Rønnestad BR, Ellefsen S. Irisin in blood increases transiently after
single sessions of intense endurance exercise and heavy strength
training. PLoS One. 2015;10(3):e0121367.
34. Forner F, Kumar C, Luber CA, Fromme T, Klingenspor M, Mann
M. Proteome differences between brown and white fat mito-
chondria reveal specialized metabolic functions. Cell Metab. 2009;
10(4):324–335.
35. Gburcik V, Cawthorn WP, Nedergaard J, Timmons JA, Cannon B.
An essential role for Tbx15 in the differentiation of brown and
“brite”but not white adipocytes. Am J Physiol Endocrinol Metab.
2012;303(8):E1053–E1060.
36. Christian M. Transcriptional fingerprinting of “browning”white
fat identifies NRG4 as a novel adipokine. Adipocyte. 2014;4(1):
50–54.
37. Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H,
Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White
AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH.
Clonal analyses and gene profiling identify genetic biomarkers
of the thermogenic potential of human brown and white pre-
adipocytes. Nat Med. 2015;21(7):760–768.
38. Shinoda K, Luijten IH, Hasegawa Y, Hong H, Sonne SB, Kim M,
Xue R, Chondronikola M, Cypess AM, Tseng YH, Nedergaard J,
Sidossis LS, Kajimura S. Genetic and functional characterization of
clonally derived adult human brown adipocytes. Nat Med. 2015;
21(4):389–394.
39. Clarke L, Buss DS, Juniper DT, Lomax MA, Symonds ME. Adipose
tissue development during early postnatal life in ewe-reared lambs.
Exp Physiol. 1997;82(6):1015–1027.
40. Symonds ME, Andrews DC, Johnson P. The control of thermo-
regulation in the developing lamb during slow wave sleep. J Dev
Physiol. 1989;11(5):289–298.
41. Ojha S, Fainberg HP, Wilson V, Pelella G, Castellanos M, May ST,
Lotto AA, Sacks H, Symonds ME, Budge H. Gene pathway de-
velopment in human epicardial adipose tissue during early life. JCI
Insight. 2016;1(13):e87460.
42. Atit R, Sgaier SK, Mohamed OA, Taketo MM, Dufort D, Joyner
AL, Niswander L, Conlon RA. Beta-catenin activation is necessary
and sufficient to specify the dorsal dermal fate in the mouse. Dev
Biol. 2006;296(1):164–176.
43. Loomis CA, Harris E, Michaud J, Wurst W, Hanks M, Joyner AL.
The mouse Engrailed-1 gene and ventral limb patterning. Nature.
1996;382(6589):360–363.
44. Moldes M, Zuo Y, Morrison RF, Silva D, Park BH, Liu J, Farmer
SR. Peroxisome-proliferator-activated receptor gamma suppresses
Wnt/beta-catenin signalling during adipogenesis. Biochem J. 2003;
376(3):607–613.
45. Christodoulides C, Lagathu C, Sethi JK, Vidal-Puig A. Adipo-
genesis and WNT signalling. Trends Endocrinol Metab. 2009;
20(1):16–24.
46. Cantile M, Procino A, D’Armiento M, Cindolo L, Cillo C. HOX
gene network is involved in the transcriptional regulation of in vivo
human adipogenesis. J Cell Physiol. 2003;194(2):225–236.
47. Hunt P, Krumlauf R. Deciphering the Hox code: clues to patterning
branchial regions of the head. Cell. 1991;66(6):1075–1078.
48. Wellik DM. Hox patterning of the vertebrate axial skeleton. Dev
Dyn. 2007;236(9):2454–2463.
49. Yamamoto Y, Gesta S, Lee KY, Tran TT, Saadatirad P, Kahn CR.
Adipose depots possess unique developmental gene signatures.
Obesity (Silver Spring). 2010;18(5):872–878.
50. Timmons JA, Wennmalm K, Larsson O, Walden TB, Lassmann T,
Petrovic N, Hamilton DL, Gimeno RE, Wahlestedt C, Baar K,
Nedergaard J, Cannon B. Myogenic gene expression signature
establishes that brown and white adipocytes originate from distinct
cell lineages. Proc Natl Acad Sci USA. 2007;104(11):4401–4406.
51. Macotela Y, Emanuelli B, Mori MA, Gesta S, Schulz TJ, Tseng YH,
Kahn CR. Intrinsic differences in adipocyte precursor cells from
different white fat depots. Diabetes. 2012;61(7):1691–1699.
52. Sacks HS, Fain JN, Bahouth SW, Ojha S, Frontini A, Budge H, Cinti
S, Symonds ME. Adult epicardial fat exhibits beige features. J Clin
Endocrinol Metab. 2013;98(9):E1448–E1455.
53. Lee KY, Yamamoto Y, Boucher J, Winnay JN, Gesta S, Cobb J,
Bl ¨uher M, Kahn CR. Shox2 is a molecular determinant of depot-
specific adipocyte function [published correction appears in Proc
Natl Acad Sci USA. 2016;113(16):E2347]. Proc Natl Acad Sci
USA. 2013;110(28):11409–11414.
54. Ellis JM, Bowman CE, Wolfgang MJ. Metabolic and tissue-specific
regulation of acyl-CoA metabolism. PLoS One. 2015;10(3):
e0116587.
55. Ramayo-Caldas Y, Ballester M, Fortes MR, Esteve-Codina A,
Castell ´o A, Noguera JL, Fern ´andez AI, P ´erez-Enciso M, Reverter A,
2224 Henry et al Ontogeny and Thermogenic Role for Sternal Fat Endocrinology, July 2017, 158(7):2212–2225
Folch JM. From SNP co-association to RNA co-expression: novel
insights into gene networks for intramuscular fatty acid compo-
sition in porcine. BMC Genomics. 2014;15:232.
56. Elsen M, Raschke S, Eckel J. Browning of white fat: does irisin
play a role in humans? J Endocrinol. 2014;222(1):R25–R38.
57. Albrecht E, Norheim F, Thiede B, Holen T, Ohashi T, Schering L,
Lee S, Brenmoehl J, Thomas S, Drevon CA, Erickson HP, Maak S.
Irisin: a myth rather than an exercise-inducible myokine. Sci Rep.
2015;5:8889.
58. Symonds ME, Andrews DC, Johnson P. The endocrine and met-
abolic response to feeding in the developing lamb. J Endocrinol.
1989;123(2):295–302.
59. Barquissau V, Beuzelin D, Pisani DF, Beranger GE, Mairal A,
Montagner A, Roussel B, Tavernier G, Marques MA, Moro C,
Guillou H, Amri EZ, Langin D. White-to-brite conversion in human
adipocytes promotes metabolic reprogramming towards fatty acid
anabolic and catabolic pathways. Mol Metab. 2016;5(5):352–365.
60. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M,
Okamoto Y, Iwahashi H, Kuriyama H, Ouchi N, Maeda K,
Nishida M, Kihara S, Sakai N, Nakajima T, Hasegawa K, Mur-
aguchi M, Ohmoto Y, Nakamura T, Yamashita S, Hanafusa T,
Matsuzawa Y. Plasma concentrations of a novel, adipose-specific
protein, adiponectin, in type 2 diabetic patients. Arterioscler
Thromb Vasc Biol. 2000;20(6):1595–1599.
61. Bispham J, Budge H, Mostyn A, Dandrea J, Clarke L, Keisler DH,
Symonds ME, Stephenson T. Ambient temperature, maternal
dexamethasone, and postnatal ontogeny of leptin in the neonatal
lamb. Pediatr Res. 2002;52(1):85–90.
62. Basse AL, Dixen K, Yadav R, Tygesen MP, Qvortrup K, Kristiansen
K, Quistorff B, Gupta R, Wang J, Hansen JB. Global gene ex-
pression profiling of brown to white adipose tissue transformation
in sheep reveals novel transcriptional components linked to adipose
remodeling. BMC Genomics. 2015;16:215.
63. Cao W, Daniel KW, Robidoux J, Puigserver P, Medvedev AV, Bai
X, Floering LM, Spiegelman BM, Collins S. p38 mitogen-activated
protein kinase is the central regulator of cyclic AMP-dependent
transcription of the brown fat uncoupling protein 1 gene. Mol Cell
Biol. 2004;24(7):3057–3067.
64. Rosell M, Kaforou M, Frontini A, Okolo A, Chan YW, Nikolo-
poulou E, Millership S, Fenech ME, MacIntyre D, Turner JO,
Moore JD, Blackburn E, Gullick WJ, Cinti S, Montana G, Parker
MG, Christian M. Brown and white adipose tissues: intrinsic dif-
ferences in gene expression and response to cold exposure in mice.
Am J Physiol Endocrinol Metab. 2014;306(8):E945–E964.
65. Kiskinis E, Chatzeli L, Curry E, Kaforou M, Frontini A, Cinti S,
Montana G, Parker MG, Christian M. RIP140 represses the
“brown-in-white”adipocyte program including a futile cycle of
triacylglycerol breakdown and synthesis. Mol Endocrinol. 2014;
28(3):344–356.
66. Symonds ME, Pope M, Budge H. The ontogeny of brown adipose
tissue. Annu Rev Nutr. 2015;35:295–320.
67. Symonds ME. Brown adipose tissue growth and development.
Scientifica (Cairo). 2013;2013:305763.
68. Rowland LA, Bal NC, Kozak LP, Periasamy M. Uncoupling protein
1 and sarcolipin are required to maintain optimal thermogenesis,
and loss of both systems compromises survival of mice under cold
stress. J Biol Chem. 2015;290(19):12282–12289.
69. Blessing W, Mohammed M, Ootsuka Y. Heating and eating: brown
adipose tissue thermogenesis precedes food ingestion as part of the
ultradian basic rest-activity cycle in rats. Physiol Behav. 2012;
105(4):966–974.
70. Ootsuka Y, de Menezes RC, Zaretsky DV, Alimoradian A, Hunt J,
Stefanidis A, Oldfield BJ, Blessing WW. Brown adipose tissue
thermogenesis heats brain and body as part of the brain-
coordinated ultradian basic rest-activity cycle. Neuroscience.
2009;164(2):849–861.
71. Kooijman S, van den Berg R, Ramkisoensing A, Boon MR, Kuipers
EN, Loef M, Zonneveld TC, Lucassen EA, Sips HC, Chatzispyrou
IA, Houtkooper RH, Meijer JH, Coomans CP, Biermasz NR,
Rensen PC. Prolonged daily light exposure increases body fat mass
through attenuation of brown adipose tissue activity. Proc Natl
Acad Sci USA. 2015;112(21):6748–6753.
72. Bal NC, Maurya SK, Sopariwala DH, Sahoo SK, Gupta SC, Shaikh
SA, Pant M, Rowland LA, Bombardier E, Goonasekera SA, Tupling
AR, Molkentin JD, Periasamy M. Sarcolipin is a newly identified
regulator of muscle-based thermogenesis in mammals [published
correction appears in Nat Med. 2012;18(12):1857]. Nat Med.
2012;18(10):1575–1579.
73. Clapham JC, Arch JR, Chapman H, Haynes A, Lister C, Moore GB,
Piercy V, Carter SA, Lehner I, Smith SA, Beeley LJ, Godden RJ,
Herrity N, Skehel M, Changani KK, Hockings PD, Reid DG,
Squires SM, Hatcher J, Trail B, Latcham J, Rastan S, Harper AJ,
Cadenas S, Buckingham JA, Brand MD, Abuin A. Mice over-
expressing human uncoupling protein-3 in skeletal muscle are
hyperphagic and lean. Nature. 2000;406(6794):415–418.
74. Blondin DP, Labb ´e SM, Phoenix S, Gu ´erin B, Turcotte EE, Richard
D, Carpentier AC, Haman F. Contributions of white and brown
adipose tissues and skeletal muscles to acute cold-induced meta-
bolic responses in healthy men. J Physiol. 2015;593(3):701–714.
75. Symonds ME, Clarke L. Influence of thyroid hormones and tem-
perature on adipose tissue development and lung maturation. Proc
Nutr Soc. 1996;55(1B):561–569.
doi: 10.1210/en.2017-00081 https://academic.oup.com/endo 2225