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Ontogeny and Thermogenic Role for Sternal Fat in Female Sheep

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Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in preventing hypothermia in new-born sheep but is then considered to rapidly disappear during postnatal life. The extent to which the anatomical location of fat influences postnatal development and thermogenic function, particularly following feeding, in adulthood, are not known and were both examined 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 accumulation of white fat. In adult sheep, implanted temperature probes were used to characterise the thermogenic response of fat and muscle to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant within sternal than retroperitoneal fat and was only retained 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 post-prandial rise in temperature was greater and more prolonged in sternal than 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 muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that promote thermogenesis in large mammals.
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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, Queens 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: 22122225, 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 depots 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):22122225 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 everythingexamined was able to
brownwhite 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 classicadipose
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 controlanimals
(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 mealfeeding 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 Fishers 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):22122225
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):22122225
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 markergene 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):22122225
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. (ad) 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):22122225
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. (bg) Representative photomicrographs of hematoxylin
and eosinstained 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):22122225
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, Queens 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
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... The interscapular BAT of mice maintains the characteristics of brown adipose and possesses thermogenic capacity in adulthood. A previous study has demonstrated that UCP1 is prominently enriched in the sternal adipose tissue of sheep, suggesting its potential contribution to the thermogenic response associated with feeding [16]. However, the roles of BAT function in adults are still unclear. ...
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Brown adipose tissue (BAT) is the main site of adaptive thermogenesis, generates heat to maintain body temperature upon cold exposure, and protects against obesity by promoting energy expenditure. RNA-seq analysis revealed that FGF11 is enriched in BAT. However, the functions and regulatory mechanisms of FGF11 in BAT thermogenesis are still limited. In this study, we found that FGF11 was significantly enriched in goat BAT compared with white adipose tissue (WAT). Gain- and loss-of-function experiments revealed that FGF11 promoted differentiation and thermogenesis in brown adipocytes. However, FGF11 had no effect on white adipocyte differentiation. Furthermore, FGF11 promoted the expression of the UCP1 protein and an EBF2 element was responsible for UCP1 promoter activity. Additionally, FGF11 induced UCP1 gene expression through promoting EBF2 binding to the UCP1 promoter. These results revealed that FGF11 promotes differentiation and thermogenesis in brown adipocytes but not in white adipocytes of goats. These findings provide evidence for FGF11 and transcription factor regulatory functions in controlling brown adipose thermogenesis of goats.
... It is, therefore, possible that the fate of these multipotent stem cells, or their ability to differentiate into other cell types, could be regulated by specific growth factors supporting normal physiological development or enabling them to respond to disease 24,43 . Our study confirms that the perirenal and sternal depots follow different development patterns 20 . Although UCP1 expression ceases in both depots with age, only perirenal fat exhibits the major hallmarks of white adipose tissue development 7 . ...
... Several factors, such as sex hormones, aging, hereditary factors and epigenetic effects, influence the fat distribution (Frank et al., 2019). In sheep models, the perirenal adipose tissue (PAT) depot has been shown to be rich in brown adipocytes during the early stages of life (Henry et al., 2017;Symonds et al., 2015). In rodents and humans, it has been confirmed that females have more brown adipose tissue than males (Karastergiou et al., 2012). ...
... Exposure to cold leads to an expected cascade of events (Figure 6), starting from an increase in catecholamines such as norepinephrine that stimulate the various subtypes of β-adrenergic receptors (ADRBs) normally found on the surface of brown adipocytes in BAT [58][59][60]. More importantly, it stimulates the adrenergic-B3-receptor, which can activate lipolysis and release fatty acids in BAT [14,61,62]. ...
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During the early postnatal period, lambs have the ability to thermoregulate body temperature via non-shivering thermogenesis through brown adipose tissue (BAT), which soon after birth begins to transform into white adipose tissue. An RNA seq approach was used to characterize the transcriptome of BAT and thyroid tissue in newborn lambs exposed to cold conditions. Fifteen newborn Romney lambs were selected and divided into three groups: group 1 (n = 3) was a control, and groups 2 and 3 (n = 6 each) were kept indoors for two days at an ambient temperature (20–22 °C) or at a cold temperature (4 °C), respectively. Sequencing was performed using a paired-end strategy through the BGISEQ-500 platform, followed by the identification of differentially expressed genes using DESeq2 and an enrichment analysis by g:Profiler. This study provides an in-depth expression network of the main characters involved in the thermogenesis and fat-whitening mechanisms that take place in the newborn lamb. Data revealed no significant differential expression of key thermogenic factors such as uncoupling protein 1, suggesting that the heat production peak under cold exposure might occur so rapidly and in such an immediate way that it may seem undetectable in BAT by day three of life. Moreover, these changes in expression might indicate the start of the whitening process of the adipose tissue, concluding the non-shivering thermogenesis period.
... ADRB3 was also down-regulated in the hypothalamus and tail-fat of the −5 • C Altay lambs compared to Hu lambs, and up-regulated in the tail-fat of −5 • C compared to 20 • C Hu lambs. The ADRBs are normally expressed on the surface of brown adipocytes in BAT and they mediate mitochondrial biogenesis and thermogenesis [48][49][50]. In the study of hibernating mammals, the ADRB3 gene was expressed greater at the end of the hibernation period [51]. ...
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Thermogenesis plays an important role in the survival of sheep exposed to low temperatures; however, little is known about the genetic mechanisms underlying cold adaptation in sheep. We examined 6 Altay (A) and 6 Hu (H) six-month-old ewe lambs. Altay sheep are raised in northern China and are adapted to dry, cold climates, while Hu sheep are raised in southern China and are adapted to warm, humid climates. Each breed was divided into two groups: chronic cold sheep, exposed to −5 °C for 25 days (3 Ac; 3 Hc), and thermo-neutral sheep, maintained at 20 °C (3 Aw; 3 Hw). The transcriptome profiles of hypothalamus, tail-fat and perirenal fat tissues from these four groups were determined using paired-end sequencing for RNA expression analysis. There are differences in cold tolerance between Hu and Altay sheep. Under cold exposure of the lambs: (1) UCP1-dependent thermogenesis and calcium- and cAMP-signaling pathways were activated; and (2) different fat tissues were activated in Hu and Altay lambs. Several candidate genes involved in thermogenesis including UCP1, ADRB3, ADORA2A, ATP2A1, RYR1 and IP6K1 were identified. Molecular mechanisms of thermogenesis in the sheep are discussed and new avenues for research are suggested.
... Therefore, decreased fat deposition at the end of the experiment in the Vaccine group may be also the result of transient periods of anorexia. Sternal fat deposits play an important role in thermogenesis in sheep [30]. There were no other gross abnormalities in any of the treatment groups apart from those previously described [15]. ...
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Aluminum (Al) hydroxide is an effective adjuvant used in sheep vaccines. However, Al-adjuvants have been implicated as potential contributors to a severe wasting syndrome in sheep—the so-called ovine autoimmune-inflammatory syndrome induced by adjuvants (ASIA syndrome). This work aimed to characterize the effects of the repetitive injection of Al-hydroxide containing products in lambs. Four flocks (Flocks 1–4; n = 21 each) kept under different conditions were studied. Three groups of seven lambs (Vaccine, Adjuvant-only, and Control) were established in each flock. Mild differences in average daily gain and fattening index were observed, indicating a reduced growth performance in Vaccine groups, likely related to short-term episodes of pyrexia and decreased daily intake. Clinical and hematological parameters remained within normal limits. Histology showed no significant differences between groups, although there was a tendency to present a higher frequency of hyperchromatic, shrunken neurons in the lumbar spinal cord in the Adjuvant-only group. Although Al-hydroxide was linked to granulomas at the injection site and behavioral changes in sheep, the results of the present experimental work indicate that injected Al-hydroxide is not enough to fully reproduce the wasting presentation of the ASIA syndrome. Other factors such as sex, breed, age, production system, diet or climate conditions could play a role.
... In sheep, BAT is recruited in perirenal adipose tissue at birth and then rapidly decreases at 30 postnatal days [13]. Furthermore, adult sheep retain BAT in both sternal and retroperitoneal fat, resulting in the conversion of stored energy into heat and nonshivering thermogenesis [14]. However, less is known about the cellular transition from BAT to WAT in goat perirenal adipose tissue. ...
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Long noncoding RNAs (lncRNAs) play an important role in the thermogenesis and energy storage of brown adipose tissue (BAT). However, knowledge of the cellular transition from BAT to white adipose tissue (WAT) and the potential role of lncRNAs in goat adipose tissue remains largely unknown. In this study, we analyzed the transformation from BAT to WAT using histological and uncoupling protein 1 (UCP1) gene analyses. Brown adipose tissue mainly existed within the goat perirenal fat at 1 day and there was obviously a transition from BAT to WAT from 1 day to 1 year. The RNA libraries constructed from the perirenal adipose tissues of 1 day, 30 days, and 1 year goats were sequenced. A total number of 21,232 lncRNAs from perirenal fat were identified, including 5393 intronic-lncRNAs and 3546 antisense-lncRNAs. Furthermore, a total of 548 differentially expressed lncRNAs were detected across three stages (fold change ≥ 2.0, false discovery rate (FDR) < 0.05), and six lncRNAs were validated by qPCR. Furthermore, trans analysis found lncRNAs that were transcribed close to 890 protein-coding genes. Additionally, a coexpression network suggested that 4519 lncRNAs and 5212 mRNAs were potentially in trans-regulatory relationships (r > 0.95 or r < −0.95). In addition, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that the targeted genes were involved in the biosynthesis of unsaturated fatty acids, fatty acid elongation and metabolism, the citrate cycle, oxidative phosphorylation, the mitochondrial respiratory chain complex, and AMP-activated protein kinase (AMPK) signaling pathways. The present study provides a comprehensive catalog of lncRNAs involved in the transformation from BAT to WAT and provides insight into understanding the role of lncRNAs in goat brown adipogenesis.
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During cold exposure, brown adipose tissue (BAT) holds the key mechanism in the generation of heat, thus inducing thermogenic adaptation in response to cooler environmental changes. This process can lead to a major lipidome remodelling in BAT, where the increase in abundance of many lipid classes plays a significant role in the thermogenic mechanisms for heat production. This study aimed to identify different types of lipids, through liquid chromatography–mass spectrometry (LC-MS), in BAT and plasma during a short-term cold challenge (2-days), or not, in new-born lambs. Fifteen new-born Romney lambs were selected randomly and divided into three groups: Group 1 (n = 3) with BAT and plasma obtained within 24 h after birth, as a control; Group 2 (n = 6) kept indoors for two days at an ambient temperature (20–22 °C) and Group 3 (n = 6) kept indoors for two days at a cold temperature (4 °C). Significant differences in lipid composition of many lipid categories (such as glycerolipids, glycerophospholipids, sphingolipids and sterol lipids) were observed in BAT and plasma under cold conditions, compared with ambient conditions. Data obtained from the present study suggest that short-term cold exposure induces profound changes in BAT and plasma lipidome composition of new-born lambs, which may enhance lipid metabolism via BAT thermogenic activation and adipocyte survival during cold adaptation. Further analysis on the roles of these lipid changes, validation of potential biomarkers for BAT activity, such as LPC 18:1 and PC 35:6, should contribute to the improvement of new-born lamb survival. Collectively, these observations help broaden the knowledge on the variations of lipid composition during cold exposure.
Chapter
Epicardial adipose tissue (EAT) is the fat depot located between the myocardium and the epicardium including the surroundings of the epicardial coronary vessels while the pericardial fat is located externally. Epicardial and intra-abdominal fat both evolve from brown adipose tissue. EAT is supplied by branches of the coronary arteries, whereas pericardial fat is supplied by branches of non-coronary arteries. In the adult human heart, EAT is more abundant in the atrioventricular and interventricular grooves. Microscopically, EAT not only is mainly composed of adipocytes but also contains nerve tissues, inflammatory, stromovascular, and immune cells. EAT is generally considered a white adipose tissue, albeit it displays also brown fatlike or beige fat features. No muscle fascia divides EAT and myocardium; therefore, the two tissues share the same microcirculation. This allows a direct interaction and crosstalk between the EAT and the myocardium. Under pathological circumstances, epicardial adipocytes display an intrinsic pro-inflammatory and atherogenic profile. A dense inflammatory infiltrate, mainly represented by macrophages, is commonly detected in epicardial fat of subjects with coronary artery disease.
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Prenatal testosterone (T)-treated female sheep exhibit an enhanced inflammatory and oxidative stress state in the visceral adipose tissue (VAT) but not in the subcutaneous (SAT), while surprisingly maintaining insulin sensitivity in both depots. In adult sheep adipose tissue is predominantly composed of white adipocytes which favor lipid storage. Brown/beige adipocytes that make up the brown adipose tissue (BAT) favor lipid utilization due to thermogenic uncoupled protein 1 expression and are interspersed amidst white adipocytes, more so in epicardiac (ECAT) and perirenal (PRAT) depots. The impact of prenatal T-treatment on ECAT and PRAT depots are unknown. As BAT imparts a metabolically healthy phenotype, the depot-specific impact of prenatal T-treatment on inflammation, oxidative stress, differentiation and insulin sensitivity could be dictated by the distribution of brown adipocytes. This hypothesis was tested by assessing markers of oxidative stress, inflammation, adipocyte differentiation, fibrosis and thermogenesis in adipose depots from control and prenatal T (100 mg T propionate twice a week from days 30-90 of gestation) -treated female sheep at 21 months of age. Our results show prenatal T-treatment induces depot-specific changes in inflammation, oxidative stress state, collagen accumulation, and differentiation with changes being more pronounced in the VAT. Prenatal T-treatment also increased thermogenic gene expression in all depots indicative of increased browning with effects being more prominent in VAT and SAT. Considering that inflammatory and oxidative stress are also elevated, the increased brown adipocyte distribution is likely a compensatory response to maintain insulin sensitivity and function of organs in the proximity of respective depots.
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Introduction Brown adipose tissue (BAT) is a thermogenic organ with substantial metabolic capacity and has important roles in the maintenance of body weight and metabolism. Regulation of BAT is primarily mediated through the β-adrenoceptor (β-AR) pathway. The in vivo endocrine regulation of this pathway in humans is unknown. The objective of our study was to assess the in vivo BAT temperature responses to acute glucocorticoid administration. Methods We studied 8 healthy male volunteers, not pre-selected for BAT presence or activity and without prior BAT cold-activation, on two occasions, following an infusion with hydrocortisone (0.2 mg.kg− 1.min− 1 for 14 h) and saline, respectively. Infusions were given in a randomized double-blind order. They underwent assessment of supraclavicular BAT temperature using infrared thermography following a mixed meal, and during β-AR stimulation with isoprenaline (25 ng.kg fat-free mass− 1.min− 1 for 60 min) in the fasting state. Results During hydrocortisone infusion, BAT temperature increased both under fasting basal conditions and during β-AR stimulation. We observed a BAT temperature threshold, which was not exceeded despite maximal β-AR activation. We conclude that BAT thermogenesis is present in humans under near-normal conditions. Glucocorticoids modulate BAT function, representing important physiological endocrine regulation of body temperature at times of acute stress.
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Context: Patients with pheochromocytoma (pheo) show presence of multilocular adipocytes that express uncoupling protein (UCP) 1 within periadrenal (pADR) and omental (OME) fat depots. It has been hypothesized that this is due to adrenergic stimulation by catecholamines produced by the pheo tumors. Objective: To characterize the prevalence and respiratory activity of brown-like adipocytes within pADR, OME and subcutaneous (SC) fat depots in human adult pheo patients. Design: This was an observational cohort study. Setting: University hospital. Patients: We studied 46 patients who underwent surgery for benign adrenal tumors (21pheos and 25 controls with adrenocortical adenomas). Main outcome measure: We characterized adipocyte browning in pADR, SC, and OME fat depots for histological and immunohistological features, mitochondrial respiration rate, and gene expression. We also determined circulating levels of catecholamines and other browning-related hormones. Results: 11 of 21 pheo pADR adipose samples, but only 1 of 25 pADR samples from control patients, exhibited multilocular adipocytes. The pADR browning phenotype was associated with higher plasma catecholamines and raised UCP1. Mitochondria from multilocular pADR fat of pheo patients exhibited increased rates of coupled and uncoupled respiration. Global gene expression analysis in pADR fat revealed enrichment in β-oxidation genes in pheo patients with multilocular adipocytes. No SC or OME fat depots exhibited aspects of browning. Conclusion: Browning of the pADR depot occurred in half of pheo patients and was associated with increased catecholamines and mitochondrial activity. No browning was detected in other fat depots, suggesting that other factors are required to promote browning in these depots.
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Studies in rodents and newborn humans demonstrate the influence of brown adipose tissue (BAT) in temperature control and energy balance and a critical role in the regulation of body weight. Here, we obtained samples of epicardial adipose tissue (EAT) from neonates, infants, and children in order to evaluate changes in their transcriptional landscape by applying a systems biology approach. Surprisingly, these analyses revealed that the transition to infancy is a critical stage for changes in the morphology of EAT and is reflected in unique gene expression patterns of a substantial proportion of thermogenic gene transcripts (~10%). Our results also indicated that the pattern of gene expression represents a distinct developmental stage, even after the rebound in abundance of thermogenic genes in later childhood. Using weighted gene coexpression network analyses, we found precise anthropometric-specific correlations with changes in gene expression and the decline of thermogenic capacity within EAT. In addition, these results indicate a sequential order of transcriptional events affecting cellular pathways, which could potentially explain the variation in the amount, or activity, of BAT in adulthood. Together, these results provide a resource to elucidate gene regulatory mechanisms underlying the progressive development of BAT during early life.
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Objective: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes. Methods: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a β3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue. Results: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response. Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.
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Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.
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Significance Increased light exposure has been associated with obesity in both humans and mice. In this article, we elucidate a mechanistic basis of this association by performing studies in mice. We report that prolonging daily light exposure increases adiposity by decreasing energy expenditure rather than increasing food intake or locomotor activity. This was caused by a light-exposure period-dependent attenuation of the noradrenergic activation of brown adipose tissue that has recently been shown to contribute substantially to energy expenditure by converting fatty acids and glucose into heat. Therefore, we conclude that impaired brown adipose tissue activity may mediate the relationship between increased light exposure and adiposity.
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The nature of the brown adipose tissue in humans is presently debated: whether it is classical brown - or of brite/beige nature. The dissimilar developmental origins and proposed distinct functions make it essential to ascertain the identity of human depots, with the perspective of recruiting and activating them for the treatment of obesity and type 2 diabetes. For identification of the tissues, a number of marker genes have been proposed, but the validity of the markers has not been well documented. We use here the established brown (interscapular), brite (inguinal) and white (epididymal) mouse adipose tissues and corresponding primary cell cultures as validators. We examined the informative value of a series of suggested markers, earlier used in the discussion considering the nature of human brown adipose tissue. Most of these markers unexpectedly turned out to be non-informative concerning tissue classification (Car4, Cited1, Ebf3, Eva1, Fbxo31, Fgf21, Lhx8, Hoxc8 and Hoxc9). Only Zic1 (brown), Cd137, Epsti1, Tbx1, Tmem26 (brite) and Tcf21 (white) proved to be informative in these three tissues. However, the expression of the brite markers was not maintained in cell culture. In a more extensive set of adipose depots, these validated markers provide new information about depot identity. Principal component analysis supported our single gene conclusions. Furthermore, Zic1, Hoxc8, Hoxc9 and Tcf21 displayed anteroposterior expression patterns, indicating a relationship between anatomical localization and adipose tissue identity (and possibly function). Together, the observed expression patterns of these validated marker genes necessitates reconsideration of adipose depot identity in mice and humans. Copyright © 2015, American Journal of Physiology - Endocrinology and Metabolism.
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Large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT). We analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time. Using global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.
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p>An inability of adipose tissue to expand consequent to exhausted capacity to recruit new adipocytes might underlie the association between obesity and insulin resistance. Adipocytes arise from mesenchymal precursors whose commitment and differentiation along the adipocytic lineage is tightly regulated. These regulatory factors mediate cross-talk between adipose cells, ensuring that adipocyte growth and differentiation are coupled to energy storage demands. The WNT family of autocrine and paracrine growth factors regulates adult tissue maintenance and remodelling and, consequently, is well suited to mediate adipose cell communication. Indeed, several recent reports, summarized in this review, implicate WNT signalling in regulating adipogenesis. Manipulating the WNT pathway to alter adipose cellular makeup, therefore, constitutes an attractive drug-development target to combat obesity-associated metabolic complications. © 2008 Elsevier Ltd. All rights reserved.</p
Article
There are three different types of adipose tissue (AT), brown, white and beige, that differ with stage of development, species and anatomical location. Of these, brown AT (BAT) is the least abundant but has the greatest potential impact on energy balance. BAT is capable of rapidly producing large amounts of heat through activation of the unique uncoupling protein (UCP)1 located within the inner mitochondrial membrane. White AT is an endocrine organ and site of lipid storage, whilst beige AT is primarily white but contains some cells that possess UCP1. BAT first appears in the fetus around mid-gestation and is then gradually lost through childhood, adolescence and adulthood depending We focus on the inter-relationships between adipocyte classification, anatomical location and impact of diet in early life together with the extent to which this process differs between the major species examined. Ultimately this may enable novel dietary interventions designed to reactivate BAT.